Journal of Ethnopharmacology ] (]]]]) ]]]–]]]

Contents lists available at SciVerse ScienceDirect

Journal of Ethnopharmacology

0378-87

http://d

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Leuven,nn Cor

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luoga@t

PleasEthn

journal homepage: www.elsevier.com/locate/jep

Antifungal activity in plants from Chinese traditional and folk medicine

Qingfei Liu a,b, Walter Luyten c,n, Klaartje Pellens d, Yiming Wang e, Wei Wang f, Karin Thevissen d,Qionglin Liang e, Bruno P.A. Cammue d, Liliane Schoofs b, Guoan Luo e,nn

a School of Medicine, Tsinghua University, Beijing 100084, Chinab Department of Biology, KU Leuven, 3000 Leuven, Belgiumc Medical School, KU Leuven, 3001 Leuven, Belgiumd Centre of Microbial and Plant Genetics, KU Leuven, B-3001 Heverlee, Belgiume Department of Chemistry, Tsinghua University, Beijing 100084, Chinaf School of Pharmacy, Hunan University of Chinese Medicine, Changsha 410208, China

a r t i c l e i n f o

Article history:

Received 27 August 2011

Received in revised form

11 June 2012

Accepted 12 June 2012

Keywords:

Traditional Chinese Medicine

Folk medicine

Ethnobotanical

Antifungal

Mycosis

Vaginitis

41/$ - see front matter & 2012 Published by

x.doi.org/10.1016/j.jep.2012.06.019

espondence to: Zoological Institute, Naams

Belgium. Tel.: þ32 16 323912; fax: þ32 16 3

responding author. Tel./fax: þ86 10 6278168

ail addresses: Walter.Luyten@med.kuleuven.b

singhua.edu.cn (G. Luo).

e cite this article as: Liu, Q., et aopharmacology (2012), http://dx.do

a b s t r a c t

Ethnopharmacological relevance: From over 100 Chinese clinical trial publications, we retrieved 22

commercial preparations and 17 clinical prescriptions used as Traditional Chinese Medicine (TCM) for

treating mycotic vaginitis, typically caused by Candida albicans. The 8 most frequently used plants as

well as another 7 TCM and 18 folk medicinal plants used in the South of China for antifungal therapy

were investigated for in vitro antifungal activity.

Materials and methods: For each plant we tested 4 extracts prepared with different solvents (water,

ethanol, acetone, and n-hexane) for inhibition of Candida albicans and Saccharomyces cerevisiae growth

in liquid culture.

Results: Some plants have quite strong antifungal activity, such as Tujinpi (Pseudolarix kaempferi Gord.),

of which each extract could significantly inhibit the growth of both tested fungi. In addition, the

acetone extract of Kushen (Sophora flavescens Ait.), the ethanol, acetone, and hexane extracts of

Guanghuoxiang (Pogostemon cablin (Blanco) Benth.) and Gaoliangjiang (Alpinia officinarum Hance), the

hexane extract of Dingxiang (Eugenia caryophyllata Thunb.), and the ethanol and acetone extracts of

Kulianpi (Melia toosendan Sieb. et Zucc.) and Laliao (Polygonum hydropiper L.), all inhibited Candida

albicans growth by more than 50%. In some cases growth inhibition was even comparable to that by the

clinically used antifungal miconazole, which we used as our positive control.

Conclusions: The majority of plants, whose clinical use for antifungal treatment is well supported

within TCM or Chinese folk medicine, show in vitro antifungal activity against Candida albicans. Since

Candida species represent the most common fungal pathogen of humans, these results provide more

scientific evidence supporting the clinical application of these plants, and can serve as a starting point

for new drug discovery from TCM and Chinese folk medicine.

& 2012 Published by Elsevier Ireland Ltd.

1. Introduction

Traditional Chinese Medicine (TCM), with its clinical practicefor thousands of years in China and the wider Eastern Asian area,continues to play a significant role in the Chinese health caresystem even today. Extracting and isolating bio-active com-pounds from TCM have also proved an attractive strategy fordrug discovery. In China many plants are still used as folkmedicine to treat various diseases. Especially in Southern China,

Elsevier Ireland Ltd.

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l., Antifungal activity in pi.org/10.1016/j.jep.2012.06.0

where the weather is warm and humid, fungal infections arecommon. Numerous plants are used there by the inhabitants asfolk medicines to treat fungal infections. After use for manydecades or even centuries, the efficacy of some TCM preparations,prescriptions and folk medicine has been firmly established.

Recently, Tripathi et al. (2008) screened 26 essential oilsagainst Botrytis cinerea (a necrotrophic fungus that affects manyplant species) and 10 oils were found to have absolute fungitoxicactivity (i.e. 100% growth inhibition). Zhou and Li (2008) investi-gated the in vitro antifungal activity of 23 kinds of TCM againstMicrosporum gypseum and Trichophyton rubrum, 2 fungi from thegenera that constitute the main cause of superficial dermatophy-toses, and some TCM showed strong antifungal activity againstone or several species of these fungi. Gong et al. (2002) screenedfor antifungal activity against Candida albicans (Robin) Berkh.,

lants from Chinese traditional and folk medicine. Journal of19

Q. Liu et al. / Journal of Ethnopharmacology ] (]]]]) ]]]–]]]2

Saccharomyces cerevisiae Hansen GL-7, and Prototheca wickerhamii

in the 95% ethanol extracts of 164 plants used as TCM in China,and the extracts from 22 plants exhibited potent antifungalactivity. From Alpinia officinarum Hance, Gui et al. (2005) reportedthat the volatile oil from its rhizome showed strong in vitro

antifungal effects against 16 strains of dermatophytes. Someactive components such as flavones, alkaloids (Rao et al., 2009),volatile oils (Zhang et al., 2009; Gong et al., 2009; Yang et al.,1996), phenols (Guha et al., 2005), and resins (Wang et al., 1995),as well as metabolites from endophytic fungi (Wang et al., 2010;Li et al., 2005; Huang et al., 2001) have been isolated andidentified, which have strong potential for development asclinical drugs.

Although there are many reports of antifungal activity in TCMpreparations, few systematic surveys exist, and those mostlyneglect the rich tradition of folk medicine in China. Moreover,many surveys do not take full advantage of evidence of clinicalefficacy when selecting their plants. Based on published clinicaltrial data as well as advice from TCM practitioners, we selected 33TCM and Chinese folk medicine plants, whose efficacy for thetreatment of mycoses was strongly supported, for a systematicstudy of in vitro antifungal activity.

The objective of the present paper is therefore to evaluate thein vitro antifungal activities of these 33 plants in a systematic andconsistent manner. The results can provide more scientific evi-dence supporting the clinical application of these plants and canserve as a starting point for new drug discovery from TCM andChinese folk medicine. Since Candida albicans remains the mostfrequent cause of fungal infections in an expanding population ofimmunocompromised patients, and candidiasis is now the thirdmost common infection in US hospitals (Pierce et al., 2010), thisspecies was employed in the present study. In addition, the modelorganism Saccharomyces cerevisiae was used as well to confirm orcompare the activity so that later mechanistic studies would befacilitated.

2. Methods and materials

2.1. Chemicals and reagents

Absolute ethanol (SZBA0290) and acetone (S76776-259) ofanalytical grade were purchased from Sigma-Aldrich Co. (Bornem,Belgium). N-hexane (09953580) of analytical grade was pur-chased from Acros Co. (Acros, Belgium). Sterile deionised waterwas produced by a water purification system (Milli-Q ReagentWater System, MA, USA). Yeast extract and peptone were pur-chased from Lab M Ltd. (Lancashire, UK). Dextrose and glucosewere purchased from Fluka—Sigma-Aldrich (MO, US). Yeastnitrogen base without amino acids and complete syntheticmedium were purchased from BD Diagnostics (Sparks, MD, US).

2.2. Botanical materials

The information on these materials such as the Chinese(Pinyin) and official (Latin) taxonomic name (according to theInternational Plant Names Index), medicinal part, origin (Chineseprovince where the plant was collected), and batch number isshown in Table 1. All the botanical materials were identifiedaccording to the Chinese Pharmacopeia (2005 version) or the localFlora, such as Flora of Guangdong (volume 1–9, GuangdongScience and Technology Press, 1987–2009) and Flora Yunnanica(volume 1–16, Science Press, 1977–2006).

Please cite this article as: Liu, Q., et al., Antifungal activity in pEthnopharmacology (2012), http://dx.doi.org/10.1016/j.jep.2012.06.

2.3. Preparation of the extract samples for antifungal test

The dried raw botanical material was ground to a fine powder.Five gram of powder was transferred into each of four 100 mLflasks, and 50 mL of sterile water, absolute ethanol, acetone, andhexane were added, respectively. The flasks were left standing for24 h at ambient temperature. The flasks were then placed in abath sonicator for 4 times 15 min each. After each sonication,there was an interval of 15 min to let the suspension cool toambient temperature. Each sample was transferred into a 100 mLpolyvinyl chloride bottle and centrifugated at 530 g (2000 rpm)for 20 min. The supernatant was transferred to a fresh bottle. Onemillilitre was transferred into a 2.5 mL polyvinyl chloride tubeand the solvent was evaporated in a Savant Speedvac Concen-trator (SVC 200H, Stratech Scientific, London, UK). For the sampleextracted with water, the residue was re-dissolved in 0.5 mL ofwater using a vortex shaker. For the samples extracted withorganic solvents (ethanol, acetone, and hexane), the residue wasdissolved in 0.5 mL of DMSO. The samples were stored at 4 1C tillthe antifungal test.

2.4. Antifungal test

2.4.1. Fungal strains

Candida albicans (SC5314) (American Type Culture Collection,Manassas, Virginia, USA) (Fonzi and Irwin, 1993) and Sacchar-

omyces cerevisiae (BY4741) (Invitrogen, Merelbeke, Belgium) wereused for the antifungal test. The colonies were inoculated on agarplates and stored in a cold room (4 1C).

2.4.2. Preparation of pre-culture

A single colony of Candida albicans and Saccharomyces cerevi-

siae was inoculated from an agar plate in 5 mL of YPD medium(1% yeast extract, 2% peptone, and 2% dextrose) in a reaction tubeunder aseptic conditions. The reaction tubes were incubatedovernight while shaking at 200 rpm at 30 1C.

2.4.3. Preparation of minimal medium (MM)

MM was prepared according to the following formulation (for1 L medium): 100 mL of yeast nitrogen base without amino acids(6.7 g/100 mL), 100 mL of complete synthetic medium (0.8 g/100 mL), 50 mL of glucose (40 g/100 mL), and 750 mL of demi-neralised H2O. All the ingredients were prepared separately andmixed after sterilisation by autoclave, except for the glucose stockwhich was filter (0.45 mm) sterilised.

2.4.4. Antifungal test

Twenty microlitre of the test sample was transferred into thewells of a 96-well plate, as well as the positive control (micona-zole, 2 mg/mL in DMSO) and blank (solvent) controls (DMSO andwater). All the samples were diluted with 20 mL H2O. Fivemicrolitre of each diluted sample was transferred into another96-well plate. Forty and 100 mL of a Candida albicans or Saccharo-

myces cerevisiae pre-culture, respectively, were diluted in 10 mLof MM. Ninety five microlitre of this suspension was added to thewells with the test sample, yielding a final volume of 100 mL. Theplate was incubated for 24 h, at 37 1C for Candida albicans and30 1C for Saccharomyces cerevisiae. The OD was then measured at awavelength of 595 nm. The test was carried out in duplicate andthe average OD value was calculated. The relative inhibition (%) ofthe test sample was calculated by dividing the OD value of thetest sample by the average OD of the solvent control, and multi-plying by 100.

lants from Chinese traditional and folk medicine. Journal of019

Table 1Information on 15 TCM plants and 18 folk medicinal plants used for antifungal testing.

Chinese name Taxonomic name Medicinal part Origin (Province) Batch no. Supplier

TCM plants Baibu Stemona sessilifolia (Miq.) Franch. & Sav. Radix Stemonae Hubei 081201 a

Baixianpi Dictamnus dasycarpus Turcz. Cortex Dictamni Neimenggu 081101

Chuanjiao Zanthoxylum bungeanum Maxim. Pericarpium Zanthoxyli Sichuan 090301

Danpi Paeonia suffruticosa Andr. Cortex Moutan Henan 081008

Difuzi Kochia scoparia (L.) Schrad. Fructus Kochiae Hebei 090301

Dingxiang Eugenia caryophyllata Thunb. Flos Caryophylli Guangdong 090301

Gaoliangjiang Alpinia officinarum Hance Rhizoma Alpiniae officinarum Guangdong 080911

Huangbo Phellodendron chinense C. K. Schneid. Cortex Phellodendri chinensis Sichuan 080801

Guanghuoxiang Pogostemon cablin (Blanco) Benth. Herba Pogostemonis Guangdong 090301

Kushen Sophora flavescens Ait. Radix Sophorae flavescentis Henan 090301

Longkui Solanum nigrum L. Herba Solani nigri Fujian 090301

Qianliguang Senecio scandens Buch.-Ham. Herba Senecionis scandentis Anhui 090306

Shechuangzi Cnidium monnieri (L.) Spreng. Fructus Cnidii Hebei 090301

Tufuling Smilax glabra Roxb. Rhizoma Smilacis glabrae Sichuan 090301

Tujinpi Pseudolarix kaempferi Gord. Cortex Pseudolaricis Zhejiang 081101

Folk plants Baixiaoniang Mallotus apelta (Lour.) Muell.-Arg. Folium Malloti apeltae Fujian 20090328 b

Gangbangui Polygonum perfoliatum L. Herba Polygoni perfoliati Jiangxi 2005032

Heimainshen Breynia fruticosa (L.) Hook. f. Folium et Cacumen Breyniae fruticosae Guangdong 20090408

Huercao Saxifraga stolonifera Curt. Herba Saxifragae Hebei 20090108

Huotanmu Polygonum chinense L. Herba Polygoni chinensis Fujian 20090308

Jiubiying Ilex rotunda Thunb. Cortex Ilicis rotundae Anhui 20090208

Jingangteng Smilax china L. Rhizoma Smilacis chinensis Gansu 20090109

Jishiteng Paederia scandens (Lour.) Merr. Herba Paederiae Jiangsu 20090106

Kulianpi Melia toosendan Sieb. et Zucc. Cortex Meliae Kulianpi 20090106

Laliao Polygonum hydropiper L. Herba Polygoni hydropiperis Anhui 20090205

Mujinpi Hibiscus syriacus L. Cortex hibisci. Guangxi 20090102

Niaotama Acalypha australis L. Herba Acalyphae Guangdong 20091008

Qidagu Glochidion eriocarpum Champ. ex Benth. Folium Glochidii eriocarpi Guangxi 20090101

Sanchaku Euodia lepta (Spreng.) Merr. Radix Euodiae leptae Yunnan 20090109

Wujue Stenoloma chusanum (L.) Ching. Herba Stenolomae Zhejiang 20090207

Xiaofeiyang Euphorbia thymifolia L. herba Euphorbiae thymifoliae Sichuan 20090408

Yangshupi Populus davidiana Dode Cortex Populi davidianae Anhui 20090301

Yizhihuanghua Solidago decurrens Lour. Herba Solidaginis Fujian 20090207

a Bozhou Qiancao Pharm. Co. Ltd. (Bozhou, Anhui Province, China).b Ningbo Dekang Biochem Co., Ltd. (Ningbo, Zhejiang Province, China).

Q. Liu et al. / Journal of Ethnopharmacology ] (]]]]) ]]]–]]] 3

3. Results and discussion

Our strategy consists of first selecting the most promisingChinese plants based on clinical evidence, then preparing extractsof these plants in different solvents, and testing those extracts forin vitro antifungal activity.

3.1. Selection of Chinese plants

Most herbal treatments in China do not use a single plant, buta combination of medicinal herbs. Such a combination can beprescribed by a TCM practitioner (clinical prescription). Alterna-tively, a number of companies market preparations based on TCMplants (commercial preparations). Such preparations are typicallybased on powdered forms or extracts from a combination ofmedicinal plants.

In order to select effective TCM preparations based on clinicaltrial information, we used Chinese E-journal databases (http://www.cnki.net, http://www.wanfangdata.com.cn, http://www.imicams.ac.cn, http://pharm.cqvip.com/channel/zhongyizy, and soon) to search the relevant Chinese clinical study literature. Thetraditional Chinese medical terms, e.g. ‘Yin Yang’ (genital itching),‘Dai Xia’ (vaginal discharge) and ‘Bai Zhuo’ (morbid leucorrhoea)were selected as keywords for searching, as well as ‘antifungal’.Hence, from over 100 Chinese clinical trial publications retrieved,we investigated 22 preparations as well as 17 clinical prescrip-tions commonly used in China for the treatment of mycoticvaginitis. Such preparations and prescriptions typically containmultiple plants, so that it is not easy to deduce whichone(s) contribute to the antifungal activity. We assumed that

Please cite this article as: Liu, Q., et al., Antifungal activity in pEthnopharmacology (2012), http://dx.doi.org/10.1016/j.jep.2012.06.0

the plants most consistently present in clinically beneficial TCMpreparations or prescriptions would be most likely to haveantifungal activity. The most frequently used constituent herbsare shown in Table 2 (for the commercial preparations) andTable 3 (for the clinical prescriptions), and the frequency withwhich these plants occurred in the preparation is shown in Fig. 1.

From these data, we concluded that Kushen (Radix Sophorae

flavescentis), Shechuangzi (Fructus Cnidii), Huangbo (Cortex Phel-

lodendri chinensis), Baixianpi (Cortex Dictamni), Tufuling (RhizomaSmilacis glabrae), Baibu (Radix Stemonae), Chuanjiao (PericarpiumZanthoxyli), and Difuzi (Fructus Kochiae) should be preferablyselected as antifungal plant candidates since they were mostconsistently present in prescriptions or preparations used to treatmycotic vaginitis.

Based on the recommendations of TCM practitioners, theplants selected based on our extensive literature analysis (seeabove) were supplemented by several other TCM plants: Tujinpi(Cortex Pseudolaricis), Guanghuoxiang (Herba Pogostemonis),Dingxiang (Flos Caryophylli), Qianliguang (Herba Senecionis scan-

dentis), Danpi (Cortex Moutan), Longkui (Herba Solani nigri), andGaoliangjiang (Rhizoma Alpiniae officinarum), as well as 18 folkmedicinal plants used in Southern China for the treatment ofmycoses.

3.2. Testing of plant extracts for antifungal activity

In order to cover the maximum chemical diversity of compo-nents, plant extracts were prepared in 4 different solvents: water,ethanol, acetone and n-hexane. The antifungal activity of each ofthese extracts, prepared from the same amount of dried raw

lants from Chinese traditional and folk medicine. Journal of19

Table 3Constituent analysis on 17 clinical antifungal formulae for treating mycotic vaginitis.

Formula Kushen Shechuangzi Huangbo Baixianpi Tufuling Baibu Chuanjiao

1 þ � þ � þ � �

2 þ þ þ þ � � �

3 þ þ þ – – þ þ

4 þ þ – þ � � �

5 – – � þ � � �

6 þ þ þ � þ þ �

7 þ þ þ þ � þ �

8 þ þ þ þ þ � �

9 þ þ þ � � þ �

10 þ þ þ þ þ � þ

11 þ þ þ þ � � þ

12 þ þ þ þ � þ þ

13 þ þ þ � � � þ

14 � � � � þ � �

15 þ þ þ � þ � �

16 þ þ þ � � � �

17 þ � � þ � þ �

Cumulative number 15 13 13 9 6 6 5

Frequency (%) 88 76 76 53 35 35 29

This table lists the presence (þ) or absence (�) of the 7 Chinese medicinal plants most commonly found in 17 clinical prescriptions for the treatment of (i.a.) mycotic

vaginitis. For each plant the cumulative frequency is listed in absolute numbers and in % (frequency (%)¼cumulative number/17�100).

Kushen: Radix Sophorae flavescentis; Shechuangzi: Fructus Cnidii; Huangbo: Cortex Phellodendri chinensis; Baixianpi: Cortex Dictamni; Tufuling: Rhizoma Smilacis glabrae;

Baibu: Radix Stemonae; Chuanjiao: Pericarpium Zanthoxyli; Difuzi: Fructus Kochiae.

Table 2Constituent analysis on 22 commercial preparations used for treating mycotic vaginitis.

Market product Kushen Shechuangzi Huangbo Baixianpi Tufuling Baibu Chuanjiao Difuzi

Fujiebao granule þ þ � þ þ þ � �

Caojueming decoction � � � � � � � �

Qumei capsule þ � � � � � þ �

Erhuang lotion þ þ þ � þ � � �

Fuyanping capsule þ þ � � � � � �

Fufang Kusheng þ þ � � � þ � �

Huoxiangjian lotion � þ � � þ � � �

Jiawei Kusheng þ þ þ � þ þ þ þ

Jiemeiyin þ � � þ � � � þ

Jieyi Shuan þ þ � þ � � � �

Kangmei capsule þ � þ þ � � � �

Kushengbaibu ext þ þ þ þ � þ � �

Kusheng decoction þ þ þ þ þ � � þ

Sanhuang lotion þ þ � þ � þ þ �

Shengjie lotion þ þ þ þ � � � þ

Yanyangjing þ þ þ � � � � �

Zhimeiling lotion þ þ þ � þ � � �

Fuyanling capsule þ � � � � þ � �

Ziqin Yougao þ � � � þ þ � �

Kusheng Baibu lotion þ þ þ þ � þ þ �

Yixishu lotion þ þ � � � � � �

Zinijiuwei decoction þ þ þ þ þ � � �

Cumulative number 20 16 10 10 8 8 4 4

Frequency (%) 91 73 45 45 36 36 18 18

This table lists the presence (þ) or absence (�) of the 8 Chinese medicinal plants most commonly found in 22 commercial preparations marketed in China for the

treatment of (i.a.) mycotic vaginitis. For each plant the cumulative frequency is listed in absolute numbers and in % (frequency (%)¼cumulative number/22�100).

Kushen: Radix Sophorae flavescentis; Shechuangzi: Fructus Cnidii; Huangbo: Cortex Phellodendri chinensis; Baixianpi: Cortex Dictamni; Tufuling: Rhizoma Smilacis glabrae;

Baibu: Radix Stemonae; Chuanjiao: Pericarpium Zanthoxyli; Difuzi: Fructus Kochiae.

Q. Liu et al. / Journal of Ethnopharmacology ] (]]]]) ]]]–]]]4

material (0.2 g/mL) of the 33 selected plants, was tested andcompared. The results are shown in Table 4 (% inhibition sur-passing 50 are underlined).

Over half of the TCM plants show some activity (definedarbitrarily as growth inhibition by more than 25%) on Candida

albicans, and almost three-quarter show some activity on Sac-

charomyces cerevisiae, whereas for the folk medicine plants thecorresponding numbers are over 80 and over 90% for Candida

albicans, and Saccharomyces cerevisiae, respectively. Thus, folkmedicine plants would appear to be a more promising source ofantifungals than TCM plants. However, when active, the degree of

Please cite this article as: Liu, Q., et al., Antifungal activity in pEthnopharmacology (2012), http://dx.doi.org/10.1016/j.jep.2012.06.

growth inhibition appears stronger in TCM compared to folkmedicine plants. This suggests that the former may contain higherconcentrations of, or more potent antifungal substances.

The activity profile of extracts on Candida albicans and Sac-

charomyces cerevisiae was usually comparable, and if a differencewas observed, the activity on the latter was mostly stronger. Thisconfirms the idea that pathogenic fungi in general, and Candida

albicans in particular, are more difficult to inhibit compared tonon-pathogenic ones.

Over half of the active plants show activity in more than 1extract, and sometimes even in 3 or all 4 extracts. This suggests that

lants from Chinese traditional and folk medicine. Journal of019

Q. Liu et al. / Journal of Ethnopharmacology ] (]]]]) ]]]–]]] 5

different active compounds are present, which may show additive orsynergistic effects when the complete plant is used for treatment.

Some extracts showed quite strong antifungal activity. Theacetone extract of Kushen (Radix Sophorae flavescentis), each extractof Tujinpi (Cortex Pseudolaricis), the ethanol, acetone, and hexane

Table 4Antifungal activity of the extracts of 15 TCM plants and 18 folk medicinal plants (%, re

Plants Candida albicans

Water extract Ethanol extract Acetone extract H

TCM plants Baibu 101 106 93 1

Baixianpi 109 101 94 1

Chuanjiao 101 80 104 1

Danpi 97 123 101 1

Difuzi 71 95 85 9

Dingxiang 91 56 71 6

Gaoliangjiang 115 31 20 3

Huangbo 105 82 88 8

Guanghuoxiang 77 22 10 9

Kushen 83 76 33 9

Longkui 127 95 128 1

Qianliguang 116 81 74 9

Shechuangzi 77 69 62 7

Tufuling 92 108 102 1

Tujinpi 14 12 12 1

Folk Plants Baixiaoniang 104 66 89 8

Gangbangui 90 46 56 9

Heimainshen 72 72 67 9

Huercao 89 92 90 8

Huotanmu 98 68 117 1

Jiubiying 113 83 77 9

Jingangteng 81 75 73 1

Jishiteng 103 85 72 8

Kulianpi 119 46 37 7

Laliao 99 24 37 9

Mujinpi 68 69 51 8

Niaotama 79 93 70 9

Qidagu 120 85 68 9

Sanchaku 77 86 74 1

Wujue 122 100 104 9

Xiaofeiyang 65 75 55 7

Yangshupi 111 58 51 9

Yizhihuanghua 51 57 76 1

Solvent control Water 100 – – –

DMSO – 100

Positive control Miconazole 11.7

Fig. 1. Kushen: Radix Sophorae flavescentis; Shechuangzi: Fructus Cnidii; Huangbo:

Cortex Phellodendri chinensis; Baixianpi: Cortex Dictamni; Tufuling: Rhizoma

Smilacis glabrae; Baibu: Radix Stemonae; Chuanjiao: Pericarpium Zanthoxyli;

Difuzi: Fructus Kochiae.

Please cite this article as: Liu, Q., et al., Antifungal activity in pEthnopharmacology (2012), http://dx.doi.org/10.1016/j.jep.2012.06.0

extracts of Guanghuoxiang (Herba Pogostemonis) and Gaoliangjiang(Rhizoma Alpiniae officinarum), the hexane extract of Dingxiang (FlosCaryophylli), and the ethanol and acetone extracts of Kulianpi(Cortex Meliae) and Laliao (Herba Polygoni hydropiperis) all inhibitedCandida albicans growth by more than 50%. In some cases inhibitionwas even similar to that of the positive control miconazole.Although we took great care to test comparable amounts of dryweight of each plant, we cannot at this stage distinguish thepresence of potent antifungals at low concentrations from highconcentrations of less potent compounds.

Some extracts showed very weak or even no activity againstCandida albicans, but strong activity against Saccharomyces cere-

visiae, such as the ethanol extracts of Longkui (Herba Solani nigri)and Heimianshen (Folium et Cacumen Breyniae fruticosae), thewater and ethanol extracts of Huercao (Herba Saxifragae), theethanol and acetone extracts of Mujinpi (Cortex Hibisci.), San-chaku (Radix Euodiae leptae), and Yangshupi (Cortex Populi

davidianae), and the ethanol extract of Xiaofeiyang (HerbaEuphorbiae thymifoliae). This may be due to the presence inCandida of membrane proteins that pump the antifungal com-pounds out of the cytoplasm (Morschhauser, 2010).

Tujinpi (Cortex Pseudolaricis) showed fairly strong antifungalactivity, but is not so widely used as other plants in publishedpreparations or formulae. Its main antifungal components havebeen reported as pseudolaric acids, of which pseudolaric acid B isthe strongest (Zhou et al., 1983; Li et al., 1995; Yang et al., 2003;Liu et al., 2006) and can be synthesised (Trost et al., 2007, 2008).For Kushen (Radix Sophorae flavescentis), it has been reported that

lative growth compared to the solvent, OD at 595 nm).

Saccharomyces cerevisiae

exane extract Water extract Ethanol extract Acetone extract Hexane extract

06 101 118 100 147

04 107 74 76 122

06 75 116 82 69

14 76 70 75 81

0 48 137 76 100

69 82 71 12

2 50 20 16 74

4 84 76 78 98

68 9 11 11

1 70 31 23 138

20 65 12 88 94

8 110 80 78 100

9 89 79 75 92

07 113 124 108 124

4 39 31 22 115

9 76 60 60 80

2 95 27 29 89

3 85 26 52 91

8 46 39 59 83

05 66 59 55 101

2 116 80 70 107

09 84 79 47 88

8 74 72 75 91

6 62 52 55 82

8 91 15 10 86

8 73 28 34 92

1 117 57 58 95

8 140 97 76 82

03 114 50 22 92

8 75 74 102 97

5 106 69 34 87

2 81 49 26 95

00 95 66 81 70

100 – – –

– 100

10.4

lants from Chinese traditional and folk medicine. Journal of19

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flavones are its main antifungal components: norkurarinone,kurarinone, kurarinol, methylkushenol C, L-maackiain, and trifo-lirhizin (Akira et al., 1989; Woo et al., 1998; Kang et al., 2000;Sohn et al., 2004). For Gaoliangjiang (Rhizoma Alpiniae offici-

narum), flavones, diarylheptanoids, and volatile oils are its prin-cipal antifungal components (Ray and Majumdar, 1976; Kiuchiet al., 1992; Gui et al., 2005; An et al., 2006a,b; Zhao et al., 2007).Volatile oils are also the main antifungal constituents of Guan-ghuoxiang (Herba Pogostemonis) and Dingxiang (Flos Caryophylli)(Yang and Xie, 1977; Kong et al., 2004; Mo et al., 2005). The keyreason that these botanical medicines have antifungal activity to adifferent degree is presumably that they contain different types ofantifungal compounds at different concentrations. In TCM, com-plex formulations consisting of several plants are often used. Onereason is that the activity of a single plant is often rather weak.Another significant reason is that each plant may be active againsta different range of microorganisms. Therefore, combined appli-cation of several medicinal plants is likely to be more effectivethan a single one. For the treatment of fungal infections, combi-nation therapy can be more powerful, especially for infectionscaused by antifungal-resistant fungi (Shi et al., 2010).

However, some plants did not show any activity, such asBaixianpi (Cortex Dictamni), Tufuling (Rhizoma Smilacis glabrae),and Baibu (Radix Stemonae), which are amongst the most fre-quently used in TCM for antifungal treatment. Of course ourresults, solely based on growth inhibition by plant extracts in anin vitro liquid culture, do not preclude clinical efficacy of theplant. If the concentration of the extract is increased, or severalextracts are combined, or different methods for measuring anti-fungal activity are used, such as growth in biofilm (which mayapproximate better the real environment of the fungi (Miceliet al., 2009; Martinez et al., 2010), the results may be differentfrom ours. Another potential explanation for the observed dis-crepancy is that the plants are inactive on both tested fungi, butmay be active on other fungi. For instance, Zapata et al. (2010)found that the oils from the plants Achyrocline alata DC. andBaccharis latifolia Pers. were active against the pathogenic fungusAspergillus fumigatus but not against Candida species.

Based on the screening results, further purification, isolation,identification, structure modification, and even synthesis of activecompounds can be carried out. With the aid of research onmechanism of action, cytotoxicity evaluation, and antifungalresistance investigation, some active compounds may becomecandidates for new drug development and clinical application.

4. Conclusion

In the present study, the antifungal activity of 15 TCM plants and18 folk Chinese medicinal plants was investigated against Candida

albicans and Saccharomyces cerevisiae. Some extracts showed quitestrong antifungal activity. The in vitro antifungal activity againstCandida albicans of the hexane extract of Dingxiang (Flos Caryophylli),the acetone and hexane extracts of Guanghuoxiang (Herba Pogoste-

monis), the acetone and hexane extracts of Tujinpi (Cortex Pseudo-

laricis), the ethanol and acetone extracts of Kulianpi (Cortex Meliae),and the ethanol and acetone extracts of Laliao (Herba Polygoni

hydropiperis) are reported here for the first time. Our results providemore scientific evidence and support for clinical use, and suggestavenues for new drug discovery.

Acknowledgements

The project was supported by the Bilateral Scientific Coopera-tion between Tsinghua University and the KU Leuven (project

Please cite this article as: Liu, Q., et al., Antifungal activity in pEthnopharmacology (2012), http://dx.doi.org/10.1016/j.jep.2012.06.

BIL08/06/ BOF-BWS). Luc Vanden Bosch is acknowledged fortechnical assistance. We are grateful for the advice of TCMpractitioners from the First Affiliated Hospital, Guangzhou Uni-versity of TCM, Guangzhou 510405, China, and in particular forthe advice of Dr. Yun Shi. Karin Thevissen acknowledges thereceipt of a postdoctoral fellowship from the Industrial ResearchFund (KU Leuven).

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