Bioacumulação de podofilotoxina em raízes in vitro e isolamento de fungos endofíticos de Hyptis suaveolens (L.) Poit

Abstract

Hyptis suaveolens contains podophyllotoxin (PTOX) in its roots, a lignan used by the pharmaceutical industry in the production of chemotherapy drugs. Secondary metabolites of economic interest can be sustainably obtained via plant biotechnology and endophytic fungi. Thus, the objective was to study elicitors in micropropagation and in root culture of H. suaveolens, aiming at increasing PTOX biosynthesis, as well as identifying endophytic fungi that produce PTOX in the species. Three experiments were carried out: 1) Apical segments of H. suaveolens were inoculated in MS medium containing different concentrations of serotonin or melatonin (0; 0.25; 0.50; 1.00; 5.00 and 10.00 μM) and phloroglucinol (0 and 50 mg/L). After 40 days, in vitro growth and biomass accumulation were evaluated. The design was DIC in a factorial scheme (6 x 2). PTOX was characterized and quantified in the roots by UHPLC-UVMS/MS and HPLC-DAD. Supplementation with 0.5 μM melatonin and 50 mg/L of phloroglucinol increased in vitro rooting. The use of 1.0 μM of melatonin or serotonin associated with phloroglucinol increased PTOX production in the roots of H. suaveolens by 2.07 and 1.61 times, respectively. The association between melatonin or serotonin and phloroglucinol favors the production of biomass and PTOX biosynthesis in the micropropagated roots of H. suaveolens. 2) Roots of H. suaveolens seedlings (400 mg) were cultivated in flasks containing 50 mL of MS culture medium and different elicitors: light quality, melatonin, serotonin, methyl-jasmonate, salicylic acid and chitosan. DIC was used, with one experiment for each elicitor. After 42 days in a shaker (100 rpm) in the dark, the fresh and dry matter of the roots and the production of PTOX in the roots and in the culture medium were evaluated. The best results regarding the accumulation of biomass in the roots were observed in the treatments with blue LED, 50 mg/L of chitosan, 10 μM of melatonin, 30 μM of serotonin, 5 and 10 mM of methyl jasmonate and 25 mM of salicylic acid. The production of PTOX was favored with the use of 100 mg/L of chitosan, 30 μM of melatonin, 10 μM of serotonin and 10 mM of methyl jasmonate. The use of elicitors is indicated in order to increase the production of biomass and PTOX via H. suaveolens root culture. 3) Endophytic fungi were isolated and cultivated in BD liquid medium for 30 days (100 r.p.m.). The fungi obtained underwent screening with application of mycelium extract and supernatant in thin layer chromatography (TLC) for potential PTOX production. Of the 21 endophytic fungi isolated, only Plectosphaerella cucumerina was identified as a PTOX producer, being confirmed by UHPLC-UVMS/MS and HPLC-DAD, producing 12.48 μg PTOX/L of BD culture medium. PTOX was not found in the mycelium. The P. cucumerina species found in the root of H. suaveolens produces and exudes PTOX into the culture medium.