Fingerprint establishment of Huangqin decoction and study on spectrum-effect relationship of its antidermatophytic activity in different phase states / 中国药房

OBJECTIVE To establish the fingerprint of Huangqin decoction （HQD）， to separate the phase states and screen the active phase states of antidermatophytic activity so as to study the spectrum-effect relationship. METHODS HPLC method was adopted using baicalin as reference， the fingerprints of 10 batches of HQD were drawn and the similarity evaluation was carried out using the Similarity Evaluation System of Chromatographic Fingerprint of TCM （2012 edition） to determine the common peak； the phase states of HQD were separated and characterized by high-speed centrifugation and membrane dialysis. The minimum inhibitory concentrations （MIC） of HQD and its different phase states against Trichophyton mentagrophytes were determined simultaneously. Using the peak area of 37 common peaks as independent variable， MIC as dependent variable， Pearson correlation analysis was performed by using SPSS 21.0 software. RESULTS A total of 37 common peaks were obtained in HPLC fingerprints of 10 batches of HQD， with the similarity higher than 0.99. Ten components were identified， such as albiflorin， paeoniflorin， liquiritin apioside， baicalin， melaleuca glycoside A， wogonoside， baicalein， glycyrrhizic acid， wogonin and oroxylin A. HQD was split into 3 phase states， such as precipitation phase （HQD-P）， solution phase （HQD-S） and nano phase （HQD-N）. The morphology of HQD-P was irregular granular， and the average particle size was 4.670-91.522 μm. The morphology of HQD-S was uniform flakes， and no particle size was detected. HQD-N was spherical in shape and the particle size was （129.0±12.9） nm. MIC values of each phase state of HQD against T. mentagrophytes in different phase states were HQD-N （4.64 mg/mL） ＜HQD （5.85 mg/mL） ＜HQD-P （7.37 mg/mL） ＜HQD-S （12.89 mg/mL） at the same dosage. Pearson correlation analysis showed that the peak area of 25 of the 37 common peaks （including identified components） was significantly negatively correlated with MIC （absolute values of correlation coefficient＞0.95 and P＜0.05）. CONCLUSIONS The chemical composition of 10 batches of HQD is consistent； HQD-N is the active phase state of HQD. Ten components such as paeoniflorin， liquiritin apioside and baicalin may be the main active components of HQD. The antidermatophytic effect of HQD is closely related to its component content and physical phase state.

Antifungal and Antioxidant Activities of Vernonia Chalybaea Mart. ex DC. Essential Oil and their Major Constituent β -caryophyllene

Abstract: This article describes the chemical composition of Vernonia chalybaea essential oil, and investigates its antimicrobial, antioxidant and hemolytic activities. The evaluation of the antifungal activity was performed by the broth microdilution method using strains of yeasts and dermatophytic fungi. The checkerboard technique to find antimicrobial modulatory effects was performed using ketoconazole as standard drug. The antioxidant activity was evaluated by DPPH scavenging assay and β-carotene/linoleic-acid system. The toxicity was characterized by the brine shrimp lethality test and hemolysis bioassays. The essential oil was obtained by hydrodistillation and analyzed by GC-MS method, showing to be rich in the sesquiterpenes β-caryophyllene (39.06%) and bicyclogermacrene (19.69%), and also demonstrated a relevant antifungal activity against strains of Trichophyton rubrum. In the modulatory activity assay, the essential oil of V. chalybaea and β-caryophyllene demonstrated a synergistic interaction with ketoconazole, with increasing of its antifungal action. The antioxidant activity was evidenced mainly by β-carotene/linoleic acid system, with IC50 value of 35.87 ± 0.32 µg/mL. The results suggest that V. chalybaea essential oil and β-caryophyllene are valuable natural medicinal agents with antioxidant and antimicrobial activities.

Susceptibility of dermatophytic fungi to commonly used disinfectants / Suscetibilidade de fungos dermatófitos a desinfetantes comumente utilizados

This study aimed evaluate the antidermatophytic activity of three commercial disinfectants commonly used for environmental control of microorganisms in veterinary medicine. Sodium hypochlorite at 40 µL/mL, chloro-phenol derived at 30 µL/mL and chlorhexidine digluconate at 66.7 µL/mL were tested against 14 strains of dermatophytes, identified as Microsporum canis (n: 3) and Microsporum gypseum (n: 11). The tests was performed in accordance with guidelines of the Clinical and Laboratory Standards Institute (CLSI), documents M38-A2 and M51-A, adapted to disinfectants. In the microdilution broth test, chlorhexidine digluconate had MIC values (Minimum Inhibitory Concentration) of 4.16 µL/mL and MCF (Minimum Fungicidal Concentration) from 4.16 to 8.33 µL/mL, while chloro-phenol derived obtained MIC and MCF of 1.87 µL/mL, and both disinfectants had fungicidal activity at concentrations below the recommended. Sodium hypochlorite obtained MIC from 10 to 80 µL/mL and MFC of 40 to 80 µL/mL, requiring at most isolates twice the recommended concentration to achieve same activity. In the disc diffusion test, the mean inhibition zones for chlorhexidine digluconate was 10.53 mm, for chloro-phenol of 9.9 mm and for sodium hypochlorite was 6.2 mm. Chlorhexidine digluconate and chloro-phenol presented a significant reduction in the growth of dermatophytes, while sodium hypochlorite in concentration recommended showed a low antifungal activity against tested isolates.

O objetivo do estudo foi avaliar a atividade antidermatofítica de três desinfetantes comerciais frequentemente utilizados no controle ambiental de micro-organismos em medicina veterinária. Hipoclorito de sódio a 40 µL/mL, derivado de clorofenol a 30 µL/mL e digluconato de clorexidine a 66.7 µL/mL foram testados contra 14 cepas de dermatófitos, identificados como Microsporum canis (n: 3) e Microsporum gypseum (n: 11). Foram utilizadas as diretrizes do Clinical and Laboratory Standard Institute (CLSI), documentos M38-A2 e M51-A, com adaptações para desinfetantes. Na microdiluição em caldo, digluconato de clorexidine apresentou valores de CIM (Concentração Inibitória Mínima) de 4.16 µL/mL e CFM (Concentração Fungicida Mínima) entre 4.16 a 8.33 µL/mL; derivado de clorofenol obteve CIM e CFM de 1.87 µL/mL, demonstrando que ambos os desinfetantes apresentaram atividade fungicida em concentrações inferiores às recomendadas. O hipoclorito de sódio demonstrou CIM entre 10 a 80 µL/mL e CFM de 40 a 80 µL/mL, requerendo duas vezes a concentração recomendada pelo fabricante para obter atividade fungicida frente a maioria dos isolados fúngicos testados. No teste de disco-difusão, a média das zonas de inibição do digluconato de clorexidine foi de 10.53 mm; do derivado clorofenol 9.9 mm e do hipoclorito de sódio 6.2 mm. O digluconato de clorexidine e o derivado cloro-fenol apresentaram redução significante no crescimento dos dermatófitos testados, enquanto o hipoclorito de sódio, na concentração recomendada, demonstrou baixa atividade antifúngica contra os dermatófitos testados.

In-vitro antidermatophytic and preliminary phytochemical studies of petroleum ether and Inter-polar methanolic leaf extracts of Thevetia nerrifolia.

Antidermatophytic activity (Agar well diffusion technique) of petroleum ether and 98% methanolic leaf extracts of Thevetia nerrifolia (Apocynaceae) was evaluated against mycotic fungi namely, Trichophyton rubrum, Trichophyton tonsurans, Trichophyton mentagrophytes, Microsporum gypseum, dimorphic fungi such as, Candida albicans and pathogenic bacteria like, Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Brevibacillus spp. The significant antidermatophytic activity was shown by 98% methanolic extract, which inhibited all the test fungi and bacteria with time and dose dependent activity. The 98% methanolic extract retarded the growth of all the organisms at 40 mg/ml up to three weeks and beyond. The MIC’s, MFC’s and MBC’s were determined against all the test strains. Preliminary Phytochemical tests were carried out using both the crude extracts. This study provides basis for the isolation and purification of antidermatophytic inter polar compounds from the leaves of T. nerrifolia.

Anti-dermatophytic activity of some traditionally used medicinal plants of North Karnataka Region.

To find the potentiality of the following medicinal plants for their anti dermatophytic property. Agar and broth dilution methods, Leaf extracts of Cassia occidentalis, Cassia tora, Lawsonia inermis, Xanthium strumarium and Caesalpinia bonducella with various solvents viz., methanol, alcohol, acetone, acetone, petroleum ether, chloroform and ethyl acetate were evaluated for antidermatophytic activity against human pathogenic fungi. In agar and broth dilution methods, all extracts showed antifungal activity even at minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs). The most biologically active extract was found to be ethyl acetate leaf extract of C. bonducella, which inhibited all test fungi with time and dose dependent activity. This plant extract retarded the growth of all the organisms at 10000 μg/ml up to 30 days and beyond. It is further suggested for detail investigation of active constituents of the plants used in the present.

The antidermatophytic activity of Zingiber officinale (ginger): In vitro study of ginger rhizome extract in ethanol using various dermatophytes and Candida albicans, and double-blind, randomized controlled trial comparing commercially prepared ginger rhizome powder 25mg/g cream and ketoconazole (Nizoral) 20mg/g cream

BACKGROUND: In vitro studies reported the antifungal activity of Zingiber officinale (ginger) rhizome extract against certain dermatophytes and Candida albicans. However, no in vivo studies had been made.OBJECTIVES: (1) To determine in vitro the antifungal activity of commercially prepared ginger rhizome powder against common dermatophytes and Candida albicans.(2) To determine the Minimum Inhibitory Concentration (MIC) of commercially prepared ginger rhizome powder against Trichophyton rubrum and Candida albicans.(3) To compare the efficacy and safety of ginger 25 mg/g cream versus ketoconazole (Nizoral) 20mg/g cream on patients with tinea.METHODS: Patients (n=24) with tinea corporis or tinea cruris were randomly allocated to two groups. They were instructed to apply either ginger or ketoconazole cream twice daily on lesions for eight weeks. Follow-up consultations were done every two weeks for a total of eight weeks. Photographs, KOH and recording of parameters (erythema, papules, scaling and pruritus) per lesion were used as outcome measures. An investigator's global response assessment was done at the end to determine the improvement of each lesion.RESULTS: The disk diffusion method revealed that Trichophyton mentagrophytes,Trichophyton rubrum and Candida albicans were inhibited by the ginger powder at 2 mg/10?L. The MIC for C. albicans as 12.5 mg/ml and 25 mg/ml for T. rubrum. The age, gender distribution, and the severity index of the two treatment groups were comparable at baseline. There were significant improvements in erythema, papules, pruritus and scaling scores of patients in each group across different time points (Friedman's ANOVA p95% conversion rate to negative KOH at the fourth and eighth week of treatment for the ginger group respectively, while 100% conversion to negative KOH was noted at the second week of treatment for ketoconazole. One patient in the ginger group experienced increased erythema, pruritus and thickness of her lesions during the second week of treatment and the medication was discontinued.CONCLUSION: While ketaconazole 20mg/g cream treatment resulted in faster clearance of fungal lesions, ginger 25mg/g cream yielded progressive improvement of tinea corporis and tinea cruris when used over an eight week period. Ginger cream may be an affordable alternative antifungal treatment. Future studies using a higher concentration and larger sample size are recommended although a possible case of contact dermatitis is seen using 25mg/g ginger cream.