Evaluation of the Effectiveness of Crotoxin as an Antiseptic against Candida spp. Biofilms.

The growing number of oral infections caused by the Candida species are becoming harder to treat as the commonly used antibiotics become less effective. This drawback has led to the search for alternative strategies of treatment, which include the use of antifungal molecules derived from natural products. Herein, crotoxin (CTX), the main toxin of Crotalus durissus terrificus venom, was challenged against Candida tropicalis (CBS94) and Candida dubliniensis (CBS7987) strains by in vitro antimicrobial susceptibility tests. Minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC), and inhibition of biofilm formation were evaluated after CTX treatment. In addition, CTX-induced cytotoxicity in HaCaT cells was assessed by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) colorimetric assay. Native CTX showed a higher antimicrobial activity (MIC = 47 µg/mL) when compared to CTX-containing mouthwash (MIC = 750 µg/mL) and nystatin (MIC = 375 µg/mL). Candida spp biofilm formation was more sensitive to both CTX and CTX-containing mouthwash (IC100 = 12 µg/mL) when compared to nystatin (IC100 > 47 µg/mL). Moreover, significant membrane permeabilization at concentrations of 1.5 and 47 µg/mL was observed. Native CTX was less cytotoxic to HaCaT cells than CTX-containing mouthwash or nystatin between 24 and 48 h. These preliminary findings highlight the potential use of CTX in the treatment of oral candidiasis caused by resistant strains.

Camelid Single-Domain Antibodies (VHHs) against Crotoxin: A Basis for Developing Modular Building Blocks for the Enhancement of Treatment or Diagnosis of Crotalic Envenoming.

Toxic effects triggered by crotalic envenoming are mainly related to crotoxin (CTX), composed of a phospholipase A2 (CB) and a subunit with no toxic activity (CA). Camelids produce immunoglobulins G devoid of light chains, in which the antigen recognition domain is called VHH. Given their unique characteristics, VHHs were selected using Phage Display against CTX from Crotalus durissus terrificus. After three rounds of biopanning, four sequence profiles for CB (KF498602, KF498603, KF498604, and KF498605) and one for CA (KF498606) were revealed. All clones presented the VHH hallmark in FR2 and a long CDR3, with the exception of KF498606. After expressing pET22b-VHHs in E. coli, approximately 2 to 6 mg of protein per liter of culture were obtained. When tested for cross-reactivity, VHHs presented specificity for the Crotalus genus and were capable of recognizing CB through Western blot. KF498602 and KF498604 showed thermostability, and displayed affinity constants for CTX in the micro or nanomolar range. They inhibited in vitro CTX PLA2 activity, and CB cytotoxicity. Furthermore, KF498604 inhibited the CTX-induced myotoxicity in mice by 78.8%. Molecular docking revealed that KF498604 interacts with the CA–CB interface of CTX, seeming to block substrate access. Selected VHHs may be alternatives for the crotalic envenoming treatment.

Use of medicinal fauna in Mexican traditional medicine.

ETHNOPHARMACOLOGICAL RELEVANCE: Mexico has great biodiversity of fauna. The use of fauna with medicinal properties is a common practice since pre-Hispanic times. In the last decade, there has been an interest in ethnozoological studies in Mexico. Therefore, more studies are needed in order to gather information regarding the use of fauna with medicinal properties in México. Ethnozoological studies are necessary in order to discover new medications for human health. This review presents current information in terms of ethnozoological, conservation status, trade, toxicological and pharmacological effects of fauna used for medicinal purposes in Mexican traditional medicine (MTM), based on scientific literature. Future prospects for research with medicinal fauna are discussed. MATERIALS AND METHODS: Bibliographic investigation was carried out by analyzing recognized books and peer-reviewed papers, consulting worldwide accepted scientific databases from the last five decades. Reports included in this review complied with the three criteria cited as follows: (i) used in Mexican traditional medicine for medicinal and/or magical-religious purposes, (ii) with experimental studies regarding the toxicological or medicinal effects and/or with studies exploring mechanisms of medicinal effects, and (iii) with information obtained from a clear source. RESULTS: A total of 163 animal species, belonging to 79 families and 4 taxonomic categories, used for medicinal purposes are reported in this review. Medicinal fauna used in MTM come from birds (48), fishes (3), insects (22), mammals (49) and reptiles (41). The most versatile species which had the greatest number of medicinal properties were Mephitis macroura (21 uses), Crotalus atrox (17 uses), Dasypus novemcinctus (13 uses) and Didelphis virginiana (13 uses). However, 14 of the 161 species listed in this review are classified as endangered. Animal species are mainly used for the treatment of inflammatory, respiratory and gastrointestinal diseases. Furthermore, insects and reptiles are the animal groups with more pharmacological studies. Approximately, 11% and 5% of medicinal fauna have been tested in terms of their pharmacological and toxicological effects, respectively. CONCLUSION: Despite the use of medicinal fauna in MTM, during centuries, there are a very limited number of scientific studies published on this topic. This review highlights the need to perform pharmacological, toxicological and chemical studies with medicinal fauna used in MTM.