山陽学園短期大学紀要 第 45 巻(2014)

原著論文

Summary of the Results of the Natural Products
Chemistry Research Project at the Department of Foodand Nutrition of Sanyo Gakuen University-College
山陽学園大学・山陽学園短期大学における天然物化学研究プロジェクトの業績一覧
Kenny Kuchta
ケニー・クフタ 1
私は、ドイツのライプチヒ大学で、日本の漢方医学を基にした植物化学及び薬用植物の薬理学について、博士課程の研究プロジェクトを完了した後、（この研究で私はベルリンの日本大使館
によってドイツ語日本語友好賞 2011 を受賞した）。当時の山陽学園大学学長で医学博士である
赤木忠厚先生によって、補完代替医学の教育・研究を目的として 2012 年春に新たに設立された
山陽学園大学補完・代替医療教育研究センターの専任講師として招待された。
2013 年春に新学長實成文彦先生によって、山陽学園大学の教育及び研究政策が再編成され、私の研究部署は山陽学園短期大学、食物栄養学科、天然物化学研究に変更になった。
ライプチヒ大学と山陽学園のどちらにおいても、私は継続的に、互いに学びあう西洋と日本の両方で医師を支援し、東と西の両方で患者の健康と福祉を強化するため、日本の伝統的な漢方
医学とヨーロッパの伝統的な薬用植物学の間のギャップを埋めようとに努力してきた。
過去 3 年の間に、私は日本の中でも世界でも、山陽学園と多数の大学間の国内及び国際的な学術協力を確立することができた。私の母校ライプチヒ大学（ドイツ）に加えて、岡山理科大学（岡山）、明治国際医療大学（京都）、長崎国際大学（長崎）、及び長崎大学（長崎）日本における島根大学（出雲）だけでなく、ヌエボ・レオン州立自治大学（メキシコ）、広州中山大学（中国）、広州済南大学（中国）、浙江省農業・林業大学（中国）、ネゲブのベングリオン大学（イスラエル）、コ
ペンハーゲン大学（デンマーク）、ミュンスター大学（ドイツ）など世界中の大学で活動を行うことができた。
これらのプロジェクトの大半はまだ進行中であるが、過去 3 年間、すでに私の研究は国際薬用植物研究雑誌における様々な雑誌に論文を受理され、数々の国際科学会議で研究データの多数を日本語、英語、ドイツ語で口頭発表した。
――――――――――――――――――――――――――

1 山陽学園短期大学食物栄養学科
Department of Food and Nutrition, Sanyo Gakuen College-11-クフタ : 山陽学園大学・山陽学園短期大学における天然物化学研究プロジェクトの業績一覧

昨年 2014 年に、サンクトペテルブルク（ロシア）の薬用植物に関する国際会議とギマランイス（ポルトガル）における国際薬用植物天然産物研究学会（ＧＡ）の年次会議における研究結果のプレゼンテーションは、この年の私の研究で、最も重要なものであった。
これらの国際会議で、私は山陽学園大学での天然物化学研究プロジェクトの成果を説明し、サンクトペテルブルクでのロシア科学アカデミー薬学部門ポスター発表賞と、薬用植物及び天然産物の研究学会のエゴン・スタール賞メダルをそれぞれ受賞した。
以下のページでは、私は私の山陽学園で最も成功したプロジェクトと、それに対応する研究成果の簡単な説明が含まれている。これらの研究活動のほかに、私は、ヨーロッパでは日本の伝統的な漢方医学の使用と、欧州における伝統薬用植物学の使用を広めるためにも、時間を費やした[1]。
特にこの後者の点では、私は、西洋医学で治療する際のハーブの使用方法について、長崎国際大学では客員教授、金沢大学と京都府立医科大学では客員講師として、一連の講義を受け持っている。
After completing my doctoral research project on the phytochemistry andpharmacology of medicinal plants from Japanese Kampo medicine at Leipzig
University in Germany – for which I was awarded the German Japanese FriendshipAward 2011 by the Japanese embassy in Berlin – I was approached by the chancellorof Sanyo Gakuen University – Prof. Dr. T. Akagi – to join the newly established“Teaching and Research Center for Complementary and Alternative Medicine”（補完代 替 医 療 教 育 研 究 セ ン タ ー ） starting in Spring 2012. After a reorientation of theteaching and research policy at Sanyo Gakuen by the new chancellor Prof. Dr. F.
Jitsunari in spring 2013, my research positon was reassigned to the department offood and nutrition of Sanyo Gakuen University-College as the project for “Natural
Products Chemistry Research”（山陽学園短期大学、食物栄養学科、天然物化学研究）.
Both during my time at Leipzig University and at Sanyo Gakuen I havecontinuously strived to bridge the gap between traditional Japanese Kampo medicine
and European Traditional Medicinal Herbalism in order to help doctors both in theWest and in Japan to learn from each other and therefore to enhance the health andwellbeing of the patients in both East and West.
During the past three years, I was able to establish national and international
academic cooperation between Sanyo Gakuen and numerous universities both inside
Japan and around the world. Besides my alma mater Leipzig University (Germany),
these are Shimane University (Izumo), Okayama University of Science (Okayama),
Meiji University of Integrative Medicine (Kyoto), Nagasaki International University(Nagasaki), and Nagasaki University (Nagasaki) in Japan, as well as Universidad
Autónoma de Nuevo León (Mexico), Sun Yat-sen University Guangzhou (China),[1] フォルカー・フィンテルマン、ルードルフ・フリッツ・ヴァイス、三浦於菟、林真一郎、ケニー・クフタ、田中耕一郎:

植物療法フィトセラピー事典:産調出版株式会社,2012.

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Jinan University Guangzhou (China), Zhejiang Agricultural & Forestry University(China), Ben-Gurion University of the Negev (Israel), University of Copenhagen(Denmark), and University of Münster (Germany) around the world.
Whereas the majority of these projects are still ongoing, the past three years havealready resulted in a multitude of research data which I published in a series ofarticles in international medicinal plant research journals and presented in oral
communications in Japanese, English, and German language at numerous
international scientific conferences.
During the past year 2014 the presentations of these results at the international
conference PHYTOPHARM in St. Petersburg (Russia) and at the annual conferenceof the Society for Medicinal Plant and Natural Product Research in Guimarães
(Portugal) formed the highlights of my academic year. At these conferences, thebelow described results of my “Natural Products Chemistry Research” project at
Sanyo Gakuen University-College were awarded the poster presentation price of theRussian academy of Pharmacy at St-Petersburg and the Egon Stahl Award Medal of
the Society for Medicinal Plant and Natural Product Research, respectively.
On the following pages, I have included short descriptions of my most successfulprojects here at Sanyo Gakuen and the corresponding research results.
Besides these research activities, I have also tried to use some of my time to spreadthe use of traditional Japanese Kampo medicine in Europe and the use of European
Traditional Medicinal Herbalism in Japan [1]. Especially in this latter respect, I was
made Visiting Professor at Nagasaki International University and Visiting Lecturer
at both Kanazawa University and Kyoto Prefectural University of Medicine, where I
have given a series of lectures on the therapeutic use herbs of Western medicine.

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Egon Stahl Award ceremony, 1st of September 2014, Guimarães, Portugal

Egon Stahl Medal-14-

山陽学園短期大学紀要 第 45 巻(2014)

Open-label clinical pilot-study on an herbal multi-component topical TCM therapy
for atopic dermatitis and comorbid conditions
We recently reported the efficacy of a novel TCM therapy – consisting of both oral
and topical medications – against atopic dermatitis (AD) [1,2,3].
Building on these results, we developed improved galenic formulations of two
multi-component TCM extracts (overall 13 drugs, see Table 1) for purely topicalapplication.
All drugs were powdered and extracted in boiling water for 5 h. Extract 1 wasformulated as an herbal soap (1) with anti-inflammatory activity and used forwashing the affected areas. For extract 2, three distinct galenic formulations weredeveloped, namely a lotion (2A) with high skin penetrating activity and fast-acting
antipruritic effect; a gelatinous jelly (2B) favorable for the treatment of scratching
and rupture scars; and a Vaseline based ointment (2C).
In this open-label clinical pilot-study 129 AD patients were continuously treatedwith these topical TCM preparations. For evaluating their clinical efficacy,
standardized scores were used for the severities of both AD (clinical severity 0-4) andpruritus (pruritus score 0-4).
Both scores had significantly improved at the end of treatment after two months.
Additionally, empirical clinical data on the therapeutic efficacy concerning relatedcomorbid conditions like psoriasis, acne, alopecia, as well as fungal, bacterial, andviral skin infections were collected, demonstrating high therapeutic potentials in allthese conditions. None of the tested preparations did display any significant adverse
effects, facilitating even prolonged application on newborn infants.
The presented topical TCM preparations can be used safely and effectively in bothclinical AD therapy, self-medication of skin disorders, or even as cosmetics.

Li S, Kuchta K, Tamaru N, Lin Y, Iwasaki S, Wang R, Kobayashi Y, Rauwald HW, Kamei T:
Efficacy of a Novel Herbal Multicomponent Traditional Chinese Medicine Therapy Approach inPatients with Atopic Dermatitis. Forsch Komplementmed 2013; 20: 189-196[3] 李頌華、田丸直美、林宇、王如偉、ケニー・クフタ、佐藤直人、岩崎純夫、小林裕太：興味深い臨床報告 アト
ピー性皮膚炎 102 例における複方苦参製剤の外用効果。中医臨床 35: 46-50, 2014.
[2]

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クフタ : 山陽学園大学・山陽学園短期大学における天然物化学研究プロジェクトの業績一覧

Before onset of therapy, both face and neck were covered with striking erythemaand eczema, accompanied by ulceration of large skin areas. After 2 months oftherapy, the patient’s condition has remarkably improved.

Before treatment, the face displayed severe erythema and ulceration accompanied
by abundant bleeding.
After 2 months of treatment, remarkable improvement is obvious.

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山陽学園短期大学紀要 第 45 巻(2014)

TCM plant drugs used in extract 1, extract 2, or both of them, given together with their traditional indications for external use
according to Chin.Ph.; Luo HS, Luo DH. Immune Chinese Medicine. Beijing Medical University and China Union Medical University
Press, Beijing 1999; and Huang KT, Ding ZZ, Zhao SX. Modern Compendium of Materia Medica. Chinese Medical Science andTechnology Press, Beijing 2001.

2

Angelica sinensis

root

1

Chrysanthemum
indicum

flowers2

Citrus x limon

fruit
juice

1

Coptis chinensis

rhizomeJapanese- Chinese Name
トウキ
当帰
キクカ
菊花
レモン
檸檬
オウレン
黄連

2

Dryobalanops
aromatica

resin

ヒョウヘン
氷片

2

Glycyrrhiza
uralensisroot2

Isatis tinctoria

leaves

カンゾウ
甘草
ダイセイヨウ
大青葉

1Paris polyphylla

rhizome

シチヨウイッシカ
七叶一枝花

anti-inflammatory, detumescent,strongly antibacterial, anti-infective

2

Polygonumcuspidatum

rhizome1

Punica granatum

pericarp

コジョウ
虎杖
セキリュウヒ
石榴皮
オウゴン
黄芩
クジン
苦参
ビャクブコン百部根

heals ulcer, haemostatic,
antibacterial, anti-infective
astringent, heals ulcer, skin protective,
antimicrobial, antifungal, antiviralanti-inflammatory, antiallergenic,
antibacterial, antiviral

Extract

1+2
1+2
1

Scientific
Name

Scutellaria
baicalensis
Sophoraflavescens
Stemonasessilifolia

Plant
Part

root
root
root

Pharmacological Effect (TCM)

TCM Indications (External)Drugmaterial
per litre

improves blood circulationeczema, frostbite

10 ganti-inflammatory
skin protective, cosmetic, antiseptic,
antibiotic, deodorizing
anti-inflammatory, heals ulcer,
antibacterial
anti-inflammatory, antibacterial,
analgesic, relieves itching, promotes skinpermeation of other extracts
anti-inflammatory, heals ulcer,
antiallergenic
antipyretic, antibacterial, antiviral

antiallergenic, antipyretic, antifungal
relieves itching, antibacterial, antiviraleczema, skin infection,
cervicitis, mumps
skin protective, frostbite, cosmetic,
insect repellent
eczema, skin infection,
otitis externa, aphthous ulcer

5 ml

pruritus, skin ulcers, eczema,
periodontitis, otitis, hemorrhoids

10 geczema, psoriasis, skin ulcer

10 gchronic eczema,
epidemic mumps
eczema, bruising, swellings,
mumps, mastitis, snake and insect
bites
suppurative dermatitis, bleeding
wounds, scalding, snake bites
suppurative otitis media,
skin ulcer
eczema, psoriasis, hemorrhoids
eczema, skin infection,
pruritus vulvae
skin infection,
trichomoniasis, lice

Clinical score was quantitatively defined according to the guidelines of theJapanese Dermatological Association.

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12 g

12 g

20 g
12 g
20 g
12 g
12 g / 20g
12 g / 20g
12 g

クフタ : 山陽学園大学・山陽学園短期大学における天然物化学研究プロジェクトの業績一覧

A phytosterol enriched refined extract of Brassica campestris L. pollen significantly
improves benign prostatic hyperplasia (BPH) in a rat model [4]In Qinghai Province, the Brassica campestris L. pollen preparation Qianlie Kang
Pule'an Tablets (QKPT) is traditionally used for benign prostatic hyperplasia (BPH)therapy. However, in QKPT the content of supposedly active phytosterols is
relatively low at 2.59%, necessitating high doses for successful therapy.
A phytosterol enriched (4.54%) refined extract of B. campestris pollen (PE) was
developed and compared with QKPT in a BPH rat model.
Six groups of rats (n=8 each), namely sham operated distilled water control,
castrated distilled water control, castrated QKPT 2.0g/kg, castrated PE 0.1g/kg,
castrated PE 0.2g/kg, and castrated PE 0.4g/kg were intragastrically treated withthe respective daily dose. Testosterone propionate (0.3mg/day) was administered to
all castrated rats, while the sham operated group received placebo injections. After30 days, the animals were sacrificed and prostates as well as seminal vesicles excised
and weighted in order to calculate prostate index (PI) and seminal vesicle index(SVI), defined as organ weight in g per 100 g body weight.
Compared with sham-operated controls, both PI (p<0.01) and SVI (p<0.01) weresignificantly increased in all castrated rats. After treatment with PE at 0.4 and 0.2
g/kg or QKPT at 2.0 g/kg per day, both indices were significantly reduced (p<0.01) ascompared to the castrated distilled water control. For PE at 0.1 g/kg per day only PI
was significantly reduced (p<0.05). At the highest PE concentration of 0.4 g/kg per
day both PI and SVI were also significantly reduced when compared to the QKPT
group (p<0.05).
Both PE and QKPT demonstrated curative effects against BPH in the applied
animal model. In its highest dose at 0.4 g/kg per day, PE was clearly superior to
QKPT.

[4] Ruwei Wang, Yuta Kobayashi, Yu Lin, Hans Wilhelm Rauwald, Ling Fang, Hongxiang Qiao,

Kenny Kuchta: A phytosterol enriched refined extract of Brassica campestris L. pollen
significantly improves benign prostatic hyperplasia (BPH) in a rat model as compared to theclassical TCM pollen preparation Qianlie Kang Pule’an Tablets. Phytomedicine 22: 145-152,
2015.

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山陽学園短期大学紀要 第 45 巻(2014)

Flowering Brassica campestris fields in Chengdong, Qinghai Province (青海省),
China (left); this region is also famous for bee farming. The pollen is collected fromthe flowers by the bees (right) and later harvested from their hives.

青海省
Qinghai

Histomorphologic changes in the rat prostates stained with haematoxylin and eosin(magnification: x400). Sham operated distiled water control group (A), castrated
distilled water control group (B), castrated QKPT 2.0g/kg group (C), castrated highdose PE 0.4g/kg group (D), castrated mid-dose PE 0.2g/kg group (E), and castrated
low-dose PE 0.1g/kg group (F).

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クフタ : 山陽学園大学・山陽学園短期大学における天然物化学研究プロジェクトの業績一覧

Left: Bar chart of the measured values for the prostate index (PI) among theindividual groups of experimental animals (n = 8) in this study, displaying highreproducibility of the physiological effects of the examined pollen preparations. ##p <0.01 as compared with sham-operated distilled water control group; *p < 0.05; **p <0.01 as compared with castrated distilled water control group; $p < .05 as com-paredwith castrated QKPT 2.0 g/kg group.
Right: Bar chart of the measured values for the seminal vesicle index (SVI) among
the individual groups of experimental animals (n = 8) in this study, displaying highreproducibility of the physiological effects of the examined pollen preparations. ##p <0.01 as compared with sham-operated distilled water control group; **p < 0.01ascompared with castrated distilled water control group; $p < 0.05 as compared withcastrated QKPT 2.0 g/kg group.
Curcumin induces apoptosis in hepatic stellate cells by modulating the expression of
apoptosis related growth factors
Turmeric (Curcuma longa L.) and its phenolic constituent curcumin exert bothpreventive and curative effects on hepatic fibrosis in rat-models, inducing apoptosis
and inhibiting proliferation of hepatic stellate cells (HSC).
Cultured rat HSCs (HSC-T6) were incubated with 10, 20, 30, and 40 µM of
curcumin for 24 h, after which apoptosis was measured by flow-cytometry incomparison to a negative-control cultured without curcumin treatment. Expressionof the pro-apoptotic factors Fas and p53b as well as the anti-apoptotic factor Bcl-2
was monitored by immunocytochemical ABC staining.
After incubation with 20, 30, or 40 µM curcumin for 24 h, the respective apoptosis
indices were 11.6±2.8%, 52.0±4.4%, and 87.1±22.6%, all significantly higher than thenegative-control group with 3.8±0.6% (p<0.01). Incubation with 10 µM yielded
6.5±1.88% thus not exhibiting the same level of statistical significance. Afterincubation with 20 µM curcumin for 24 h, the expression rates of the pro-apoptotic
factors Fas and p53 were increased to 87.4±2.8% and 43.1±7.3%, as compared to therespective negative-control values of 53.1±5.1% and 20.6±7.2%. The expression rate-20-

山陽学園短期大学紀要 第 45 巻(2014)of the anti-apoptotic factor Bcl-2 was decreased to 28.7±5.9% as compared to thenegative-control with 95.4±3.6%. All these effects of 20 µM curcumin were highly
reproducible (p<0.05), in contrast to those of the 10 µM sample. In the case of the 30
and 40 µM samples, apoptosis occurred so rapidly that the ABC staining could not be
carried out properly.
Curcumin has an up-regulating effect on pro-apoptotic factors like Fas and p53 as
well as a down-regulating one of the anti-apoptotic factor Bcl-2, thus inducing
apoptosis in HSC.

クルクミン / Curcumin

Expression of Fas (200 X). (A) HSCs of the control group received no treatment. (B)
HSCs treated with 20 µM curcumin. Brown gains, which reveal expression of Fas inboth membrane and cytosol (arrows), are significantly more numerous in B, proving
an increase in expression of Fas as compared to the control group.

Expression of p53 (200 X). (A) HSCs of the control group received no treatment. (B)
HSCs treated with 20 µM curcumn. Brown grains, which reveal expression of p53 in
the nuclei (arrows), are significantly more numerous in B, proving an increase in
expression of p53 as compared to the control group.

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クフタ : 山陽学園大学・山陽学園短期大学における天然物化学研究プロジェクトの業績一覧

Expression of Bcl-2 (200 X). (A) HSCs of the control group received no treatment.
(B) HSCs treated with 20 µM curcuin. Brown grains, which reveal expression of Bcl-2
at the cell membrane (arrows), are significantly more numerous in A, proving an
decrease in expression of Bcl-2 as compared to the control group.
Expression rates of Fas, p53, and Bcl-2 following curcumin treatment (20 µM).
* = P < 0.05 as compared with the control group
GroupsControl group
Curcumin group

Expression rate
Fas

p53

Bcl-2

33.1 ± 3.1 %87.4 ± 2.8 %*20.6 ± 7.2 %43.1 ± 7.3 %*95.4 ± 3.6 %28.7 ± 5.9 %*Randomized cross-over trial on mood enhancing effects of bergamot (Citrus bergamia(Risso) Wright & Arn.) volatile oil vapor, also regarding personality and lifestyle
related changes in salivary cortisol levels [5,6]
Bergamot essential oil (BEO) is widely used in aromatherapy for chronic stressand anxiety.
As clinical data for these indications are still insufficient, we conducted arandomized cross-over trial in human subjects.
Data were collected under three testing conditions – rest (R), rest + water vapor(RW), rest + water vapor + BEO (RWB) – for 15 min each in 42 healthy women.
Heart rate variability was recorded continuously, calculating its high-frequency
component (HF), and saliva samples were collected for determination of salivary[5] 渡邉映理、木村真理、ケニー・クフタ、亀井勉、今西二郎：ベルガモット精油による芳香浴の自律神経系および情動に及ぼす効果。Aroma Research 54: 150-154, 2013.
[6] Eri Watanabe, Kenny Kuchta, Mari Kimura, Hans Wilhelm Rauwald, Tsutomu Kamei, Jiro

Imanishi: Effects of bergamot (Citrus bergamia (Risso) Wright & Arn.) essential oil
aromatherapy on mood states, parasympathetic nervous system activity, and salivary cortisol
levels in 41 healthy females. Research in Complementary Medicine 22: 43-49, 2015.

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山陽学園短期大学紀要 第 45 巻(2014)

cortisol (CS) levels via ELISA after each test, while the subjects were given 10 min ofrest and an additional 10 min to fill out psychological questionnaires.
In comparison to the pure water placebo, the BEO vapor data show a significant
increase in HF heart rate components (p=0.009), indicating increased activity of theautonomous nervous system, as well as a decrease in confusion (p=0.035) and fatigue(p=0.042), paired with enhanced vigor of mood (p=0.017). CS levels decreased in 32
subjects (CS-D group) but increased in the remaining 10 (CS-I group). When
comparing the lifestyles of this CS-I group with those of the of the CS-D group, theformer were significantly more ordered (t=-2.08, p=0.044) and values for extraversion(t=1.92, p=0.048), and optimism (t=2.17, p=0.035) were higher, while their feelings offatigue (t=-2.61, p=0.012) were lower.
BEO, when inhaled after dispersion in water vapor, exhibits swift psychologicaland physiological effects favorable for the therapy of chronic fatigue, stress, andanxiety. These effects are influenced by the individual lifestyle of the subject.

Bergamot (Citrus bergamia (Risso) Wright & Arn.): Photography (left) andillustration from a 19th century German herbal (Köhler's Medizinal-Pflanzen Atlas,1887) (middle). In the present randomized cross-over trial, we used Bergamot
essential oil (BEO), which was dispersed in the air together with water vapour using
an ultrasound diffusor from “Seikatsu no Ki” (right).

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クフタ : 山陽学園大学・山陽学園短期大学における天然物化学研究プロジェクトの業績一覧

Effects of Leonurus japonicus Houtt. and its N-containing constituents leonurine andstachydrine on the activity of PPARα, β/δ, and γ in a newly developed in vitro
luciferase reporter gene assay

Leonurus japonicus (Yimucao; Chin.Ph., DAB) has been used in TCM since earliest
times for conditions presently referred to as the “metabolic syndrome”. Here, theagonistic activity of aqueous Yimucao extracts – both from China and from German
TCM plant cultivation – and their dominant constituents leonurine and stachydrine[7,8] onthe metabolic syndrome related peroxisome proliferator-activated receptors[7] Kuchta K, Ortwein J, Rauwald HW (2012) Leonurus japonicus, Leonurus cardiaca,

Leonotis leonurus: A novel HPLC study on the occurrence and content of thepharmacologically active guanidino derivative leonurine. Pharmazie 67: 973-979.
[8] Kuchta K, Ortwein J, Hennig L, Rauwald HW (2014) 1H-qNMR for Direct Quantification of
Stachydrine in Leonurus japonicus and L. cardiaca. Fitoterapia 96: 8-17.

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山陽学園短期大学紀要 第 45 巻(2014)(PPAR) α, β/δ, and γ was investigated using a novel luciferase reporter gene assay.
COS-1 cells were co-transfected with the luciferase reporter plasmid p17m2G,
containing a GAL4 upstream activating sequence in the promoter region, an
expression vector for the human PPARβ/δ ligand-binding domain fused to the GAL4
DNA-binding domain pPPARβ/δ-GAL4, and the secreted alkaline phosphatase
control vector pSEAP. The results are relative to the luciferase expression levels,
which were normalized using secreted alkaline phosphatase (SEAP) activity. The two

L. japonicus extracts and the two isolated constituents (all at 6.25, 25, and 100
μg/ml), and GW0742 (positive control, 0.1 nM) were dissolved in DMSO and added tothe medium of the transfected cells. The same approach was used for PPARα andPPARγ in which case the COS-1 cells were transfected with pPPARα or γ-GAL4 andp17m2G, respectively (positive controls 50 μM WY14643 / 10 μM troglitazone).
Whereas significant activity was measured for the Chinese Yimucao extract in all
three PPAR assays at both 100 and to a lesser extent 25 μg/ml, no activity above
DMSO vehicle negative control levels could be detected for the sample from German
cultivation or for the two N-containing compounds. Thus, the agonistic activity ofChinese Yimucao in all three PPARs indicates its potential for diabetes andmetabolic syndrome therapy. However, the still unidentified active constituents
seem to be absent from the German cultivated drug.

O

Leonuri

O

NH

NH

O

H 3C

O

HO
N

O
H 3C

H 3C

+

Stachydr
CH 3

OH

-25-

NH 2

クフタ : 山陽学園大学・山陽学園短期大学における天然物化学研究プロジェクトの業績一覧

Ginkgolic acids in Ginkgo biloba L. leaf TCM extracts: Simultaneous HPLC
quantification of all five derivatives using ginkgoneolic acid as a single marker
compound [9]A new HPLC quantification method for allergenic ginkgolic acids (GA) in Ginkgo

biloba leaf extracts (GBE) was developed, using an acetonitrile-water gradientelution system with an Agilent-SB C18 column. With ginkgoneolic acid (13:0 GA) as
a marker, the relative correlation factors of the four other GAs - 15:0, 15:1, 17:1, and17:2 GA – to 13:0 GA were determined by HPLC and subsequently used for
calculating their contents in 10 G. biloba leaf samples from Shandong, which wereextracted according to the Chin.Ph. monograph for TCM GBE (not identical to thecorresponding Ph.Eur. monograph). In other words, the content of 13:0 GA in the[9] Ruwei Wang, Yuta Kobayashi, Yu Lin, Hans Wilhelm Rauwald, Jianbiao Yao, Ling Fang,
Hongxiang Qiao, Kenny Kuchta: HPLC Quantification of All Five Ginkgolic Acid Derivatives inGinkgo biloba Extracts using 13 : 0 Ginkgolic Acid as a Single Marker Compound. Planta Med
81: 71-78, 2015.

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山陽学園短期大学紀要 第 45 巻(2014)

extracts was determined using the pure compound as an external standard.
Subsequently the now known concentration of this compound functioned as aninternal standard for the quantification of the other four GA derivatives via therelative correlation factors. The new HPLC method was validated by control
measurements with external standards for each individual GA. The results did not
differ significantly for any of the 10 leaf samples. Additionally, 10 commercial GBE(9 Chin.Ph., 1 Ph.Eur.) preparations were tested with the new method, incomparison to the current Chin.Ph. protocol. Although all 9 TCM extracts were inaccordance with the regulation of a maximal content of 10 ppm GA when tested with
the Chin.Ph. HPLC method, only two of these were below this concentration whentested with the new protocol (2.41 vs 9.76 ppm / 2.49 vs 9.52 ppm), demonstratingthat in many cases TCM GBE preparations on the Chinese market containconsiderable amounts of GA that are not detected by the present Chin.Ph. HPLC.
For the Ph.Eur. extract, 0.81 vs 4.87 ppm were measured, both in agreement withthe maximal content of 5 ppm GA according to Ph.Eur.. The newly developed
protocol is therefore simple, reproducible, and can be used to determine the total
contents of GA derivatives in G. biloba leaf extracts.

The five tested ginkgolic acids (1 was also used as marker compound) which are also
known by the trivial names ginkgoneolic acid (C13:0) (1), hydroginkgolic acid (C15:0)(2), ginkgolic acid I (C15:1) (3), ginkgolic acid II (C17:1) (4), and anacardic acid c(C17:2) (5). All double bonds in Z configuration.

3
1

4

5
2

1

3
2

5

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4

クフタ : 山陽学園大学・山陽学園短期大学における天然物化学研究プロジェクトの業績一覧

Representative chromatograms of both the mixed reference standards solution (A)and a typical GBE test sample (lot No. 120418) (B), using the most favourable
Agilent SB-C18 column with the respective Agilent 1260 instrument and an Agilent
SB C18 (150 mm × 4.6 mm, 5 μm) column (column temperature: 30°C) as well as a
gradient elution system with a 0.1% solution of trifluoroacetic acid (TFA) in
acetonitrile as component A and a 0.1% solution of TFA in water as component B.
The flow rate was kept constant at 1 mL min-1 while the gradient developed overtime as follows: t = 0 min: A = 75%; B = 25% / t = 30 min: A = 90%; B = 10% / t = 35
min; A = 90%; B = 10%. The injection volume was set at 50 μL and the detection
wavelength at 210 nm. The peaks of the individual tested GA constituents (C13:0) (1),
(C15:1) (3), (C17:2) (5), (C15:0) (2), (C17:1) (4) are marked by the respective numbers.

Contents of GAs in the examined GBE samples, measured via individual external
standards (ES), and by using 13:0 ginkgolic acid (C13:0) as a single marker
compound (SMC). ● C17:1, ● C15:0, ● C17:2, ● C15:1, and ● C13:0. The upper line
represents the limit of 10 ppm GAs according to Chin.Ph. while the lower line
represents the limit of 5 ppm GAs according to Ph.Eur..

Ginkgo biloba L. leaves and fruits-28-