135

IntroductionGlycation is the non-enzymatic reaction between a protein

and a reducing sugar, such as glucose and fructose 1). Glycation stress is one of the risk factor for aging inside and outside of the body 2). Advanced glycation end products (AGEs) are formed after a series of complex reactions. Hundreds of AGEs have been discovered, including 3-deoxyglucosone (3DG), which is an intermediate to AGEs, pentosidine, and N ε-(carboxymethyl)lysine (CML).

In skin, glycation of collagen type I has been linked to the development of skin dullness and a decrease in skin elasticity 2). Thus, in recent years, research has been directed to inhibiting AGE formation to treat aging, health promotion, and lifestyle-related disease.

Skin conditions greatly affect an individual’s appearance, and many cosmetic and food companies develop skin-care products or anti-skin aging treatments with the goal of maintaining a youthful skin and preventing pre-mature skin aging.

One recent approach is to seek ways of inhibiting AGE formation, and the cosmetic and food industries have researched many glycation-inhibiting ingredients, found in plant extracts,

Original Article

Inhibition of Advanced Glycation End Product Formation by Herbal Teas and Its Relation to Anti-Skin Aging

Mio Hori 1), Masayuki Yagi 1), Keitaro Nomoto 2), Akihiko Shimode 2), Mari Ogura 3), Yoshikazu Yonei 2)

1) Glycation Stress Research Center, Graduate School of Life and Medical Sciences, Doshisha University 2) Anti-Aging Medical Research Center, Graduate School of Life and Medical Sciences, Doshisha University3) Faculty of Human Life and Science, Doshisha Women’s College of Liberal Arts

AbstractAims: Advanced glycation end product (AGE) accumulation in the body has been linked to the progression of aging and age-related diseases. In skin, glycation of collagen type I has been linked to development of skin dullness and a decrease in skin elasticity. Here, we evaluated the inhibition of glycation by herbal teas and investigated the potential of herbal teas as an Anti-Aging treatment for skin and body.Methods: Two in vitro models of glycation, glucose and bovine collagen type I, and glucose and human serum albumin (HSA), were used to test if herbal teas inhibited AGEs formation. Extracts from the models containing fluorescent AGEs, 3-deoxyglucosone (3DG), pentosidine, and N ε-(carboxymethyl)lysine (CML) were analyzed by fluorescence spectroscopy, high pressure liquid chromatography (HPLC), and enzyme linked immunoassay (ELISA). Screening was conducted from 32 tea (Camella sinensis) samples and 81 herbal tea samples. In addition, the effects of the topical application of herbal teas on skin elasticity, moisture, melanin, erythema, AGE, and CML levels were evaluated. Results: Glycation was inhibited by teas prepared from Japanese persimmon leaf (Diospyros kaki), banabá (Lagerstroemia speciosa), kuma bamboo (Sasa veitchii), and Chinese blackberry (Rubus suavissimus). The topical application of extracts made from these four herbal teas prevented the increase of fluorescent AGEs in skin after four weeks, and increased skin elasticity after eight weeks.Conclusion: The results suggest that extracts of Japanese persimmon leaf and banabá, strongly inhibit HSA and bovine collagen type I glycation in vitro, and the inhibition is stronger when the herbal teas are blended. Topical application of these extracts increased skin elasticity and reduced AGEs accumulation in the epidermis. These herbal teas may be useful for developing Anti-Aging products that could be included in health foods and cosmetics for skin and body.

KEY WORDS: collagen, Camella sinensis, persimmon (Diospyros kaki), banabá (Lagerstroemia speciosa), kuma bamboo (Sasa veitchii)

Received: Apr 13, 2012 Accepted: Aug. 13, 2012Published online: Oct. 31, 2012

Anti-Aging Medicine 9 (6) : 135-148, 2012(c) Japanese Society of Anti-Aging Medicine

Prof. Yoshikazu Yonei, M.D., Ph.D.Anti-Aging Medical Research Center, Graduate School of Life and Medical Sciences Doshisha University

1-3, Tatara Miyakodani, Kyotanabe city, Kyoto Prefecture 610-0321, JapanTel & Fax: +81-774-65-6394 / E-mail: yyonei@mail.doshisha.ac.jp

such as herbal teas. Previous studies reported that green tea extracts inhibited the formation of AGEs in myocardial collagen in rats 3,4). A clinical trial in patients with pre-diabetes found that a mixture of herbal extract (MHE) (containing: chamomile (Anthemis nobilis), dokudami (Houttuynia cordata), hawthorn (Crataegus laevigata), grape (Vitis vinifera)) extracted with hot water, exhibited strong anti-glycation effects, improved skin elasticity, and lowered CML levels in the blood 5).

Anti-glycation cosmetics are gaining popularity in the cosmetics industry. For instance, Pola Corporation (Shinagawa-ku, Tokyo, Japan) has developed products containing plant extracts which degrades AGEs in the dermis, and are sold as skin care products to prevent sagging and decrease in skin elasticity. Other companies are developing functional foods and cosmetics containing other plant-derived anti-glycation ingredients.

Here, we evaluated the anti-glycation effects of herbal teas and assessed their potential as anti-glycation products. In vitro models of glycation using glucose and bovine collagen type I, and glucose and human serum albumin (HSA) as reported previously 6) were used to test the inhibition of AGEs formation by herbal teas. Also, the effects of topical application of herbal tea extracts on skin elasticity, moisture, melanin, erythema, AGE accumulation, and CML levels were evaluated.

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Inhibition of AGEs by Herbal Teas

Methods

Screening of herbal teasSample preparation and taste evaluation

Samples of 32 tea (Camella sinensis) and 81 herbal tea samples provided by Hikawa (Shimane, Japan) and Tea Life (Shimada, Shizuoka, Japan) were prepared following recommendations from the Japan Tea Association: 150ml of water heated to 80 °C was added to a beaker containing 3.75g of the test sample and incubated in a water bath set to 80 °C; a 10-ml sample was taken after a 3-minute extraction and assessed for taste, smell, and color. Taste was evaluated on a 5-point scale in three categories: bitterness, sweetness, and astringency.

The remainder of each sample was left to extract in the water bath for one hour. The mass concentration of each tea was measured by taking 5ml of each 3-minute and 1-hour extract samples and drying in aluminum trays at 120 °C for 3 hours. The mass concentration was calculated from the weight difference before and after incubation.

Previous studies found that a mixed herbal extract (MHE, ARKRAY, Nakagyo-ku, Kyoto, Japan) inhibited glycation both in vitro and in vivo 7). The effect of MHE was compared with the other herbal tea samples by dissolving 10mg of MHE in 10ml of distilled water and shaking until the solid dissolved.

Inhibitory activity of sample teas and herbal teas against fluorescent AGE formation in bovine collagen type I

A glucose-collagen model previously reported 6) was used to evaluate the inhibitory activity of sample teas and herbal teas. Fluorescent AGEs and CML formed by glucose and collagen type I derived from bovine skin were measured.

Inhibitory activity of sample herbal teas against fluorescent AGE formation in HSA model

A glucose-HSA model previously reported 6) was used to evaluate the inhibitory activity of sample teas and herbal teas. Similarly, we measured fluorescent AGEs, 3DG and pentosidine which were formed from the reaction of glucose and HSA.

Determination of polyphenol concentration in herbal teasBriefly, 100µL of Folin-Ciocalteu’s phenol reagent (MP

Biomedicals, CA, USA) diluted 1:1 with water, 200µL of 1-hour extract tea samples diluted 20-fold with 50% ethanol, and 1,000µL of 0.4M Na2CO3 were combined in a test tube. A standard curve was constructed from samples of serially diluted (+)catechin (0.0124-0.6 mg/ml) and distilled water (control); 200µL from each sample were dispensed into a microplate and incubated at 30 °C for 30 minutes; absorbance was measured at 660nm. The polyphenol concentration in each tea sample, expressed as (+)catechin equivalent, was determined from the standard curve.

Antioxidant activity of herbal teasThe antioxidant activity of the herbal tea samples was

measured using an Antioxidant Activity Measuring Kit Radical Catch (Hitachi Aloka Medical, Mitaka, Tokyo, Japan). Fifty micro litters of cobalt chloride solution, 50µL of luminol solution, and 20µL of 1-hour extract tea sample (three serial dilutions of each herbal tea) were dispensed into a test tube and mixed for two seconds; water was used as a control. The test tubes were incubated at 37 °C for five minutes and 50µL of H2O2 were added to the test tube, mixed for two seconds, and immediately measured for luminescence using an AccuFlexLumi 400 (Hitachi Aloka Medical, Tokyo, Japan). The relative light unit (RLU) was

calculated by taking the sum of values from 80 seconds to 120 seconds after mixing. The elimination rate was calculated from:

Elimination rate (%) = ((control RLU – sample RLU) / control RLU) × 100

The IC50 (50% inhibitory concentration) of antioxidant activity was determined from a regression curve of the elimination rate at three concentrations.

Glycation inhibition by blended herbal tea extractExtract preparation

A blended herbal tea was prepared from equal weights of persimmon leaf (Diospyros kaki), banabá (Lagerstroemia speciosa), kuma bamboo grass (Sasa veitchii), and Chinese blackberry (Rubus suavissimus). The blend was extracted in 600ml water at 80 °C in a water bath for one hour, and the mass concentration was determined as described above.

Inhibitory activity of blended extract (1-hour extraction) against formation of glycated products

The inhibition by blended extracts against the formation of fluorescent AGEs in the collagen and HSA models, pentosidine and 3DG in the HSA models, and CML in the collagen model was evaluated as described above.

Polyphenol concentration and antioxidant activity of blended extract

Polyphenol concentration and antioxidant activity of blended extract was determined by the method described above.

Clinical Study – Topical application of herbal tea extractsPreparation of test product

The blended herbal tea was diluted (0.03% W/V) with water so that the color was clear with a slight tint of yellow.

Subjects and study designFive female volunteers (27 ± 10 years) were instructed to

apply the blended herbal tea extract solution to their arm twice daily for eight weeks, between November 16, 2011 and January 11, 2012. A piece of cotton (5cm × 6cm) sprayed with the test product (3-5 ml) was wiped over the lower inner part of the left upper arm, located 5-10cm above the elbow twice a day (morning and night) after bathing. The upper inner part of the left upper arm was used to take control measurements. Subjects were instructed to avoid excessive exercise or food intake, and lack of sleep, and were prohibited from applying cosmetics in the area. In addition, subjects were asked to record each application and to note any major changes in lifestyle. Skin was examined three times during the observation period; all volunteers completed the trial with an average extract application rate of 96%.

Writ ten informed consent was obtained f rom each participant after full explanation of the purpose and methods of the study, participants’ rights, and that no penalty was associated with dropout.

Volunteers completed a questionnaire on their skin condition, changes (on a five-point scale) in subjective symptoms (skin moisture, softness, stiffness, whiteness, darkness, dampness, elasticity, and smoothness), at the beginning of the trial, and the following measurements were taken at 0, 4, and 8 weeks: CML

137

Inhibition of AGEs by Herbal Teas

137

concentration in the stratum corneum, skin AGEs accumulation, skin elasticity, skin moisture, melanin, and erythema levels. The percentage change in each measurement over the examination period compared to the initial measurement was determined.

Measurement of skin moisture, melanin, and erythema levelsSkin moisture was measured with a Corneometer (CM825;

Courage + Khazaka Electronic GmbH, Koln, Germany) 8), which functions on the principle that electric capacitance changes according to moisture level. Melanin and erythema were measured with a Mexameter (MX18; Courage + Khazaka Electronic GmbH) 9,10), which irradiates the skin with a specific wavelength light and measures the reflected light to calculate the melanin and erythema indices. Moisture, melanin, and erythema levels were measured five times at the same point, and the results were expressed as the mean of three values.

Measurement of skin elasticitySkin elasticity was evaluated using a Cutometer (MPA580;

Courage + Khazaka Electronic, GmbH) 11,12). Briefly, a probe was placed on the skin surface, and an area of skin was drawn up into the probe using negative pressure; the length of skin drawn into the probe was then measured using a glass prism. The R2 index is the ratio of skin length recovery after elongation and constriction (Ua1/Uf1), an ideal elastic material has an R2 value of 1.00, and normal skin has values between 0.3-0.5. The R7 index is the ratio of skin elasticity while constricted (Ur1/Uf1); the most elastic skin has a R7 value close to 1.00.

The R2 and R7 indices are the most reliable indices derived from a Cutometer, and previous authors have reported that R2 and R7 indices decline with age 13). R2 and R7 were measured three times at the same point, and the results were expressed as the mean of three values.

Measurement of AGE accumulation in skinAn AGE Reader™ was used to measure AGE accumulation

in skin 14,15). In human skin, the instrument mostly detects the AGEs existing in the epidermis. Examination of skin biopsies from diabetic and dialysis patients has confirmed that skin auto fluorescence (AF) is correlated with the accumulation of typical AGEs in the skin, such as pentosidine and CML. In addition, AF is high in type 2 diabetes patients and increases in healthy persons with age 16).

Subjects were asked to rest their elbow on the AGE Reader™. Measurements were taken at the inner part of the upper left arm, 10cm and 20cm (control) from the edge of the elbow 17). The inner part of the arm was chosen to minimize the effects of suntan on the measurements. The measurement area was wiped using a cotton swab and alcohol, AF intensity and reflectance were measured three times at the same point, and the results were expressed as the mean of three values.

Extraction of protein in stratum corneumProtein in stratum coreum was extracted by the tape

stripping method 18-20). Briefly, the skin was washed and dried, and a Corneum Checker (Asahi Biomed, Chiyoda-ku, Tokyo, Japan) was placed on the skin for five seconds, peeled off, and sealed back onto the sheet; the process was repeated twice with a new sticker and the second sticker retrieved and cut into a 2.5 × 2.5cm square with scissors sterilized with 70% alcohol 21). The sticker was peeled off the sheet with sterilized tweezers, and placed into a 2ml tube with the sticky side facing inward. 600µL of extraction buffer (50mM Tris-HCl (pH 7.5), 12.0mM NaCl, 1mM Na3VO4, 0.1% sodium dodecyl sulfate (SDS)) was

added into the tube, and the mixture homogenized with a micro homogenizer for two minutes at 9,000rpm. The sticker was removed from the tube, and the tube was centrifuged at 3,000rpm for five minutes. The supernatant was placed into a new tube and assayed for protein concentration.

Determination of protein concentration in stratum corneumThe protein content in the stratum Corneum was measured

using a DC Protein Assay kit (Bio-Rad Laboratories, CA, USA) 21). All reagents were supplied in the kit. First, 40µL of standards (HSA) or the supernatant extracted from Corneum checker stickers in the previous step were dispensed into a 96-well black microplate; 20µL reagent A solution (prepared following the manufacturer’s instructions) was dispensed into each well followed by 160µL of Reagent B. The microplate was shaken lightly and left at room temperature for 30minutes. The f luorescence at 750nm was measured with a microplate reader (ARVO MX 1420 ARVO series Multilabel Counter). The protein content in each supernatant sample was determined by constructing a standard curve.

Determination of CML concentration in stratum corneumTo remove SDS in supernatant samples prepared as

described above, 80µL of perchloric acid (PCA, 14%) was added to 400μl µL of the supernatant, stirred, and left at room temperature for 2 minutes; centrifuged for 15 minutes at 10,000rpm and the supernatant removed; 400μl of Tris-HCl (50mM, pH 8.0) was added and stirred. The CML content in the prepared supernatants was measured using the N ε-(carboxymethyl)lysine ELISA Kit, as described in above and the results are expressed CML/protein (ng/μg) 6).

Ethical ConsiderationsThis study was carried out after a briefing session on the

enforcement of the examination was held and written consent from the subjects were obtained.

Herbal tea blend as a beverage for skin Anti-AgingInhibitory activity against f luorescent AGEs in HSA by

3-minute extracts of herbal teas Inhibitory activity against f lorescent AGE formation in HSA by 3-minute extracts of Chinese blackberry, kuma bamboo, and persimmon leaf were assessed as described above.

Sample preparationFour 3-minute extractions of tea blends were prepared

with different ratios of four herbal teas (banabá, kuma bamboo grass, Chinese blackberry, and persimmon leaf) in the following ratios: blend 1 - 2:10:1:7; blend 2- 7:15:2:6; blend 3- 3:19:2:6; blend 4- 3:20:1:6. Each blend was prepared by adding the herbal tea at a concentration approximately ten times the IC50 against fluorescent AGE formation in the collagen model. Seven subjects were asked to sip and spit out each tea and blended tea, and rate the beverage for bitterness, sweetness, astringency, good taste, and easiness to drink on a five point scale from very good (5) to very bad (1).

Statistical analysisResults were expressed as mean values ± standard deviation.

138

Inhibition of AGEs by Herbal Teas

Differences in mean values between the control and test area were tested by the Wilcoxon sign ranked test. Differences between samples were determined by the Kruskal-Wallis test. In the analysis, p< 0.05 was considered significant.

Results

Screening of herbal teasInhibitory activity of sample herbal teas against fluorescent AGE formation in collagen in bovine collagen type I

The IC50 of all 114 tea and herbal tea samples (1-hour and 3-minute extractions) against f luorescent AGEs formation in collagen were ranked (Table 1). Samples with IC50 20-fold less than that of aminoguanidine (IC50= 0.40mg/ml) for both 3-minute and 1-hour extract were selected, and classified under Camellia sinensis derived teas and non-Camellia sinensis derived teas (herbal teas) (Fig. 1). Six herbal teas, banabá, brown rice (Oryza sativa), Chinese blackberry, hama tea (Chamaecris tanomame), kuma bamboo grass, and persimmon leaf were further investigated.

Teas prepared from pumpkin (Cucurbita), young barley grass (Hordeummurinum subsp. Leporinum), and black soybean (Glycine max) exhibited lower inhibitory activity against fluorescent AGE formation in the collagen model (IC50 > 1 mg/ml) than the other samples. Thus, these three teas were used as negative controls in the next analysis.

Inhibitory activity of sample herbal teas against CML formation in bovine collagen type I

Inhibitory activity against CML formation in the collagen model by 1-hour extracted herbal tea samples is summarized in Table 2. The inhibitory activity against CML formation was greatest in brown rice, hama tea, persimmon leaf, Chinese blackberry, and banabá, and lowest in kuma bamboo grass, pumpkin, young barley grass, and black soybean.

Glycation inhibitory activity of sample herbal teas against fluorescent AGE formation in HSA

Inhibitory activity against f luorescent AGE formation in the HSA model by 1-hour extracted herbal tea samples is summarized in Table 2. Inhibitory activity was greater in Chinese blackberry and banabá than aminoguanidine (IC50 = 0.07 mg/ml). While hama tea, kuma bamboo grass, and persimmon leaf had strong inhibitory activity (IC50 < 0.15 mg/ml), brown rice, which had the highest inhibitory activity against fluorescent AGE formation in the collagen model, exhibited no inhibitory activity against fluorescent AGE formation in the HSA model (IC50 > 1 mg/ml). The IC50 of pumpkin, young barley grass, and black soybean (negative controls) was ≥ 1 mg/ml.

Inhibitory activity of sample herbal teas against pentosidine formation in HSA

The inhibitory activity and IC50 against pentosidine formation in the HSA model were less than 1 mg/mL in persimmon leaf and Chinese blackberry (Table 2). The other samples showed little inhibitory activity against pentosidine formation compared aminoguanidine (IC50 > 1 mg/ml).

Table 1 Inhibitory activity by teas and herbal teas (1-hour and 3-minute extracts) against formation of fluorescent AGEs. The samples are ranked by inhibitory activity (expressed as IC50) in 1-hour extracted samples. IC50 values are expressed in mg/ml. IC50; 50% inhibitory concentration.

74275068121356T696T331297849999379573898388100261012021

Brown riceBlack teaChinese blackberryGreen teaDokudamiJasmine teaHama teaChinese blackberryGreen teaPu-erh teaGreen teaOolong teaOolong teaOolong teaRoasted green teaGreen teaPersimmon leaf Green teaMacchaOolong teaGreen teaGreen teaGreen teaPu-erh teaGreen teaPu-erh teaGreen tea

Oryza sativaCamellia sinensisRubus suavissimusCamellia sinensisHouttuynia cordataCamellia sinensisChamaecrista nomameRubus suavissimusCamellia sinensisCamellia sinensisCamellia sinensisCamellia sinensisCamellia sinensisCamellia sinensisCamellia sinensisCamellia sinensisDiospyros kakiCamellia sinensisCamellia sinensisCamellia sinensisCamellia sinensisCamellia sinensisCamellia sinensisCamellia sinensisCamellia sinensisCamellia sinensisCamellia sinensis

Sample Number

CrushedLeaf

Grown in Japan (Yutakamidori)

Grown in Japan

Grown in Japan (benifuki)

For tea bags

Grown in TaiwanFor tea bagsLeafGrown in Japan (yabukita)ChoppedGrown in JapanGrown in JapanGrown in China, stemGrown in Japan, second picking

Grown in Japan (saemidori)LeafGrown in Japan, third picking

Leaf

Common Name Scientific Name Details IC50 (1-hr ext)

0.002 0.002 0.003 0.003 0.004 0.005 0.006 0.006 0.007 0.007 0.008 0.008 0.009 0.009 0.009 0.009 0.010 0.010 0.010 0.011 0.011 0.011 0.013 0.013 0.014 0.014 0.014

0.002 0.013 0.001 0.517 0.027 0.009 0.003 0.009 0.010 0.014 0.016 0.007 0.014 0.002 0.013 0.014 0.017 0.013 0.019 0.025 0.021 0.005 0.014 0.015 0.018 0.022 0.008

IC50 (3-min ext)

139

Inhibition of AGEs by Herbal Teas

139

939490210443992T748714172T815283360227754197935691T17632416452346365525704102515343428786651138052178166T930865932846761T4

Green teaGreen teaHojichaSenchaGuavaHojichaShisoGreen teaBanabáKuma bamboo grassDokudamiRooibosDokudamiRooibosEvening primroseJiaogulanHama teaNikko mapleLemon grassNaked barleyDokudamiJapanese mugwortGreen teaField horsetailDokudamiJob's tearsSalaciaLoquatChamomileGymnemaEucommiaMulberry leafKuma bamboo grassAloeJapanese hawthorn fruitDokudamiLigzhi mushroomDokudamiBlueberrySweet potatoEucommiaWolfberry leafChrysanthemumTartary buckwheatRose hipMulukhiyahMulberry leafHatomugiAzuki beanJapanese roseSafflowerGlycyrrhizaBrown rice teaSprouted brown riceHatomugiChinese sennaBlack soybean AshitabaBeach silvertopPomegranate AshitabaBarleyTangerine peelDandelion root

Camellia sinensisCamellia sinensisCamellia sinensisCamellia sinensisPsidium guajavaCamellia sinensisPerilla frutescens var. crispaCamellia sinensisLagerstroemia speciosaSasa veitchiiHouttuynia cordataAspalathus linearisHouttuynia cordataAspalathus linearisOenothera tetrapteraGynostemma pentaphyllumChamaecrista nomameAcer maximowiczianumCymbopogon citratesHordeumvulgare var. nudumHouttuynia cordataArtemisia princepsCamellia sinensisEquisetum arvenseHouttuynia cordataCoixlacryma-jobiSalacia reticulateEriobotrya japonicaMatricariarecutitaGymnema sylvestreEucommia ulmoidesMorus australisSasa veitchiiAloe arborescensCrataegus cuneataHouttuynia cordataGanoderma lucidumHouttuynia cordataVaccinium corymbosumIpomoea batatasEucommia ulmoidesLycium chinenseChrysanthemum morifolium Ramat.Fagopyrum tataricumRosa caninaCorchorus olitoriusMorusaustralisCoix lacryma-jobiVigna angularisRosa rugosaCarthamus tinctoriusGlycyrrhizaCamellia sinensis & Oryza sativaOryza sativaCoixlacryma-jobiSenna obtusifoliaGlycine maxAngelica keiskeiGlehnial ittoralisPunica granatumAngelica keiskeiHordeum vulgareCitrus tangerineTaraxacum officinale

Sample Number

Grown in JapanGrown in Kagoshima prefecture, JapanGrown in Japan, stem

For tea bags

Grown in Japan, fall-winter picking

Grown in China, leaf

Grown in China, leaf

Grown in Japan

Grown in Japan

Grown in Shizuoka prefecture, Japan

Grown in China, stem

Grown in Japan

Circular leaf

Grown in China, stem

Grown in Japan

Grown in Shimane prefecture, Japan

No flower

CrushedPowder

Grown in JapanGrown in Shimane prefecture, JapanChopped

Common Name Scientific Name Details IC50 (1-hr ext)

0.014 0.015 0.015 0.017 0.017 0.017 0.017 0.018 0.018 0.019 0.019 0.019 0.021 0.025 0.027 0.028 0.029 0.032 0.033 0.038 0.040 0.041 0.043 0.044 0.044 0.044 0.044 0.045 0.046 0.046 0.047 0.047 0.051 0.052 0.058 0.060 0.064 0.065 0.073 0.078 0.079 0.083 0.086 0.094 0.097 0.102 0.107 0.126 0.136 0.182 0.191 0.202 0.206 0.216 0.232 0.241 0.246 0.258 0.291 0.293 0.308 0.338 0.352 0.365

0.017 0.020 0.014 0.018 0.026 0.016 0.033 0.020 0.011 0.007 0.022 0.032 0.025 0.023 0.005 0.028 0.098 0.002 0.033 0.006 0.134 0.048 0.037 0.047 0.101 0.000 0.030 0.049 0.017 0.061 0.067 0.062 0.057 0.028 0.143 0.048 6.614 0.252 0.068 0.063 0.054 0.122 0.083 0.226 >100.102 0.397 >0.1>100.034 0.457 0.404 >10>100.691 0.062 0.621 0.269 0.207 >101.095 0.004 1.992 0.219

IC50 (3-min ext)

140

Inhibition of AGEs by Herbal Teas

Sample Number Common Name Scientific Name Details IC50

(1-hr ext)IC50 (3-min ext)

103

5T26476104

98916247T54097388218853475145758

Ginger AminoguanidineChicoryBrown rice teaBarley grassDandelionGlutinous rice/brown riceMixed Herbal Extract KukoshiEdible burdockSobaChili pepperBlack soybean Bitter MelonCarrotBarley grassPumpkinAsian ginsengGingerHabu teaGreen teaRoasted soybeansCoarse teaCorn

Zingiber officinale

Cichorium intybusCamellia sinensis/Oryza sativaHordeum murinum subsp. LeporinumTaraxacum officinaleOryza glutinosa/Oryza sativa

Lycium chinenseArctium lappaFagopyrum esculentumCapsicumGlycine maxMomordica charantia var. pavelDaucus carotaHordeum murinum subsp. LeporinumCucurbitaPanax ginsengZingiber officinaleSenna obtusifoliaCamellia sinensisGlycine maxCamellia sinensisZea mays

Grown in Japan

Grown in Japan

Grown in Shimane prefecture, JapanGrown in JapanGrown in Japanm stem

0.401 0.403 0.403 0.406 0.411 0.425 0.510 0.558 0.629 0.751 0.778 0.848 0.991 1.035 1.367 1.455 2.254 2.638 3.473 5.689 4257>10>10

0.113 -0.601 0.627 0.295 6.597 23.075 -0.607 >10>100.557 5.787 0.328 >106.133 >101.867 0.131 0.562 98>10>100.024

Fig. 1. Inhibitory activity of selected teas and herbal teas (1-hour and 3-minute extracts) against fluorescent AGE formation in collagen. IC50 values are expressed in mg/ml. The samples are categorized as Camellia sinensis-derived teas or non-Camellia sinensis-derived teas (herbal teas). AGE; advanced glycation end product. IC50; 50% inhibitory concentration.

141

Inhibition of AGEs by Herbal Teas

141

Table 2 Inhibitory activity of 1-hour extracted herbal tea samples against formation of fluorescent AGEs and CML in collagen, and fluorescent AGEs, pentosidine, and 3DG in HSA. IC50 values are expressed in mg/ml.

Banabá

Brown rice

Chinese blackberry

Hama tea

Kuma bamboo grass

Persimmon leaf

Barley grass

Black soybean

Pumpkin

Aminoguanidine

AGEs; advanced glycation end products. CML; Nε-(carboxymethyl)lysine. 3DG; 3-deoxyglucosone. HSA; human serum albumin. Col: bovine skin collagen type I, HSA: human serum albumin. IC50; 50% inhibitory concentration.

CML(Col)

FluorescentAGEs (Col)Sample Fluorescent

AGEs (HSA)

0.018

0.002

0.010

0.006

0.019

0.010

>1

0.991

>1

0.400

0.0003

0.150

0.011

0.026

0.249

0.049

0.553

0.710

0.668

0.180

0.049

>1

0.046

0.146

0.140

0.091

0.957

>1

>1

0.068

Pentosidine(HSA)

>1

>1

0.141

>1

>1

0.005

>1

>1

>1

>1

3DG (HSA)

0.020

>1

0.021

0.057

0.112

0.049

>1

>1

>1

0.320

Inhibitory activity of sample herbal teas against 3DG formation in HSA

One-hour extracts of hama tea, banabá, Chinese blackberry, persimmon leaf, and kuma bamboo had higher inhibitory activity against 3DG formation in the HSA model compared to aminoguanidine (IC50 = 0.32 mg/ml; Table 2). The IC50 of brown rice, pumpkin, young barley grass, and black soybean was > 1, which indicates low inhibitory activity against 3DG formation.

Final selection of herbal teasA summary of inhibitory activity by 1-hour extracted herbal

tea samples is given in Table 2, compared in each case with aminoguanidine. Of the six selected herbal teas, only Chinese blackberry and persimmon leaf had higher inhibitory activity than aminoguanidine. Banabá had a higher inhibitory activity against the formation of all AGEs tested except pentosidine in the HSA model. Hama tea had no inhibitory activity against the formation of pentosidine in the HSA model, slightly lower inhibitory activity in the HSA model, and higher inhibitory activity against the formation of other AGEs. Kuma bamboo had no inhibitory activity against pentosidine formation in the HSA model, a slightly lower inhibitory activity against the formation of f lorescent AGEs in the collagen and HSA models, and a higher inhibitory activity against the formation of 3DG in the HSA model. Brown rice had higher inhibitory activity against the formation of fluorescent AGEs in the collagen model, but no significant inhibitory activity against any other AGE. Chinese blackberry, persimmon leaf, and banabá were determined to have the highest glycation inhibitory activities in the collagen and HSA models. Hama tea and kuma bamboo had similar glycation inhibitory activities. Given that previous studies indicated kuma bamboo was different to the other three herbal teas and that this species inhibits glycation, it was selected for further analysis with banabá, Chinese blackberry, and persimmon leaf.

Polyphenol concentration in herbal teasPolyphenol, expressed as (+) catechin equivalent, was

present in banabá and Chinese blackberry, but not in persimmon leaf and kuma bamboo grass (Fig. 2).

Antioxidant activity of herbal teasFor the 1-hour extraction samples, antioxidant activity was

highest in banabá (IC50 = 0.03mg/ml) and lowest in persimmon leaf was the (IC50 = 0.23mg/ml) (Fig. 2). The four herbal teas had weaker antioxidant activities than green tea, which is known to be strong in antioxidants (IC50 = 0.005mg/ml).

Glycation inhibition by blended herbal tea extractInhibitory activity of blended herbal tea (1-hour extraction) against the formation of glycated products

Inhibitory activity of the blended extract of the four test herbs against the formation of fluorescent AGEs and CML in the collagen model (IC50 = 0.002mg/mL) was more than five times greater than the inhibition by each herbal tea on its own (Fig. 3). Inhibitory activity of the blended extract against the formation of fluorescent AGEs in the HSA model (IC50 = 0.045 mg/ml) and CML in the collagen model (IC50 = 0.002mg/ml) was also greater than the inhibition by each individual tea. Inhibitory activity of the blended extract against the formation of pentosidine in the HSA model (IC50 = 0.007 mg/ml) was greater than that by banabá, Chinese blackberry, and kuma bamboo grass, and nearly equal to persimmon leaf. Inhibitory activity of the blended extract against the formation of 3DG in the HSA model (IC50

= 0.002mg/ml) was greater than that by persimmon leaf and kuma bamboo grass, and nearly equal to Chinese blackberry and banabá.

Polyphenol concentration of blended herbal teaThe polyphenol concentration of blended herbal tea was

equivalent to that of Chinese blackberry, with a catechin equivalent concentration of 0.06 mg/mL (diluted by 20 fold, for measurements could not be taken at high concentrations) (Fig. 2).

Antioxidant activity of blended herbal teaBlended herbal tea showed a strong antioxidant activity (IC50

= 0.03mg/ml), equivalent to banabá (Fig. 2).

Clinical study – topical application of herbal tea extracts

Skin AF at the extract applied area increased at a slower rate than the control area at four weeks (p= 0.043), but no significant difference was observed in melanin, and erythema levels, or skin elasticity (R2, R7) and CML concentration between the treated and untreated areas (Table 3, Fig. 4). Measurements of R7 (p= 0.043) differed between the beginning and end of trial, and measurements of R2 (p=0.080) barely missed achieving statistical significance, but no other significant differences in skin condition were observed (Fig. 5). In regards to skin moisture,a significant change was observed at week 4 (p=0.043) and the change at week 8 barely missed achieving statistical significance (p=0.08) (Fig.6).

No change in subjective symptoms was reported over the eight week period. No adverse events that can be attributed to the application of the test product were reported during and after the examination, and we suggest tea extracts are safe when applied to human skin.

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Inhibition of AGEs by Herbal Teas

Fig. 2. Polyphenol concentration and antioxidant activity of herbal teas (1-hour extraction) and blended herbal tea. Polyphenol concentrations are expressed in mg polyphenol/ml herbal tea sample. IC50 values are expressed in mg/ml. IC50; 50% inhibitory concentration.

Fig. 3. Inhibitory activity of blended herbal tea against the formation of fluorescent AGEs and CML in collagen, and fluorescent AGEs, pentosidine, and 3DG in HSA. Values are expressed as IC50 (mg/ml) against the formation of each glycated product. IC50 values are expressed in mg/ml. AGEs; advanced glycation end products. CML; Nε-(carboxymethyl)lysine. 3DG; 3-deoxyglucosone. HSA; human serum albumin. IC50; 50% inhibitory concentration.

143

Inhibition of AGEs by Herbal Teas

143

AF: auto-fluorescence, Ref: reflectance. CML; Nε-(carboxymethyl)lysine.

Table 3 Skin condition over the 8-week examination period. Excluding CML (units: ng CML/µg protein), units for measurements are arbitrary units determined by each manufacturer. (n=5, average±standard deviation [SD]).

MoistureMelaninErythemaR2R7AFRefCML

Control (n=5)

Measurements mean SD

37186245

0.930.721.310.134.36

±

±

±

±

±

±

±

±

94579

0.020.060.310.052.52

Week 0

mean SD

38163215

0.930.711.450.147.29

±

±

±

±

±

±

±

±

53639

0.020.070.280.061.71

Week 4

mean SD

31173199

0.930.711.320.143.81

±

±

±

±

±

±

±

±

85269

0.030.090.230.060.99

Week 8

mean SD

1068892

10098

112106219

±

±

±

±

±

±

±

±

214

19369

16138

W4 - W0

Percent Change(%)

mean SD

829282

10198

102110127

±

±

±

±

±

±

±

±

169

1638

1024

105

W8 - W0

MoistureMelaninErythemaR2R7AFRefCML

Test (n=5)

Measurements mean SD

37191247

0.930.721.310.134.66

±

±

±

±

±

±

±

±

94466

0.020.050.250.074.00

Week 0

mean SD

34171185

0.910.681.360.167.67

±

±

±

±

±

±

±

±

74259

0.050.080.270.073.14

Week 4

mean SD

35183230

0.950.771.440.134.55

±

±

±

±

±

±

±

±

53754

0.000.040.280.052.00

Week 8

mean SD

9489749894

104125233

±

±

±

±

±

±

±

±

1458577

37127

W4 - W0

Percent Change(%)

mean SD

1059794

103106110110134

±

±

±

±

±

±

±

±

176

11396

2282

W8 - W0

Fig. 4. Percent change in skin auto-fluorescence (AF) in the control and test area over an eight week examination period. Difference between two groups compared to week zero was determined by Wilcoxon’s signed ranked test (n=5, average ± standard deviation [SD]). A significant difference between the two groups was observed at week four (p< 0.05); *p<0.05.

144

Inhibition of AGEs by Herbal Teas

Fig. 5. Percent change in skin elasticity (R7) in the control and test areas over an eight week examination period. Difference between two groups compared to week zero was determined by Wilcoxon’s signed ranked test (n=5, average ± standard deviation [SD]). A significant difference between the two groups was observed at week eight (p<0.05); *p<0.05.

Fig. 6. Percent change in moisture level in the control and test area over an eight week examination period. Difference between two groups compared to week zero was determined by Wilcoxon’s signed ranked test (n=5, average ± standard deviation [SD]). A significant difference between the two groups was observed at week four (p< 0.05); *p<0.05.

145

Inhibition of AGEs by Herbal Teas

145

Herbal tea blend as a future beverage for skin Anti-AgingInhibitory activity against f luorescent AGEs in HSA by herbal teas (3-minute extracts)

In Chinese blackberry and kuma bamboo, the inhibitory activity against fluorescent AGEs formation in HSA was higher when extraction time was shorter, and in persimmon leaf inhibitory activity was higher with longer extraction time (Fig. 7). The inhibitory activity of banabá did not seem to depend on extraction time. However, extraction time did not appear to affect the inhibitory activities of herbal teas in the collagen model.

Fig. 7. Comparison of inhibitory activity of herbal teas (3-minute extracts) against the formation of fluorescent AGEs in HSA and bovine collagen type I.IC50 values are expressed in mg/ml. AGEs; advanced glycation end products. HSA; human serum albumin. IC50; 50% inhibitory concentration.

Fig. 8. Taste evaluation of herbal tea blends. The taste was rated on a five point scale, 5: very good, 1: very poor. Differences between samples were determined by the Kruskal-Wallis test and p< 0.05 was considered significant (n=7, average ± standard deviation [SD]). The difference in taste scores of teas were not statistically significant.

Taste evaluationThe results of the taste evaluation are summarized in Fig. 8.

In the initial tests, taste scores of each tea were not statistically significant (p= 0.079, Kruskal-Wallis test). When the significance level was reset to p= 0.1, small differences were detected in the taste of banabá and kuma bamboo grass (p= 0.011), persimmon leaf and kuma bamboo grass (p= 0.017), banabá and blend 1 (p= 0.026), banabá and blend 3 (p= 0.026), persimmon leaf and blend 1 (p= 0.038) and persimmon leaf and blend 3 (p= 0.038).

146

Inhibition of AGEs by Herbal Teas

Discussion

Glycation inhibition by herbal teasThese findings of this study suggest that banabá, Chinese

blackberry, kuma bamboo, and persimmon leaf are strong inhibitors of collagen and HSA glycation. While all four herbal teas showed high inhibitory activity against the formation of f luorescent AGEs and CML in the collagen model, and fluorescent AGEs and 3DG in the HSA model, only Chinese blackberry and persimmon leaf inhibited pentosidine formation in the HSA model. We suggest that some unidentified ingredients in Chinese blackberry and persimmon leaf may block the conversion of Amadori product to pentosidine. All four herbal teas suppressed the formation of 3DG in HSA, suggesting that all four inhibit the conversion of Amadori products to 3DG or fructose-3-phospate to 3DG. All herbal teas inhibited CML formation in the collagen model, suggesting that the formation of CML via Amadori products or glyoxal was inhibited. However, AGE formation pathways are extremely complex, and more analysis on AGE formation inhibition by herbal teas is required to understand the inhibition mechanism.

Inhibitory activity of the blended extract against fluorescent AGEs in the collagen model (IC50 = 0.002mg/ml) was higher than that of aminoguanidine and MHE (IC50 = 0.01mg/ml), both of which have high glycation inhibitory properties 7). While inhibition of skin collagen by aminoguanidine has been reported in vitro 22), in vivo 23,24) studies reported that administration of aminoguanidine had no effect on the glycation of collagen in rat skin. However, the four herbal teas investigated here were stronger inhibitors of collagen glycation than aminoguanidine, which suggests these teas may have a positive effect on skin collagen in vivo. In the HSA model, inhibitory activity against fluorescent AGEs formation of blended extract (IC50 = 0.045 mg/ml) was greater than that of both aminoguanidine and MHE (IC50 = 0.1mg/ml). These results suggest that combining the four herbal teas increased the overall inhibition of glycation in the collagen and HSA models.

Antioxidant activity and polyphenol concentration in herbal teas

Some AGEs, such as CML and pentosidine, are formed by glycation and oxidation reactions 25,26), thus samples with high antioxidant activity are likely to inhibit the formation of some AGEs. The high inhibition of CML and pentosidine formation by banabá, Chinese blackberry, kuma bamboo grass, and persimmon leaf may partly result from their antioxidant properties. Previous studies have reported that polyphenols in teas also inhibit oxidation and glycation 27,28), and banabá and Chinese blackberry both contain polyphenol. Persimmon leaf and kuma bamboo grass do not contain polyphenol, which suggests these plants may contain other glycation inhibitors.

Topical application of blended herbal tea extractMelanin, erythema levels, R2, and CML levels in stratum

corneum did not change significantly during the eight-week examination period. It is unlikely that the extract would permeate into the dermis, therefore the results of this examination reflects the changes in the epidermis. Compared to the control, skin moisture dropped significantly in the test area after four weeks, but improved slightly after eight weeks. However, it is unlikely

that the change was related to the anti-glycation effects of the extract, for AGE levels increased over the period of eight weeks in the test area. Auto-f luorescence indicating AGE levels, increased in both the test and control areas after four weeks, and this change might have resulted from lifestyle changes (i.e. year-end parties) during the winter season. While the AGEs levels increased in both areas, there was significantly less change in the test area compared to the control, suggesting that the extract may have suppressed the formation of f luorescent AGEs in skin, primarily the fluorescent AGEs formed by the glycation of proteins in the epidermis. After eight weeks, a significant improvement in R7 was observed in the test compared to the control skin, and a small improvement in R2 was observed. The accumulation of AGEs in the epidermis is known to reduce flexibility of skin, thus, improved elasticity may have been due to the inhibition of glycation of proteins in the epidermis, such as keratin.

Additional information would be gained by studying the effects of application of teas over a longer time, in a larger variety of subjects including male subjects, selected from a wider age range and in healthy and non-healthy individuals, such as diabetic patients with high glycation risks.

Herbal tea blend as a beverage for skin Anti-Aging Extraction time had a drastic effect on the inhibitory

activities of the herbal teas. Prolonged heating of Chinese blackberry and kuma bamboo during extraction might have damaged some of the inhibiting ingredients, but a longer extraction period may be required to extract active ingredients from persimmon leaf.

Although irrelevant to topical application, taste is an important if teas are to be consumed as a beverage. Despite health benefits, people are unlikely to consume unpleasant teas regularly.

Banabá tea and persimmon leaf were given low ratings for taste when brewed individually, but a blend of all four herbs improved overall taste. MHE, which had lower glycation inhibitory activity compared to the blended extract, did improve skin elasticity in pre-diabetic patients when consumed as a supplement 5), and it is possible that the consumption of the tested herbal tea blend might also improve skin condition, especially pre-diabetic and diabetic patients. A clinical trial should evaluate the anti-skin aging potential of herbal tea blends.

While the topical application of herbal tea extracts most likely affected glycation only in the epidermis, consumption of these herbal teas, either as beverages or supplements, if absorbed by the body, may affect glycation in both in the epidermis and the dermis. As such, it may inhibit glycation of collagen, thereby affecting other characteristics of skin.

Summary of herbal teasFor hundreds of years, banabá, Chinese blackberry, kuma

bamboo, and persimmon leaf have been drunk for health benefits.Banabá contains calcium, magnesium, potassium, and

zinc, and is rich in corosolic, which is known to prevent blood sugar level increases 29). Banabá is often consumed to treat diabetes mellitus (type II) and impaired glucose tolerance 30). In the present study, banabá was found to possess high glycation inhibitory activity in both the collagen and HSA models. Glycated HSA is known to aggravate diabetic conditions through the activation of RAGE. We suggest that the efficacy of banabá

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Inhibition of AGEs by Herbal Teas

147

consumption for the prevention and treatment of diabetes is partly due to the inhibition of HSA glycation.

Chinese blackber ry contains rubusoside, calcium, magnesium, and polyphenol. This herb is well known for its anti-allergic action 31), and can be used to prevent and treat allergy related conditions, such as hay fever. The anti-allergic action may be triggered by the polyphenol 32), which prevents the secretion of histamine, but might also be because this herb’s high glycation inhibition reduces AGEs activation of RAGE, which cause inflammation.

Kuma bamboo grass is rich in calcium, vitamin B1, B2, C, K, benzoin acid, bamfolin, and chlorophyll. It has been reported to posess anti-inflammatory 33,34), antioxidant 35), and antiviral, antibacterial 36) activities. Previous studies reported that kuma bamboo contains “Absolutely Hemicellulose Senanesis” (AHSS), which is a strong antioxidant 35). The present study confirmed the antioxidant activity of kuma bamboo grass 37), and this activity might have contributed to the high glycation inhibition observed in kuma bamboo grass samples.

Persimmon leaf contains pro-vitamin C, tannins, flavonoids, rutin, choline carotenoids, amino acids, and the elements magnesium, manganese, titanium, calcium, phosphorous. Pro-vitamin C, which is resistant to damage by heat, is converted to vitamin C in the body, is essential in the synthesis of collagen, and inhibits the increase in melanin caused by UV rays. Previous studies indicated that persimmon leaves contain f lavonoids possess antioxidative activities 38). As vitamin C is a strong antioxidant, we suggest that the combination of flavonoids and vitamin C probably enables the antioxidant activity of persimmon leaf observed in the present study.In addition, persimmon leaf is known to contain ingredients that inhibit enzymatic activities of collagenase and elastase 39), which may be effective in preventing the formation of wrinkles. Persimmon leaf also inhibited collagen glycation and might therefore help maintain collagen in the body.

ConclusionIn the present study, we evaluated the inhibition of

fluorescent AGEs and CML in collagen and fluorescent AGEs, 3DG, and pentosidine in HSA by herbal teas, and the effect of topical application of herbal tea extracts on skin elasticity, moisture, melanin, erythema, AGE, and CML levels.

The results indicated that glycation was inhibited by extracts of banabá (Lagerstroemia speciosa), Chinese blackberry (Rubus suavissimus), kuma bamboo grass (Sasa veitchii), and persimmon leaf (Diospyros kaki). A topical application of an extract containing all four herbal teas for four weeks prevented the increase of fluorescent AGEs, and increased skin elasticity after eight weeks. Further investigation is necessary to determine if oral consumption of herbal teas effects skin. These herbal teas might be used in health foods and cosmetics designed as Anti-Aging products for skin and body.

Conflict of interest statementThe authors declare no financial or other conflicts of interest

in the writing of this paper.

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