Kaurenoic Acid Research Articles

Evaluation of Ursolic Acid as the Main Component Isolated from <i>Catharanthus roseus</i> against Hyperglycemia

Ursolic acid with large amount (0.67% of dried plant weight) along with 7 compounds, namely as spatozoate (1), kaurenoic acid (2), ursonic acid (3), 3-hydroxy-11-ursen-28,13-olide (4), ursolic acid (5), vindoline (6) and mixture of β-sitosterol and stigmasterol were isolated from dichloromethane and ethyl acetate extracts which have shown anti-glucosidase activity of the whole plant of C.roseus. Some isolated compounds and their derivatives were also tested for anti-glucosidase and cytotoxicity.Ursolic acid was examined for hypoglycemic activity inalloxan-induced diabetic micewith dose of 200 and 300 mg/kg/day, respectively. The results have shown that the blood glucose level were reduced by 45.75% and 51.31% to compare with the control group. This study has confirmed that the main component of Vietnamese C. roseus has had significant anti-hyperglycemia activity.

Kaurenoic acid, a Diterpene Derived from , Alleviates Lipogenesis in HepG2 Cells.

Kaurenoic acid, a Diterpene Derived from , Alleviates Lipogenesis in HepG2 Cells.

In vitro cytotoxicity study of ent-kaurenoic acid derivatives against human breast carcinoma cell line

Kaurenoic acid is a diterpene isolated from copaiba oleo resin. Based on the reported moderate cytotoxic activity of kaurenoic acid, a series of amides and diols derivatives of kaurenoic acid were synthesized and their abilities to inhibit the growth of MCF-7 cells were evaluated. Among the derivatives, compound 2f was the most active with an IC50 of 7.84 µM.

Antimicrobial activity of Aspilia latissima (Asteraceae).

We evaluated the antimicrobial activity of Aspilia latissima - an abundant plant from the Brazilian Pantanal region - against Candida albicans, Candida parapsilosis, Candida krusei, Candida tropicalis, Pseudomonas aeruginosa, Enterococcus faecalis, Escherichia coli and Staphylococcus aureus. The crude extracts and fractions showed activity in all tested microorganisms. The chloroform fraction of the leaves and roots showed the most antimicrobial activity against S. aureus, with an MIC of 500 μg/mL. This fraction was submitted to bioautographic assays to characterize the activity of the compounds. Two bands from the leaves (L-A and L-B) and three bands from the roots (R-C, R-D and R-E) were bioactive. Within the root-derived bands, the terpene derivatives stigmasterol, kaurenoic acid and kaura-9(11), 16-dien-18-oic acid were identified. Antibiotic activity of A. latissima is reported for the first time.

Semi-Synthesis of Kaurenoic Acid Derivatives and Their In Vitro Cytotoxic Activities

The cytotoxic activities of the diterpene kaurenoic acid ( 1 ) and its 15 semi-synthesis derivatives were assessed on human cell cultures. The human tumor cells used comprised colon (SW620 and SW480), pancreatic (PANC-1 and BxPC-3), stomach (SGC-7901), esophageal (Eca-109), and leukemia (K562 and HL-60). Kaurenoic acid was inactive against the tumor cell lines; however, its derivatives which contain α,β -unsaturated ketone rendered compounds with cytotoxic activity. Compounds 5 – 14, 17 – 19 , and 24 with a substitution at the C-4 position showed significant inhibitory activity against the tested cell lines, while compound 3 , without a substitution at the C-4 position, was slightly less active in these cell lines. The SW620 colon cancer cell was highly susceptible to all of the tested derivatives.

New Non-Toxic Semi-Synthetic Derivatives from Natural Diterpenes Displaying Anti-Tuberculosis Activity.

We report herein the synthesis of six diterpene derivatives, three of which are new, generated through known organic chemistry reactions that allowed structural modification of the existing natural products kaurenoic acid (1) and copalic acid (2). The new compounds were fully characterized using high resolution mass spectrometry, infrared spectroscopy, 1H- and 13C-NMR experiments. We also report the evaluation of the anti-tuberculosis potential for all compounds, which showed some promising results for Micobacterium tuberculosis inhibition. Moreover, the toxicity for each of the most active compounds was also assessed.

Effects of Kaurenoic Acid and Arginine on Random Skin Flap Oxidative Stress, Inflammation, and Cytokines in Rats.

Kaurenoic acid (KA), a diterpene extracted from copaíba oil-resin, is known to have potent antioxidant and anti-inflammatory properties. L-Arginine (LA) is an amino acid and a nitrogenous precursor for the synthesis of nitric oxide (NO). NO paper in wound healing has already been well documented. The aim of this study was to investigate the protective effects of LA and KA against ischemia reperfusion injury in a randomized skin flap model in rats. A modified McFarlane flap model measuring 2.5 wide × 8 cm long was established in 36 anesthetized rats and evaluated within 3 groups: group control, group L-arginine, and group kaurenoic acid. Each group was subdivided into two subgroups (T1 and T2, n = 6 each). Samples were collected 24 h (T1)/48 h (T2) postoperatively for oxidative stress (glutathione), as non-protein thiols, malondialdehyde (MDA), NO2, inflammation [myeloperoxidase (MPO)], and cytokines TNF-α and IL-1β assays. KA promoted a significant decrease of TNF-α and IL-1 expression and MPO activity at T1/T2 time points. NSGH levels increased significantly in KA-treated rats, while MDA levels decreased significantly in the same rats. Arginine promoted a significant decrease in MDA levels at the T1 time point and a significant increase in non-protein thiols concentrations at T1/T2 time points. NO2 concentration also decreased at the T1 time point. KA may attenuate the oxidative stress and the inflammation, thereby reducing tissue damage induced by ischemia/reperfusion in rats subjected to dorsal skin flaps. This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors http://www.springer.com/00266.

A strategy for the separation of diterpenoid isomers from the root of Aralia continentalis by countercurrent chromatography: The distribution ratio as a substitute for the partition coefficient and a three-phase solvent system

Aralia continentalis (Araliaceae) is widely used as a medicinal plant in East Asia. Previous studies have indicated that diterpenoid isomers (kaurenoic acid, continentalic acid, and ent-continentalic acid) are the major bioactive compounds of this plant. A new strategy was developed to alleviate difficulties in the separation of these isomers from this plant. A three-phase solvent system was applied to separate the isomers, and furthermore, the distribution ratio (Kc) was introduced as a substitute for the partition coefficient (KD). For compounds exhibiting a single equilibrium, their distributions in two immiscible phases were only affected by the partition coefficient of each solute. However, compounds that have a dissociating functional group (e.g., –COOH) are involved in two types of equilibrium in the two-phase system. In this case, the partitioning behaviors of the solutes are greatly affected by the pH of the solution. A mathematical prediction was applied for adjusting the solutions to the proper pH values. To prevent non-used phase (medium phase) waste, both the stationary phase (upper phase) and mobile phase (lower phase) were prepared on-demand without pre-saturation with the application of 1H NMR. Each fraction obtained was collected and dried, yielding the following diterpenoid isomers from the 50mg injected sample: kaurenoic acid (19.7mg, yield: 39%) and ent-continentalic acid (21.3mg, yield: 42%).

A kinetic study of the main guaco metabolites using syrup formulation and the identification of an alternative route of coumarin metabolism in humans.

For decades guaco species have been empirically used for the treatment of respiratory diseases. However, studies have shown that the toxic and therapeutic effects of the main guaco metabolites are dose-dependent, and none clinical study was done to evaluate the behavior of these substances in humans. In this work, a pilot study measuring the kinetic profile of the main guaco metabolites was performed leading to the knowledge of an alternative route of coumarin metabolism in humans. Initial screenings demonstrated that the administration of 60 mL of guaco syrup (single dose) did not provide sufficient levels of coumarin (COU), 7-hydroxycoumarin (7-HCOU), o-coumaric acid (OCA) and kaurenoic acid (KAU). The pharmacokinetic parameters were calculated by orally administering 60 mL of guaco syrup spiked with 1500 mg of COU. The kinetic study demonstrated that the plasmatic levels of 7-HCOU (considered the main metabolite of COU) were 10 times lower than the levels of COU, and the kinetic profile of 7-HCOU suggests sequential metabolism in the liver with low access of 7-HCOU to the systemic circulation. The study also demonstrated that OCA is one of the main bioavailable metabolites of COU. Therefore, the hydrolysis of the lactone ring forming a carboxylated compound is one of the possible routes of COU metabolism in humans. The half-lives of COU, 7-HCOU and OCA were approximately 4.0, 1.0 and 3.0 h, respectively and there was evidence that the recommended dosage of guaco syrup did not provide sufficient levels of COU, 7-HCOU or OCA to obtain a bronchodilation effect. Clinical studies are necessary to prove the efficacy and safety of products based on guaco.

Copaifera langsdorffii supercritical fluid extraction: Chemical and functional characterization by LC/MS and in vitro assays

Copaifera langsdorffii is a native Brazilian plant containing important amounts of polyphenols and diterpenes with biological activity. To obtain extracts enriched in these two types of compounds, supercritical fluid extraction (SFE) conditions have been optimized by using a response surface methodology (RSM) and considering as factors: extraction pressure and temperature and percentage of co-solvent (ethanol). The response variables selected were the concentration of the two most representative compounds in C. langsdorffii, kaurenoic acid (diterpenic acid) and quercitrin (glycosylated flavonol), total flavonoids content, and antioxidant activity (measured by DPPH and TEAC assays). According to the statistical analysis of the experimental design, extraction temperature and ethanol percentage were the main factors influencing the selectivity towards the extraction of the target compounds. Two optimized extracts were obtained: (1) containing high amount of total flavonoids, high concentration of quercitrin and low EC50 for the DPPH assay, named HQE—High Quercitrin Extract (100bar, 70°C and 50% of ethanol, in the region of gas-expanded liquids, GXLs) and (2) containing high value of kaurenoic acid extract, named HKAE-High Kaurenoic Acid Extract, (390bar, 70°C and pure CO2). Chemical characterization by LC/MS of HQE showed the presence of galloylquinic acids and flavonoids while, on the other hand, HKAE presented only nonpolar compounds such as kaurenoic acid. Moreover, a solid–liquid extraction (SLE) with methanol was carried out as a benchmark protocol for comparative purposes. SFE presented approximately 10-fold higher content of total flavonoids than the extracts obtained by SLE.

Enhanced production of steviol glycosides in mycorrhizal plants: A concerted effect of arbuscular mycorrhizal symbiosis on transcription of biosynthetic genes

Stevia rebaudiana (Bertoni) produces steviol glycosides (SGs) – stevioside (stev) and rebaudioside-A (reb-A) that are valued as low calorie sweeteners. Inoculation with arbuscular mycorrhizal fungi (AMF) augments SGs production, though the effect of this interaction on SGs biosynthesis has not been studied at molecular level. In this study transcription profiles of eleven key genes grouped under three stages of the SGs biosynthesis pathway were compared. The transcript analysis showed upregulation of genes encoding 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway enzymes viz.,1-deoxy-d-xylulose 5-phospate synthase (DXS), 1-deoxy-d-xylulose 5-phospate reductoisomerase (DXR) and 2-C-methyl-D-erytrithol 2,4-cyclodiphosphate synthase (MDS) in mycorrhizal (M) plants. Zn and Mn are imperative for the expression of MDS and their enhanced uptake in M plants could be responsible for the increased transcription of MDS. Furthermore, in the second stage of SGs biosynthesis pathway, mycorrhization enhanced the transcription of copalyl diphosphate synthase (CPPS) and kaurenoic acid hydroxylase (KAH). Their expression is decisive for SGs biosynthesis as CPPS regulates flow of metabolites towards synthesis of kaurenoid precursors and KAH directs these towards steviol synthesis instead of gibberellins. In the third stage glucosylation of steviol to reb-A by four specific uridine diphosphate (UDP)-dependent glycosyltransferases (UGTs) occurs. While higher transcription of all the three characterized UGTs in M plants explains augmented production of SGs; higher transcript levels of UGT76G1, specifically improved reb-A to stev ratio implying increased sweetness. The work signifies that AM symbiosis upregulates the transcription of all eleven SGs biosynthesis genes as a result of improved nutrition and enhanced sugar concentration due to increased photosynthesis in M plants.

Functional characterization of ent-kaurene oxidase, MtKO, from Montanoa tomentosa (Zoapatle)

Kaurene oxidases are P450 proteins that catalyze the conversion of ent-kaurene into kaurenoic acid, the final enzymatic product with a wide range of pharmacological properties. We describe the functional characterization of an ent-kaurene oxidase (EC 1.14.13.78) isolated from Montanoa tomentosa after heterologous expression in Saccharomyces cerevisiae, as well as the detection of the enzymatic activity in the plant itself. In the presence of NADPH and FAD, the microsomal fraction from transformed INVSc1 cells, ent-kaurene produced ent-kaurenoic acid, which was confirmed by GC-MS analyses. The kinetic parameters for ent-kaurene using 0.5 mg of microsomal protein were Km app= 80.63±1.2 μM and V max app= 31.80±1.8 μmol-1mg-1h-1. Optimal temperature and pH were 30°C and 7.6, respectively. Similar kinetic parameters were observed when leaf microsomes from M. tomentosa were assayed under the same conditions as for yeast microsomes. This result strongly suggests that ent-kaurene oxidase activity is present in leaf microsomes. The enzymatic activity was competitively inhibited by paclobutrazol, with IC50=43.9 μM, implying that MtKO is resistant to inhibition by azolic-type compounds. This study confirmed the biochemical detection of ent-kaurene oxidase activity in the plant, and the heterologous functionality of a cDNA with an ent-kaurene oxidase identity from M. tomentosa (zoapatle).

Kaurenoic Acid Possesses Leishmanicidal Activity by Triggering a NLRP12/IL-1β/cNOS/NO Pathway.

Leishmania amazonensis (L. amazonensis) infection can cause severe local and diffuse injuries in humans, a condition clinically known as American cutaneous leishmaniasis (ACL). Currently, the therapeutic approach for ACL is based on Glucantime, which shows high toxicity and poor effectiveness. Therefore, ACL remains a neglected disease with limited options for treatment. Herein, the in vitro antiprotozoal effect and mechanisms of the diterpene kaurenoic acid [ent-kaur-16-en-19-oic acid] (KA) against L. amazonensis were investigated. KA exhibited a direct antileishmanial effect on L. amazonensis promastigotes. Importantly, KA also reduced the intracellular number of amastigote forms and percentage of infected peritoneal macrophages of BALB/c mice. Mechanistically, KA treatment reestablished the production of nitric oxide (NO) in a constitutive NO synthase- (cNOS-) dependent manner, subverting the NO-depleting escape mechanism of L. amazonensis. Furthermore, KA induced increased production of IL-1β and expression of the inflammasome-activating component NLRP12. These findings demonstrate the leishmanicidal capability of KA against L. amazonensis in macrophage culture by triggering a NLRP12/IL-1β/cNOS/NO mechanism.

Stimulation of in vitro morphogenesis, antioxidant activity and over expression of kaurenoic acid 13-hydroxylase gene in Stevia rebaudiana Bertoni by chlorocholine chloride

Phytoconstituents from medicinal plants are considered as important source of raw materials of drugs for pharmaceutical industries. Biotechnology has become an inevitable approach in the area of research and development of medicinal plants for many decades. The present work has been carried out to ascertain the role of chlorocholine chloride (CCC) on in vitro morphogenesis, antioxidant activity and expression level of kaurenoic acid 13-hydroxylase (KA13H) gene in Stevia rebaudiana. To fulfill these purposes chlorocholine chloride was applied in the Murashige and Skoog (Physiol Plant 15(3):473–497, 1962) medium in combination with other plant growth regulators such as 1-naphthalene acetic acid, kinetin and thidiazuron. Chlorocholine chloride was found to contribute significant role on in vitro morphogenesis of S. rebaudiana as evidenced by the formation of embryogenic calli and increase in callusing and microshooting efficiency of explant, i.e., cotyledonary leaf. Moreover, antioxidant enzyme activity as well as ascorbic acid content of the calli and leaves was also stimulated after application of chlorocholine chloride. Q-PCR amplification using gene-specific primers revealed that CCC also promoted the expression level of KA13H gene in S. rebaudiana leaves. The overall study highlighted the promising role of chlorocholine chloride on regeneration efficiency of cotyledonary leaf, significant promotion in antioxidant potential and expression of KA13H gene in S. rebaudiana.

Ent-Kaurane diterpenes from the stem bark of Annona vepretorum (Annonaceae) and cytotoxic evaluation

This work describes a novel ent-kaurane diterpene, ent-3β-hydroxy-kaur-16-en-19-al along with five known ent-kaurane diterpenes, ent-3β,19-dihydroxy-kaur-16-eno, ent-3β-hydroxy-kaur-16-eno, ent-3β-acetoxy-kaur-16-eno, ent-3β-hydroxy-kaurenoic acid and kaurenoic acid, as well as caryophyllene oxide, humulene epoxide II, β-sitosterol, stigmasterol and campesterol from the stem bark of Annona vepretorum Mart. (Annonaceae). Cytotoxic activities towards tumor B16-F10, HepG2, K562 and HL60 and non-tumor PBMC cell lines were evaluated for ent-kaurane diterpenes. Among them, ent-3β-hydroxy-kaur-16-en-19-al was the most active compound with higher cytotoxic effect over K562 cell line (IC50 of 2.49μg/mL) and lower over B16-F10 cell line (IC50 of 21.02μg/mL).

Immunomodulatory and Antioxidant Properties of Kaurenoic Acid on Macrophages of BALB/c <i>in Vitro</i>

Kaurenoic acid has been displaying anti-inflammatory effect described in different models. However, the per se immunomodulatory effects of kaurenoic acid remain to be investigated. Thus, the immunomodulatory and antioxidant effects of kaurenoic acid were investigated in vitro on peritoneal macrophages from BALB/c mice. Kaurenoic acid induced per se the production of pro-inflammatory cytokines such as TNFI±, IL-1I² and IFN-I³ while also increased the levels of IL-10. There was also reduction of NO production and induction of anti-oxidant profile. Therefore, in addition to inhibiting inflammation, kaurenoic acid presents immunomodulatory effects per se.

Overexpression of SrUGT85C2 from Stevia reduced growth and yield of transgenic Arabidopsis by influencing plastidial MEP pathway

The transcript expression of a gene SrUGT85C2 has been documented for direct relation with steviol glycoside content in Stevia plant. Steviol glycoside and gibberellin biosynthetic routes are divergent branches of methyl erythritol-4 phosphate (MEP) pathway. So, SrUGT85C2 might be an influencing gibberellin content. Hence in the present study, transgenic Arabidopsis thaliana overexpressing SrUGT85C2 cDNA from Stevia rebaudiana was developed to check its effect on gibberellin accumulation and related plant growth parameters. The developed transgenics showed a noteworthy decrease of 78-83% in GA3 content. Moreover, the transgenics showed a gibberellin deficient phenotype comprising stunted hypocotyl length, reduced shoot growth and a significant fall in relative water content. Transgenics also showed 17-37 and 64-76% reduction in chlorophyll a and chlorophyll b contents, respectively. Reduction in photosynthetic pigments could be responsible for the noticed significant decrease in plant biomass. Like steviol glycoside and gibberellin biosynthesis, chlorophyll biosynthesis also occurs from the precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) of MEP pathway in the plastids. The observed downregulated expression of genes encoding MEP pathway enzymes geranyl geranyl diphosphate synthase (GGDPS), copalyl diphosphate synthase (CDPS), kaurenoic acid oxidase (KAO), chlorophyll synthetase and chlorophyll a oxygenase in transgenics overexpressing SrUGT85C2 might be responsible for the reduction in gibberellins as well as chlorophyll. This study has documented for the first time the regulatory role of SrUGT85C2 in the biosynthesis of steviol glycoside, gibberellins and chlorophyll.

Ovipositional Preference and Larval Performance of the Banded Sunflower Moth (Lepidoptera: Tortricidae) and Its Larval Parasitoids on Resistant and Susceptible Lines of Sunflower (Asterales: Asteraceae)

Banded sunflower moth, Cochylis hospes Walsingham, is one of the most destructive seed-feeding insect pests of sunflowers, causing significant economic yield losses in the northern Great Plains. In an attempt to understand host-plant resistance mechanisms for this pest, we field-tested, over several years, the effects of seven sunflower accessions, rated as resistant to C. hospes in previous screening trials, and a susceptible control (Par 1673-2), on the ovipositional preference and larval performance of C. hospes and its larval parasitoids. Of the resistant accessions, PI 494859 was the most preferred for oviposition, receiving a significantly greater number of eggs per head than did the susceptible Par 1673-2 in 2 of 3 yr. However, the numbers of larvae, and consequently the rate of seed infestation, found in PI 494859 heads were significantly lower than those in Par 1673-2 heads over all 3 yr. Female moths laid relatively few eggs on accessions PI 170385, 291403, and 251902, compared with on Par 1673-2, resulting in lower numbers of larvae per head and lower percentages of seed damaged. No association was observed between the concentrations of two diterpenoid alcohols or two diterpenoid acids in sunflower bracts and the numbers of eggs laid on the heads of the accessions. The number of banded sunflower moth larvae and the proportion of seeds damaged were positively correlated with kaurenoic acid concentrations and negatively correlated with kauranol concentrations. A positive association between resistance to larval feeding and parasitism was found in years 2006 and 2008, with resistant accessions having significantly greater proportions of parasitized larvae than did the susceptible Par 1673-2.

Kaurenoic Acid Isolated from the Root Bark of Annona senegalensis Induces Cytotoxic and Antiproliferative Effects against PANC-1 and HeLa cells

Aims: Cancer is one of the leading causes of death worldwide with an estimated 6.7 million deaths and 24.6 million people living with cancer in 2002. Presently, there is a global increase in prevalence, mortality and health burden of various malignancies. World Health Organization (WHO) report projected that cancer prevalence rates could further increase by 50% to 15 million new cases in the year 2020. The bioactivity guided isolation of the bioactive constituent and its characterization, responsible for the anticonvulsant effects of the root bark extract of

Gibberellin oxidase activities in Bradyrhizobium japonicum bacteroids

Bradyrhizobium japonicum bacteroids isolated from root nodules of soybean (Glycine max.) plants converted the gibberellin (GA) precursor [14C1]GA12 into several products identified by combined gas chromatography–mass spectrometry as [14C1]GA24, [14C1]GA9, [14C1]GA15, GA9 17-nor-16-one and unidentified products. The oxidation of GA12, catalyzed by the GA 20-oxidase, was present in symbiotic bacteroids from plants around flowering, but not in bacteroids from plants at either an early vegetative stage or at late growth stages. Expression of cps and ks genes, involved in ent-kaurene biosynthesis, was also demonstrated in bacteroids from soybean plants around flowering. Earlier precursors of the GA pathway, ent-[14C1]kaurenoic acid or [14C4]GA12-aldehyde, were efficiently utilized by B. japonicum bacteroids to give labelled GA9 plus intermediates partially oxidized at C-20, as well as GA9 17-nor-16-one and an unidentified product. No 3β or 13-hydroxylated [14C]GAs were detected in any of the incubations. Moreover the C19-GAs [14C1]GA4 or [14C1]GA20 were recovered unconverted upon incubation with the bacteroids which supports the absence of GA 3β-hydroxylase activity in B. japonicum. The bacterial 20-oxidase utilized the 13-hydroxylated substrates [14C1]GA53, [14C1]GA44 or [14C1]GA19, although with less efficiency than [14C1]GA12 to give [14C1]GA20 as final product, while the 3β-hydroxylated substrate [14C1]GA14 was converted to [14C1]GA4 to a very small extent. Endogenous GA9 and GA24 were identified by GC–MS in methanolic nodule extracts. These results suggest that B. japonicum bacteroids would synthesize GA9 under the symbiotic conditions present in soybean root nodules.