Effect of Uncaria tomentosa aqueous extract on the response to palmitate-induced lipotoxicity in cultured skeletal muscle cells.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is frequently associated with dyslipidemia, which corresponds to the increase in the triglycerides and fatty acid concentrations in tissues, such as the skeletal muscle. Also, T2DM molecular mechanism involves increasing in reactive oxygen species (ROS) production and oxidative stress. The use of herbal medicines such as Uncaria tomentosa (Ut) has been proposed as an auxiliary treatment for patients with T2DM. In this study, it was evaluated the effect of Ut aqueous extract on cell viability and ROS production, in skeletal myoblasts from C2C12 lineage exposed to the free fatty acid palmitate (PA). METHODS: Cells were incubated with PA in different concentrations ranging from 10 to 1000 µM, for 24 or 48 h, for cytotoxicity assay. Cell death, DNA fragmentation and ROS production assays were performed in cell cultures incubated with PA for 24 h, in the pre (preventive condition) or post treatment (therapeutic condition) with 250 µg/ml Ut aqueous extract, for 2 or 6 h. Cell death was evaluated by MTT method or flow cytometry. ROS generation was measured by fluorescence spectroscopy using the DCFDA probe. RESULTS: Cell viability was reduced to approximately 44% after the incubation with PA for 24 h from the concentration of 500 µM. In the incubation of cells with 500 µM PA and Ut extract for 6 h, in both conditions (preventive or therapeutic), it was observed an increase of 27 and 70% in cell viability respectively, in comparison to the cultures incubated with only PA. Also, the incubation of cultures with 500 µM PA, for 24 h, increased 20-fold the ROS formation, while the treatment with Ut extract, for 6 h, both in the preventive or therapeutic conditions, promoted decrease of 21 and 55%, respectively. CONCLUSION: The Ut extract was efficient in promoting cell protection against PA lipotoxicity and ROS generation, potentially preventing oxidative stress in C2C12 skeletal muscle cells. Since T2DM molecular mechanism involves oxidative stress condition and it is often associated with dyslipidemia and fatty acid accumulation in muscle tissue, these results open perspectives for the use of Ut as an auxiliary strategy for T2DM management.

Glucoindole alkaloid accumulation induced by yeast extract in Uncaria tomentosa root cultures is involved in defense response.

OBJECTIVE: To evaluate the induction of monoterpenoid indole alkaloids (MIA) and phenolic compound production by yeast extract (YE) and its relationship with defense responses in Uncaria tomentosa (Rubiaceae) root cultures. RESULTS: Root cultures were elicited by YE at three concentrations. The 0.5 mg YE ml-1 treatment did not affect cell viability but increased the hydrogen peroxide concentration by 5.7 times; guaiacol peroxidase activity by twofold; and the glucoindole alkaloid 3α-dihydrocadambine (DHC) content by 2.6 times (to 825.3 ± 27.3 µg g-1). This treatment did not affect the contents of monoterpenoid oxindole alkaloids or chlorogenic acids. In response to 0.5 mg YE ml-1 treatment, the transcript levels of MIA biosynthetic genes, TDC and LAMT, increased 5.4 and 1.9-fold, respectively, that of SGD decreased by 32%, and that of STR did not change. The transcript levels of genes related to phenolic compounds, PAL, CHS and HQT, increased by 1.7, 7.7, and 1.2-fold, respectively. Notably, the transcript levels of Prx1 and Prx encoding class III peroxidases increased by 1.4 and 2.5-fold. CONCLUSION: The YE elicitor induced an antioxidant defense response, increased the transcript levels of genes encoding enzymes related to strictosidine biosynthesis precursors and class III peroxidases, and decreased the transcript level of SGD. Thus, YE could stimulate antifungal DHC production in root cultures of U. tomentosa.

Unnatural spirocyclic oxindole alkaloids biosynthesis in Uncaria guianensis.

Spiro-oxindole scaffolds have been studied due to their promising therapeutic potential. In the Amazon rainforest there are two important Uncaria species known as "cat's claw", which biosynthesize spirocyclic oxindole alkaloids; Uncaria tomentosa (Willd. ex Schult.) DC. and Uncaria guianensis (Aublet) Gmell. We carried out a precursor-directed biosynthesis approach with U. guianensis and successfully obtained oxindole alkaloid analogues with molecular mass corresponding to the addition of a methyl or fluorine group on the oxindole ring using tryptamine analogue precursors. Two of these novel oxindole alkaloid analogues (3b-7-methyl-isomitraphylline and 3c-6-fluoro-isomitraphylline) were isolated and characterized by NMR spectroscopy and ESI-QTOF-MS/MS. Having established a substrate feeding protocol for these plantlets, the biosynthetic route for mitraphylline (1), rhynchophylline (2), isomitraphylline (3) and isorhynchophylline (4) was also investigated using 13C-precursors (1-13C-D-glucose, 2-13C-tryptophan, 1-13C-DL-glyceraldehyde, and methyl-13C-D-methionine).

The Amazon rain forest plant Uncaria tomentosa (cat's claw) and its specific proanthocyanidin constituents are potent inhibitors and reducers of both brain plaques and tangles.

Brain aging and Alzheimer's disease both demonstrate the accumulation of beta-amyloid protein containing "plaques" and tau protein containing "tangles" that contribute to accelerated memory loss and cognitive decline. In the present investigation we identified a specific plant extract and its constituents as a potential alternative natural solution for preventing and reducing both brain "plaques and tangles". PTI-00703 cat's claw (Uncaria tomentosa from a specific Peruvian source), a specific and natural plant extract from the Amazon rain forest, was identified as a potent inhibitor and reducer of both beta-amyloid fibrils (the main component of "plaques") and tau protein paired helical filaments/fibrils (the main component of "tangles"). PTI-00703 cat's claw demonstrated both the ability to prevent formation/aggregation and disaggregate preformed Aß fibrils (1-42 and 1-40) and tau protein tangles/filaments. The disaggregation/dissolution of Aß fibrils occurred nearly instantly when PTI-00703 cat's claw and Aß fibrils were mixed together as shown by a variety of methods including Thioflavin T fluorometry, Congo red staining, Thioflavin S fluorescence and electron microscopy. Sophisticated structural elucidation studies identified the major fractions and specific constituents within PTI-00703 cat's claw responsible for both the observed "plaque" and "tangle" inhibitory and reducing activity. Specific proanthocyanidins (i.e. epicatechin dimers and variants thereof) are newly identified polyphenolic components within Uncaria tomentosa that possess both "plaque and tangle" reducing and inhibitory activity. One major identified specific polyphenol within PTI-00703 cat's claw was epicatechin-4ß-8-epicatechin (i.e. an epicatechin dimer known as proanthocyanidin B2) that markedly reduced brain plaque load and improved short-term memory in younger and older APP "plaque-producing" (TASD-41) transgenic mice (bearing London and Swedish mutations). Proanthocyanidin B2 was also a potent inhibitor of brain inflammation as shown by reduction in astrocytosis and gliosis in TASD-41 transgenic mice. Blood-brain-barrier studies in Sprague-Dawley rats and CD-1 mice indicated that the major components of PTI-00703 cat's claw crossed the blood-brain-barrier and entered the brain parenchyma within 2 minutes of being in the blood. The discovery of a natural plant extract from the Amazon rain forest plant (i.e. Uncaria tomentosa or cat's claw) as both a potent "plaque and tangle" inhibitor and disaggregator is postulated to represent a potential breakthrough for the natural treatment of both normal brain aging and Alzheimer's disease.

Uncaria tomentosa improves insulin sensitivity and inflammation in experimental NAFLD.

We investigated the effect of the crude herbal extract from Uncaria tomentosa (UT) on non-alcoholic fatty liver disease (NAFLD) in two models of obesity: high fat diet (HFD) and genetically obese (ob/ob) mice. Both obese mouse models were insulin resistant and exhibited an abundance of lipid droplets in the hepatocytes and inflammatory cell infiltration in the liver, while only the HFD group had collagen deposition in the perivascular space of the liver. UT treatment significantly reduced liver steatosis and inflammation in both obese mouse models. Furthermore, serine phosphorylation of IRS-1 was reduced by 25% in the HFD mice treated with UT. Overall, UT treated animals exhibited higher insulin sensitivity as compared to vehicle administration. In conclusion, Uncaria tomentosa extract improved glucose homeostasis and reverted NAFLD to a benign hepatic steatosis condition and these effects were associated with the attenuation of liver inflammation in obese mice.

Long-term response on growth, antioxidant enzymes, and secondary metabolites in salicylic acid pre-treated Uncaria tomentosa microplants.

OBJECTIVE: To obtain micro propagated Uncaria tomentosa plantlets with enhanced secondary metabolites production, long-term responses to salicylic acid (SA) pre-treatments at 1 and 100 µM were evaluated after propagation of the plantlets in a SA-free medium. RESULTS: SA pre-treatments of single node cuttings OF U. tomentosa produced long-term responses in microplants grown for 75 days in a SA-free medium. Reduction in survival rate, root formation, and stem elongation were observed only with 100 µM SA pre-treatments with respect to the control (0 + DMSO).Both pre-treatments enhanced H2O2 and inhibited superoxide dismutase and catalase activities, while guaiacol peroxidase was increased only with 1 µM SA. Also, both pre-treatments increased total monoterpenoid oxindole alkaloids by ca. 55 % (16.5 mg g(-1) DW), including isopteropodine, speciophylline, mitraphylline, isomitraphylline, rhynchopylline, and isorhynchopylline; and flavonoids by ca. 21 % (914 µg g(-1) DW), whereas phenolic compounds were increased 80 % (599 µg g(-1) DW) at 1 µM and 8.2 % (359 µg g(-1) DW) at 100 µM SA. CONCLUSION: Pre-treatment with 1 µM SA of U.tomentosa microplants preserved the survival rate and increased oxindole alkaloids, flavonoids, and phenolic compounds in correlation with H2O2 and peroxidase activity enhancements, offering biotechnological advantages over non-treated microplants.

Strictosidine-related enzymes involved in the alkaloid biosynthesis of Uncaria tomentosa root cultures grown under oxidative stress.

The activity and gene expression of strictosidine-related enzymes in Uncaria tomentosa root cultures exposed to oxidative stress were studied. Elicitation with 0.2 mM hydrogen peroxide (H2 O2 ) or a combination of 0.8 mM buthionine sulfoximine and 0.2 mM jasmonic acid (BSO-JA) increased peroxidase activities by twofold at Day 8 and glutathione reductase by 1.4-fold at Day 5 in H2 O2 elicited cultures respect to the control. Production of monoterpenoid oxindole alkaloids (MOA), 3α-dihydrocadambine, and dolichantoside was stimulated after H2 O2 elicitation, reaching levels of 886.4 ± 23.6, 847.7 ± 25.4, and 87.5 ± 7.2 µg/g DW, at Day 8 which were 1.7-, 2.1-, and 2.3-fold higher relative to control. BSO-JA elicited cultures produced about twice alkaloids than H2 O2 -treated cultures, following a biphasic pattern with maxima at 0.5 and 8 days. Alkaloid production was preceded by increase in strictosidine synthase (STR) and strictosidine glucosidase (SGD) activities. After elicitation with H2 O2 or BSO-JA, the STR activity (pKat/mg protein) increased by 1.9-fold (93.8 ± 17.8 at 24 h) or 2.5-fold (102.4 ± 2.2 at 6 h) and the SGD activity (pKat/mg protein) by 2.8-fold (245.2 ± 14.4 at 6 h) or 4.2-fold (421.2 ± 1.8 at 18 h) relative to control. STR and SGD transcripts were upregulated after elicitation. H2 O2 -treated roots showed higher levels of STR at 48-192 h and SGD at 24-48 h, while BSO-JA treatments showed STR increased at 12 h and SGD at 24 h. Also, LC/ESI-MS confirmed the biosynthesis of dolichantoside from N-ω-methyltryptamine and secologanin by U. tomentosa protein extracts.

Evaluation of biological activity of Uncaria tomentosa (Willd.) DC. using the chicken embryo model.

The biological activity of Uncaria tomentosa (Willd.) DC. (cat's claw) was evaluated by application of the chicken embryo model. Among three groups of eggs (n = 360) with twelve-day old embryos, two were injected with different doses of cat's claw extracts (0.0492 and 0.492 mg/200 lambda). To the third control group 200 lambda of physiological salt was applied. All eggs were incubated in conventional forced-air apparatus until hatched. Hatchability, chicken weight and wholesomeness were analyzed. Selected parameters of blood including number of erythrocytes (RBC), number of leukocytes (WBC), mean red cell volume (MCV), hematocrit (HCT), hemoglobin concentration (HGB), mean amount of cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC) and embryo weight (MAS) were assayed and compared. Significant differences with ANOVA were observed for MCV (P = 0.002), MCHC (P = 0.00001) and MCH (P = 0.02). Applying the chicken embryo model brought new information about the biological activity of U. tomentosa showing an unfavourable effect on some morphological blood parameters.

Induced accumulation of oleanolic acid and ursolic acid in cell suspension cultures of Uncaria tomentosa.

Increasing sucrose from 20 to 50 g l(-1) in Uncaria tomentosa cell suspension cultures enhanced ursolic acid and oleanolic acid production from 129 +/- 61 to 553 +/- 193 microg g(-1) cell dry wt. The maximal concentration of both triterpenes (1680 +/- 39 microg g(-1) cell dry wt) was 8 days after elicitation by jasmonic acid, while yeast extract or citrus pectin treatments produced 1189 +/- 20 or 1120 +/- 26 microg g(-1) cell dry wt, respectively. The ratio of ursolic acid:oleanolic acid was constant at 70:30.

Biosynthesis of sterols and triterpenes in cell suspension cultures of Uncaria tomentosa.

Pectin administered to Uncaria tomentosa cell suspension cultures, was found to increase the production of triterpene acids (ursolic and oleanolic acid), however, neither growth nor sterol accumulation were affected. Cell cultures showed that pectin treatment caused a rapid threefold increase in the activities of enzymes involved in the biosynthesis of C(5) and C(30 )isoprenoid, such as isopentenyl diphosphate isomerase and squalene synthase. The activity of a farnesyl diphosphatase, which could divert the flux of farnesyl diphosphate to farnesol, was two times lower in elicited than in control cells. Elicited cells also transformed more rapidly a higher percentage of [5-(3)H]mevalonic acid into triterpene acids. Interestingly, addition of terbinafine, an inhibitor of squalene epoxidase, to elicited cell cultures inhibited sterol accumulation while triterpene production was not inhibited. These results suggest that in U. tomentosa cells, both the previously mentioned enzymes and those involved in squalene 2,3-oxide formation play an important regulatory role in the biosynthesis of sterols and triterpenes.