Controlled Production of Carnosic Acid and Carnosol in Cell Suspensions of Lepechinia meyenii Treated with Different Elicitors and Biosynthetic Precursors.

Lepechinia meyenii is a medicinal plant specialized in the biosynthesis of different types of antioxidants including the diterpenes carnosic (CA) acid and carnosol (CS). Herein we present the results of plant tissue culture approaches performed in this medicinal plant with particular emphasis on the generation and evaluation of a cell suspension system for CA and CS production. The effect of sucrose concentration, temperature, pH, and UV-light exposure was explored. In addition, diverse concentrations of microbial elicitors (salicylic acid, pyocyanin, Glucanex, and chitin), simulators of abiotic elicitors (polyethylene glycol and NaCl), and biosynthetic precursors (mevalonolactone, geranylgeraniol, and miltiradiene/abietatriene) were evaluated on batch cultures for 20 days. Miltiradiene/abietatriene obtainment was achieved through a metabolic engineering approach using a recombinant strain of Saccharomyces cerevisiae. Our results suggested that the maximum accumulation (Accmax ) of CA and CS was mainly conferred to stimuli associated with oxidative stress such as UV-light exposure (Accmax , 6.2 mg L-1 ) polyethylene glycol (Accmax , 6.5 mg L-1 ) NaCl (Accmax , 5.9 mg L-1 ) which simulated drought and saline stress, respectively. Nevertheless the bacterial elicitor pyocyanin was also effective to increase the production of both diterpenes (Accmax , 6.4 mg L-1 ). Outstandingly, the incorporation of upstream biosynthetic precursors such as geranylgeraniol and miltiradiene/abietatriene, generated the best results with Accmax of 8.6 and 16.7 mg L-1 , respectively. Optimized batch cultures containing 100 mg L-1 geranylgeraniol, 50 mg L-1 miltiradiene/abietatriene (95 : 5 %) and 5 g L-1 polyethylene glycol treated with 6 min UV light pulse during 30 days resulted in Accmax of 26.7 mg L-1 for CA and 17.3 mg L-1 for CS on days 18-24. This strategy allowed to increase seven folds the amounts of CA and CS in comparison with batch cultures without elicitation (Accmax , 4.3 mg L-1 ).

Rosmarinic Acid Present in Lepechinia floribunda and Lepechinia meyenii as a Potent Inhibitor of the Adenylyl Cyclase gNC1 from Giardia lamblia.

Giardiasis is a parasitosis caused by Giardia lamblia with significant epidemiological and clinical importance due to its high prevalence and pathogenicity. The lack of optimal therapies for treating this parasite makes the development of new effective chemical entities an urgent need. In the search for new inhibitors of the adenylyl cyclase gNC1 obtained from G. lamblia, 14 extracts from Argentinian native plants were screened. Lepechinia floribunda and L. meyenii extracts exhibited the highest gNC1 inhibitory activity, with IC50 values of 9 and 31 µg/mL, respectively. In silico studies showed rosmarinic acid, a hydroxycinnamic acid present in both mentioned species, to be a promising anti-gNC1 compound. This result was confirmed experimentally, with rosmarinic acid showing an IC50 value of 10.1 µM. Theoretical and experimental findings elucidate the molecular-level mechanism of rosmarinic acid, pinpointing the key interactions stabilizing the compound-enzyme complex and the binding site. These results strongly support that rosmarinic acid is a promising scaffold for developing novel compounds with inhibitory activity against gNC1, which could serve as potential therapeutic agents to treat giardiasis.

Screening and Evaluation of Active Compounds in Polyphenol Mixtures by a Novel AAPH Offline HPLC Method and Its Application.

In this study, we developed a novel offline high-performance liquid chromatography (HPLC) method based on 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) radicals for antioxidant screening in 20 polyphenolic compounds and used the Trolox equivalent antioxidant capacity assay to evaluate their antioxidant activity. Compared to the existing offline HPLC methods based on 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH), the offline HPLC method based on the AAPH radical is more sensitive. Additionally, we applied this method to Lepechinia meyenii (Walp.) Epling extract and screened out seven antioxidants, caffeic acid, hesperidin, rosmarinic acid, diosmin, methyl rosmarinate, diosmetin, and n-butyl rosmarinate, which are known antioxidants. Therefore, this study provides new insights into the screening of antioxidants in natural extracts.

Separation and Identification of Antioxidants and Aldose Reductase Inhibitors in Lepechinia meyenii (Walp.) Epling.

We previously reported that Lepechinia meyenii (Walp.) Epling has antioxidant and aldose reductase (AR) inhibitory activities. In this study, L. meyenii was extracted in a 50% MeOH and CH2Cl2/MeOH system. The active extracts of MeOH and 50% MeOH were subjected to fractionation, followed by separation using high-speed counter-current chromatography (HSCCC) and preparative HPLC. Separation and identification revealed the presence of caffeic acid, hesperidin, rosmarinic acid, diosmin, methyl rosmarinate, diosmetin, and butyl rosmarinate. Of these, rosmarinic acid, methyl rosmarinate, and butyl rosmarinate possessed remarkable antioxidant and AR inhibitory activities. The other compounds were less active. In particular, rosmarinic acid is the key contributor to the antioxidant and AR inhibitory activities of L. meyenii; it is rich in the MeOH extract (333.84 mg/g) and 50% MeOH extract (135.41 mg/g) of L. meyenii and is especially abundant in the EtOAc and n-BuOH fractions (373.71-804.07 mg/g) of the MeOH and 50% MeOH extracts. The results clarified the basis of antioxidant and AR inhibitory activity of L. meyenii, adding scientific evidence supporting its traditional use as an anti-diabetic herbal medicine. The HSCCC separation method established in this study can be used for the preparative separation of rosmarinic acid from natural products.

Inhibitory effects of compounds isolated from Lepechinia meyenii on tyrosinase.

To contribute enzymatic browning inhibitors to the food industry and also extend knowledge about the phytochemical profile of the anti-tyrosinase plant Lepechinia meyenii, its ethanol extract was subjected to bioguided fractionation. Three hydroxycinnamic acids, p-coumaric acid (1), caffeic acid (2) and rosmarinic acid (3), were isolated as mainly responsible for its activity. Compounds 1, 2 and 3 showed themselves highly effective for inhibiting tyrosinase with IC50 values of 0.30, 1.50 and 4.14 µM, respectively, for monophenolase activity and 0.62, 2.30 and 8.59 µM, respectively for diphenolase activity. This is the first report describing the isolation of the compounds causing the tyrosinase inhibitory activity of L. meyenii extract. The inhibitory kinetics of 1-3 using both L-tyrosine and L-DOPA as substrates was investigated and the results obtained were discussed at molecular level by docking analysis. The resulting compounds 1-3 and a phenolic-enriched fraction of the extract, 2.9-fold more active than the starting material, may be suitable as non-toxic and inexpensive alternatives for the control of deleterious enzymatic darkening.