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Journal of Ethnopharmacology 132 (2010) 438–442

Contents lists available at ScienceDirect

Journal of Ethnopharmacology

journa l homepage: www.e lsev ier .com/ locate / je thpharm

he relation between clinical effects of Tokishakuyakusan and the identity ofaeonia lactiflora materials

ayoko Shimadaa, Masaya Kawaseb, Naotoshi Shibaharac, Yuto Nakamuraa,adashi Saitod, Kyoko Takahashia,e,∗

Graduate School of Pharmaceutical Sciences, Osaka University, 1-6, Yamada-oka, Suita, Osaka 565-0871, JapanNagahama Institute of Bio-Science and Technology, 1266, Tamura-cho, Nagahama, Shiga 526-0829, JapanInstitute of Natural Medicine, University of Toyama, 2630, Sugitani, Toyama, Toyama 930-0194, JapanRadioisotope Research Center, Osaka University 2-4, Yamada-oka, Suita, Osaka 565-0871, JapanThe Museum of Osaka University 1-16, Machikaneyama-cho, Toyonaka 560-0043, Japan

r t i c l e i n f o

rticle history:eceived 25 January 2010eceived in revised form 6 August 2010ccepted 7 August 2010vailable online 14 August 2010

eywords:okishakuyakusanronampouality

a b s t r a c t

Aim of the study: To investigate the relation between the clinical effects and the quality of crude drugs,we focused on Tokishakuyakusan (TS), consisted of 6 crude drugs.Materials and methods: We prepared two kinds of TS containing either medicinal cultivar of Paeonialactiflora (MTS) or ornamental one (OTS). Other components were the same. First, we assessed the clinicaleffects of two TS formulations by cross-over study among the anemia patients. Second, we investigatedthe chemical differences between them by using Inductively Coupled Plasma Mass Spectrometry (ICP-MS)and Mössbauer analysis.Results: The clinical effects of these formulations (3 g/day for 8 weeks) were tested in the cross-overstudy consisted of 12 women patients who were diagnosed as having anemia (Hb ≤ 11 g/dl) and con-sented to participate to this study. Both TS formulations were effective for anemia symptoms as shown

össbauer by the improvement of several hematological parameters, whereas their comprehensive effects weredistinguishable by Genetic Algorithm Partial Least Squares (GA-PLS) analysis. There were no significantdifferences in organic ingredients and Fe content measured by ultra performance liquid chromatography(UPLC) and ICP-MS, respectively. Interestingly, Mössbauer spectra of Fe ion were remarkably differentbetween two formulations. Fe ion in MTS was only one form, but that in OTS was at least two forms.

ugges

Conclusions: This study sRadix.

. Introduction

Kampo medicines are widely used in clinical setting in Japan.okishakuyakusan (TS) is traditionally used for the patients withoketsu” syndrome, described in ancient Chinese medical texts asdisorder of the blood circulation causes various symptoms, such

s stasis, reduction and cessation of blood flow (Matsumoto et al.,

008). Recently, from the scientific view, “oketsu” is suggested toe related to the blood viscosity, platelet functions and peripherallood flow and TS has been revealed to improve them (Toriizuka etl., 1987; Ueda et al., 2004; Shen et al., 2005).

∗ Corresponding author at: Department of Applied Pharmacognosy, Graduatechool of Pharmaceutical Sciences, Osaka University, 1-6, Yamada-oka, Suita, Osaka65-0871, Japan. Tel.: +81 6 6879 8160; fax: +81 6 6879 8160.

E-mail addresses: k-simada@phs.osaka-u.ac.jp (K. Shimada),kawase@nagahama-i-bio.ac.jp (M. Kawase), shiba1@inm.u-toyama.ac.jp

N. Shibahara), otouchan@phs.osaka-u.ac.jp (Y. Nakamura), saito@rirc.osaka-u.ac.jpT. Saito), kyokot@phs.osaka-u.ac.jp (K. Takahashi).

378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2010.08.023

ted that clinical effects of TS formulation reflect the quality of Paeoniae

© 2010 Elsevier Ireland Ltd. All rights reserved.

It is important to build modern quality assurance standards forKampo formulas, since Kampo medicines are all-natural and arebased upon ancient wisdom. Identity and quality information onclinical application is needed for each individual ingredient andthe final product. In traditional health cultures, standards do exist.At that time, the criteria for botanical ingredients include the bestseason and stage of growth for the plant to be most therapeuti-cally active; the best area for gathering them, in order to peak theirmedicinal potency.

Paeoniae Radix (Latin name for crude drugs) is the dried root ofPaeonia lactiflora Pallas (: peony in English, Shakuyaku in Japanese)that belongs to the family of Paeoniaceae and has been selectivelybred for its medicinal activity. It is a main botanical ingredientfor Kampo medicines and has been cultivated as Japanese peony

in Nara, Hokkaido and Tottori prefectures in Japan. ‘Bon-ten’ is amedicinal cultivar strain of Paeonia lactiflora (MP) and cultivatedwithout blooming for 3–5 years before harvesting to get enrichedroots (Ministry of Health and Welfare, 1994). Although Europeanpeony of garden plant is an ornamental cultivar of Paeonia lactiflora

K. Shimada et al. / Journal of Ethnopharmacology 132 (2010) 438–442 439

Table 1Crude-drugs composing Tokishakuyakusan and their ratio.

Crude drugs Ratio crude-drugs component

Herbal name (Japanese name) Scientific name Amount/22 g (g) w/w (%)

Angelicae Radix (Toki) Angelica acutiloba Kitagawa 3.0 13.6Cnidii Rhizoma (Senkyu) Cnidium officinale Makino 3.0 13.6Paeoniae Radix (Shakuyaku) Paeonia lactiflora Pallas 4.0 18.2Poria (Bukuryo) Poria cocos Wolf 4.0 18.2Atractylodis Rhizoma (Byakujutsu) Atractylodes japonica Koidzumi ex Kitamura 4.0 18.2Alismatis Rhizoma (Takusha) Alisma orientale Juzepczuk 4.0 18.2

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edicinal part of Angelicae Radix and Paeoniae Radix is root, Cnidii Rhizoma and Atrude drugs were powdered and mixed by the ratio showed above. The ratio is shoeals three times a day in the clinical study.

OP) for well-formed flower, it is also used as a medicinal ingredientow.

We prepared two kinds of TS. One contained MP (MTS) andnother contained OP (OTS), but other ingredients were same. Theim of this study was to determine whether the quality of Paeo-iae Radix would act on clinical effects of TS formulation. First, wessessed the clinical effects of two TS formulations by crossovertudy among the anemia patients. Second, we investigated thehemical differences between them by using Inductively Coupledlasma Mass Spectrometry (ICP-MS) and Mössbauer analysis.

. Material and methods

.1. Clinical study

.1.1. DrugsTS, in-hospital formulation, consisted of six crude drugs mixed

n the ratio shown in Table 1. All ingredients (powdered cruderugs) provided by Uchida Wakanyaku Co., Ltd., Tokyo, Japan. MPLot number: YA352122) or OP (Lot number: OJ303213) were cul-ivated in Toyama or Ohgata, respectively. In our study, MP andP were both Paeonia lactiflora which fit the criteria of Paeoniaeadix in the Japanese Pharmacopoeia (Ministry of Health, Labournd Welfare, 2006), but their strains and methods of cultivatingere quite different. Rest crude drugs (Angelicae Radix, Atracty-

odis Rhizoma, Alismatis Rhizoma, Poria and Cnidii Rhizoma) werelso based on the criteria of the Japanese Pharmacopoeia.

Ultra performance liquid chromatography (UPLC) charts of TSontaining MP (MTS) or OP (OTS) are shown in Fig. 1. MTS and

ig. 1. Typical chromatograms of Tokishakuyakusan containing medicinal cultivarf Paeonia lactiflora (MTS) and ornamental cultivar of Paeonia lactiflora (OTS). Datas shown in UV-TIC (total ion current) chromatograms showing the integrated UVbsorbance of 200–400 nm at each retention time.

odis Rhizoma is Rhizome, Alismatis Rhizoma is tuber, and Poria is sclerotium. Thesey the amount included in 22 g of the prescription, and 1 g of that was taken before

OTS powder (10 mg) were extracted with MeOH/H2O (90/10, v/v)containing vincamine as internal standard. The extractions werewashed with hexane. The polar phase was dried in a vacuum cen-trifugal dryer. Afterward, the residue was dissolved with 50 �l ofMeOH and H2O (5/95, v/v). The mixture was filtered and appliedto LC analysis. LC analyses were performed on a Waters ACQUITYUPLC system (Waters, Milford, MA). LC separations were car-ried out at 40 ◦C with an Acquity UPLC BEH C18 column, 1.7 �m,2.1 mm × 100 mm (Waters). Solvent A was 0.1% (v/v) formic acid,and solvent B was acetonitrile. The initial composition of the binarysolvent was 0% B from 0 to 2.5 min. Solvent B was increased from 0to 35% over 17.5 min, and to 95% in 10 min. The composition of sol-vent remained for 10 min at 95% B. A flow rate was set at 0.3 mL/min.5.0 �l of sample solution was applied to LC analysis. Photodiodearray detector was used for monitoring. The acquired wavelengthwas from 200 to 400 nm. The data at one run was acquired for40 min.

2.1.2. Subjects and study designThe patients enrolled in this study were diagnosed as suffer-

ing from anemia (Hb ≤ 11 g/dl) and suitable to use TS by Kampophysician at the Department of Japanese Oriental Medicine, Hoku-sei Hospital in Toyama, Japan. In total, 12 patients were enrolled.They were all women, and mean (±SE) age was 31.7 ± 4.2.

The study was carried out as 8 weeks trials in a randomizedcrossover, two period design. Experimental periods were separatedby a 4-week washout period. Subjects were randomly assigned toMTS treatment group or OTS treatment group using the concealedenveloped method. Subjects were administrated each TS (3.0 g/day)before meals three times a day. Clinical and laboratory assessmentswere performed before and after each period. All patients providedwritten informed consent in order to participate in the study, whichwas conducted in accordance with the World Medical AssociationDeclaration of Helsinki (Helsinki, Finland, 1964) and subsequentamendments (Tokyo, Japan, 1975; Venice, Italy, 1983; Hong Kong,1989; and Somerset West, Republic of South Africa, 1996) and wasapproved by the Ethics Committee of the Hokusei Hospital.

2.1.3. Clinical and laboratory assessmentBefore and after each experimental period, hematological test

was performed by the physician to determine the red blood cellcount (RBC), hemoglobin (Hb), hematocrit (Ht), mean corpuscu-lar volume (MCV), mean corpuscular hemoglobin (MCH), meancorpuscular hemoglobin concentration (MCHC), serum iron con-centration (Fe), the total white blood cell count (WBC), platelet

count (PLT), the serum levels of total protein (TP), aspartateaminotransferase (AST), alanine aminotransferase (ALT), alkalinephosphatase (ALP), lactate dehydrogenase (LDH), total cholesterol(TC), triglyceride (TG), blood urea nitrogen (BUN), and creatinine(Crea).

440 K. Shimada et al. / Journal of Ethnopharmacology 132 (2010) 438–442

Table 2Variation of the blood tests after MTS or OTS treatment for iron-deficient anemia.

MTS OTS

Baseline At 8 weeks Baseline At 8 weeks

RBC (104/�l) 397.1 ± 41.1 402.0 ± 40.0* 396.8 ± 38.8 398.1 ± 37.4Hb (g/dl) 10.0 ± 0.5 10.4 ± 0.5** 10.1 ± 0.5 10.2 ± 0.4*

Ht (%) 30.7 ± 3.2 31.8 ± 2.6** 30.9 ± 2.9 31.2 ± 2.7MCV (fl) 77.5 ± 5.2 79.4 ± 4.0** 78.1 ± 5.1 78.7 ± 5.3*

MCH (pg) 25.4 ± 2.0 25.9 ± 1.9** 25.6 ± 1.9 25.8 ± 2.0*

MCHC (%) 32.9 ± 2.7 32.7 ± 2.1 32.9 ± 2.4 32.9 ± 2.3Fe (�g/dl) 9.7 ± 5.5 13.8 ± 5.0** 10.7 ± 4.8 11.8 ± 5.0*

WBC (/�l) 5142 ± 944 5138 ± 1024 4845 ± 1078 5246 ± 1269PLT (104/�l) 22.2 ± 2.7 22.1 ± 3.0 21.3 ± 3.8 21.6 ± 4.1TP (g/dl) 7.5 ± 0.5 7.9 ± 0.7 7.8 ± 0.7 7.9 ± 1.3AST (IU/l) 13.7 ± 5.7 14.2 ± 6.7 14.1 ± 6.5 14.2 ± 7.1ALT (IU/l) 12.5 ± 6.3 14.3 ± 9.4 13.5 ± 7.4 13.8 ± 7.9ALP (IU/l) 229.4 ± 44.5 245.8 ± 43.6* 243.8 ± 60.7 242.0 ± 61.0LDH (IU/l) 201.0 ± 39.8 210.5 ± 43.5 212.2 ± 39.8 207.0 ± 39.3TC (mg/dl) 206.9 ± 24.8 212.9 ± 24.7 211.4 ± 33.9 221.1 ± 46.4TG (mg/dl) 119.3 ± 40.2 126.6 ± 43.8* 124.5 ± 44.9 127.9 ± 50.4BUN (mg/dl) 12.4 ± 1.4 13.1 ± 1.8* 12.9 ± 2.2 13.1 ± 2.5Crea (mg/dl) 0.42 ± 0.08 0.42 ± 0.08 0.43 ± 0.11 0.47 ± 0.11

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Anemia is one of the most popular hematological diseases.Among the anemia, iron deficiency anemia is most frequently seen,especially among women (Tsujioka and Tohyama, 2008). Anemia isespecially concerned among pregnant women, and it is said to beassociated with fatigue, decreased work capacity, and poor preg-

he effects of MTS and OTS were assessed by crossover study. Each formulation was2 women subjects.

* P < 0.05 compared with the baseline by Wilcoxon signed-ranks test.** P < 0.01 compared with the baseline by Wilcoxon signed-ranks test.

.1.4. Statistical analysisWilcoxon signed-ranks test was used to compare the hemato-

ogical parameters before and after each period. Relevance betweenhe hematological parameters and the prescriptions were analyzedy using Two-factor factorial ANOVA on at 8 weeks/baseline ratiosf each parameter. Genetic Algorithm Partial Least Squares (GA-LS) analysis was performed in order to distinguish the effect ofTS and OTS using Chemish Software (Tanada et al., 2000). P < 0.05as considered statistically different.

.2. The analysis of Fe

.2.1. ICP-MS spectrometryMTS and OTS powder (4 mg) was added to 1 mL of HNO3 (Nacalai

esque, Kyoto, Japan), vortexed and stood overnight at room tem-erature. Then, 200 �l of samples were diluted with 9.8 mL of waternd filtered through 0.45 �m pore size hydrophilic PTFE membranelter (Millipore, Billerica, MA). ICP-MS analysis was performed onhe Agilent 7500 Series ICP-MS (Agilent Technologies, Inc., Santalara, CA).

Among the attained data, the elements which were detectedore than 1 �g/l in the solvent was selected. Each data in the chartas shown as relative concentration. Relative concentration was

alculated by setting the average concentration of each elementontained in MTS and OTS as 1.

.2.2. Mössbauer analysis57Fe Mössbauer spectra were measured using 57Co (925MBq)

n Rh as a �-ray source at room temperature. The attained spec-ra were analyzed using MossWinn software (Klencsár et al., 1996;lencsár, 1997).

. Results and discussions

.1. The clinical effects of TS

The clinical effects of TS were assessed by crossover studyTable 2). First, we analyzed whether each parameters wasmproved after the treatment. In MTS group, significant improve-

ents were seen in RBC (397.1 ± 41.1 versus 402.0 ± 40.0), Hb10.0 ± 0.5 versus 10.4 ± 0.5), Ht (30.7 ± 3.2 versus 31.8 ± 2.6), MCV

3.0 g/day before each meal for 8 weeks, respectively. Results are means ± S.D. from

(77.5 ± 5.2 versus 79.4 ± 4.0), MCH (25.4 ± 2.0 versus 25.9 ± 1.9),and Fe (9.7 ± 5.5 versus 13.8 ± 5.0). Also in OTS group, Hb(10.1 ± 0.5 versus 10.2 ± 0.4), MCV (78.1 ± 5.1 versus 78.7 ± 5.3),MCH (25.6 ± 1.9 versus 25.8 ± 2.0) and Fe (10.7 ± 4.8 versus11.8 ± 5.0) were significantly increased compared with the base-line. ALP (229.4 ± 44.5 versus 245.8 ± 43.6), TG (119.3 ± 40.2 versus126.6 ± 43.8) and BUN (12.4 ± 1.4 versus 13.1 ± 1.8) were also sig-nificantly increased by MTS treatment, but the parameters onlyshifted within the normal range and there are no clinical benefitsin that change.

Next, we calculated the at 8 weeks/baseline ratio of each param-eters, and investigated whether there are differences in the effectsamong MTS and OTS. We found the interaction between parametersand prescriptions by two-factor factorial ANOVA. Then, we tried toreveal the synthetic difference between MTS and OTS by GA-PLS.The effects of the two prescriptions were distinguished (Fig. 2), sothe hematological effects of two kinds of TS seemed to be different.

Fig. 2. The result of GA-PLS (Genetic Algorithm Partial Least Squares) analysis aboutthe efficacy of MTS or OTS on the hematological values. The hematological param-eters (shown in Table 1) were analyzed by GA-PLS.

K. Shimada et al. / Journal of Ethnopharmacology 132 (2010) 438–442 441

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the regulations of such herbal medicines vary widely among coun-tries, so there is a movement to provide the accurate and up-to-datescientific information of common herbal medicines. WHO pre-sented the “WHO Monographs of Selected Medicinal Plants” with aview to promote the proper use of herbal medicines throughout the

Table 3Mössbauer parameter of MTS and OTS.

ig. 3. Typical chart of ICP-MS (inductively coupled plasma mass spectrometry) ofTS and OTS. Elements which were detected more than 1 �g/l in the sample are

hown. The data is shown as the relative concentration which calculated by settinghe average concentration of each element contained in MTS and OTS as 1 (n = 3).

ancy outcomes (Seck and Jackson, 2007). For anemia patients, ironupplementation is commonly performed (Kanamaru, 2008), butts side effects, mainly of the gastrointestinal system are suggestedo be leading to poor compliance (Melamed et al., 2007).

Akase et al. have been studied about the anemia-amelioratingffect of TS. They compared the effects of iron preparation andS in rats with iron deficiency anemia. They reported that TS hadhe anemia-ameliorating effect without the side effects of the ironreparation (Akase et al., 2004, 2007). They also tried the compar-tive study of TS and an oral iron preparation among patients whoere diagnosed as having hypochromic anemia associated withenorrhagia attributed to uterine myoma. They reported that TS

mproved the signs and symptoms of anemia without side effectshereas iron preparation improved both hematological parame-

ers and symptoms but triggered the side effects in 80% of patients.o, they promoted the combined therapy using TS with an oralupplement for anemia (Akase et al., 2003).

Our study also suggested the therapeutic effect of TS on anemia.kase et al. showed the clinical effects of TS by using extract formu-

ation made from decoction of TS. In present study, we used “san”,ne of the traditional Kampo forms. “San” means powder, and con-ists of directly powdered crude drug. So in these forms, the qualityf the components would more directly affect on the clinical effectshan extract formulation. Both MTS and OTS showed clinical effectsn anemia, but they were distinguishable. These differences woulde derived from the differences of the quality of Paeoniae Radix. So,hen, we tried to reveal what makes their clinical effects different.

.2. The differences of Fe state

The amounts of paeoniflorin contained in both Paeoniae Radixere met the criteria of the Japanese Pharmacopoeia 15th edition.lso, the UPLC chromatograms of MTS and OTS shown in Fig. 1eemed to be quite similar. So we focused on Fe, the quite relevantrace element to anemia.

First, the amounts of Fe in MTS and OTS were measured semi-uantitatively by ICP-MS. The major elements detected in MTS andTS are shown in Fig. 3. Fe contents of two prescriptions were

imilar.Then, we focused on the state of Fe. We found the Mössbauer

pectra of Fe contained in both formulations were apparentlyifferent; the spectrum of MTS was symmetric and OTS was asym-etric (Fig. 4). The Mössbauer effect refers to the process of

ecoil-free emission and resonant absorption of nuclear gammaays in solids. We obtained two useful parameters from Mössbauer

Fig. 4. Mössbauer spectra of MTS (a) and OTS (b).

spectra; the isomer shift (I.S.) and quadrupole splitting (Q.S.). I.S.indicates the valence state of Fe and Q.S. reflects the local surround-ings of Fe ions (Taylor et al., 1968). However, it is impossible todetermine the exact ligands of Fe only from these parameters. Inthis study, Mössbauer parameter suggested that Fe of OTS took atleast two different forms whereas the state of Fe in MTS would besingle (Table 3). From this analysis, it was indicated that the state ofFe in MTS and OTS were apparently different. Moreover, as shownin Fig. 3, the contents of some of trace elements were also differentbetween MTS and OTS. Only Paeonia Radix was different betweenthe components of two formulations, so such differences wouldrepresent the differences of the quality of two Paeonia Radix. Someof such qualitative differences seem to affect on the clinical effectsshown in this study.

We aimed to reveal the importance of the quality identity onPaeonia lactiflora materials in TS. The original dosage form of TS isnot decoction but powders called ‘san’, which crushed the ingre-dients directly. ‘Bon-ten’ is a medicinal strain of Japanese made.The production of quality raw materials can only be assured byemploying good agricultural practices. The quality of MP reflectedthe traditional knowledge on efficacy, safety and cultivation tech-niques for therapeutic use, whereas OP is without much knowledgeor experience with herbal medicines. A lot of studies have beenrevealed interspecific differences of the herbal medicines, but onlya few studies focused on intraspecific differences. We have demon-strated that the quality of crude drugs is diverse even if they arecalled by the same scientific name and they would affect to the clini-cal effects. The global importance of herbal medicines has increasedrecently with both medical and economic implications. However,

I.S. (mm s−1) Q.S. (mm s−1) Ratio of area (%)

MTS 0.121 0.596 100OTS 0.339 0.984 27.9

0.13 0.567 72.1

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Tsujioka, T., Tohyama, K., 2008. The prevalence of anemia in Japan. Japanese Journal

42 K. Shimada et al. / Journal of Eth

orld (Mahady, 2001). In order to take advantages of the “ancientisdom”, we should reveal the efficacy of traditional medicines and

he importance of the quality of natural medicines in the light ofhe modern science.

. Conclusion

In this study, we revealed that Tokishakuyakusan improved somef the hematological parameters in iron deficiency anemia. Andhere were significant differences between two prescriptions whichontains the same ingredients except Paeoniae Radix. We foundhe difference in Fe state contained in two prescriptions, and theossibility of the participation of the other trace elements was also

ndicated.

cknowledgements

We thank Dr. Katsuko Komatsu and Dr. Hirotoshi Fushimi forhe support of the materials, and Dr. Kazuo Harada for help withPLC. Also, we are grateful to Dr. Xiao-Long Hou for overseeing the

ranslation into English.

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