Printer Friendly

Antifungal Activity of the Ethanol Extract from Flos Rosae Chinensis with Activity against Fluconazole-Resistant Clinical Candida.

1. Introduction

In the modern society, with the increasing use of cancer chemotherapy, organ transplantation, and hematopathy and the increased incidence of diabetes and diseases of aging, broad-spectrum antibiotics, adrenal cortical hormone, cytotoxic drugs, and immunosuppressants have been clinically applied in an unreasonable manner for a long time. The morbidity and mortality of fungal infections (mainly caused by C. albicans) have been on the rise [1-3]. FCZ is the most widely used azole drug in clinical settings. With the increasing use of FCZ, drug-resistant strains are emerging rapidly [4-6]. How to exploit new antifungal drugs or to identify non-resistance-forming methods of killing C. albicans remains one of the hottest issues in antifungal research. It is known that a variety of Chinese herbal medicines exert activities against pathogenic microorganisms [7-11]. Moreover, antifungal activities of extracts from Morus mesozygia [12], Toddalia asiatica [13], Wrightia tinctoria [14], Vismia rubescens [15], and many other herbs have also been reported by other labs. Studies at our research center have shown that baicalein [16], berberine [17], and tetrandrine [18] could enhance the antifungal activity of FCZ against FCZ-resistant C. albicans. Our study was conducted to identify new compounds from Chinese herbal medicines that can synergistically potentiate the inhibitory effects of FCZ on the growth of C. albicans.

2. Materials and Methods

2.1. Ethics Statement. All mice were obtained from SLAC Laboratory Animal Co. Ltd., Shanghai, China. They were housed under controlled temperature (23 to 25[degrees]C) and lighting (8:00 a.m. to 8:00 p.m. light, 8:00 p.m. to 8:00 a.m. dark) and with free access to standard food and drinking water. All animal experiments were approved by the Administrative Committee of Experimental Animal Care and Use of the Second Military Medical University and were performed strictly in accordance with the National Institutes of Health guidelines on the ethical use of animals. All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.

2.2. Plant Material Extraction. Flos Rosae Chinensis as a commonly used Chinese traditional medicine is the dry flower of Rosa chinensis Jacq. in Rosaceae. It was bought from having drug supply certificate pharmacy. Approximately 5 g of dried and smashed crude drug was extracted two times with an excess 100 ml of 70% ethanol, after which the extract was filtered. After removal of the solvent by rotary evaporation under reduced pressure at 50[degrees]C, the residue was dissolved with distilled water to approximately 0.5 g/ml FRC. So the amount of 10 mg/ml of FRC is equivalent to 1 g of crude drug. The FRC mentioned in the article refers to the hydroalcoholic extract. The final extract was dark brown liquid and stored at 4[degrees]C for further use.

2.3. Strains and Media. Thirteen clinical isolates of FCZ-resistant C. albicans, three clinical isolates of FCZ-sensitive C. albicans, one international calibration strain (SC5314), two ATCC-typed Candida strains (C. albicans ATCC10231 and C. parapsilosis ATCC90018), C. parapsilosis 160, C. krusei 4996, and C. tropicalis 2718 were utilized. All clinical isolates and other fungi were provided by the Shanghai Hospital (Shanghai, China) and their drug resistance was identified by the hospital clinical laboratory microbial group. SC5314 was kindly provided by William A. Fonzi (Department of Microbiology and Immunology, Georgetown University, Washington, DC). Strains were cultured at 30[degrees]C under constant shaking (200 rpm) in complete liquid medium (yeast extract peptone dextrose--YPD) consisting of 1% (w/v) yeast extract, 2% (w/v) peptone, and 2% (w/v) dextrose.

2.4. Antifungal Susceptibility Test. An antifungal susceptibility test was performed on all strains according to CLSI (formerly NCCLS) methods (M27-A) [19, 20]. C. parapsilosis ATCC22019 was considered a quality control strain and was tested in each assay. The final concentration of fungus suspended in RPMI 1640 medium was [10.sup.3] colony-forming units (CFU)/ml, and the final concentration ranged from 0.125 to 64 [micro]g/ml for FCZ and [10.sup.4] to 19.53 [micro]g/ml for FRC. The Candida plates were incubated at 35[degrees]C for 24 h. Optical density (OD) was measured at 630 nm, and the background OD was subtracted from that of each well. Each strain was tested in triplicate. [MIC.sub.80] refers to the concentration at which 80% of the tested strain was unable to grow. The fractional inhibitory concentration (FIC) index was defined as the sum of the [MIC.sub.80] of each drug when the drug used in combination was divided by the [MIC.sub.80] of the drug used alone. Synergy and antagonism were defined by FIC indices of [less than or equal to]0.5 and >4, respectively. An FIC index result of >0.5, but [less than or equal to] 4 was considered indifferent [21].

2.5. Agar Disk Diffusion Assay. C. albicans 103 (an FCZ-resistant isolate with an [MIC.sub.80] of 19.53 [micro]g/ml for FRC) was further tested by performing an agar diffusion assay [17]. A 100 [micro]l aliquot of [10.sup.6] CFU/ml suspension was spread uniformly onto YPD agar plates with or without 64 [micro]g/ml FCZ. Then, 6 mm paper disks impregnated with FRC and FCZ alone or in combination were placed onto the agar surface. Control disks contained 5 [micro]l of saline. Inhibition zones were measured after incubation at 35[degrees]C for 48 h. Assays were performed in duplicate.

2.6. Time-Kill Curve Studies. C. albicans 103 in RPMI 1640 medium was prepared at a starting inoculum of 103 CFU/ml [22]. FRC and FCZ were added at respective concentrations of 10 mg/ml and 10 [micro]g/ml for (in vivo achievable concentration of FCZ) [23]. At predetermined time points of 0, 12, 24, 36, and 48 h after incubation with agitation at 35[degrees]C, a 100 [micro]l cell suspension was withdrawn from every solution and serially diluted 10-fold in sterile water. A 100 [micro]l aliquot from each dilution was then streaked on a Sabouraud dextrose agar plate. Colony counts were determined after incubation for 48 h at 35[degrees]C. The experiment was performed in triplicate. Synergism and antagonism were defined as a respective increase or decrease of [greater than or equal to] 2 [log.sub.10] CFU/ml in antifungal activity produced by the combination compared with that of the more active agent alone, while a change of <2 log10 CFU/ml was considered indifferent [24].

2.7. Infection of Mice with C. albicans. Sixty ICR mice weighing 18-20 g (4-6 weeks old) were used once in the study. The experimental procedures were performed strictly in accordance with generally accepted international rules and regulations. ICR mice were equally divided into four groups (control, FCZ at 0.5 mg/kg, FRC at 0.4 g/kg, and a combination of FCZ at 0.5 mg/kg and FRC at 0.4 g/kg) to evaluate the synergistic effects of the combination of FRC + FCZ against FCZ-resistant C. albicans 103. Each group contained 15 mice. Immunosuppression was induced by intraperitoneal treatment with 80 mg/kg cyclophosphamide (Shionogi, Osaka, Japan) 3 days before infection [25]. Then, 1 x 105 cells were inoculated in the mouse lateral tail vein. Two hours after fungal injection, the mice in the drug groups were treated with drugs in the amount of 0.2 ml/10 g of body weight [26]. Control mice were given the same volume of saline solution. Drugs were administered by gavage in liquid form once per day for 4 days. From grouping until the end of the experiment, the survival situations of mice were observed at a fixed time every day. The number of dead mice was recorded every day after treatment was given until all mice died.

2.8. Tissue Burden Assay. The kidney is the most frequent target of C. albicans, so the kidney burden assay is the most commonly used method to investigate infection with C. albicans. [27-30] Forty additional ICR mice were equally divided into four groups (control, FCZ at 0.5 mg/kg, FRC at 0.4 g/kg, and a combination of FCZ at 0.25 mg/kg and FRC at 0.2 g/kg) to evaluate the fungal burden in the kidney. First, 5 x [10.sup.4] cells were inoculated in the mouse lateral tail vein. The other operations were the same as those performed for the survival experiment. After 4 days of treatment, mice were euthanized immediately by sodium pentobarbital inhalation and processed for necropsy; the kidneys were excised via a sterile technique, weighed, and homogenized in 2 ml of sterile 0.9% saline. The homogenates were diluted 10-fold in sterile saline solution, and then 0.1 ml of each dilution and the undiluted homogenate were cultured in triplicate on Sabouraud dextrose agar (SDA) plates. Culture plates were incubated at 30[degrees]C for 48 h, and the number of CFU/g of tissue was calculated.

2.9. Sterol Analysis. C. albicans 103 was cultured overnight in YPD medium at 30[degrees]C with constant shaking (200 rpm). After 16 h of incubation, 2 ml from this suspension was subcultured for 24 h in 98 ml of YPD medium containing 0, 1 [micro]g/ml FCZ, 1 [micro]g/ml FCZ + 25 [micro]g/ml FRC, 1 [micro]g/ml FCZ + 250 [micro]g/ml FRC, 1 [micro]g/ml FCZ + 2500 [micro]g/ml FRC, or 2500 [micro]g/ml FRC. Cells were then washed three times and centrifuged. Then, 0.5 g of wet cells was weighed from each group, and 6 ml of 15% NaOH in 90% (V/V) ethanol and 2.5 ml of PBS buffer were added. The samples were heated at 80[degrees]C for 1 h. Nonsaponifiable lipids were extracted three times with 6 ml of mineral ether (30-60[degrees]C boiling) each time, and extracts were washed with 6 ml of sterile water. The washed extracts were evaporated to dryness at 60[degrees]C. The samples were dissolved in 0.5 ml of cyclohexane and stored at -20[degrees]C prior to analysis by gas chromatography-mass spectrometry (GC-MS). The operating condition used for GC/MS were as described by Zhang et al. [31].

2.10. Cytotoxic Assay. The capacity of FRC to inhibit cell growth was determined in vitro in human umbilical vein endothelial cells (HUVECs). The cytotoxic assay was performed as Chiesi et al. [32] The cells, cultured in Dulbecco's Modified Eagle's Medium (supplemented with 10% fetal calf serum, 2% penicillin, and streptomycin) in an incubator at 37[degrees]C under 5% C[O.sub.2], were redistributed into 96-well microtiter plates at 8000 cells/well. After 24 h incubation, solutions containing FRC and FCZ were added to obtain final concentrations ranging from 160 [micro]g/ml to 0.625 [micro]g/ml and 32 [micro]g/ml to 0.125 [micro]g/ml, respectively. After 48 h of incubation, the solutions were discarded. Cells were incubated in 100 [micro]l MTT solution (90 [micro]l of DMEM + 10 [micro]l 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)) for an additional 4 hours. At this time, the succinodehydrogenase in the mitochondria of living cells can reduce the MTT to a water-insoluble blue-violet crystalline formazan and deposit in the cells, whereas dead cells do not. Then the MTT solution was aspirated carefully; 100 [micro]l of dimethyl sulfoxide (DMSO) solution was added to dissolve the formazan. After shaking for 10 minutes in the dark, the absorbance value was measured at OD 570 nm/630 nm, which can indirectly reflect the living cell number. The assay was performed in triplicate. [LD.sub.50] refers to the concentration at which 50% of the tested cells died.

3. Results

3.1. FRC Inhibits the Growth of Part Fungi. The results of the checkerboard test are summarized in the tables. The FCZ-FRC combination markedly reduced the [MIC.sub.80] required for FCZ-resistant C. albicans (Table 1). Synergism (100%) was observed for all 13 isolates of FCZ-resistant C. albicans. The corresponding mean FIC index was 0.165 (range 0.016-0.312). Indifference (100%) was observed for 5 isolates of FCZ-sensitive C. albicans (Table 2). In addition to C. albicans, synergism was observed for C. parapsilosis 160. Indifference was also observed for C. krusei 4996, C. tropicalis 2718, and C. parapsilosis 90018 (Table 3). FRC inhibited the growth of 3 isolates of FCZ-resistant C. albicans in combination with other azole drugs. Indifference was observed for 3 isolates of FCZ-resistant C. albicanswhen FRC was combined with flucytosine or amphotericin (Table 4). Regardless of the [MIC.sub.80] endpoints, antagonism was not observed in the combination group. In addition, FRC alone was efficient against C. albicans within an [MIC.sub.80] range of 20-40 [micro]g/ml. From these results, FRC can inhibit the growth of most fungi [33].

Agar diffusion test can facilitate the visualization of synergistic interactions. FRC exerted no antifungal activity at any concentration (Figure 1(a)) and FCZ at 10 [micro]g showed only weak inhibition (Figure 1(c)). In contrast, on the agar plate containing 64 [micro]g/ml FCZ, FRC demonstrated a powerful antifungal effect (Figure 1(b)). The mean diameters of the inhibitory zones for 312.5, 625, 1250, and 2500 [micro]g FRC increased to 6, 7, 13, and 18 mm, respectively. In addition, the FCZ and FRC combination yielded significantly clearer and larger zones than the zones of either drug alone on the plain agar plate (Figure 1(c)). The sizes of the inhibition zone increased to 7 and 14 mm around the disks impregnated with 10 [micro]g of FCZ plus 625 [micro]g and 1250 [micro]g of FRC, respectively.

To confirm the synergism, the interaction was also detected with a time-kill curve assay (Figure 2). The results indicate that 10 [micro]g/ml FCZ alone exerted a weak antifungal effect, but 10 mg/ml FRC alone demonstrated a better antifungal effect after 24 h. More specifically, the antifungal effect of FCZ was improved by the addition of FRC. Given the initial inoculum of [10.sup.3] CFU/ml, the combination of FCZ and FRC caused a 2.25, 2.65, and 3.05-[log.sub.10] CFU/ml decrease compared with 10 [micro]g/ml FCZ alone 24 h, 36 h, and 48 h later (Table 5). The synergistic antifungal effect was produced at 24 hours and the effect was stronger at 48 hours. These results suggested that FRC exerted a major time-dependent antifungal effect on the synergism of FCZ and FRC.

3.2. FRC and FCZ Combination Increases the Lifespan in a Mouse Model of Systemic Candidiasis. FRC showed antifungal activity in vitro; therefore, we examined this activity in vivo. For this purpose, a mouse model of systemic fungal infection was established. All mice were infected with the resistant C. albicans isolate 103 and were orally administered drugs. Four days later, all mice died in the control and FRC groups. Thus, administration of C. albicans 103 to the other two groups was stopped. Twelve days later, the other two groups of mice all died. We observed that the combination of FRC and FCZ increased the lifespan of the infected mice, indicating that this combination is protective during infection of a live animal. Survival analysis was performed using SPSS 17.0 (Figure 3). FRC was ineffective against C. albicans 103 (P > 0.05 versus control), while FCZ could prolong the survival time of the mice (P < 0.05 versus control). In addition, the combination of FRC and FCZ increased the lifespan of the infected mice, indicating that this combination is protective during the infection of live mice. The increased lifespan in the combination group was statistically significant as confirmed by Kaplan-Meier statistical analysis (P < 0.05). Therefore, consistent with its in vitro activities, FRC is an antifungal agent in this mouse model of systemic fungal infection. Figure 4 presents the fungal burdens in the mouse kidneys. The combination of FCZ (0.25 mg/kg) and FRC (0.2 g/kg) reduced the number of CFU/g in the kidneys of mice infected by C. albicans 103, but there was no difference between the combination and FCZ alone groups. FRC at the dose of 0.4 g/kg daily was ineffective.

3.3. FRC Low Toxicity to Cells. The cytotoxic activities of FRC and FCZ were analyzed on a HUVEC cell line and showed [LD.sub.50] at a concentration of 320 [micro]g/ml and 64 [micro]g/ml for FRC and FCZ, respectively. According to the checkerboard results, the [MIC.sub.80] values for FRC were much lower than the [LD.sub.50]. When FRC was combined with FCZ, the [LD.sub.50] of FCZ decreased from 64 [micro]g/ml to 4 [micro]g/ml (Table 6). FRC exerts a low toxicity to cells and can reduce the cytotoxic dose of FCZ.

3.4. FRC Decreases Content of Ergosterol. Given that synergistic antifungal activity was demonstrated in the studies described above, we then investigated the synergistic mechanism of FCZ and FRC against C. albicans. The sterol profile was studied by GC-MS using strain 103. As summarized in Table 7, ergosterol was the major sterol in strain 103, which accounted for 87.80% of the total sterols extracted from the control group. However, the ergosterol contents decreased to 8.93%, 8.38%, 1.81%, 1.72%, and 21.75% in the groups administered 1 [micro]g/ml FCZ, 1 [micro]g/ml FCZ + 25 [micro]g/ml FRC, 1 [micro]g/ml FCZ + 250 [micro]g/ml FRC, 1 [micro]g/ml FCZ + 2500 [micro]g/ml FRC, and 2500 [micro]g/ml FRC, respectively. The decreased contents of ergosterol were more significant when FCZ was combined with 250 [micro]g/ml or 2500 [micro]g/ml FRC. The consumption of ergosterol induced an increase in the eburicol fraction and a decrease in lanosterol. The sterol content changes were consistent with the FCZ inhibition activity of Erg11 in the ergosterol biosynthetic pathway.

4. Discussion

C. albicans is one of the most important opportunistic human fungal pathogens and causes diseases such as thrush and vaginitis [34]. Amphotericin B is widely used in the treatment of many systemic mycoses [35]. However, given their severe side effects, the azole drugs, especially FCZ, are widely used to fight C. albicans infections. Not surprising, repeated FCZ therapy for fungal infections in patients leads to emergence of serious FCZ-resistant C. albicans. Thus it is very important to identify new antifungal drugs.

FRC is included in the Chinese Pharmacopoeia for the treatment of menstrual disorders. For the first time, we have identified the antifungal capacity of FRC. In our work, the synergistic action of FCZ and FRC was shown in vitro and was confirmed by animal experiments in vivo. The results of FRC in combination with FCZ indicate its high biological activity. Moreover, FRC has low toxicity to the cell and can reduce the [LD.sub.50] of FCZ in HUVEC cell line. The synergistic effect of FRC and FCZ against C. albicans in vitro and in vivo indicates that FRC is a promising Chinese herbal medicine for C. albicans resistant to FCZ.

In vitro experiments showed that the effective concentrations of FRC in different experiments were different. We found that as little as 20-40 [micro]g/ml can exert activity against C. albicans in the checkerboard test. However, up to 2500 [micro]g in the disk diffusion test was required to exert antifungal effects. However, in time-kill curve studies, the FRC can spread normally in a liquid environment and did not require high drug concentration to exhibit synergistic fungicidal activity. The agar diffusion test results were affected by drug diffusion. Agar and filter paper, which have network structures, can hinder macromolecular diffusion. Additionally, the FRC extract is a mixture. Considering these influencing factors, the diffusion capacity of FRC in agar cannot be equated to diffusion in liquid; therefore, the antifungal activity was significantly weaker than in the microdilution assay.

The main compounds in FRC are flavonoids, phenolic acid, ethereal oils, and tannin [36]. The flavonoids include kaempferol, kaempferol-3-O-a-L-arabinoside, kaempferol-3-O-a-L-rhamnoside, quercetin, and quercetin-3-O-a-Larabinoside, in which kaempferol has antifungal effect [37]. It has also been reported that many phenolic compounds exert potent anti-Candida activities, and some of these compounds work synergistically or additively with FCZ [38-44]. We have simply separated different components from the hydroalcoholic extract. The checkerboard results showed that not only flavonoids exert the strongest antifungal effect, but also phenols. However, the antifungal effects of the separated components from the hydroalcoholic extract were weaker than that of the hydroalcoholic extract. We speculate that certain components of the extract of FRC hydroalcoholic have antifungal effect; all components mixed together can exert a stronger antifungal effect. Therefore more experiments must be performed to identify the specific effective ingredients.

The sterol profile analysis results show that FRC strengthens the FCZ-mediated inhibition of ergosterol biosynthesis. Ergosterol is an important sterol presented in the yeast cell membrane that controls membrane integrity and fluidity, and it is an important target for some antifungal drugs [45]. The antifungal mechanism of FCZ is inhibition of the activity of cytochrome P450 (Erg11p) responsible for the lanosterol 14[alpha]-demethylase (CYP51) to suppress ergosterol biosynthesis [46]. It can be seen from Table 7 that FRC can reduce the ergosterol synthesis, but the effect is weaker than FCZ. When FRC used with FCZ, the conversion of lanosterol to eburicol is significantly increased, leading to almost no synthesis of ergosterol. We conclude that FRC and FCZ get through different ways to change the normal metabolism direction of lanosterol: the effect of FCZ on the accumulation of 14[alpha]-methylsterols [47]. FCZ promotes the accumulation of 14[alpha]-methylsterols to reduce lanosterol; FRC reduces lanosterol by promoting the synthesis of eburicol. Both of them inhibit the synthesis of lanosterol to ergosterol. The synergistic effect is to further reduce the synthesis of ergosterol, interfering with the functions of ergosterol. Eventually, the fungus died due to cell membrane structure and function damage.

5. Conclusion

In this study we showed that FRC displayed the higher activity against FCZ-resistant clinical C. albicans and the lower cytotoxicity to cells when compared with azoles. The synergism showed the Flos Rosae Chinensis potential antifungal activity. As a traditional Chinese medicine, FRC antifungal activity suggests that we should find more effective substances for the treatment of fungal diseases and further explore the new use of traditional Chinese medicine.

http://dx.doi.org/10.1155/2017/4780746

Competing Interests

The authors declare that they have no competing interests.

Authors' Contributions

Lulu Zhang and Hui Lin contributed equally to this work.

Acknowledgments

This study was supported by the Natural Science Foundation of China (Grants 81173100 to YongBing Cao and 81173635 to Yuan-Ying Jiang) and the Science and Technology Development Fund of Shanghai (Grant 11JC1415400 to YongBing Cao).

References

[1] J. Maertens, M. Vrebos, and M. Boogaerts, "Assessing risk factors for systemic fungal infections," European Journal of Cancer Care, vol. 10, no. 1, pp. 56-62, 2001.

[2] S. A. Turner and G. Butler, "The Candida pathogenic species complex," Cold Spring Harbor Perspectives in Medicine, vol. 4, no. 9, Article ID a019778, 2014.

[3] D. R. Snydman, "Shifting patterns in the epidemiology of nosocomial Candida infections," Chest, vol. 123, no. 5, pp. 500S-503S, 2003.

[4] M. Niimi, N. A. Firth, and R. D. Cannon, "Antifungal drug resistance of oral fungi," Odontology, vol. 98, no. 1, pp. 15-25, 2010.

[5] J. Wilkerson, C. McPherson, and A. Donze, "Fluconazole to prevent systemic fungal infections in infants: reviewing the evidence," Neonatal Network : NN, vol. 29, no. 5, pp. 323-333, 2010.

[6] A. Gullo, "Invasive fungal infections: the challenge continues," Drugs, vol. 69, supplement 1, pp. 65-73, 2009.

[7] A. Vollekova, D. Kost'Alova, V. Kettmann, and J. Toth, "Anti-fungal activity of Mahonia aquifolium extract and its major protoberberine alkaloids," Phytotherapy Research, vol. 17, no. 7, pp. 834-837, 2003.

[8] D. Yang, H. Hu, S. Huang, J. P. Chaumont, and J. Millet, "Study on the inhibitory activity, in vitro, of baicalein and baicalin against skin fungi and bacteria," Zhong Yao Cai, vol. 23, no. 5, pp. 272-274, 2000.

[9] B.-D. Dai, Y.-Y. Cao, S. Huang et al., "Baicalein induces programmed cell death in Candida albicans," Journal of Microbiology and Biotechnology, vol. 19, no. 8, pp. 803-809, 2009.

[10] J.-D. Zhang, Y.-B. Cao, Z. Xu et al., "In Vitro and in Vivo antifungal activities of the eight steroid saponins from Tribulus terrestris L. with potent activity against fluconazole-resistant fungal," Biological and Pharmaceutical Bulletin, vol. 28, no. 12, pp. 2211-2215, 2005.

[11] X.-M. Xie and Y. Xu, "Advances in study on anti-fungal mechanism of Chinese herbal medicines," Zhongguo Zhong Yao Za Zhi, vol. 29, no. 3, pp. 200-202, 2004.

[12] V. Kuete, D. C. Fozing, W. F. G. D. Kapche et al., "Antimicrobial activity of the methanolic extract and compounds from Morus mesozygia stem bark," Journal of Ethnopharmacology, vol. 124, no. 3, pp. 551-555, 2009.

[13] V. Duraipandiyan and S. Ignacimuthu, "Antibacterial and antifungal activity of Flindersine isolated from the traditional medicinal plant, Toddalia asiatica (L.) Lam," Journal of Ethnopharmacology, vol. 123, no. 3, pp. 494-498, 2009.

[14] K. Ponnusamy, C. Petchiammal, R. Mohankumar, and W. Hopper, "In vitro antifungal activity of indirubin isolated from a South Indian ethnomedicinal plant Wrightia tinctoria R. Br.," Journal of Ethnopharmacology, vol. 132, no. 1, pp. 349-354, 2010.

[15] J. D. D. Tamokou, M. F. Tala, H. K. Wabo, J. R. Kuiate, and P. Tane, "Antimicrobial activities of methanol extract and compounds from stem bark of Vismia rubescens," Journal of Ethnopharmacology, vol. 124, no. 3, pp. 571-575, 2009.

[16] S. Huang, Y. Y. Cao, B. D. Dai et al., "In vitro synergism of fluconazole and Baicalein against clinical isolates of Candida albicans resistant to fluconazole," Biological and Pharmaceutical Bulletin, vol. 31, no. 12, pp. 2234-2236, 2008.

[17] H. Quan, Y.-Y. Cao, Z. Xu et al., "Potent in vitro synergism of fluconazole and berberine chloride against clinical isolates of Candida albicans resistant to fluconazole," Antimicrobial Agents and Chemotherapy, vol. 50, no. 3, pp. 1096-1099, 2006.

[18] F. X. Li and H. Zhang, "In vitro study of the synergistic effect of tetrandrine and fluconazole against Candida albicans," Chinese Journal of Dermatology, vol. 39, no. 8, pp. 454-456, 2006.

[19] W. Craig and S. Gudmundsson, Antibiotics in Laboratory Medicine, Williams & Wilkins, Baltimore, MD, USA, 1996.

[20] J. Waitz, M. Bartlett, M. Ghannoum et al., Reference Method of Broth Dilution Antifungal Susceptibility Testing of Yeasts, Approved standard M27-A National Committee for Clinical Laboratory Standards, Wayne, Pa, USA, 1997.

[21] F. C. Odds, "Synergy, antagonism, and what the chequerboard puts between them," Journal of Antimicrobial Chemotherapy, vol. 52, no. 1, p. 1, 2003.

[22] M. E. Klepser, E. J. Wolfe, R. N. Jones, C. H. Nightingale, and M. A. Pfaller, "Antifungal pharmacodynamic characteristics of fluconazole and amphotericin B tested against Candida albicans," Antimicrobial Agents and Chemotherapy, vol. 41, no. 6, pp. 1392-1395, 1997.

[23] O. Marchetti, P. Moreillon, M. P. Glauser, J. Bille, and D. Sanglard, "Potent synergism of the combination of fluconazole and cyclosporine in Candida albicans," Antimicrobial Agents and Chemotherapy, vol. 44, no. 9, pp. 2373-2381, 2000.

[24] G. M. Eliopoulos and R. C. Moellering, "Antimicrobial combinations," in Antibiotics in Laboratory Medicine, V. Lorian, Ed., pp. 330-396, 1996.

[25] N. Tsuchimori, R. Hayashi, N. Kitamoto et al., "In vitro and in vivo antifungal activities of TAK-456, a novel oral triazole with a broad antifungal spectrum," Antimicrobial Agents and Chemotherapy, vol. 46, no. 5, pp. 1388-1393, 2002.

[26] V. Amareshwar, S. J. Patil, and N. Goudgaon, "Synthesis, in vitro and in vivo antifungal activity of 5-phenylthio-2, 4-bisbenzyloxypyrimidine: a novel nucleobase," Indian Journal of Pharmaceutical Sciences, vol. 72, no. 6, pp. 778-781, 2010.

[27] J. Y. Ju, C. Polhamus, K. A. Marr, S. M. Holland, and J. E. Bennett, "Efficacies of fluconazole, caspofungin, and amphotericin B in Candida glabrata-infected p47phox-/- knockout mice," Antimicrob Agents Chemother, vol. 46, no. 5, pp. 1240-1245, 2002.

[28] K. Maki, A. R. Holmes, E. Watabe et al., "Direct comparison of the pharmacodynamics of four antifungal drugs in a mouse model of disseminated candidiasis using microbiological assays of serum drug concentrations," Microbiology and Immunology, vol. 51, no. 11, pp. 1053-1059, 2007.

[29] O. Marchetti, J. M. Entenza, D. Sanglard, J. Bille, M. P. Glauser, and P. Moreillon, "Fluconazole plus cyclosporine: a fungicidal combination effective against experimental due to Candida albicans," Antimicrobial Agents and Chemotherapy, vol. 44, no. 11, pp. 2932-2938, 2000.

[30] L. Pitzurra, R. Fringuelli, S. Perito et al., "A new azole derivative of 1,4-benzothiazine increases the antifungal mechanisms of natural effector cells," Antimicrobial Agents and Chemotherapy, vol. 43, no. 9, pp. 2170-2175, 1999.

[31] J. D. Zhang, M. H. Li, L. Yan et al., "DNA microarray analysis of fluconazole resistance in a laboratory Candida albicans strain," Acta Biochimica et Biophysica Sinica (Shanghai), vol. 40, no. 12, pp. 1048-1060, 2008.

[32] C. Chiesi, C. Fernandez-Blanco, L. Cossignani, G. Font, and M. J. Ruiz, "Alternariol-induced cytotoxicity in Caco-2 cells. Protective effect of the phenolic fraction from virgin olive oil," Toxicon, vol. 93, pp. 103-111, 2015.

[33] S. C. Tripathi and S. N. Dixit, "Fungitoxic properties of Rosa chinensis Jacq," Experientia, vol. 33, no. 2, pp. 207-209, 1977.

[34] D.-D. Li, Y. Xu, D.-Z. Zhang et al., "Fluconazole assists berberine to kill fluconazole-resistant Candida albicans," Antimicrobial Agents and Chemotherapy, vol. 57, no. 12, pp. 6016-6027, 2013.

[35] M. An, H. Shen, Y. Cao et al., "Allicin enhances the oxidative damage effect of amphotericin B against Candida albicans," International Journal of Antimicrobial Agents, vol. 33, no. 3, pp. 258-263, 2009.

[36] P. Zhang, Y. Xue, L. S. Qing, J. F. An, X. Liao, and L. S. Ding, "The main chemical composition of Rosa Chinensis Jacq.," Chinese Traditional and Herbal Drug, no. 10, pp. 1616-1618, 2010.

[37] D. Seleem, V. Pardi, and R. M. Murata, "Review of flavonoids: a diverse group of natural compounds with anti-Candida albicans activity in vitro," Archives of Oral Biology, vol. 76, pp. 76-83, 2017.

[38] S. J. Sun, H. F. Guo, H. X. Luo et al., "Antifungal activities of phenolic compounds against Candida albicans and their structureactivity relationship," Food and Drug, vol. 8, no. 3A, pp. 30-36, 2006.

[39] M. Zuzarte, L. Vale-Silva, M. J. Goncalves et al., "Antifungal activity of phenolic-rich Lavandula multifida L. Essential oil," European Journal of Clinical Microbiology and Infectious Diseases, vol. 31, no. 7, pp. 1359-1366, 2012.

[40] A. M. S. Pereira, C. Hernandes, S. I. V. Pereira et al., "Evaluation of anticandidal and antioxidant activities of phenolic compounds from Pyrostegia venusta (Ker Gawl.) Miers," ChemicoBiological Interactions, vol. 224, pp. 136-141, 2014.

[41] A. Manayi, S. Saeidnia, M. A. Faramarzi et al., "A comparative study of anti-Candida activity and phenolic contents of the calluses from Lythrum salicaria L. in different treatments," Applied Biochemistry and Biotechnology, vol. 170, no. 1, pp. 176-184, 2013.

[42] X.-C. Li, M. R. Jacob, D. S. Pasco et al., "Phenolic compounds from Miconia myriantha inhibiting Candida aspartic proteases," Journal of Natural Products, vol. 64, no. 10, pp. 1282-1285, 2001.

[43] D. G. Lee, Y. Park, M.-R. Kim et al., "Anti-fungal effects of phenolic amides isolated from the root bark of Lycium chinense," Biotechnology Letters, vol. 26, no. 14, pp. 1125-1130, 2004.

[44] C. T. Alves, I. C. F. R. Ferreira, L. Barros, S. Silva, J. Azeredo, and M. Henriques, "Antifungal activity of phenolic compounds identified in flowers from North Eastern Portugal against Candida species," Future Microbiology, vol. 9, no. 2, pp. 139-146, 2014.

[45] N. N. R. Cardoso, C. S. Alviano, A. F. Blank et al., "Synergism effect of the essential oil from Ocimum basilicum var. Maria Bonita and its major components with fluconazole and its influence on ergosterol biosynthesis," Evidence-based Complementary and Alternative Medicine, vol. 2016, Article ID 5647182, 12 pages, 2016.

[46] D. Sanglard, F. Ischer, T. Parkinson, D. Falconer, and J. Bille, "Candida albicans mutations in the ergosterol biosynthetic pathway and resistance to several antifungal agents," Antimicrobial Agents and Chemotherapy, vol. 47, no. 8, pp. 2404-2412, 2003.

[47] A. Lupetti, R. Danesi, M. Campa, M. D. Tacca, and S. Kelly, "Molecular basis of resistance to azole antifungals," Trends in Molecular Medicine, vol. 8, no. 2, pp. 76-81, 2002.

Lulu Zhang, Hui Lin, Wei Liu, Baodi Dai, Lan Yan, YongBing Cao, and Yuan-Ying Jiang

New Drug Research and Development Center, School of Pharmacy, Second Military Medical University, Shanghai 200433, China

Correspondence should be addressed to YongBing Cao; ybcao@vip.sina.com and Yuan-Ying Jiang; 13761571578@163.com

Received 10 October 2016; Revised 22 November 2016; Accepted 1 December 2016; Published 20 February 2017

Academic Editor: Letizia Angiolella

Caption: Figure 1: Agar disk diffusion assay to visualize the synergism of FCZ with FRC against clinical FCZ-resistant C. albicans isolate 103. (a) and (c) are plain agar plates; (b) is an agar plate containing 64 [micro]g/ml FCZ. (a) and (b) according to the description of (d); (c) as indicated in (e). C and F in the circle are controls.

Caption: Figure 2: Time-kill curves of C. albicans isolate 103 (a clinical FCZ-resistant isolate) obtained using an initial inoculum of 103 CFU/ml. The isolate was exposed to 10 [micro]g/ml FCZ or 10 mg/ml FRC alone, or in combination. CFUs were determined after 0, 12, 24, 36, and 48 h of incubation. The mean values for [log.sub.10] CFU/ml versus time in three separate experiments are shown in the plots. * Synergism, [log.sub.10] CFU/ml [greater than or equal to] 2 antifungal activity produced by the combination compared with that of FCZ alone.

Caption: Figure 3: Effects of the combination of FRC and FCZ against systemic infection caused by C. albicans 103 in mice.

Caption: Figure 4: Fungal burdens in the kidneys of mice infected with C. albicans 103.
Table 1: Activity of FRC alone and in combination with FCZ against
FCZ-resistant C. albicans ([micro]g/ml)a.

                    Alone       Combination (b)

C. albicans      FRC     FCZ      FRC     FCZ

100             39.06    >64      4.88   0.125
904             19.53    >64      4.88   0.125
953             19.53    >64      4.88   0.125
901             312.5    >64      2.44     1
103             19.53    >64      2.44   0.125
J5              19.53    >64      4.88     2
32              19.53    >64      4.88     2
842             19.53    >64      4.88     8
557             19.53    >64      1.22     8
J28             39.06    >64      4.88     1
J38             39.06    >64      4.88   0.125
112678#         19.53    >64      1.22     2
502             39.06    >64      2.44     1

                 FIC index for     Model of
C. albicans     combination (c)   interaction

100                  0.126            syn
904                  0.25             syn
953                  0.25             syn
901                  0.016            syn
103                  0.126            syn
J5                   0.265            syn
32                   0.265            syn
842                  0.312            syn
557                  0.125            syn
J28                  0.133            syn
J38                  0.126            syn
112678#              0.078            syn
502                  0.070            syn

(a) FCZ, fluconazole; FRC, Flos Rosae Chinensis; FIC index, fractional
inhibitory concentration index; syn, synergism.

(b) [MIC.sub.80] s values for combinations are expressed as
[MIC.sub.80] of FCZ/[MIC.sub.80] of FRC. High off-scale [MIC.sub.80]
values were converted to the next highest concentration.

(c) Synergy and antagonism were defined by FIC indices of <0.5 and >4,
respectively. An FIC index result of >0.5 but <4 was considered
indifferent.

Table 2: Activity of FRC alone and in combination with FCZ against
FCZ-sensitive C. albicans ([micro]g/ml)a.

                  Alone        Combination (b)

C. albicans     FRC    FCZ      FRC     FCZ

SC5314         19.53    1      2.44      1
AT10231        19.53   0.5     4.88    0.25
465            39.06   0.5     4.88    0.25
113187#        19.53    2      2.44      1
112721#        19.53    2      4.88      1

                FIC index for     Model of
C. albicans    combination (c)   interaction

SC5314              1.125          indiff
AT10231             0.750          indiff
465                 0.625          indiff
113187#             0.625          indiff
112721#             0.750          indiff

(a) FCZ, fluconazole; FRC, Flos Rosae Chinensis; FIC index, fractional
inhibitory concentration index; indiff, indifference.

(b) [MIC.sub.80] s values for combinations are expressed as
[MIC.sub.80] of FCZ/[MIC.sub.80] of FRC. High off-scale [MIC.sub.80]
values were converted to the next highest concentration.

(c) Synergy and antagonism were defined by FIC indices of <0.5 and >4,
respectively. An FIC index result of >0.5 but <4 was considered
indifferent.

Table 3: Activity of FRC alone and in combination with FCZ against
other fungi ([micro]g/ml)a.

                                  Alone       Combination (b)

Fungi                          FRC     FCZ     FRC      FCZ

C. krusei 4996                19.53    64      4.88     >64
C. tropicalis 2718            78.125   64      4.88    0.25
C. parapsilosis 90018          4.88    0.5     2.44    0.25
C. parapsilosis 160           156.25   0.5    19.53    0.125
Aspergillus fumigatus 7544    >10000   >64    156.25    >64
M. gypseum                     625     32     19.53    0.125
T. rubrum                     312.5    16     19.53    0.125

                               FIC index for     Model of
Fungi                         combination (c)   interaction

C. krusei 4996                     2.25           indiff
C. tropicalis 2718                 0.066          indiff
C. parapsilosis 90018                1            indiff
C. parapsilosis 160                0.375            syn
Aspergillus fumigatus 7544         1.008          indiff
M. gypseum                         0.035            syn
T. rubrum                          0.07             syn

(a) FCZ, fluconazole; FRC, Flos Rosae Chinensis; FIC index, fractional
inhibitory concentration index; syn, synergism; indiff, indifference.

(b) [MIC.sub.80] values for combinations are expressed as [MIC.sub.80]
of FCZ/[MIC.sub.80] of FRC. High off-scale [MIC.sub.80] values were
converted to the next highest concentration.

(c) Synergy and antagonism were defined by FIC indices of <0.5 and >4,
respectively. An FIC index result of >0.5 but <4 was considered
indifferent.

Table 4: Activity of FRC alone and in combination with other antifungal
drugs against FCZ-resistant C. albicans ([micro]g/ml)a.

                     Alone           Combination (b)

                         Positive            Positive
Positive drug     FRC      drug      FRC       drug

MCZ              39.06      64       2.44     0.125
MCZ              312.5      64       2.44     0.125
MCZ              39.06      64       2.44     0.125
KCZ              39.06      32       2.44     0.125
KCZ              312.5      32       2.44     0.125
KCZ              39.06      32       2.44     0.125
ICZ              39.06     >64       2.44     0.125
ICZ              312.5     >64       2.44      0.5
ICZ              39.06     >64       2.44      0.25
5-FLU            39.06    0.016      2.44     0.016
5-FLU            312.5     0.25     39.06     0.125
5-FLU            19.53     0.25      1.22      0.25
AMB              39.06     0.5       4.88      0.5
AMB              312.5     0.25     78.125    0.125
AMB              19.53     0.5       4.88      0.25

                  FIC index for     Model of
Positive drug    combination (c)   interaction   C. albicans

MCZ                   0.064            syn           J5
MCZ                   0.01             syn           901
MCZ                   0.064            syn           103
KCZ                   0.066            syn           J5
KCZ                   0.012            syn           901
KCZ                   0.066            syn           103
ICZ                   0.063            syn           J5
ICZ                   0.012            syn           901
ICZ                   0.064            syn           103
5-FLU                 1.062          indiff          J5
5-FLU                 0.625          indiff          901
5-FLU                 1.062          indiff          103
AMB                   1.125          indiff          J5
AMB                   0.75           indiff          901
AMB                   0.75           indiff          103

(a) KCZ, ketoconazole; ICZ, itraconazole; MCZ, miconazole; 5-Fc,
5-fluorocytosine; AMB, amphotericin B; FRC, Flos Rosae Chinensis;
FIC index, fractional inhibitory concentration index; syn, synergism;
indiff, indifference.

(b) [MIC.sub.80] values for combinations are expressed as [MIC.sub.80]
of FCZ/[MIC.sub.80] of FRC. High off-scale [MIC.sub.80] values were
converted to the next highest concentration.

(c) Synergy and antagonism were defined by FIC indices of <0.5 and >4,
respectively. An FIC index result of >0.5 but <4 was considered
indifferent.

Table 5: FRC combination with FCZ inhibited the growth of clinical
isolate 103 of FCZ-resistant C. albicans (CFU) (a).

                                   Time (h)

                           0          12          24
Control
  average              4966.667    676666.7   1.67E + 08
  lg(avg)              3.696065    5.830375    8.221849
  SD                   0.7409601   0.06247    0.5773503
FCZ 10 [micro]g/mL
  average              4966.667    22133.33    6183333
  lg(avg)              3.696065    4.345047    6.791223
  SD                    0.74096    0.767858    0.654104
FRC 10mg/mL
  average              4966.667     110400     293333.3
  lg(avg)              3.696065    5.042969    5.467361
  SD                    0.74096    0.422549    0.952448
FCZ 10 [micro]g/mL +
FRC 10 mg/mL
  average              4966.667    17066.67     35000
  lg(avg)              3.696065    4.232149   4.544068 *
  SD                    0.74096    0.400816    0.795532

                             Time (h)

                           36            48
Control
  average                2E + 08     2.83E + 08
  lg(avg)               8.301753      8.452298
  SD                   0.867533187    0.578173
FCZ 10 [micro]g/mL
  average               37000000      39866667
  lg(avg)               7.568202      7.60061
  SD                    1.024609      0.890279
FRC 10mg/mL
  average               753333.3       720000
  lg(avg)               5.876987      5.857332
  SD                     0.54554      0.745681
FCZ 10 [micro]g/mL +
FRC 10 mg/mL
  average               81666.67      35166.67
  lg(avg)              4.912045 *    4.546131 *
  SD                    0.654104      0.957788

(a) FCZ, fluconazole; FRC, Flos Rosae Chinensis; avg, average.
* Synergism, [greater than or equal to] 2 [log.sub.10] CFU/ml in
antifungal activity produced by the combination compared with
that of FCZ alone.

Table 6: Effects of FRC and FCZ on cell toxicity ([micro]g/ml).

              FCZ   FRC   FCZ + FRC

[LD.sub.50]   64    320     4/160

Table 7: Sterol compositions of C. albicans strain 103 following FCZ
or FRC treatment, as analyzed by gas chromatography-mass
spectrometry (a).

                                                              FCZ
                                               Control   1 [micro]g/ml

Ergosterol                                      87.8         8.93
14a-Methyl-5a-ergosta-8,24(28)-dien-3a-ol        ND           3.2
Ergosta-8,24(28)-dien-3-ol,4,14-dimethyl        1.76         6.45
Stigmast-5-en-3-ol                               ND           ND
Eburicol                                         ND          55.54
Cholest-4-en-3-one                               ND           ND

Lanosterol                                       ND          25.22
Ergosta-7,22-dien-3-ol                          1.27          ND
Ergosta-5,8-dien-3-ol                           1.81          ND
Cholest-7-en-3-one,4,4-dimethyl-                 ND           3.2
Cholesta-8,24-dien-3-ol,(3a,5a)-(zymosterol)    1.26          ND
Ergosta-5,24(28)-dien-3-ol                      1.12         0.65
Cholest-7-en-3-ol,4,4-dimethyl-                 1.05          ND
Unknown                                         3.93          ND
Ergost-5-en-3-ol                                 ND           ND

                                               FCZ 1 [micro]g/ml +
                                               FRC 25 [micro]g/ml

Ergosterol                                            8.38
14a-Methyl-5a-ergosta-8,24(28)-dien-3a-ol             2.87
Ergosta-8,24(28)-dien-3-ol,4,14-dimethyl              27.12
Stigmast-5-en-3-ol                                    1.21
Eburicol                                              55.9
Cholest-4-en-3-one                                     ND

Lanosterol                                             ND
Ergosta-7,22-dien-3-ol                                 ND
Ergosta-5,8-dien-3-ol                                  ND
Cholest-7-en-3-one,4,4-dimethyl-                       ND
Cholesta-8,24-dien-3-ol,(3a,5a)-(zymosterol)           ND
Ergosta-5,24(28)-dien-3-ol                            3.97
Cholest-7-en-3-ol,4,4-dimethyl-                        ND
Unknown                                                ND
Ergost-5-en-3-ol                                       ND

                                               FCZ 1 [micro]g/ml +
                                               FRC 250 [micro]g/ml

Ergosterol                                            1.81
14a-Methyl-5a-ergosta-8,24(28)-dien-3a-ol              ND
Ergosta-8,24(28)-dien-3-ol,4,14-dimethyl              2.36
Stigmast-5-en-3-ol                                    12.28
Eburicol                                              70.83
Cholest-4-en-3-one                                     0.7

Lanosterol                                            9.05
Ergosta-7,22-dien-3-ol                                 ND
Ergosta-5,8-dien-3-ol                                  ND
Cholest-7-en-3-one,4,4-dimethyl-                       ND
Cholesta-8,24-dien-3-ol,(3a,5a)-(zymosterol)           ND
Ergosta-5,24(28)-dien-3-ol                            2.07
Cholest-7-en-3-ol,4,4-dimethyl-                        ND
Unknown                                                ND
Ergost-5-en-3-ol                                       0.9

                                               FCZ 1 [micro]g/ml +
                                                  FRC 2.5 mg/ml

Ergosterol                                            1.72
14a-Methyl-5a-ergosta-8,24(28)-dien-3a-ol              ND
Ergosta-8,24(28)-dien-3-ol,4,14-dimethyl              2.02
Stigmast-5-en-3-ol                                    11.26
Eburicol                                              72.93
Cholest-4-en-3-one                                    0.34

Lanosterol                                            9.99
Ergosta-7,22-dien-3-ol                                 ND
Ergosta-5,8-dien-3-ol                                  ND
Cholest-7-en-3-one,4,4-dimethyl-                       ND
Cholesta-8,24-dien-3-ol,(3a,5a)-(zymosterol)           ND
Ergosta-5,24(28)-dien-3-ol                            1.14
Cholest-7-en-3-ol,4,4-dimethyl-                        ND
Unknown                                                ND
Ergost-5-en-3-ol                                       0.6

                                                  FRC
                                               2.5 mg/ml

Ergosterol                                       21.75
14a-Methyl-5a-ergosta-8,24(28)-dien-3a-ol         ND
Ergosta-8,24(28)-dien-3-ol,4,14-dimethyl         8.78
Stigmast-5-en-3-ol                               8.05
Eburicol                                         56.03
Cholest-4-en-3-one                                ND

Lanosterol                                        ND
Ergosta-7,22-dien-3-ol                           0.69
Ergosta-5,8-dien-3-ol                            2.35
Cholest-7-en-3-one,4,4-dimethyl-                  ND
Cholesta-8,24-dien-3-ol,(3a,5a)-(zymosterol)      ND
Ergosta-5,24(28)-dien-3-ol                       0.86
Cholest-7-en-3-ol,4,4-dimethyl-                  0.95
Unknown                                           ND
Ergost-5-en-3-ol                                 0.55

(a) FCZ, fluconazole; FRC, Flos Rosae Chinensis; ND, not detected.
COPYRIGHT 2017 Hindawi Limited
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2017 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Title Annotation:Research Article
Author:Zhang, Lulu; Lin, Hui; Liu, Wei; Dai, Baodi; Yan, Lan; Cao, YongBing; Jiang, Yuan-Ying
Publication:Evidence - Based Complementary and Alternative Medicine
Article Type:Report
Date:Jan 1, 2017
Words:7293
Previous Article:Response Surface Optimisation for the Production of Antioxidant Hydrolysates from Stone Fish Protein Using Bromelain.
Next Article:Modernization Trends of Infertility Treatment of Traditional Korean Medicine.
Topics:

Terms of use | Privacy policy | Copyright © 2021 Farlex, Inc. | Feedback | For webmasters |