Abstract:Hernandulcin (HE) is a non‐caloric sweetener synthesized by the Mexican medicinal plant Phyla scaberrima. Herein we present the results of HE production through cell suspensions of P. scaberrima as well as the influence of pH, temperature, biosynthetic precursors and potential elicitors to enhance HE accumulation. The incorporation of mevalonolactone (30–400 mg L−1) farnesol (30–400 mg L−1), AgNO3 (0.025–0.175 M), cellulase (5–60 mg L−1; 0.3 units/mg), chitin (20–140 mg L−1) and (+)‐epi‐α‐bisabolol (300‐210 mg… Show more
“…The addition of high concentrations of mevalonolactone (>300 mg L −1 ) did not exert a noteworthy change in the concentration of CA and CS nor in biomass accumulation ( Figure 4 C ). This result coincided with that reported in the cell suspension system of Phylla scaberrima feed with the same precursor [12] . Since mevalonate can be distributed in the biosynthesis of several terpenes including mono‐, sesqui‐, and triterpenes, small quantities of the precursor are probably destined for the biosynthesis of CA [12] .…”
Section: Resultssupporting
confidence: 90%
“…Concentrations of 50–150 mg L −1 , produced a significant accumulation of CA and CS but concentrations over 200 mg L −1 were not linked with a clear increase of these diterpenes. This effect was also coincident with that observed in cell suspension of P. scaberrima in which the acyclic precursor farnesol was transformed into hernandulcin [12] . The absorption of these substances may be facilitated by the amphipathic character of geranylgeraniol and its ability to cross cell membranes [33] …”
Section: Resultssupporting
confidence: 73%
“…On the other hand, pH over 8 caused a significant inhibition of cell accumulation. The toxic effect of acidic and alkaline pH has been previously demonstrated in the cells suspensions of S. officinalis and Phyla scaberrima [12,20] . This effect is probably associated with the oxidation of vitamins and other nutrients found in the culture media [20] .…”
Section: Resultsmentioning
confidence: 84%
“…Mevalonolactone is a water‐soluble form of mevalonic acid which is tagged as a general precursor of terpenes [12] . The addition of high concentrations of mevalonolactone (>300 mg L −1 ) did not exert a noteworthy change in the concentration of CA and CS nor in biomass accumulation ( Figure 4 C ).…”
Section: Resultsmentioning
confidence: 97%
“…This fact facilitates recovering bioactive metabolites with high purity avoiding the use of complex and expensive protocols of purification. Since cell suspensions are comprised of independent cells, elicitation can be efficiently applied in this system and the responses are usually quicker than those observed in the whole plant [12] . To the best of our knowledge, little is known about the ecological role of CA and possible elicitors able to stimulate its accumulation.…”
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).
“…The addition of high concentrations of mevalonolactone (>300 mg L −1 ) did not exert a noteworthy change in the concentration of CA and CS nor in biomass accumulation ( Figure 4 C ). This result coincided with that reported in the cell suspension system of Phylla scaberrima feed with the same precursor [12] . Since mevalonate can be distributed in the biosynthesis of several terpenes including mono‐, sesqui‐, and triterpenes, small quantities of the precursor are probably destined for the biosynthesis of CA [12] .…”
Section: Resultssupporting
confidence: 90%
“…Concentrations of 50–150 mg L −1 , produced a significant accumulation of CA and CS but concentrations over 200 mg L −1 were not linked with a clear increase of these diterpenes. This effect was also coincident with that observed in cell suspension of P. scaberrima in which the acyclic precursor farnesol was transformed into hernandulcin [12] . The absorption of these substances may be facilitated by the amphipathic character of geranylgeraniol and its ability to cross cell membranes [33] …”
Section: Resultssupporting
confidence: 73%
“…On the other hand, pH over 8 caused a significant inhibition of cell accumulation. The toxic effect of acidic and alkaline pH has been previously demonstrated in the cells suspensions of S. officinalis and Phyla scaberrima [12,20] . This effect is probably associated with the oxidation of vitamins and other nutrients found in the culture media [20] .…”
Section: Resultsmentioning
confidence: 84%
“…Mevalonolactone is a water‐soluble form of mevalonic acid which is tagged as a general precursor of terpenes [12] . The addition of high concentrations of mevalonolactone (>300 mg L −1 ) did not exert a noteworthy change in the concentration of CA and CS nor in biomass accumulation ( Figure 4 C ).…”
Section: Resultsmentioning
confidence: 97%
“…This fact facilitates recovering bioactive metabolites with high purity avoiding the use of complex and expensive protocols of purification. Since cell suspensions are comprised of independent cells, elicitation can be efficiently applied in this system and the responses are usually quicker than those observed in the whole plant [12] . To the best of our knowledge, little is known about the ecological role of CA and possible elicitors able to stimulate its accumulation.…”
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).
Herein we report on the generation of hairy root lines of P. scaberrima able to produce hernandulcin (HE), a non‐caloric sweetener with nutraceutical properties. From ten different lines analyzed, three synthesized up to 100 mg·L‐1 HE under the batch culture conditions standardized in this investigation. Adding elicitors (salicylic acid, chitin, Glucanex, polyethylene glycol) and biosynthetic precursors (farnesol and (+)‐epi‐alpha‐bisabolol) significantly altered HE accumulation. Chitin and Glucanex enhanced HE production from 130 to 160 mg·L‐1, whereas farnesol and (+)‐epi‐alpha‐bisabolol from 165 to 200 mg·L‐1 without dependence on biomass accumulation. Improved batch cultures containing liquid Murashige & Skoog medium (MS; pH 7), added with 4 % sucrose, 0.5 mg·L‐1 naphthaleneacetic acid, 100 mg·L‐1 Glucanex, 150 mg·L‐1 chitin, 250 mg·L‐1 farnesol, and 150 mg·L‐1 (+)‐epi‐alpha‐bisabolol at 25 °C (12h light/12h darkness), triggered HE accumulation to 250 mg·L‐1 in 25 days. The efficiency of each recombinant line is discussed.
Resveratrol (RV), carnosic acid (CA) and hernandulcin (HE; a non-caloric sweetener) are envisioned as promising nutraceuticals to designnew functional foods for improving lipid and carbohydrate metabolism. This study aimed to investigate the in vitro inhibitory effectof these molecules on specific enzyme targets and their capacity to improve distinctive markers associated to carbohydrate and lipidmetabolism in murine model. The enzymes explored were alpha-amylase, alpha-glucosidase, and pancreatic lipase whereas ICR malemice were used for in vivo testing. Saturation curves (10-200 µM mL-1) and Lineweaver-Burk regressions suggested that RV, CA andHE exerts non-competitive inhibition on pancreatic lipase, alpha-glucosidase and alpha-amylase but, CA produced a strong competitiveactivity on alpha-amylase. RV was more effective to inhibit alpha-glucosidase (IC50, 22.1 µM) whereas CA was the most effective toinhibit both alpha-amylase (IC50, 11.7 µM) and pancreatic lipase (IC50, 31.5 µM). The effects of the oral administration of RV (300 mg/kg) HE (100 mg/kg) and CA (100 mg/kg) as well as the simultaneous administration of the three compounds at the same concentration was also explored in normoglycemic and diabetic mice. In addition, the prolonged administration of these substances combined with hypercaloric/atherogenic diet for 30 days was performed. Our results revealed a clear modulatory activity in both postprandial glucose and triglyceride levels as well an improvement in biochemical markers of mice treated with hypercaloric/atherogenic diet. The administration of HE produced a notable change (p < 0.01) in postprandial glucose assimilation at 60 min post-treatment in diabetic mice, whereas the other two compounds exerted a stronger depletion of glucose levels from 30 to 120 min post-treatment. A similar trend was recorded by RV and CA in postprandial triglyceride content, however, the latter compound was more effective (p < 0.05) at lower doses than RV. The simultaneous administration of the three compounds produced a significant improvement (p < 0.01) in biochemical parameters associated to carbohydrate (insulin and glucose) and lipid metabolism (total cholesterol, LDL-c, HDL-c, triglycerides, leptin, and adiponectin).Outstandingly, the mixture of the three compounds was more effective (p < 0.01) than the administration of sole compounds to amelioratethe side effects of the hypercaloric/atherogenic diet. Finally, the body weight of treated mice significantly decreased (from 5 to 20%; p < 0.05) in comparison with mice only fed with hypercaloric/atherogenic diet. Our results suggest that mixtures of RV, HE and CA may work better than their sole administration in mice and part of their biological activity could be associated with their inhibitory properties on the enzyme targets evaluated in this investigation.
Keywords: Carnosic acid; Hernandulcin; Resveratrol; Hypoglycemic; Hypolipidemic; Murine model; Target enzymes
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