Chemical composition, potential toxicity, and quality control procedures of the crude drug of Cyrtopodium macrobulbon.

Ethnopharmacological Relevance: Cyrtopodium macrobulbon ("cañaveral") has been long used in Mexican traditional medicine for the treatment of painful urinary ailments ("mal de orin") in men. This study was conducted (i) to establish the potential acute toxicity and the antinociceptive activity of some preparations of Cyrtopodium macrobulbon, in order to demonstrate its preclinical efficacy for treating symptoms of "mal de orin"; and (ii) to determine the chemical composition and quality control parameters of this medicinal orchid.

Materials And Methods: The antinociceptive effect was assessed using the acetic acid-induced writhing and the hot-plate tests. Investigation of the acute toxicity was accomplished by the Lorke method. The organic extract (OE) was subjected to conventional phytochemical study using chromatographic conventional procedures. The volatile components profile of the species was accomplished via GC-MS analysis of HS-SPME-adsorbed compounds. Furthermore, an HPLC method to quantify ephemeranthol B (10) was developed and validated according to the International Conference on Harmonization Guidelines. Microscopic anatomy studies were performed using light and scanning electron microscopies. Finally, a potential distribution map was generated using the MaxEnt modeling method.

Results: AE and OE were not toxic to mice since the LD50 was higher than 5000 mg/kg. OE was only active in the acetic acid-induced writhing assay at the doses of 100 and 316 mg/kg. Conventional phytochemical analysis of OE led to the isolation and characterization of n-hexacosyl-trans-p-coumarate (1), n-octacosyl-trans-p-coumarate (2), n-triacontyl-trans-p-coumarate (3), 4-methoxy-benzyl alcohol (4), 4-hydroxybenzaldehyde (5), 1,5,7-trimethoxy-9,10-dihydrophenanthrene-2,6-diol (6), confusarin (7), gigantol (8), batatasin III (9), and ephemeranthol B (10). The major volatile components identified by HS-SPME analysis were 6,10,14-trimethyl-2-pentadecanone, eucalyptol (11), and isobornyl formate. An HPLC analytical method for the quantification of compound 10 in the plant was developed and fully validated for selectivity, accuracy, and precision. The microscopic studies revealed that the epidermal tissue displayed a layer of enlarged, crenate and cell thin-walled cells with a thickened cuticle; these cells are described for first time for this species. The potential distribution map generated revealed that this species is widespread in Mexico from Sinaloa to Merida states.

Conclusions: The results of the pharmacological studies tend to support the traditional use of Cyrtopodium macrobulbon for "mal de orin"; the presence of compounds 8, 9, and 11 with known antinociceptive activity might be related with the pharmacological effect demonstrated. The HPLC and microscopic analyses developed in this work will be valuable tools for quality control purposes for this plant.