Continentalic Acid Research Articles

Naturally Derived Terpenoids Targeting the 3Dpol of Foot-and-Mouth Disease Virus: An Integrated In Silico and In Vitro Investigation.

Foot-and-mouth disease virus (FMDV) belongs to the Picornaviridae family and is an important pathogen affecting cloven-hoof livestock. However, neither effective vaccines covering all serotypes nor specific antivirals against FMDV infections are currently available. In this study, we employed virtual screening to screen for secondary metabolite terpenoids targeting the RNA-dependent RNA polymerase (RdRp), or 3Dpol, of FMDV. Subsequently, we identified the potential antiviral activity of the 32 top-ranked terpenoids, revealing that continentalic acid, dehydroabietic acid (abietic diterpenoids), brusatol, bruceine D, and bruceine E (tetracyclic triterpenoids) significantly reduced cytopathic effects and viral infection in the terpenoid-treated, FMDV-infected BHK-21 cells in a dose-dependent manner, with nanomolar to low micromolar levels. The FMDV minigenome assay demonstrated that brusatol and bruceine D, in particular, effectively blocked FMDV 3Dpol activity, exhibiting IC50 values in the range of 0.37-0.39 µM and surpassing the efficacy of the antiviral drug control, ribavirin. Continentalic acid and bruceine E exhibited moderate inhibition of FMDV 3Dpol. The predicted protein-ligand interaction confirmed that these potential terpenoids interacted with the main catalytic and bystander residues of FMDV 3Dpol. Additionally, brusatol and bruceine D exhibited additive effects when combined with ribavirin. In conclusion, terpenoids from natural resources show promise for the development of anti-FMD agents.

Design and Evaluation of Continentalic Acid Encapsulated Transfersomal Gel and Profiling of its Anti-arthritis Activity.

Rheumatoid arthritis restricts the physical ability of patients and increases the disease burden; therefore, research has always been focused on evaluating better therapeutic options. The present research aimed to design Continentalic acid (CA)-loaded transfersomes (CA-TF) embedded in Carbopol gel containing permeation enhancer (PE) for the treatment of rheumatoid arthritis. CA-TF was developed via a modified thin film hydration method and incorporated into Carbopol 934 gel containing Eucalyptus oil (EO) as PE. The fabricated CA-TF showed particle size of < 140nm with spherical geometry, optimal encapsulation efficiency (EE), and sustained drug release pattern. CA-TF-gel along with PE (CA-TF-PE-gel) showed better ex vivo skin penetration than plain CA gel and CA-TF-gel without PE. In vivo evaluation supported improved therapeutic outcomes of CA-TF-PE-gel in terms of behavioral findings, arthritic index, and histological findings whereas biochemical assays and pro-inflammatory cytokines (TNF-α and IL-1β) showed a significant decrease in their levels. Furthermore, immunohistochemistry assay for Nrf2 and HO-1 signaling pathways showed significant improvement in the expression of the Nrf2, and HO-1 proteins to depict improvement in arthritic condition in the animal model. CA-TF-PE-gel significantly delivered CA to the diseased target site via a topical route with promising therapeutic outcomes displayed in the CFA-induced arthritic model.

Identification of the antibacterial action mechanism of diterpenoids through transcriptome profiling.

Effective antibacterial substances of Aralia continentalis have anti-biofilm and bactericidal activity to the oral pathogen Streptococcus mutans. In this study, three compounds extracted from A. continentalis were identified as acanthoic acid, continentalic acid, and kaurenoic acid by NMR and were further investigated how these diterpenoids affect the physiology of the S. mutans. When S. mutans was exposed to individual or mixed fraction of diterpenoids, severe growth defects and unique morphology were observed. The proportion of unsaturated fatty acids in the cell membrane was increased compared to that of saturated fatty acids in the presence of diterpenoids. Genome-wide gene expression profiles with RNA-seq were compared to reveal the mode of action of diterpenoids. Streptococcus mutans commonly enhanced the expression of 176 genes in the presence of the individual diterpenoids, whereas the expression of 232 genes was considerably reduced. The diterpenoid treatment modulated the expression of genes or operon(s) involved in cell membrane synthesis, cell division, and carbohydrate metabolism of S. mutans. Collectively, these findings provide novel insights into the antibacterial effect of diterpenoids to control S. mutans infection, which causes human dental caries.

Anticancer Activity of Continentalic Acid in B-Cell Lymphoma.

Aralia continentalis has been used in Korea as a folk remedy for arthralgia, rheumatism, and inflammation. However, its anti-lymphoma effect remains uncharacterized. Here, we demonstrate that A. continentalis extract and its three diterpenes efficiently kill B-lymphoma cells. Our in vitro and in vivo results suggest that the cytotoxic activities of continentalic acid, a major diterpene from A. continentalis extract, are specific towards cancer cells while leaving normal murine cells and tissues unharmed. Mechanistically, continentalic acid represses the expression of pro-survival Bcl-2 family members, such as Mcl-1 and Bcl-xL. It dissociates the mitochondrial membrane potential, leading to the stimulation of effector caspase 3/7 activities and, ultimately, cell death. Intriguingly, this agent therapeutically synergizes with roflumilast, a pan-PDE4 inhibitor that has been successfully repurposed for the treatment of aggressive B-cell malignancies in recent clinical tests. Our findings unveiled that A. continentalis extract and three of the plant’s diterpenes exhibit anti-cancer activities. We also demonstrate the synergistic inhibitory effect of continentalic acid on the survival of B-lymphoma cells when combined with roflumilast. Taken in conjunction, continentalic acid may hold significant potential for the treatment of B-cell lymphoma.

RETRACTED: Pharmacological evaluation of continentalic acid for antidiabetic potential

Diabetes is a complex endocrine and metabolic disorder. Continentalic acid is a natural drug product found in roots of Aralia continentalis (family Araliaceae), which used in traditional medicine for treatment of rheumatic arthritis, lumbag, lameness, inflammation, gastritis, nephritis and diabetes mellitus. This study is aim to investigate the continentalic acid anti-diabetic potential. In-silico, in-vitro, in-vivo and molecular techniques were used to investigate various effects of continentalic acid by Auto Doc Vina, α-amylase and α-glucosidase inhibitory assay and alloxan-induced diabetes rats model. In-silico results revealed that continentalic acid exhibits binding energy values of -5 to -9.3Kcal/mol against selected targets. In-vitro assay showed that continentalic acid caused α-amylase and α-glucosidase enzymes inhibition. In-vivo finding exhibits that continentalic acid (50mg/kg) decreased blood glucose level, body weight, oral glucose tolerance overload, glycosylated hemoglobin, triglycerides, total cholesterol, low density lipoprotein, aspartate transaminase, aspartate aminotransferase, alkaline phosphate, total bilirubin and increased high density lipoprotein (P<0.05, P<0.01, P<0.001 vs. diabetic control group). In animals pancreas and liver tissues, continentalic acid enhanced glutathione-s-transferase, reduced glutathione, catalase and decreased lipid hydroperoxide level, improved cellular architecture in histopathological examination and decrease expression of inflammatory markers: cyclooxygenase 2, tumor necrosis factor alpha, phosphorylated-nuclear factor kappa B, prostaglandins E2, interleukin-18 and increased heme oxygenase-1, as evidenced in immunohistochemistry and enzyme-linked immunosorbent assay molecular investigations. This study shows that continentalic acid exhibited binding affinities against the different targets and anti-diabetic action, mediated possibly through α-amylase and α-glucosidase inhibition, anti-hyperlipidemic, hepatoprotection, antioxidant and anti-inflammatory pathways.

Attenuation of LPS-induced acute lung injury by continentalic acid in rodents through inhibition of inflammatory mediators correlates with increased Nrf2 protein expression

BackgroundAcute lung injury (ALI) together with acute respiratory distress syndrome (ARDS) are associated with high rate of mortality and morbidity in patients. In the current study, the anti-inflammatory effects of continentalic acid (CNT) in LPS-induced acute lung injury model was explored.MethodsThe acute lung injury model was established by administering LPS (5 mg/kg) intraperitonealy. Following LPS administration, the survival rate, temperature changes and lung Wet/Dry ratio were assessed. The antioxidants (GSH, GST, Catalase and SOD) and oxidative stress markers (MDA, NO, MPO) were evaluated in all the treated groups. Similarly, the cytokines such as IL-1β, IL-6 and TNF-α were analyzed using ELISA assay. The histological changes were determined using H and E staining, while Nrf2 and iNOS level were determined using immunohistochemistry analysis. The molecular docking analysis was performed to assess the pharmacokinetics parameters and interaction of the CNT with various protein targets.ResultsThe results showed that CNT dose dependently (10, 50 and 100 mg/kg) reduced mortality rate, body temperature and lungs Wet/Dry ratio. CNT post-treatment significantly inhibited LPS-induced production of pro-inflammatory cytokines such as IL-1β, IL-6 and TNF-α. The CNT post-treatment markedly improved the hematological parameters, while significantly reduced the MPO (indicator of the neutrophilic infiltration) activity compared to the LPS treated group. Furthermore, the CNT (100 mg/kg) post-administration remarkably inhibited the lung Wet/Dry ratio. The CNT (100 mg/kg) treated group showed marked reduction in the oxidative stress markers such as malonaldehyde (MDA) and Nitric oxide (NO) concentration, while induced the level of the anti-oxidant enzymes such as GST, GSH, Catalase and SOD. Similarly, the CNT markedly reduced the iNOS expression level, while induced the Nrf2 protein expression. Additionally, the molecular docking study showed significant binding interaction with the Nrf2, p65, Keap1, HO-1, IL-1β, IL-6, TNF-α and COX-2, while exhibited excellent physicochemical properties.ConclusionThe CNT showed marked protection against the LPS-induced lung injury and improved the behavioral, biochemical and histological parameters. Furthermore, the CNT showed significant interaction with several protein targets and exhibited better physicochemical properties.

Optimization of Extraction Conditions of Continentalic and Kaurenoic Acids from Aralia continentalis by HPLC/UV and Their Validation.

Aralia continentalis (AC) is a perennial herb that has long been used as a traditional medicine for many diseases. Continentalic acid (CA) and kaurenoic acid (KA) are major diterpenoids in AC, which are known to exert various pharmacological activities. This study focuses on the optimization of the extraction of CA and KA from dried AC roots by evaluating the influence of different extraction conditions on their yield. Five extraction variables were examined: sample weight, solvent concentration, extraction time, solid matrix and the number of repeated extractions. The analytical method used in this study was also validated in terms of linearity, limit of detection, limit of quantification, precision and accuracy. The CA and KA yields were measured by high-performance liquid chromatography analysis. The results show that CA and KA were the highest when unpulverized samples (3.75g) were subjected to a single extraction for 5h using 50% ethanol (300mL) as the solvent. These conditions are proposed for the optimization of the extraction of CA and KA from AC.

Continentalic acid exhibited nephroprotective activity against the LPS and E. coli-induced kidney injury through inhibition of the oxidative stress and inflammation

The present study investigated the effect of the continentalic acid (CNT) isolated from the Aralia Continentalis against the LPS and E. coli-induced nephrotoxicity. The LPS and E. coli administration markedly altered the behavioral parameters including spontaneous pain, tail suspension and survival rate. However, the treatment with CNT dose dependently improved the behavioral parameters. The CNT treatment significantly improved the renal functions test (RFTs) and hematological parameters following LPS and E. coli-induced kidney injury. Furthermore, the LPS and E. coli administration markedly compromised the anti-oxidant enzymes and enhanced the oxidative stress markers. However, the CNT treatment markedly enhanced the anti-oxidants enzymes such as GSH, GST, Catalase and SOD, while attenuated the oxidative stress markers such as MDA and POD. The MPO enzyme is widely used marker for the neutrophilic infiltration, the LPS and E. coli administration markedly increased the MPO activity. However, the CNT treatment markedly attenuated the MPO activity in both LPS and E. coli-induced kidney injury. Furthermore, the CNT treatment markedly attenuated the NO production compared to the LPS and E. coli-induced kidney injury group. Additionally, the CNT treatment improved the histological parameters markedly (H and E, PAS and Masson’s trichome staining) and protect the kidney from the inflammatory insult of the LPS and E. coli evidently. The comet assay revealed marked DNA damage, however, the CNT treatment markedly prevented the LPS and E. coli-induced kidney damage. The CNT treatment markedly enhanced the expression of Nrf2, while attenuated the iNOS expression in both models of kidney injury.

Simultaneous determination of two diterpenoids, continentalic acid and kaurenoic acid, in the water extract of Aralia continentalis and their wound-healing activity

Background: The roots of Aralia continentalis have been used in traditional Korean medicine to treat pain, lumbago, lameness, and rheumatism. Objective: Simultaneous determination of two diterpenoids; continentalic acid (CA) and kaurenoic acid, in the water extract of A. continentalis using the high-performance liquid chromatography (HPLC) was established for quality control, as well as evaluation of their wound-healing activity. Materials and Methods: The separation was conducted on YMC hydrosphere C18 column with isocratic elution of 65% acetonitrile. Furthermore, the isolated diterpenoids were screened for their wound-healing activity using keratinocytes (HaCaT cells). Results: The calibration curves were linear over the established range with R2 > 0.9999. The intraday and interday RSDs for each compound were 0.13%–0.89% and 0.14%–0.73%, respectively. The limits of detection and limits of quantification for the two tested diterpenoids were 0.0912, 0.0018 μg/μL, and 0.2764, 0.0056 μg/μL, respectively. In addition, the CA-treated groups showed accelerated wound closure compared to the control group. Conclusion: The HPLC method could be accomplished to the quality control and stable experiment for the preparations consisted of two diterpenoids. Compared to the control group, the CA-treated group showed that wound closure was accelerated, indicating its potential in promoting migration of skin cells which is the most important step of wound closure.

Continentalic Acid Rather Than Kaurenoic Acid Is Responsible for the Anti-Arthritic Activity of Manchurian Spikenard In Vitro and In Vivo.

The aim of this study was to identify the active compound responsible for the pharmacological activities of Manchurian spikenard (Aralia continentalis Kitag.). Interleukin (IL)-1β-stimulated human chondrocytes and monoiodoacetate (MIA)-induced osteoarthritic rats were treated with the 50% ethanolic extract of spikenard or its major components, such as continentalic acid (ent-pimara-8(14),15-diene-19-oic acid) and kaurenoic acid (ent-kaura-16-en-19-oic acid). The spikenard extract significantly inhibited IL-1β-stimulated production of IL-6, IL-8, metalloproteinase (MMP)-1, MMP-13, cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS) and prostaglandin(PG)E2 in a dose-dependent manner but not MMP-3 production. The extract also inhibited the IL-1β-induced translocation of NF-κB/p65 into the nucleus and dose-dependent phosphorylation levels of extracellular signal-regulated kinase (ERK), Jun amino-terminal kinase (JNK) and p38 mitogen-activated protein (MAP) kinase. Continentalic acid exhibited significant anti-arthritic activity corresponding exactly to that of the extract containing an equivalent amount of continentalic acid. On the other hand, kaurenoic acid exhibited a compatible activity at about a 10-times higher molar concentration than that of continentalic acid. In vitro anti-arthritic activities of the spikenard extract and continentalic acid were also confirmed in MIA-induced osteoarthritic rats. The 50% ethanolic extract of Manchurian spikenard exhibited promising anti-arthritic activities in the in vitro and in vivo osteoarthritis models, and continentalic acid, not kaurenoic acid, was most probably responsible for those activities.

Ultra-performance convergence chromatography for the quantitative determination of bioactive compounds in Aralia continentalis Kitagawa as quality control markers.

A rapid ultra-performance convergence chromatography method was developed for the quantitative determination of bioactive compounds in Aralia continentalis as quality control markers. Quantitative analysis indicated the presence of two major bioactive compounds: diterpenoid acids continentalic acid and kaurenoic acid. Using a Torus 1-aminoanthracene column, continentalic acid and kaurenoic acid were separated in less than 8min. The method was validated with respect to precision, accuracy, and linearity according to the International Conference on Harmonization guidelines. The optimized method exhibited a good linear correlation (r2 > 0.996), excellent precision (RSD<1.0%), and acceptable recoveries (99.97-100.26%). Limits of detection for continentalic acid and kaurenoic acid were 0.068and 0.097μg/mL, respectively, while their corresponding limits of quantitation were 0.207 and 0.295μg/mL. The system performance of ultra-performance convergence chromatography was compared with that of conventional high-performance liquid chromatography with respect to analysis time and efficiency. The proposed method was found to be reliable and convenient for the quantitative analysis of continentalic acid and kaurenoic acid in A. continentalis from South Korea and A. pubescens from China. This study is expected to serve as a guideline for the quality control of Aralia continentalis.

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%).

In vitro Cytotoxicity of Methanol Extract from Aerial Parts of Aralia cachemirica and Purified Continentalic Acid.

The present study was designed to evaluate the in vitro cytotoxic effect of methanol extract of aerial parts including stems, leaves and twigs of Aralia cachemirica and purified continentalic acid isolated from this extract against a panel of human cancer cell lines of varied tissues. Percentage of growth inhibition was evaluated by sulphorhodamine B assay. Purified continentalic acid showed moderate cytotoxicity against all the cell lines used. In contrast, the extract exhibited significant concentration dependant cytotoxicity against A-549 (lung), THP-1 (leukemia) and MCF-7 (breast) cell lines. This work highlights cytotoxic potential of this extract, which can further be explored for different constituents for their possible use autonomously or in combined manner in cancer therapy. The detailed analysis of their cytotoxicity has been presented in this paper.

Optimization of ultrasonic-assisted extraction of continentalic acid from the root of Aralia continentalis by using the response surface methodology

In order to optimize the extraction conditions of continentalic acid, the main compound from Aralia continentalis root, we developed a model using the response surface methodology. The continentalic acid yield was analyzed and quantified by high-performance liquid chromatography coupled with UV detection. The extraction solvent, temperature, and time the three main factors for ultrasound-assisted extraction were optimized using the central composite design. Analysis of variance showed a good model fit (R (2) = 0.9323). The maximum extraction of continentalic acid obtained experimentally was 1.0138 % under an extraction temperature of 33 °C and extraction time of 28 min when 100 % ethanol was used as the solvent. The experimental value was in good agreement (the yield 1.0103 %) with those predicted values. The results clearly showed that quality by statistical design could be effectively applied to optimize extraction of continentalic acid.

New phytoconstituents from the roots of Aralia cachemirica Decne

Aralia species are used in traditional medicine to treat rheumatic arthritis, nephritis, lumbago, lameness, gastritis, inflammation and diabetes mellitus. Phytochemical investigation of the roots of Aralia cachemirica Decne (Araliaceae) collected from the Aharbal region of Kashmir afforded four new phytoconstituents identified as n-tetracont-19-enoic acid (1), 4α,4β,8β,10β,13β,17β-hexamethyl perhydrophenanthrenyl-3β-n-decanoate (3), tetrahydrocontinentalic acid (4) and 1β,4α,4β-trimethyl-6-(10,14,18-trimethyl-tridec-6-enyl)-cyclohexane-4β-ol (5) along with the known compounds continentalic acid (2), maltose (6) and sucrose (7). The structures of these phytoconstituents have been elucidated on the basis of spectral data analysis.

Anti-inflammatory activity of the constituents of the roots of Aralia continentalis

To assess the anti-inflammatory activity of the constituents of the roots of Aralia continentalis, ent-pimara-8(14),15-diene-19-oic acid (continentalic acid, pimaradienoic acid, compound I), 7beta-hydroxy-ent-pimara-8(14),15-diene-19-oic acid (compound II), 7-oxo-ent-pimara-8(14),15-diene-19-oic acid (compound III), 15alpha,16alpha-epoxy-17-hydroxy-ent-kauran-19-oic acid (compound IV) and ent-kaura-16-en-19-oic acid (kaurenoic acid, compound V), their inhibitory effects against cyclooxygenase-2 (COX-2)-catalyzed PGE(2) and inducible nitric oxide synthase (iNOS)-catalyzed NO production by lipopolysaccharide-treated RAW 264.7 cells were examined. Among the compounds tested, compound III and V moderately inhibited NO production. In addition, compound III weakly inhibited PGE2 production, while treatment with compounds II and IV at concentrations of up to 100 microM had no significant effects. Conversely, compound I only weakly inhibited PGE2 and NO production. To elucidate the mechanism by which these changes occurred, the iNOS down-regulating capacity of compound III was investigated. Western blot analysis and an electrophoretic mobility shift assay demonstrated that compound III weakly inhibited COX-2 and iNOS expression at 50-100 microM, and inhibited NF-kappaB activation. When in vivo anti-inflammatory activities of compounds I, III and V were examined, intraperitoneal injection of 4-100 mg/kg of compound I and V significantly inhibited carrageenan-induced paw edema in mice, whereas compound III did not. Taken together, the results of this study suggest that some constituents of A. continentalis, especially compounds I, III and V, exert significant anti-inflammatory activity, which suggests that these constituents contribute, at least in part, to the anti-inflammatory action of the roots of A. continentalis.

Continentalic acid from Aralia continentalis induces growth inhibition and apoptosis in HepG2 cells

In this study, we investigated the effects of continentalic acid (CA, (-)-pimara-8(14), 15-diene-19-oic acid), a diterpenic acid, isolated from Aralia continentalis, on the proliferation and apoptosis induction of HepG2 cells. In 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, the inhibitory effect became gradually stronger with the passage of time, 24, 48 and 72 h after treatment with CA, and the most significant effect was observed at 72 h. CA treatment for 72 h induced DNA fragmentation in a dose-dependent manner. Furthermore, flow cytometric analysis of HepG2 cells exposed to CA showed that apoptotic cells increased in a dose-dependent manner. The induction of apoptosis in HepG2 cells by CA was mediated through the activation of caspase-3, Bak, and Bax, and then through the cleavage of peroxisome proliferator-activated receptor (PARP) and the down-regulation of Bcl-2. These results demonstrate that CA efficiently induces apoptosis and is a good candidate for further evaluation as an effective chemotherapeutic agent.

Continentalic acid from Aralia continentalis shows activity against methicillin‐resistant Staphylococcus aureus

In a continuing search for compounds with antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), a chloroform extract of roots of Aralia continentalis was found to contain continentalic acid (CA, C(20)H(30)O(2)), a diterpenic acid. This compound exhibited potent activity against standard methicillin-susceptible Staphylococcus aureus (MSSA) as well as clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA). It was determined that continentalic acid had minimum inhibitory concentrations (MICs) of approximately 8-16 microg/mL against S. aureus, including the MSSA and MRSA standard strains. Therefore, the results obtained in this study suggest that continentalic acid might have potential as an adjunct in the treatment of antibiotic-resistant bacteria.