Introduction
We participated in the Cross-ministerial Strategic Innovation Promotion Program (SIP). We are a member of the Development of Functional Agricultural and Food Products for the Next Generation Program, Technologies for Creating Next-Generation Agriculture, Forestry and Fisheries of SIP. The purpose of this project is to contribute to the vitalization of agriculture, forestry and fishery, acquiring
Print edition : ISSN 2188-3602 Received : September 2, 2017 Accepted : October 20, 2017 Published online : December 31, 2017
Glycative Stress Research 2017; 4 (4): 317-328 Original article
1) Anti-Aging Medical Research Center and Glycative Stress Research Center, Faculty of Life and Medical Sciences, Doshisha University, Kyoto, Japan
2) Yomeishu Seizo Co. Ltd., Tokyo, Japan
KEY WORDS:
kuromoji (Lindera umbellata), Japanese mugwort (yomogi; Artemisia indica var. maximowiczii), advanced glycation end products (AGEs), diabetic nephropathy, cataractAbstract
Aim: We participated in the Cross-ministerial Strategic Innovation Promotion Program (SIP). SIP aims to find materials made from agricultural crops which have new functions, and to realize the social implementations to launch new products into markets. This study selected, from plant-derived materials, materials which have strong inhibitory activities against the formation of advanced glycation end products (AGEs) and high quality of intestinal absorption. Furthermore, we examined whether or not these materials exerted anti-diastatic actions in experiments with diabetic animal models.
Methods: From extracts of 536 types of plant-derived materials, this study selected materials with strong inhibitory activities against fluorescent AGE formation (“AGE inhibition”), which were produced in the reaction between human serum albumin (HSA) and glucose (comparison in each IC50). Screening was conducted for the examination of AGE inhibition in culture fluid which passed through intestinal epithelial cells (Caco-2), and furthermore, the AGE inhibition in serum of rats following oral administration. Aminoguanidine was used as a positive control. Test materials were administered to streptozotocin (STZ)- induced diabetic rats for 8 weeks. Subsequently, this study examined the metabolic index of glycolipids, diabetic nephropathy index and cataract stage.
Results: Kuromoji (Lindera umbellata) and yomogi (Artemisia indica var. maximowiczii) were selected by screening, as AGE inhibition following an absorption test was the same level as aminoguanidine. STZ treated rats (control) showed, in comparison with rats without STZ treatment, increases in blood glucose, glycoalbumin (GA), triglyceride (TG) and free fatty acid (FFA), increases in renal tissue TNF-α/IL-6 and decreases in creatinine clearance (CCr). Administration of kuromoji extract significantly improved TG and FFA. TNF-α/IL-6 in renal tissues and CCr were also significantly improved. Kuromoji and yomogi extracts both showed effects on the prevention of onset and progression of cataracts.
Conclusion: This study concluded that kuromoji extracts, which were screened and selected in terms of high anti-glycative activity and characteristic of intestinal absorption, had the effects of prevention of onset and progression of nephropathy and cataracts in the diabetic rat model. Further examination of kuromoji has been decided to continue for the purpose of social implementations.
Screening and selection of anti-glycative materials: Kuromoji (Lindera umbellata)
new next-generation functionality in agricultural, forest and fishery products and providing added value to the products.
The budget for this program is as much as 2.5 billion yen for five years, which is the largest scale in Japan as a program for the development of functional food and the survey of medical evidence. Glycative stress has been positioned as a risk factor for both. It is highly expected that new functional
Contact Address: Professor Yoshikazu Yonei, MD, PhD
Glycative Stress Research Center, Faculty of Life and Medical Sciences, Doshisha University 1-3, Tatara Miyakodani, Kyotanabe, Kyoto, 610-0394 Japan
Phone/Fax: +81-774-65-6394 E-mail: [email protected]
Co-authors: Yagi M, [email protected] ; Takabe W, [email protected] ; Matsumi S, [email protected] ; Shimode A, [email protected] ;
Masayuki Yagi 1), Wakako Takabe 1), Shigeru Matsumi 2), Akihiko Shimode 2), Tetsuya Maruyama 2), Yoshikazu Yonei 1)
food with anti-glycative activity should be developed.
Our research center has examined more than 500 types of food materials. Anti-glycative materials have been explored 1-3) to measure the inhibitory activities against the formation of advanced glycation end products (AGEs), where glycation reaction model in vitro was employed with human serum albumin (HSA) as a target protein. Therefore, this study has identified more than 100 types of materials with inhibitory activities against the AGE formation. The identified materials had the same as or higher activities than aminoguanidine than the positive control in the HSA glycation model. The SIP requires, other than experimental outcomes, the performance of the social implementation to develop and launch new products into markets. Therefore, for the development of commercial products created from these materials, this study examined the absorption property and the preventive effect of nephropathy and cataracts in experiments with animal models of diabetes mellitus.
Methods
1. Screening of anti-glycative materials
(1) MaterialsDried materials of 536 types of plants were diluted at a weight ratio of 10 : 1 (water: dried material) and were extracted by heating at 95°C for 60 minutes. These materials were filtrated and then were dried under reduced pressure by an evaporator. These extracts were examined with positive control arm using aminoguanidine (Wako Pure Chemical Industries, Ltd. Chuo-ku, Osaka, Japan).
(2) Methods of Examination
1) Human serum albumin-glucose (HSA-Glu) test system1, 2) Into the glycation solution consisting of 0.1 mol/L phosphate buffer (pH 7.4), 8.0 mg/mL HSA and 0.2 mol/L glucose, plant extracts or aminoguanidine of a positive control, which were at the stipulated concentration, and were added to reach 1/10 of volume concentration. Solutions of plant extracts and aminoguanidine were incubated at 60°C for 40 hours. For a control arm, the solution was distilled with water instead of plant extracts. After the completion of the glycation reaction, fluorescent AGEs, which were produced in the reaction fluid, were measured using a microplate reader (excitation wavelength: 370 nm, fluorescent wavelength:
440 nm). For analysis of the inhibition rate of each AGE formation, A is the measurement value of the reaction liquid where sample solution was added to glycative reaction system, B is the measurement value of the reaction liquid where distilled water was added instead of glucose solution, C is the measurement value of the reaction liquid where distilled water was added instead of extracts or aminoguanidine, and D is the measurement value of the control liquid where distilled water was added instead of glucose solution.
IC50 (inhibitory concentration 50%) was calculated in the following formula:
The inhibitory concentration of AGE formation (%) = {1− (A−B) / (C−D)} × 100
The strength of activity was evaluated by the relative values in the following formula, as IC50 of aminoguanidine was designated as one.
The activity value of extracts = aminoguanidine (IC50) / plant extracts (IC50)
2) Permeability test in intestinal epithelium absorption model The permeability test in the intestinal epithelium absorption model employed POCA® intestinal absorption kit (DS Pharma Biomedical Co., Ltd., Suita, Osaka, Japan), where cells derived from human colon cancer (Caco-2) 4-6) were disseminated on the insert side of a transwell plate.
Culture solution of D-MEM with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA) in the amount of 100 μL, which contained extracts (5 mg/mL) and aminoguanidine (5 mg/mL), was added to the insert side of the plate. After the solution was incubated for 3 hours, the inhibitory rate of the AGE formation in HSA-Glu test system was measured for culture solution which was permeated to the side of multiwell. The strength of activity was evaluated by relative values in the following formula, as the inhibitory rate of aminoguanidine was designated as 1.
The activity value of extracts = inhibitory rate of plant extracts / inhibitory rate of aminoguanidine
3) Test using blood serum of rats which extracts were administered to
SD male rats at 5 week-old (Charles River Laboratories Japan, Inc. Kohoku-ku, Yokohama, Kanagawa, Japan) were kept for one-week to be prepared for the test. Then, plant extracts (2 g/kg weight) and aminoguanidine (1 g/kg weight) were administered to the rats after 16-hour fasting. Blood sampling from jugular vein was conducted with an injector heparin sodium treated 1 hour pre, 1 hour after and 3 hours after administrations. Blood sampling was centrifuged to obtain blood plasma of 500 μL. This blood plasma was deproteinized using Nanosep® Centrifugal Devices (Pall Corporation, Port Washington, NY, USA) to obtain samples.
Furthermore, the AGE formation inhibitory rate was measured in HSA-Glu test system. The strength of activity was evaluated by relative values in the following formula, as AUC (area under curve) of aminoguanidine was designated as one.
The activity value of extracts = AUC of plant extracts / AUC of aminoguanidine
2. Streptozotocin (STZ)-induced diabetic rats
(1) MaterialsDried and ground plants of trunks and branches of Lindera umbellata (kuromoji) and leaves of Artemisia indica var. maximowiczii (yomogi) were diluted at a 10 : 1 ratio (distilled water: dried plants), which were extracted by heating at 95°C for 60 minutes. After filtrated, extracts were condensed using continuous vacuum evaporator and then were lyophilized, which were obtained as subject materials.
(2) Animal experiments 1) Animals
SD rats at 6-week old (Japan SLC, Inc., Nishi-ku, Hamamatsu, Shizuoka, Japan) arrived and were reared in a barriered facility under the following environment:
temperature: 24 ± 2°C, humidity: 50 ± 10%, lighting time:
12 hours a day (7:00-19:00). After one week of preliminary period rearing, SD rats were examined in experiments.
2) Rearing management
Through the periods of both preliminary rearing and administrating test material, SD rats were kept separately in a breeding cage, which was made from polycarbonate (W26 × D42 × H18 cm). In the cage, animal bedding made of fir tree (bedding for experimental animals soft wood chips: Japan
SLC, Inc.) was used.
3) Feed and drinking water
Labo MR Stock Powder (Nosan Corporation, Nishi-ku, Yokohama, Kanagawa, Japan) or Labo MR Stock Powder added to test materials were supplied to be freely consumed.
Also, drinking water, which was well water disinfected with chlorine, was free to drink using water supply bottles.
4) Production of diabetic animal models and construction of groups
After the period of rearing for preparation, seven-week- old rats were given intraperitoneal injections of STZ with 60 mg/kg weight. One week later, a partial blood sample was collected from the caudal vein under non-fasting conditions, followed by the measurement of blood glucose level (Simple blood sugar measuring instrument, One-touch Ultra: Johnson
& Johnson, Chiyoda-ku, Tokyo, Japan). Animals with blood glucose level 300 mg/dL or more were divided into groups without deviation of blood glucose level and weight (Statlight Grouping Soft: Yukms Co. Ltd., Asao-ku, Kawasaki, Japan).
The grouping structure was as follows:
Group without treatment: untreated rats were given Labo MR Stock Powder (n = 8).
Control group: STZ administered rats were given Labo MR Stock Powder (n = 8).
Group with the administration of yomogi: STZ administered rats were given Labo MR Stock Powder combined 2%
extracts of leaves of yomogi (n = 8).
Group with the administration of kuromoji: STZ administered rats were given Labo MR Stock Powder combined 2% extracts of kuromoji (n = 7).
Rats were maintained for 8 weeks with free access to subject materials.
5) Method of measurement
ⅰ) Weight and intake amount
Weight and intake amount were measured once a week.
ⅱ) Ophthalmological test
Eight weeks after the beginning of administration, cataract stage examinations were conducted to observe corneas and crystalline lenses of both eyes under isoflurane anesthesia using a portable slit lamp (SL-5: Kowa Company. Ltd., Naka- ku, Nagoya, Aichi, Japan). Assessment of cataract stage was as follows:
Stage 0: Normal
Stage 1: Early cataract (discrete anterior and posterior cortical opacities)
Stage 2: Immature cataract (development of opaque lenses from the cortex to the inner core)
Stage 3: Mature cataract (severely opaque lenses and white pupils)
ⅲ) Blood sample collection, hemoglobin Alc (HbA1c) measurement and biochemical examination
On the completion day of the 8-week administration, 16- hour fasting was conducted from the evening to the next morning. Then, whole blood collection was performed from abdominal aorta under isoflurane anesthesia followed by HbA1c measurement for some portion of blood (HbAlc measurement device: Siemens Healthcare Diagnostic, Shinagawa-ku, Tokyo, Japan). Blood serum sampling was separately obtained from the remainder of the blood and was kept frozen. At a later time, measurements were performed by enzymatic method in blood glucose (Glu,) glycoalbumin
(GA), urea nitrogen (BUN), creatinine (CRE), amylase (AMY), triglyceride (TG), and free fatty acid (FFA).
ⅳ) Renal function
After whole blood collection, severance of the abdominal aorta and the caudal vena cava was performed as euthanasia.
Right renal gland was collected and was measured by weight, to which was added phosphate buffer saline (PBS) 4 times as much as the weight, homogenated and centrifuged. Tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in supernatant were measured using Rat OptEIA ELISA Set (BD Pharmingen [Becton, Dickinson and Company], Franklin Lakes, NJ, USA) and protein was measured using BCA Protein Assay Kit (Takara Bio INC., Kusatsu, Shiga, Japan), which was shown as a value per protein mg. For the assessment of creatinine clearance (CCr,) urine sampling was performed (V) for 16 hours at the 8 week after administration. Cr concentration value in urine was measured (uCr.) From serum creatinine concentration (serum Cr concentration) (sCr.) CCr was calculated in the following formula and CCr was shown as a value per one minute:
CCr = (uCr / sCr) × V
6) Ethical review on animal experiments
This research abided by “Act on Welfare and Management of Animals” (Act No. 105 of October 1, 1973, Revision June 22, 2005) and “Standards relating to the Care and Keeping and Reducing Pain of Laboratory Animals” (the Ministry of Environment Notification No. 88 of April 28, 2006.) This study was given review and approval based on Guide for the Care and Use of Laboratory Animals.
7) Data analysis
Results were shown as mean ± standard deviation (SD).
To provide a statistical processing system, student’s t-test was employed for comparison between two groups and multiple comparison tests of Bonferroni corrections was employed for comparison among multiple groups; p < 0.05 is regarded as a significant difference.
Results
1. Screening of anti-glycation materials
Materials which proceeded to the next step of screening from 536 types of plant extracts were shown in Table 1, as results of examinations in inhibitory actions (IC50) to inhibit the fluorescence AGE formation in HSA-Glu reaction system in vitro. Data which had already been published was excluded.
Sample materials with stronger inhibitory effects against the formation of AGEs than aminoguanidine, which were more than 5 times larger than aminoguanidine in IC50, were the following: ([ ] shows IC50 rate to aminoguanidine).
Forsythia koreana A [11.11], Syzygium aromaticum A [9.33], Aspalathus linearis [9.33], Camellia sinensis (L.) Kuntze [7.64], Camellia sinensis A, [7.64], Rhus javanica [6.74], Camellia sinensis [6.46], Cinnamomum verum [6.31], Forsythia suspense B [6.03], kuromoji_branch [5.80], kuromoji_branch and leaf [5.69] , Origanum majorana [5.60], Castanea crenata_shell [5.60], Spatholobus suberectus Dunn [5.09], Rubus suavissimus A [5.08].
The second screening assessed the inhibitory actions against the AGE formation after permeating intestinal
Table 1. The sample list of vegetables and herbs with high anti-glycative activity: ratio against aminoguanidine. 蓬 丁子 五倍子 黒文字 独活 杜仲葉 月桂樹 普洱茶 鴨跖草 阿波番茶 楤芽 大茴香 蓼 香需 花椒 巴拿巴茶 向日葵種子 黒文字 枝葉 緑薄荷 [A] 蝦夷蛇苺 桂皮 番茶 車前草 柿葉 鬱金、欝金 茉夭刺那 アジョワン 蕺草 [B] 三香子 胡桃仁 桑葉 蕎麦 [殻] 続断
1.16 1.08 0.78 0.75 0.75 0.75 0.72 0.59 0.50 0.50 0.47 0.46 0.42 0.41 0.34 0.25 0.22 0.18 0.15 0.09 0.03 0.02 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.33 1.55 0.55 0.91 0.14 0.27 0.65 0.63 0.45 0.51 0.35 0.43 1.41 0.74 0.39 0.98 1.05 1.21 0.40 0.46 0.23 0.56 0.34 1.00 0.72 0.69 0.54 0.46 0.46 0.44 0.42 0.38 0.33
1.20 9.33 6.74 5.80 1.98 0.56 3.82 7.64 1.86 6.46 2.58 1.06 1.17 1.71 2.60 1.39 2.29 5.69 3.50 − 6.31 0.87 2.04 0.75 0.89 5.60 2.15 2.15 0.70 0.95 1.50 1.56 1.91
Japanese mugwort Clove [A], Gallnut Lindera, Kuromoji [twigg] Udo Eucommia leaf Bay leaf, Laurel Pu-erh tea [A] Tsukikusa Awa bancha Fatsia sprouts Star anise Smartweed Xiangru, Chinese mosla Sichuan pepper Banaba tea Sunflower seeds Lindera, Kuromoji [foliage] Spearmint Wild strawberry Cinnamon Coarse tea Plantain Persimmon leaves Turmeric Marjoram Ajowan Houttuynia [B] Allspice Walnut seed Mulberry leaves Buckwheat [shell] Dipsacus japonicus root
Yomogi Choji [A] Fushi, Gobaishi Kuromoji [eda] Udo Tochu ha Gekkeiju, Laure Puaru-cha [A] Tsukikusa Awa bancha Tara no me Dai-uikyo Tade Koju Kasho Banaba cha Himawari no tane Kuromoji [edaha] Spearmint [A] Wild strawberry Keihi, Cinnamon Ban-cha Shazen-so Kaki no ha Turmeric, Ukon Marjoram, Mayorana Ajowan Dokudami [B] Allspice Onikurumi [seed], hú táo rén Kuwa no ha Soba [kara] Zokudan, nabena
Artemisia indica var. maximowiczii Syzygium aromaticum Rhus javanica Lindera umbellata Angelica pubescens Maxim.f. biserrata Shan et Yuan Eucommia ulmoides Cinnamomum tamala Camellia sinensis Commelina communis L. Camellia sinensis Aralia elata Illicium verum Hook. f. Polygonum hydropiper Mosla chinensis Zanthoxylum bungeanum Lagerstroemia speciosa Helianthus annuus Lindera umbellata Mentha spicata Fragaria vesca Cinnamomum verum Camellia sinensis Plantago asiatica Diospyros kaki Curcuma longa Origanum majorana Trachyspermum ammi Houttuynia cordata Pimenta dioica Juglans regia L. Morus alba L. Fagopyrum esculentum Dipsacus asperoides C. Y. Cheng et T. M. Ai
Japanese nameEngilish nameChinese characterScientific nameHSA-Glu model (n = 3)Caco-2 test (n = 1)Rat serum test (n = 2 ~ 3)
Table 1. The sample list of vegetables and herbs with high anti-glycative activity: ratio against aminoguanidine. 荷葉 緑茶 山椒 大吉岭紅茶 梅 [果肉] 菊花 黒胡椒 檸檬草 木立薄荷 縞綱麻 花薄荷 鶏血藤 蕨 甜茶 [B] 阿列布葉 紫蘇子 五加木 薄荷 朝鮮薊 連銭草 林檎 富士 連翹葉 [B] 栃 [殻] 鵝不食草 胡椒薄荷、西洋薄荷 南非国寶茶[B] 路路通 姫茴香 茴香 迷迭香 庭常属 仙鶴草 花豆 [鞘] 半枝蓮
0.00 − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − −
0.29 0.35 0.30 0.27 0.27 0.26 0.24 0.21 0.20 0.18 0.17 0.17 0.16 0.14 0.14 0.12 0.11 0.09 0.09 0.08 0.08 0.07 0.07 0.06 0.05 0.05 0.05 0.04 0.03 0.03 0.02 0.01 0.01 0.00
1.79 1.37 1.58 3.82 − 1.53 3.00 4.20 1.68 2.10 1.00 5.09 2.79 4.20 − − 4.29 4.24 − 1.41 0.06 6.03 2.14 1.13 1.35 9.33 2.11 1.14 1.83 1.56 − 1.62 2.05 2.25
Lotus leaf Green tea Japanese pepper Darjeeling tea Plum [flesh] Chrysanthemum Black pepper Lemongrass Savory Jew's mallow Oregano Milettia reticulata Bracken Tien-cha, Chinese blackberry tea Olive leaf Sissoko, fruit of purple perilla Araliaceae Peppermint Artichoke Chinese alehoof Apple fuji Golden bell flower leaf [B] Japanese horse chestnut [shell] Centipeda, diet flower Peppermint Rooibos tea [B] Callaway Fennel Rosemary Elderberry Ryûgesô scarlet runner bean [sheath] Scutellaria barbata
Kayo Ryoku-cha Sansho Darjeeling tea Ume [kaniku] Kiku [hana] Kuro-kosho Lemongrass Savory Molokheiya Oregano, hana-hakka Keiketto Warabi Ten-cha [B] Olive [leaf] Shisoko Ukogi, okogi Hakka Artichoke Rensen-so, kakidoshi Ringo fuji Rengyo-ha [B] Tochi [kara] Gafushoku-so, ebushicao. Peppermint Rooibos tea [B] Rorotu Callaway, Hime-uikyo Fennel Rosemary Elderberry Sentsurukusa Hanamame_[saya] Hanshiren
Nelumbo nucifera Camellia sinensis Zanthoxylum piperitum Camellia sinensis (L.) Kuntze Prunus mume Chrysanthemum morifolium Ramat. Piper nigrum Cymbopogon citratus Satureja montana Corchorus olitorius Origanum vulgare Spatholobus suberectus Dunn Pteridium aquilinum Rubus suavissimus Olea europaea Perilla frutescens Acanthopanax sieboldianus Mentha x piperita L. Cynara scolymus Glechoma longituba (Naka) Kupr. Malus domestica Forsythia suspensa Aesculus turbinata Centipeda minima (L.) A. Braun et Aschers. Mentha piperita Aspalathus linearis Liquidambar formosana Hance Carum carvi Foeniculum vulgare Rosmarinus officinalis Sambucus sieboldiana Agrimonia pilosa Ledeb. Phaseolus coccineus Scutellaria barbata
Japanese nameEngilish nameChinese characterScientific nameHSA-Glu model (n = 3)Caco-2 test (n = 1)Rat serum test (n = 2 ~ 3) Beautiful sweetgum fruit, formosa sweetgum
Table 1. The sample list of vegetables and herbs with high anti-glycative activity: ratio against aminoguanidine. HSA-Glu model: inhibitory actions of each sample were examined in the reaction model between HSA and glucose. The results are expressed as ratio of IC50 when assumed aminoguanidine as 1.0. Caco-2 test: inhibitory actions of each sample which has been passed through the Caco-2 cell layer, using the HSA-Glu model. The results were expressed as ratio of percentage inhibition when assumed aminoguanidine as 1.0. Rat serum test: inhibitory actions of rat serum was examined in the HSA-Glu model after each sample has been orally applied and the serum obtained at 0-, 1- and 3-hour. Results are expressed as ratio of AUC of percentage inhibition for 3 hours when assumed aminoguanidine as 1.0. IC50, 50%inhibitory concentration; HSA, human serum albumin, Glu, glucose, AUC, area under curve.
半枝蓮 合歓皮 血竭 狗脊 連翹葉 [A] 香水薄荷 [A] 食用大黄 林檎 王林 林檎 信濃金 木瓜 薄紅葵 蕗 番紅花 果物時計草 発芽芽花野菜 目箒, 羅勒 陳皮 和蘭三葉[葉] 和蘭三葉 [茎] 西瓜 [皮] 食用菊 [A] 生姜 [A] 葉苣 [A] 屈, 草蘇鉄 [A] 馬芹 柿 [A] 銀杏葉
− − − − − − − − − − − − − − − − − − − − − − − − − − −
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
2.25 0.99 0.80 0.71 11.11 2.47 0.00 0.01 − 1.09 − 3.64 2.50 0.28 0.83 1.02 − 0.70 0.21 − 0.88 4.20 1.12 1.29 0.53 0.06 −
Scutellaria barbata Gôkanpi, Dragon's blood, xuè jié Cibotium rhizome Golden bell flower leaf [A] Lemon balm [A] Rhubarb Apple orin Apple Shinano-gold Japanese quince Blue mallow Butterbu Saffron Passion fruit Broccoli sprout Basil Citrus peel Celery [leaf] Celery [stalk] Watermelon [peel] Edible chrysanthemum 「A」 Ginger [A] Lollo rosso, Red-leaf lettuce Ostrich fern Cumin Persimmon Ginkgo biloba
Hanshiren Gokanhi Kekketsu Kuseki Rengyo-ha [A] Lemon balm [A] Rhubarb Ringo orin Ringo Shinano-gold Boke Usubeni-aoi Fuki Safuran Passion fruit Hatsuga broccoli Bajiru Chinpi Celery [ha] Celery [kuki] Suika [kawa] Shokuyo-kiku [A] Shoga [A] Sunny lettuce [A] Kogomi [A] Cumin Kaki [A] Icho ha
Scutellaria barbata Albizia julibrissin Durazz. Daemonorops draco Bl. Cibotium barometz (L.) J.Sm. Forsythia koreana Melissa officinalis Rheum rhabarbarum Malus domestica Malus domestica Chaenomeles speciosa Malva sylvestris Petasites japonicus (Siebold et Zucc.) Maxim. Crocus sativus Passiflora edulis Brassica oleracea var. italica Ocimum basilicum Citrus unshiu Apium graveolens var. dulce Apium graveolens var. dulce Citrullus lanatus Chrysanthemum morifolium Zingiber officinale Lactuca sativa var. crispa Matteuccia struthiopteris Cuminum cyminum Diospyros kaki Ginkgo biloba
Japanese nameEngilish nameChinese characterScientific nameHSA-Glu model (n = 3)Caco-2 test (n = 1)Rat serum test (n = 2 ~ 3)
0 100 200 300 400 500
0 1 2 3 4 5 6 7 8
No treat Control YO-treated KU-treated STZ-induced DM rats
Body weight (g)
Time (week)
0 1 2 3 4 5 6 7 8
0 10 20 30 40 50
Food intake (g)
No treat Control YO-treated KU-treated STZ-induced DM rats
Time (week) epithelial cells, using intestinal epithelial absorption models.
In consideration of potential commercial products, 11 sample materials were newly added: Sambucus sieboldiana, Prunus mume_flesh, Perilla frutescens, Olea europaea, Malva sylvestris, Malus domestica Shinano Gold, Ginkgo biloba, Fragaria vesca, Cynara scolymus, Citrus unshiu, and Citrullus lanatus_peel. The total number of sample materials in the second screening was 93 types of plants. Among these samples, 5 types had smaller IC50 than aminoguanidine:
Syzygium aromaticum A [1.55], Polygonum hydropiper [1.41], kuromoji_branch and leaf) [1.21], Helianthus annuus_
seed) [1.05], and Diospyros kaki_leaf [1.003]. Forsythia koreana showed strong in vitro activity to inhibit the AGE formation but extremely weak inhibition activity after permeating intestinal epithelial cells.
The third screening was the examination where 34 types of sample materials were assessed, taking into consideration absorption and metabolism of extracts in rats which test materials were administered to. Only 2 types, yomogi [1.16]
and Syzygium aromaticum-Choji_A [1.08] had stronger AGE inhibition than aminoguanidine of the materials which were absorbed and metabolized inside rat serum. Next, Rhus javanica [0.78], kuromoji_branch [0.755], Angelica pubescens Maxim.f. biserrata Shan et Yuan [0.754] and Eucommia ulmoides [0.748] were shown. Kuromoji_branch and leaf showed relatively strong activity [1.21] in intestinal epithelium absorption models but low activity [0.18] in the rat absorption test.
2. Evaluation of streptozotocin (STZ)-induced diabetic rats
To contemplate future application of functional food and the results of the screenings, yomogi and kuromoji were selected to examine the effectiveness for glycative-stress- induced disorders.
Fig. 1, 2 show the transitions of weight and intake volume following STZ treatment. There was no significant difference
Fig. 1. Change of body weight in the STZ-induced diabetic rats.
No treatment group (n = 8), STZ-treated group (n = 8), STZ & YO-treated group (n = 8) and STZ & KU-treated group (n =7).
Results are expressed as mean ± SD (shown in STZ-treated group and STZ & LU-treated group). STZ, streptozotocin; YO, yomogi (Japanese mugwort; Artemisia indica var. maximowiczii); KU, kuromoji (Lindera umbellata); SD, standard deviation.
Fig. 2. Change of food intake amount in the STZ-induced diabetic rats.
No treatment group (n = 8), STZ-treated group (n = 8), STZ & YO-treated group (n = 8) and STZ & KU-treated group (n =7).
Results are expressed as mean ± SD (shown in STZ-treated group and STZ & KU-treated group). STZ, streptozotocin; YO, yomogi (Japanese mugwort; Artemisia indica var. maximowiczii); KU, kuromoji (Lindera umbellata); SD, standard deviation.
0 200 400 600 800 1000 1200 1400 1600 1800 2000
0 2 4 6 8 10 12 14 16 18
GLU GA HbA1c AMY TG FFA
No treat Control YO-treated KU-treated
*
*
**
GLU/TG (mg/dL)・AMY (IU/L) FFA (µEq/L) GA (%) ・HbA1c (IU/L)
STZ-induced DM rats
Weight TNF-α IL-6 BUN CRE CCr
Kidney Serum
* *
*
*
0 1 2 3 4 5 6 7 8 9 10
0 10 20 30 40 50 60 70 80 90 100
Weight (g/100g bw) ・TNF-α/IL-6 (ng/mg pt） CRE (mg/dL）・ CCr (mL/min) BUN (mg/dL）
No treat Control YO-treated KU-treated STZ-induced DM rats
in weight and intake amount among three groups: the control group, the group with the administration of yomogi and the group with the administration of kuromoji.
Fig. 3 shows the results of blood tests eight weeks after the administration of test materials. There was no significant difference in Glu, GA and HbA1c between the control, the yomogi group and the kuromoji group. As for the serum AMY, the kuromoji group (1,224 ± 176 IU/L) showed significantly higher than the control (1,009 ± 134 IU/L, p < 0.05). As for the serum TG, the kuromoji group (217 ± 95 mg/dL) showed significantly lower than the control (379 ± 150 mg/dL, p <
0.01). As for serum FFA, the kuromoji group (512 ± 114 μEq/
L) showed significantly lower than the control (811 ± 126 μEq/L, p < 0.05).
Fig. 4 shows the results of the renal function tests (BUN, CRE and CCr) and TNF-α and lL-6 in renal tissues, which were conducted 8 weeks after administration of test materials.
As for renal weight, the kuromoji group (0.91 ± 0.10 g/100g body weight) was significantly lighter than the control (1.02 ± 0.06 g/100g body weight, p < 0.05). As for the serum TNF-α and lL-6 in renal tissues, the kuromoji group (TNF-α: 0.72 ± 0.20 ng/mg protein, IL-6: 5.68 ± 1.60 ng/mg protein) showed significantly lower than the control (TNF-α:
Fig. 3. The values of blood markers after 8 week treatment by kuromoji and yomogi in the STZ-induced diabetic rats.
Results are expressed as mean ± SD. No treatment group (n = 8), STZ-treated group (n = 8), STZ & YO-treated group (n = 8) and STZ &
KU-treated group (n =7). GLU, glucose; GA, glycoalbumin; AMY, amylase; TG, triglyceride; FFA, free fatty acid; STZ, streptozotocin; YO, yomogi (Japanese mugwort; Artemisia indica var. maximowiczii); KU, kuromoji (Lindera umbellata); SD, standard deviation.
Fig. 4. The renal function index after 8 week treatment by kuromoji and yomogi in the STZ-induced diabetic rats.
Results are expressed as mean ± SD. No treatment group (n = 8), STZ-treated group (n = 8), STZ & YO-treated group (n = 8) and STZ &
KU-treated group (n =7). TNFα, tumor necrosis factor-α; IL-6, interleukin-6; BUN, blood urea nitrogen; CRE, creatinine; CCr, creatinine clearance; STZ, streptozotocin; YO, yomogi (Japanese mugwort; Artemisia indica var. maximowiczii); KU, kuromoji (Lindera umbellata);
SD, standard deviation.
0%
20%
40%
60%
80%
100%
Cataract stage
Stage 3 Stage 2 Stage 1 Stage 0
Right Left Right Left Right Left
Control YO-treated KU-treated
STZ-induced DM rats
Fig. 5. Cataract prevention by kuromoji and yomogi (Japanese mugwort).
The graph shows the ratio of each stage of cataract in the STZ-treated rats. STZ-treated group (n = 8), STZ & YO-treated group (n = 8) and STZ & KU-treated group (n =7). Stages are classified as follows:
Stage 0, normal; Stage 1, early phase; Stage 2, inmatured phase; Stage 3, matured phase.
0.98 ± 0.21 ng/mg protein, IL-6: 7.51 ± 1.27 ng/mg protein, p < 0.05) and inflammatory cytokine in the tissue was kept low. There was no significant difference in serum BUN and CRE. The kuromoji group showed a significantly higher CCr value (2.57 ± 0.14 mL/min) than that of the control (2.12 ± 0.38 mL/min, p < 0.05). The renal function was maintained.
There was no significant difference in the above- mentioned index between the yomogi group and the control.
Fig. 5 shows assessments of the cataract stage 8 weeks after the administration of test material. The control had a higher ratio of cataract stage 2 (immature cataract), while the kuromoji group and the yomogi group had a higher ratio in cataract stage 0 (normal) and stage 1 (early cataract), which indicates that cataracts were light, slight or mild.
Discussion
Kuromoji
Kuromoji tree belongs to the Lauraceae, or laurel family and is indigenous to Japan, which is widely distributed from Kyushu to Hokkaido in Japan. The trunks contain an aromatic essential oil component and the branches are used as herbal medicine called Usho. Usho has various beneficial drug effects to improve the alimentary system under the following conditions: kakuran which means ictus solis or illness in summer, senki, which means abdominal pain, fukucho, which means sense of distension, and shukujiki, which means maldigestion. Also, the branches and leaves emit a refreshing fragrance and are used as a material for high- end toothpicks. Kuromoji essential oil, which is extracted by steam distillation from branches and leaves, is used as perfume and soap. Kuromoji essential oil contains linalool,
cineol and geraniol 7, 8). These oil components also contribute to forest bath effect 9,10).
It has been reported that kuromoji essential oil provides diversified effects, such as anti-tumor activity 11), anti-inflammatory activity 12), mentally and physically relaxing activity 10,13,14), stress-relief activity 15,16), anti-bacterial activity 17) on various bacteria, periodontal disease bacteria, and candida. Kuromoji essential oil has a less potent anti- oxidative activity 18) than the essential oil of cypress leaves, cypress foliage and cryptomeria leaves. It has been reported that analysis of electro cardiogram examinations revealed an increase in parasympathetic nerve system activities, a decrease in sympathetic nervous system activities, an increase in salivary secretion 14) and a significant decrease in ratio of α wave of electroencephalogram 13), which enhances the dominance of parasympathetic nerve system activities.
Kuromoji leaves contains linderatin 18) of the dihydrochalcone strain and linderatone 19) of flavanones. Kuromoji branches contain kaempferol, quercetin, afzerin, avicularin, hyperin, isoquercitrin and rutin as flavanones 20). Tannin contained by kuromoji extracts is reported to have anti-pepsin activity and anti-tumor activity 21). Hexahydrodibenzofuran derivative of kuromoji branch extracts has functions to control the production of melanin pigments 22).
This study confirmed that kuromoji has, other than the activities mentioned above, inhibitory activities against the formation of in vitro AGEs in the HSA-Glu reaction model.
Furthermore, it was shown that these activities were exerted even after the permeation of intestinal epithelium and after digestion and absorption in rats. Also, kuromoji extracts exerted inhibitory activities against the AGEs formation and showed effects resulting in prevention and progression of nephropathy and cataract formation after kuromoji extracts were administered to streptozotocin-induced diabetic rats.
It has been reported that cataract formation deeply relates
to glycative reactions of crystallin protein in the crystalline lens. Trapa japonica extracts are reported to inhibit glycation of crystalline by glyoxal treatment. The data of kuromoji extracts showed the inhibitory effects on cataract formation and there is a possibility that kuromoji may have a similar mechanism to inhibit the glycation of crystallin protein 23). Kuromoji administration elevated amylase values, which had been reduced by STZ treatments. Pancreatic amylase reflects pancreatic function. Thus, it was assumed that the administration of kuromoji could improve pancreatic functions, which were lowered by STZ treatment.
Glycative stress shows the physical states where aldehyde is likely to be produced 24,25): diabetes, dyslipidemia, metabolic syndrome, excessive alcohol intake and chronic kidney disease (CKD). STZ-induced rats showed high values in Glu, TG and FFA. Aldehyde, which resulted from these conditions, reacts with protein in the body and initial indexes of glycation reaction such as GA and HbA1c became high, which induces the formation and accumulation of AGEs. Furthermore, AGEs bond with RAGE (Receptor for AGEs) on the cell surface of macrophage system. Signal activation of AGEs/RAGE mediates the production of inflammatory cytokine. Thus, tissue injuries, such as nephropathy and cataracts, could be improved. Pocyanidin B2, which is contained in kuromoji extracts, inhibits the production of IL-6 and TNF-α in macrophage culture stimulated by lipopolysaccharide 12). This finding and view supports the results of examination where IL-6 and TNF-α were significantly lowered in the renal glands of diabetic rats of the kuromoji group. In addition of kuromoji inhibitory activities against the formation of AGEs, there was a possibility that kuromoji also inhibited the activation of AGEs/RAGE signal. Further examinations need to be conducted in the future.
Yomogi
Yomogi is a perennial of the asteraceae family and is used diversely. The leaves are used as yomogi-mochi, glutinouns rice dumpling with a green color and a characteristic flavor, and dried leaves are made into moxa for kyu, moxibustion 26, 27). Yomogi is used as shoyaku, a type of herbal medicine. The herbal medicine is called gaiyo, which has the functions of a blood coagulant and improves problematic symptoms during pregnancy. Kyukikyogaito has functional effects in the cases of continuous vaginal bleeding, hematemesis after a miscarriage and abdominal pain during pregnancy. Hakuyoto is administered for hematemesis and hemorrhage. It is reported that skin external agents containing yomogi extracts have anti-pruritic effects 28-38) and mitigating atopic dermatitis 39-42). Yomogi extracts are widely used for skin external products such as soap 43) and cosmetics.
The chemical components of yomogi were essential oil, flavonoid, polyphenol and others 27). The main components in the flavor are the following: cineol, thujone, β-caryophyllene, borneol and camphor (these 5 components are terpenoids 44)), and in addition, nonacosane, and hentriacontane (these two are hydrocarbons 45)). Apart from that, yomogi contains tannin, palmitic acid, oleic acid, and linoleic acid (these are fatty acids), and vitamin A, B1 and B2 46).
Yomogi leaf extracts have been reported to have choleretic action 47), growth stimulation of vascular endothelial cells 48), inhibitory activity against angiotensin-converting enzyme (ACE) 49), reductive activity on age-related body odor and uraroma 50), and anti-inflammatory activity 51). Our research center has confirmed that polyphenol contained in yomogi
extracts has inhibitory effect against the formation of
AGEs 3, 52). Caffeic acid and chlorogenic acid, which are isolated
as tannin from yomogi extracts, have inhibitory activities against lipid peroxidation 53-55), improving actions for lipometabolism (decrease in TG and decrease in lipid peroxide) 53, 54) and inhibitory activities against histamines disengagement 53, 55). These stabilization activities are characteristic to yomogi, which is the reason that yomogi is used for the purpose of inhibiting atopic dermatitis and skin itching.
In comparison between yomogi and kuromoji, both have the same level of inhibitory effect against the formation of AGEs. However, yomogi has an advantage in stabilizing mast cells and kuromoji exerted an anti-inflammatory effect against macrophage. It is assumed that these differences produced the results in this examination where kuromoji significantly decreased cytokine in renal tissues but yomogi did not show a significant difference.
Conclusion
As a part of the SIP Program, this study performed screenings and selections from five hundred and 36 agricultural, forest and fishery materials to evaluate plant- derived materials, focusing on the inhibitory effects against the formation in vitro fluoresce AGEs, permeability in intestinal epithelium and absorption activities in model rats.
Selection of kuromoji extracts and yomogi extracts was performed to identify materials, focusing on domestic availability of materials and application to functional food production. The resulting data of the examination of STZ- induced diabetes mellitus model rats revealed that kuromoji extracts had effects on improving TG and FFA and preventing the onset and progression of diabetic nephropathy. Furthermore, effects to prevent the onset and progression of cataracts were also recognized in kuromoji and yomogi extracts. We have decided to continue further examination of kuromoji, taking it into consideration to prepare for social implementation of commercialized products.
Acknowledgement
This work was supported by the Japanese Council for Science, Technology and Innovation, SIP (Project ID 14533567), “Technologies for creating next-generation agriculture, forestry and fisheries” (funding agency: Bio- oriented Technology Research Advancement Institution, NARO).
Conflict of Interest Statement
The present study was supported by a SIP cooperative company, Yomeishu Seizo Co. Ltd.
Reference
1) Hori M, Yagi M, Nomoto K, et al. Inhibition of advanced glycation end product formation by herbal teas and its relation to anti-skin aging. Anti-Aging Med. 2012; 9: 135- 2) Parengkuan L, Yagi M, Matsushima M, et al. Anti-glycation148.
activity of various fruits. Anti-Aging Med. 2013; 10: 70-76.
3) Ishioka Y, Yagi M, Ogura M, et al. Antiglycation effect of various vegetables: Inhibition of advanced glycation end product formation in glucose and human serum albumin reaction system. Glycative Stress Res. 2015; 2: 22-34.
4) Okawara M, Tokudome Y, Todo H, et al. Effect of β-cyclodextrin derivatives on the diosgenin absorption in Caco-2 cell monolayer and rats. Biol Pharm Bull. 2014;
37: 54-59.
5) Tomono T, Otsuka K, Yano K, et al. Recommended daily dose of sesame lignans has no influence on oral absorption of P-glycoprotein substrates in rats. Biol Pharm Bull. 2015;
38: 1960-1963.
6) Ikeda I, Tamakuni K, Sakuma T, et al. Resistant maltodextrin decreases micellar solubility of lipids and diffusion of bile salt micelles and suppresses incorporation
of micellar fatty acids into Caco-2 cells. J Nutr Sci Vitaminol (Tokyo). 2016; 62: 335-340.
7) Kuroda M, Sakurai K, Mimaki Y. Chemical constituents of the stems and twigs of Lindera umbellata. J Nat Med.
2011; 65: 198-201.
8) Kim KH, Moon E, Ha SK, et al. Bioactive lignan constituents from the twigs of Lindera glauca. Chem Pharm Bull (Tokyo). 2014; 62: 1136-1140.
9) Nonaka S, Otani K, Nitta N, et al. Study of healing by the fragrance of the forest at Kumano-Kodo. Annual report of Wakayama Prefectural Research Center of Environment and Public Health. 2009; 54: 25-28. (in Japanese)
10) Akakabe Y. The relaxation effect of aroma in forest bath (shinrinyoku) for human. Aroma Research. 2010; 11: 156- 161. (in Japanese)
11) Maeda H, Yamazaki M, Katagata Y. Kuromoji (Lindera umbellata) essential oil-induced apoptosis and differentiation in human leukemia HL-60 cells. Exp Ther Med. 2012; 3: 49-52.
12) Maeda H, Yamazaki M, Katagata Y. Kuromoji (Lindera umbellata) essential oil inhibits LPS-induced inflammation in RAW 264.7 cells. Biosci Biotechnol Biochem. 2013; 77:
482-486.
13) Akakabe Y, Kamikawa T. The relaxation effect of the bark oil of Lindera umbellata. The Japanese Journal of Taste
and Smell Research. 2011; 18: 591-594. (in Japanese) 14) Yamagami A, Imura H, Shibuya K, et al. Effects of aroma
on saliva secretion and activities of autonomic nerves and brain: Essential oil of kuromoji and bergamot. The Japanese Journal of Taste and Smell Research. 2012; 19:
453-456. (in Japanese)
15) Saito A, Sato K, Chiba R. Influence of personal preference for essential oil scents on physiological responses:
Comparison of essential oils from Cryptomeria japanica leaves and from Lindera umbellata branches and leaves Aroma Research. 2015; 16: 260-265. (in Japanese) 16) Tsutsui N, Igarashi K, Fukui K, et al. Stress relieving effect of kuromoji (Lindera umbellata) essential oil: Medical
aromatherapy for stress relief during dental treatment (part 1). The Journal of Japan Society for Dental Hygiene. 2017;
11: 73-84. (in Japanese)
17) Chiba R, Sato M, Tsuruta K, et al. Antibacterial activity of two Japanese endemic tree essential oils on oral bacteria and Candida. Aroma Research. 2016; 17: 376-380. (in Japanese)
18) Tanaka H, Ichino K, Ito K. A novel dihydrochalcone, linderatin from Lindera umbellata var. lancea. Chemical
& Pharmaceutical Bulletin. 1984; 32: 3747-3750.
19) Tanaka H, Ichino K, Ito K. A novel flavanone, linderatone, from Lindera umbellata. Chemical & Pharmaceutical Bulletin. 1985; 33: 2602-2604.
20) Takizawa N. Studies on the constituents of Lindera species: (I) On the flavonoid compounds of Lindera families. The Japanese Journal of Pharmacology. 1984;
38: 194-197. (in Japanese)
21) Kato M, Ezaki N, Tsutsumi S, et al. Pharmacological studies on Lindera umbellata ramus: (III) Effect of the crude
extracts of Lindera umbellata ramus on experimental gastric ulceration in rats. The Japanese Journal of Pharmacology. 1982; 36: 350-355. (in Japanese)
22) Mimaki Y, Kameyama A, Sashida Y, et al. A novel hexahydrodibenzofuran derivative with potent inhibitory activity on melanin biosynthesis of cultured B-16 melanoma cells from Lindera umbellata bark. Chem Pharm Bull
(Tokyo). 1995; 43: 893-895.
23) Uemura T, Takeshita S, Yamada M. The effectiveness of the peel extract of water chestnut (Trapa bispinosa Roxb.) in an α-crystallin glycation model with glyoxal. Glycative Stress Res. 2017; 4: 104-108.
24) Nagai R, Mori T, Yamamoto Y, et al. Significance of advanced glycation end products in aging-related disease.
Anti-Aging Med. 2010; 7: 112-119.
25) Ichihashi M, Yagi M, Nomoto K, et al. Glycation stress and photo-aging in skin. Anti-Aging Med. 2011; 8: 23-29.
26) Ishii R, Kuratome J, Ogino R, et al. The possible use of the Japanese mugwort in the traditional medicine. The Journal of Japan College Association of Oriental Medicine. 2010;
33: 26-30. (in Japanese)
27) Toda S. Research on antioxidative effect of yomogi. The Bulletin of Kansai University of Health Sciences. 2012; 6:
20-32. (in Japanese)
28) Hamashita Y, Mokuma M, Nishimura S. Nirsing care for “itching”: A trial of bed bith using yomogi (Japanese
mugwort). Nursing. 1982; 2: 1657-1661. (in Japanese) 29) Mokuma M, Hamashita S, Nishimura S. Continuing care
of patients complaining of being unable to sleep due to itching. Effect of bed bath with mugwort decoction for better sleep. Nursing. 1986; 6: 1068-1073. (in Japanese) 30) Mishima S, Miwa K, Komatsu H, et al. Active nursing
assistance to relieve the feeling of pruritus. Trial of cleaning with Artemisia vulgaris indica. Annual of Gifu Municipal Hospital. 1988; 8: 159-163. (in Japanese) 31) Nakashio S, Inoue K, Suganuma M, et al. Anti-itching
effect of Yomoneol on pruritus in patients with chronic renal failure. Progress in Medicine. 1989; 9: 1259-1263.
(in Japanese)
32) Urushibara Y, Yuki K, Sato S, et al. Trial application of yomogi lotion on the patients complain of itching. Zenjinkai Annual Research Report. 1990; 11: 75-77. (in Japanese) 33) Furube Y, Ikeuchi Y, Kato T, et al. On antipruritic effects
of mufwortlotion (Artemisia vulgaris indica lotion) using a soft extractive for external rse. Medicine and Drug Journal. 1990; 26: 115-118. (in Japanese)
34) Yuki W, Ikeuchi Y, Makino N, et al. Hemostatic effect of Artemisia vulgaris indica lotion for pruritus cutaneus of dialysis patient. Zenjinkai Annual Research Report. 1991;
12: 23-26. (in Japanese)
35) Tezuka T, Kusuda S, Higashida T, et al. The clinical effects of mugwort extract on pruritic skin lesions. Skin Research.
1993; 35: 303-311. (in Japanese)
36) Muroi N, Oishi R, Kimura S, et al. Antihistaminic activity of Artemisiae folium extract and its clinical effect on pruritus cutaneous. Journal of the Nippon Hospital Pharmacists Association. 1994; 20: 10-16. (in Japanese) 37) Mitsui R, Yoshii M. Preparation and clinical experience of
yomogi ointment for itching. Advances in Nursing Art and Science: ANAS. 1994; 6: 125-130. (in Japanese)
38) Matsuura K, Ogawa R, Koinuma M, et al. Antipruritic effect of mugwort lotion for postburn hypertrophic scar patients. Japan Journal of Burn Injuries. 2003; 29: 50-56.
(in Japanese)
39) Tamaki A, Muratsu M. Clinical trial of SY skin care series containing mugwort extract. Skin Research. 1994;
36: 369-378. (in Japanese)
40) Shoji A, Inoue A, Tani S, et al. Clinical trial of skin care compress containing mugwort extract. Skin Research.
1998; 40: 501-506. (in Japanese)
41) Sugiura M, Hayakawa R, Sugiura K, et al. Atopic dermatitis and chemicals: Application of yomogi-containing lotion
on dry scalp in the patients with atopic dermatitis. Skin Reserach. 2000; 42(Suppl.22): 1-4. (in Japanese)
42) Hioki C, Arai K, Arai M. Evaluation of volatile oil of Artemisia princeps for treatment of atopic dermatitis:
herbal ointment including volatile oils of four herbal medicines. Japanese Journal of Aromatherapy. 2010; 10:
25-32. (in Japanese)
43) Kanamori R. Verification of antibacterial actios of yomogi extract (Yomoneol). Infection Prevention (Kansen Boushi).
2002; 12: 35-38. (in Japanese)
44) Tsuchiya F, Yajima T, Kawaguchi T. Analysis of major constituents in essential oils stean-distilled from Chinese herbs II. The Journal of Holistic Sciences. 2012; 6: 1-8. (in Japanese)
45) Toda S, Ohnishi M, Kimura M, et al. Research on essential oil of moxa (No. 1). The Japan Society of Acupuncture and Moxibustion. 1988; 38: 330-333. (in Japanese)
46) Okuda T, Hatano T, Agata I, et al. Tannins in Artemisia montana. A. princeps and related species of plant.
Yakugaku Zasshi. 1986; 106: 894-899. (in Japanese) 47) Okuno I, Uchida K, Namba T. Choleretic activity of
Artemisia plants. Yakugaku Zasshi. 1984; 104: 384-389.
(in Japanese)
48) Kaji T, Kaga K, Miezi N, Ejiri N, Sakuragawa N. A stimulatory effect of Artemisia leaf extract on the proliferation of cultured endothelial cells. Chem Pharm Bull (Tokyo). 1990; 38: 538-540.
49) Kimoto K, Shimizu E, Kuroda H. Research on ACE inhibitors in food. Bulletin of Tokyo Kasei University.
1998; 38: 59-63. (in Japanese)
50) Osada K, Curran M, Kurachi M, et al. Effect of an orally ingested mugwort and mushroom extract mixture on urine odor from aged mice. Biosci Biotechnol Biochem. 2008;
72: 1249-1256.
51) Park JM, Han YM, Lee JS, et al. Nrf2-mediated mucoprotective and anti-inflammatory actions of Artemisia extracts led to attenuate stress related mucosal damages. J Clin Biochem Nutr. 2015; 56: 132-142.
52) Ishioka Y, Yagi M, Ogura M, et al. Polyphenol content of various vegetables: Relationship to antiglycation activity.
Glycative Stress Res. 2015; 2: 41-51.
53) Hatano T, Okuda T, Togawa Y. Tannin active ingredient of Mugwort class and its lipid peroxidation inhibitory effect.
Journal of Traditional Medicines. 1985; 2: 124-125. (in Japanese)
54) Kimura Y, Okuda H, Okuda T, et al. Studies on the activities of tannins and related compounds from medicinal plants and drugs. VII. Effects of extracts of leaves of
Artemisia species, and caffeic acid and chlorogenic acid on lipid metabolic injury in rats fed peroxidized oil. Chem Pharm Bull (Tokyo). 1985; 33: 2028-2034.
55) Kimura Y, Okuda H, Okuda T, et al. Studies on the activities of tannins and related compounds from medicinal plants
and drugs. VI. Inhibitory effects of caffeoylquinic acids on histamine release from rat peritoneal mast cells. Chem Pharm Bull (Tokyo). 1985; 33: 690-696.
