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Anti-fungal Efficacy ofPolybutylcyanoacrylate Nanoparticles ofAllicin and Comparison With Pure AllicinDong Qing Luo a , Jian Hua Guo b , Feng Jie Wang c , Zhi Xiong Jin d , XiaoLi Cheng e , Jian Cheng Zhu f , Cheng Qing Peng g & Che Zhang ha Taihe Hospital of YunYang Medical College, Shiyan, Hubei 442000, P. R.Chinab Taihe Hospital of YunYang Medical College, Shiyan, Hubei 442000, P. R.Chinac Taihe Hospital of YunYang Medical College, Shiyan, Hubei 442000, P. R.Chinad Taihe Hospital of YunYang Medical College, Shiyan, Hubei 442000, P. R.Chinae Taihe Hospital of YunYang Medical College, Shiyan, Hubei 442000, P. R.Chinaf Taihe Hospital of YunYang Medical College, Shiyan, Hubei 442000, P. R.Chinag Taihe Hospital of YunYang Medical College, Shiyan, Hubei 442000, P. R.Chinah Taihe Hospital of YunYang Medical College, Shiyan, Hubei 442000, P. R.ChinaPublished online: 02 Apr 2012.

To cite this article: Dong Qing Luo , Jian Hua Guo , Feng Jie Wang , Zhi Xiong Jin , Xiao Li Cheng ,Jian Cheng Zhu , Cheng Qing Peng & Che Zhang (2009) Anti-fungal Efficacy of PolybutylcyanoacrylateNanoparticles of Allicin and Comparison With Pure Allicin, Journal of Biomaterials Science, PolymerEdition, 20:1, 21-31, DOI: 10.1163/156856208X393473

To link to this article: http://dx.doi.org/10.1163/156856208X393473

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Journal of Biomaterials Science 20 (2009) 21–31www.brill.nl/jbs

Anti-fungal Efficacy of PolybutylcyanoacrylateNanoparticles of Allicin and Comparison With Pure Allicin

Dong Qing Luo, Jian Hua Guo, Feng Jie Wang, Zhi Xiong Jin, Xiao Li Cheng,

Jian Cheng Zhu, Cheng Qing Peng and Che Zhang ∗

Taihe Hospital of YunYang Medical College, Shiyan, Hubei 442000, P. R. China

Received 15 December 2007; accepted 17 January 2008

AbstractAlthough garlic has been used in Chinese traditional medicine for its medical properties for thousands ofyears, investigations into its mode of action are relatively recent. The purpose of this study was to evalu-ate the in vitro anti-fungal efficacy of the active principle of garlic, pure allicin and polybutylcyanoacrylate(PBCA) nanoparticles (NPs) loaded with allicin. Pure allicin was prepared by reacting synthetic alliin witha stabilized process of the garlic enzyme alliinase. PBCA NPs were prepared by emulsion polymerizationmethod and pure allicin was wrapped into it. The in vitro efficacy of pure allicin and PBCA-allicin NPs toCandida albicans, Cryputococcus neoformans, Trichophyton rubum, Microsporum gypseum, M. canis andEpidermophyton floccosum was examined and evaluated by MIC and MFC. The MIC of PBCA-allicin NPsto C. albicans (2.93 × 10−2 mg/ml), T. rubum (1.46 × 10−2 mg/ml) and E. floccosum (1.46 × 10−2 mg/ml)was significantly lower than that of pure allicin (5.86×10−2 mg/ml, 2.93×10−2 mg/ml, 2.93×10−2 mg/ml,respectively); accordingly, the MFC of PBCA-allicin NPs to C. albicans (5.86 × 10−2 mg/ml), T. rubum(2.93 × 10−2 mg/ml), E. floccosum (2.93 × 10−2 mg/ml) and M. canis (2.93 × 10−2 mg/ml) also decreaseddramatically. These favourable results indicated that pure allicin has stronger in vitro anti-fungal efficacyto six tested fungi than alliinase and alliin. Moreover, it has improved significantly after pure allicin be-ing wrapped into PBCA NP, which may be due to the NP’s good prolonged release effect and nano-scaledimensions.© Koninklijke Brill NV, Leiden, 2009

KeywordsAllicin, PBCA, nanoparticle, in vitro anti-fungal effect.

Introduction

Garlic (Allium sativum) has been used in traditional Chinese medicine for thou-sands of years [1]. It has been demonstrated that garlic is able to inhibit the growthof a variety of microorganisms, not only bacteria but also fungi and viruses [2].

* To whom correspondence should be addressed. Tel.: (86-719) 880-1136; Fax: (86-719) 880-1218; e-mail:prof.zh@163.com

© Koninklijke Brill NV, Leiden, 2009 DOI:10.1163/156856208X393473

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It is necessary to investigate whether or not some components of garlic haveantimicrobial activity, in order to identify its mode of action and its new phar-macological effect. Allicin is a major ingredient in garlic and generated by thephosphopyridoxal enzyme alliinase. The antimicrobial, anti-tumor, anti-genotoxicand inhibitory immuno-modulatory activities of allicin have been reported [3–5].

Nanoparticles (NPs) are ultra-microstructures with sizes between 1 and 100 nm[6, 7]. Many kinds of NPs have been used as gene carriers that wrap DNA, RNAand other molecules into their interior or adsorb them onto their surface. Re-cently, it has also been demonstrated that NP possesses an anti-adherent effectand poly(propylcyanoacrylate) NP was capable of adhering to C. albicans blas-tospores and reducing their subsequent adherence to buccal epithelial cells in vitro[8]. Based on these previous studies, we believed that NP, with the advantage ofnano-scale particle size which facilitates the penetration of NP through the mem-brane of micro-organisms, could enhance the efficacy of usual anti-fungal agent.However, until now few NPs have been manufactured that fulfill the above require-ments [9, 10].

In this study, pure allicin was prepared by reacting synthetic alliin with a stabi-lized process of the garlic enzyme alliinase. Then, our research group has developeda novel drug vector, polybutylcyanoacrylate (PBCA) NP by emulsion polymer-ization method and wrapped the pure allicin into it. Minimal inhibitory fungalconcentration (MIC) and minimal fungicidal concentration (MFC) of pure allicinand PBCA-allicin NPs against six fungus strains (Candida albicans, Cryputococcusneoformans, Trichophyton rubum, Microsporum gypseum, M. canis and Epidermo-phyton floccosum) in vitro were used to evaluate their anti-fungal efficacy.

Materials and Methods

Materials

Alliin and alliinase were kindly offered by the Institute of Pharmaceutical Research(Xinjiang Province, P. R. China). The garlic from this region is rich in alliin andalliinase, and has stronger pharmaceutical efficacy than garlic from other regions inChina [11]. α-Butyl-ester cyanoacrylic acid (BCA) was from Baiyun (Guangzhou,China). All chemicals, reagents and solvents in the present study were of the highestgrade available and used as directed. The distilled water used was obtained by anion-exchanging and distillation process in our laboratory.

Sabouraud Dextrose medium (SD, Cat. No. 51063) and Sabouraud DextroseAgar (SDA, Cat. No. 51077) were purchased from the Chinese Branch office ofBiomerieux.

Equipment used included a refrigerated centrifuge (Universal 16 R Centrifuge,Hettich, Tuttlingen, Germany), oscillator (THZ-8ZA; Shanghai, China), Super-clean bench (SW-CJ-IFD; Suzhou, China), incubator (Sanyo, Tokyo, Japan), Ze-tasizer 3000 Analyzer (Malvern Instruments, Southborough, MA, USA), AJ-IIIatomic force microscope (AFM, Aijian Nanotechnology Development, Shanghai,

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China) and a high-performance liquid chromatograph (HPLC, LC-10ATVP, Shi-madzu, Japan).

Organisms

Six fungus strains were employed in this study, Candida albicans (ATCC 10231),Cryputococcus neoformans (CCCC MID2α), Trichophyton rubum (CCCC MIDT1α), Microsporum gypseum (CCCC MID M20), M. canis (CCCC MID Mα),Epidermophyton floccosum (CCCC MID E1C). They were all clinical isolates andidentified using standard physiological tests. Human pathogenic fungi were grownon SDA medium. Anti-fungal activity was observed after 48 h incubation at 25◦C.

Preparation of Pure Allicin

Pure allicin was prepared using the method of Shadkchan et al. [12]. The syn-thetic substrate alliin ((+) S-2-propenyl L-cysteine S-oxide) was past through animmobilized alliinase column. The concentration of pure allicin in the solution wasregularly confirmed by sample analysis using HPLC. Pure allicin was kept in a darktightly closed flask at 4◦C and remained stable during all periods of the experiment.

Preparation of PBCA-Allicin NP

PBCA NPs were prepared by an emulsion polymerization method. Firstly, Tween-80 was dissolved in distilled water (pH 2.8). Secondly, PBCA was added into slowlyand mixed by magnetic stirring apparatus at room temperature (22–25◦C) for 5h. Thirdly, the solution was centrifuged at 5000 rpm for 15 min, and the super-natant was removed. Finally, the suspension was filtered through a poly(ethyleneterephthalate) nuclear membrane filter (diameter of pores = 0.22 µm), and PBCANPs were formed. PBCA-allicin NP was prepared and kindly offered by College ofPharmaceutical Sciences of Central South University.

Characterization of PBCA-Allicin NP

The mean size of PBCA NPs prepared by our group was analyzed using Zeta-sizer 3000 Analyzer and all samples were diluted 3-fold in 0.1 M NaCl. The size andsurface morphology of PBCA NPs were analyzed by AFM with 50-fold dilution.Zeta-potential of NPs in a phosphate-buffered saline (PBS) solution for different pHvalues was determined using a Malvern Zetasizer 3000. Quantitative determinationof allicin in PBCA-loaded NP was performed by HPLC.

In Vitro Anti-fungal Test

Alliin solution and alliinase solution with the same concentration (16 mg/ml),15 mg/ml allicin solution and 32 mg/ml PBCA NP solution (containing the samemass of allicin according to the results of HPLC) were prepared for the anti-fungalassay, using sterile distilled water to adjust their concentration.

MIC was determined by the 2-fold serial medium dilution method [13]. Fourdrug solutions mentioned above were added into test tubes 1–11 with SD medium

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in two groups. After that, 0.1 ml of one kind of tested fungus was dropped intoeach tube. Test tubes 12 and 13 in each group were controls with SD medium andfungi, respectively. The other five fungi tested were treated with the same steps andthere were 156 tubes in all with 1 ml solution. Anti-fungal activity was observedand recorded after 48–72 h incubation at 25–27◦C, except that 24–48 h incubationwas employed for C. albicans and Cr. neoformans.

MFC was determined using the 2-fold serial agar dilution method [13]. Two drugsolutions were added into test tubes 1–11 with SD medium in two groups. After that,0.1 ml of one kind of fungus tested was dropped into each tube. Test tubes 12–13in each group were controls with SD medium and fungi, respectively. The otherfive fungi tested were treated with the same steps and there were 156 tubes in all.Then, the mediums containing drugs and fungi were mixed with SDA together andpoured into plates (three plates for each tube). Finally, the plates were put into anincubator and anti-fungal activity was observed. The colony counts were recordedafter 48–72 h incubation at 25–27◦C, except that 24–48 h incubation was employedfor C. albicans and Cr. neoformans.

Statistics

Continuous variables were expressed as mean ± SD from triplicate experimentsperformed in a parallel manner unless otherwise indicated. Statistical analysis wasperformed with Fisher’s exact test for any 2×2 tables, Student’s t-test and one-wayanalysis of variance (ANOVA) for other questions. Statistical analysis was per-formed with SPSS 12.0. Differences were considered statistically significant whenP was less than 0.05.

Results

Characterization of PBCA-Allicin NP

The particle size and dispersity of PBCA-allicin NPs were examined using a Zeta-sizer 3000 Analyzer. It has been demonstrated that the mean size of NPs was 96 nm,with a size range of 69–127 nm. The particle sizes presented as a normal distrib-ution uniformly (Fig. 1). The polymerized index (degree of polymerization) of NPwas 0.159±0.028. AFM observation revealed that PBCA-allicin NPs were uniformand the diameters were between 72 and 131 nm (Fig. 2), similar to those obtainedby Zetasizer 3000. Zeta potentials of PBCA-allicin NPs were performed by a dy-namic light scattering method for different pH values, in 10 mM PBS (Fig. 3). Inthese assay conditions, the zeta potentials decreased and inversed around pH 6 forPBCA-allicin NPs, so that colloids were negatively charged at pH 7. According tothe results measured by HPLC, drug release from PBCA-allicin NP in vitro fol-lowed a double-phase kinetics model (Fig. 4).

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Figure 1. Particle size distribution of PBCA-allicin NPs measured by Zetasizer 3000 Analyzer.

Figure 2. AFM photograph of PBCA-allicin NPs. The size of the complexes ranged from 72to 131 nm.

In Vitro Anti-fungal Efficacy of PBCA-Allicin NP and Pure Allicin

In vitro anti-fungal efficacy of alliin, alliinase, pure allicin and PBCA-allicin NP islisted in Tables 1–6. The MIC and MFC value of pure allicin and PBCA-allicin NPagainst C. albicans, Cr. neoformans, T. rubum, M. gypseum, M. canis and E. floc-cosum were measured by the broth and agar dilution methods (Table 7). It wasdemonstrated that alliin could only inhibit the growth of C. albicans and alliinasehas no anti-fungal efficacy to all tested fungi. The pure allicin expressed signifi-cantly stronger inhibition than alliin and alliinase (P = 0.03 and 0.02, respectively).

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26 D. Q. Luo et al. / Journal of Biomaterials Science 20 (2009) 21–31

Figure 3. Zeta potentials of PBCA-allicin NPs with different pH values.

Figure 4. HPLC analysis of drug released from PBCA-allicin NP in vitro. (A) Allicin solution,(B) empty PBCA NP solution and (C) PBCA-allicin NP.

The MIC values against Cr. neoformans, T. rubum, M. gypseum, M. canis andE. floccosum were between 1.46×10−2 mg/ml and 2.93×10−2 mg/ml in SD brothmedium, and MFC values against these fungi were between 2.93×10−2 mg/ml and5.86 × 10−2 mg/ml on SDA agar medium. The MIC and MFC of allicin againstC. albicans were 5.86 × 10−2 mg/ml and 1.17 × 10−1 mg/ml, respectively. Whenpure allicin was carried by PBCA NP, its anti-fungal efficiency was improved sig-nificantly. The MIC value of PBCA-allicin NP against Cr. neoformans, T. rubum,M. gypseum, M. canis and E. floccosum decreased to 1.46 × 10−2 mg/ml in SD

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Table 1.In vitro anti-fungal efficacy of alliin

Tube

1 2 3 4 5 6 7 8 9 10 11 12 13

Dilution Stock 1:1 1:2 1:22 1:23 1:24 1:25 1:26 1:27 1:28 1:29 Control Controlsolution

C. albicans − − − − − + + + + + + − +Cr. neoformans + + + + + + + + + + + − +T. rubum + + + + + + + + + + + − +E. floccosum + + + + + + + + + + + − +M. gypseum + + + + + + + + + + + − +M. canis + + + + + + + + + + + − +

Note: − refers to fungi not growing and the medium was transparent, + refers to fungi growingand the medium had become turbid.

Table 2.In vitro anti-fungal efficacy of alliinase

Tube

1 2 3 4 5 6 7 8 9 10 11 12 13

Dilution Stock 1:1 1:2 1:22 1:23 1:24 1:25 1:26 1:27 1:28 1:29 Control Controlsolution

C. albicans + + + + + + + + + + + − +Cr. neoformans + + + + + + + + + + + − +T. rubum + + + + + + + + + + + − +E. floccosum + + + + + + + + + + + − +M. gypseum + + + + + + + + + + + − +M. canis + + + + + + + + + + + − +

Note: − refers to fungi not growing and the medium was transparent, + refers to fungi growingand the medium had become turbid.

broth medium, and the MFC value (2.93 × 10−2 mg/ml on SDA agar medium) wasalso dramatically lower than pure allicin (P < 0.01). The MIC and MFC of PBCA-allicin NP against C. albicans were 2.93 × 10−2 mg/ml and 5.86 × 10−2 mg/ml.

Discussion

Garlic was discovered for medicinal use about 5000 years ago and has been used inChinese medicine for at least 3000 years [14, 15]. Currently, garlic is used for reduc-ing cholesterol levels and cardiovascular risk, as well as for its anti-neoplastic andantimicrobial properties. Allicin is one of the active compounds of freshly crushedgarlic [16, 17]. Allicin possesses a variety of biological activities such as antimicro-

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Table 3.In vitro anti-fungal efficacy of allicin

Tube

1 2 3 4 5 6 7 8 9 10 11 12 13

Dilution Stock 1:1 1:2 1:22 1:23 1:24 1:25 1:26 1:27 1:28 1:29 Control Controlsolution

C. albicans − − − − − − − − + + + − +Cr. neoformans − − − − − − − − − − + − +T. rubum − − − − − − − − − + + − +E. floccosum − − − − − − − − − + + − +M. gypseum − − − − − − − − − − + − +M. canis − − − − − − − − − − + − +

Note: − refers to fungi not growing and the medium was transparent, + refers to fungi growingand the medium had become turbid.

Table 4.In vitro anti-fungal efficacy of PBCA-allicin NP

Tube

1 2 3 4 5 6 7 8 9 10 11 12 13

Dilution Stock 1:1 1:2 1:22 1:23 1:24 1:25 1:26 1:27 1:28 1:29 Control Controlsolution

C. albicans − − − − − − − − − + + − +Cr. neoformans − − − − − − − − − − + − +T. rubum − − − − − − − − − − + − +E. floccosum − − − − − − − − − − + − +M. gypseum − − − − − − − − − − + − +M. canis − − − − − − − − − − + − +

Note: − refers to fungi not growing and the medium was transparent, + refers to fungi growingand the medium had become turbid.

bial, anti-inflammatory, anti-thrombotic, anti-atherosclerotic, serum lipid loweringand anticancer activities [18, 19]. Allicin has been indicated to be produced by anenzymic reaction. The enzyme, alliinase, stored in a separate compartment in gar-lic, combines with a compound called alliin in raw garlic and produces allicin [20].Hence, in this study, we prepared pure allicin based on this principle and our prepar-ative method was good at its simplicity and the purity of its production. At the sametime, our results also indicated that pure allicin had significantly stronger in vitroinhibitory effect on the growth of six tested fungi (C. albicans, Cr. neoformans,T. rubum, M. gypseum, M. canis and E. floccosum) than alliin and alliinase.

There are different forms of allicin on the market [2, 4], such as capsulesand injectables. However, many disadvantages, such as short half-life and unsta-

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D. Q. Luo et al. / Journal of Biomaterials Science 20 (2009) 21–31 29Ta

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30 D. Q. Luo et al. / Journal of Biomaterials Science 20 (2009) 21–31

Table 7.MIC and MFC of pure allicin

Tested fungi PBCA-allicin NP Allicin

MIC (mg/ml) MFC (mg/ml) MIC (mg/ml) MFC (mg/ml)

C. albicans 2.93 × 10−2 5.86 × 10−2 5.86 × 10−2 1.17 × 10−1

Cr. neoformans 1.46 × 10−2 2.93 × 10−2 1.46 × 10−2 2.93 × 10−2

T. rubum 1.46 × 10−2 2.93 × 10−2 2.93 × 10−2 5.86 × 10−2

E. floccosum 1.46 × 10−2 2.93 × 10−2 2.93 × 10−2 5.86 × 10−2

M. gypseum 1.46 × 10−2 2.93 × 10−2 1.46 × 10−2 2.93 × 10−2

M. canis 1.46 × 10−2 2.93 × 10−2 1.46 × 10−2 5.86 × 10−2

ble physico-chemical charactistics, depress their efficacy. In order to resolve thisproblem, we have designed a novel PBCA NP as a drug vector for pure allicin.PBCA has good biodegradability, biocompatibility and non-immunogenicity [21].The preparation of PBCA-allicin NP is simple and to our knowledge there is noreport in this field, so it is a great achievement of our research group. All aspects ofPBCA-allicin NP have been investigated systematically. It possessed smooth andround appearance with good dispersion stability, which is an important factor inrelation to the long term storage of NP. The measurement of the zeta potential is auseful method for assessing the extent of PBCA coating on the surface of pure al-licin. In theory, more pronounced zeta potential values, being positive or negative,tend to stabilize particle suspension. The electrostatic repulsion between particleswith the same electric charge prevents the aggregation of the spheres. In this study,the zeta potential of PBCA-allicin NPs was positive when the pH value is below 7.The positive zeta potential of complexes is necessary to ensure nanoparticles’ up-take by cells because a positive surface charge allows an electrostatic interactionbetween negatively charged cellular membranes and positively charged complexes,which is also one of the reasons for the improvement of in vitro anti-fungal efficacyof pure allicin after loading on PBCA NPs. As the result of HPLC, we could believethat a considerable mass of allicin was wrapped into PBCA NP and drug releasedfrom PBCA-allicin NP in vitro was in accord with double-phase kinetics model.Furthermore, PBCA-allicin NP expressed a significantly stronger inhibition effectin vitro against C. albicans, T. rubum, M. gypseum, M. canis and E. floccosum thanpure allicine.

Nanoparticles (NPs), as colloidal drug-delivery system, are capable of deliver-ing a range of therapeutically active entrapped payloads [22, 23]. The mechanismby which NP exerted its effect was thought to be a physical outcome, with the NPsterically hindering a close irreversible attachment, its good prolonged release ef-fect enhancing the efficacy of drugs loaded by it and particle sizes in the submicronrange facilitating the penetration of the NPs through the cellular membrane [24].In this paper, PBCA-allicin NP has been prepared generally in a solution of sur-

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factant (Tween-80) which is adsorbed, stabilizing the suspension and preventingcoalescence. As allicin possess some innate anti-fungal properties, it is, therefore,necessary to evaluate any anti-fungal activity that is bestowed to PBCA-allicin NP.

In conclusion, this work has shown that PBCA-allicin NP has dramaticallystronger anti-fungal properties than pure allicin. Our further aim is to investigatethe exact mechanism of PBCA-allicin NP when it exerts the anti-fungal properties.

Acknowledgement

This work was supported by the Department of Education of Hubei Province, Chinagrants F2004D007.

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