Accumulation Of Oleanolic Acid Research Articles

Expression of PnSS Promotes Squalene and Oleanolic Acid (OA) Accumulation in Aralia elata via Methyl Jasmonate (MeJA) Induction.

Aralia elata is an important herb due to the abundance of pentacyclic triterpenoid saponins whose important precursors are squalene and OA. Here, we found that MeJA treatment promoted both precursors accumulation, especially the latter, in transgenic A. elata, overexpressing a squalene synthase gene from Panax notoginseng(PnSS). In this study, Rhizobium-mediated transformation was used to express the PnSS gene. Gene expression analysis and high-performance liquid chromatography (HPLC) were used to identify the effect of MeJA on squalene and OA accumulation. The PnSS gene was isolated and expressed in A. elata. Transgenic lines showed a very high expression of the PnSS gene and farnesyl diphosphate synthase gene (AeFPS) and a slightly higher squalene content than the wild-type, but endogenous squalene synthase (AeSS), squalene epoxidase (AeSE), and β-amyrin synthase (Aeβ-AS) gene were decreased as well as OA content. Following one day of MeJA treatment, the expression levels of PeSS, AeSS, and AeSE genes increased significantly. On day 3, the maximum content of both products reached 17.34 and 0.70 mg·g-1, which increased 1.39- and 4.90-fold than in the same lines without treatment. Transgenic lines expressing PnSS gene had a limited capability to promote squalene and OA accumulation. MeJA strongly activated their biosynthesis pathways, leading to enhance yield.

A suspension culture of the hormone autotrophic cell line of Aralia elata (Miq.) Seem. for production of oleanolic acid and flavonoids

The hormone autotrophic cell (HAC) line of Aralia elata grows quickly in vitro without a plant-growth regulator and is thus a potential platform cell line for high-value metabolite production. In this study, a suspension culture of this HAC line was established by optimizing the concentration of sucrose (30–90 g/L), the inoculation amount, and the strength of the Murashige and Skoog (MS) medium. Then, different concentrations of the elicitors methyl jasmonate (MJ; 0, 50, 100, 250, and 500 μmol/L) and salicylic acid (SA; 0, 10, 25, 50, 100 and 250 mg/L) were used to improve its oleanolic acid accumulation, and the precursors phenylalanine, sodium acetate, and cinnamic acid were screened to enhance flavonoid accumulation. The optimal conditions for HAC line growth were 1.0 g HACs suspended in 100 mL liquid MS medium (in a 250-mL flask) with the addition of 70 g/L sucrose, and the time-points for the highest accumulations of oleanolic acid and total flavonoids were 18 and 21 days, respectively. The treatment of HACs with 250 μmol/L MJ for 6 days or 25 mg/L SA for 3 days improved oleanolic acid accumulation efficiency by 1.597-times (12.065 μg/d) and 3.59-times (22.860 μg/d) that of the control, respectively. The most effective concentrations of the three precursors were 50 mg/L for phenylalanine and sodium acetate, and 5 mg/L for cinnamic acid. Of these, sodium acetate treatment for 14 days proved most effective, providing a total flavonoids content (4.124 mg/L) 1.87-times that of the control and an accumulation efficiency 2.37-times that of the control. Therefore, the most effective treatments for HACs after 10 days of culturing were 25 mg/L SA for 3 days and 5 mg/L sodium acetate for 14 days, which improve the accumulations of oleanolic acid and total flavonoids, respectively.

Development of putrescine anchored nano-crystalsomes bearing doxorubicin and oleanolic acid: deciphering their role in inhibiting metastatic breast cancer.

Angiogenesis driven tumor initiation and progression calls for a targeted therapy. Moreover, combined chemotherapy supplements the therapy to act on the cause of concern. In this study, we aimed to develop a targeted crystalsomes approach to delineate tumor cells against normal cells. Self-assembled crystalline monodispersed nanosized polyethylene-polyethylene glycol (PE-PEG)-based hollow crystalsomes were modified with pluronylated putrescine (Put-PF) and loaded with doxorubicin (Dox), synergistically in combination with oleanolic acid (OA) to target the glypican-1 (gp-1) receptor on tumor cells. The developed crystalsomes (Put-D + O@NCs) showed increased intracellular accumulation of Dox and OA in a synergistic combination inside the MDA-MB-231 cell lines. The developed crystalsomes marked an enhanced depolarization of the mitochondrial membrane potential and cell cycle arrest leading to apoptosis. Furthermore, the proposed therapy has a greater anti-angiogenesis activity with vascular endothelial growth factor (VEGF) dependent modulation in the proliferation, invasion, migration and tube formation of human endothelial umbilical vein cells (HUVECs) in vitro and in vivo in a BALB/c mouse model. Interestingly, the perseverance of the tumor boundary, inhibiting the expression and activity of the matrix metalloproteinase (MMPs) (>5.2-fold) with suppressed degradation of the extracellular matrix paves the way for significant inhibition of metastases. However, an intravenously administered Put-D + O@NCs showed an improved pharmacokinetic profile and exquisite inhibition of the 4T1 induced tumor with a significantly lower toxicity. In a nutshell, these findings highlight the important role of Put in the gp-1 receptor for specific targeting and synergistic delivery of Dox and OA through crystalsomes as a potential approach for the treatment of metastatic breast cancer using combined chemotherapy.

NAA and 6-BA promote accumulation of oleanolic acid by JA regulation in Achyranthes bidentata Bl.

Application of plant growth regulators has become one of the most important means of improving yield and quality of medicinal plants. To understand the molecular basis of phytohormone-regulated oleanolic acid metabolism, RNA-seq was used to analyze global gene expression in Achyranthes bidentata treated with 2.0 mg/L 1-naphthaleneacetic acid (NAA) and 1.0 mg/L 6-benzyladenine (6-BA). Compared with untreated controls, the expression levels of 20,896 genes were significantly altered with phytohormone treatment. We found that 13071 (62.5%) unigenes were up-regulated, and a lot of differentially expressed genes involved in hormone or terpenoid biosynthesis, or transcription factors were significantly up-regulated. These results suggest that oleanolic acid biosynthesis induced by NAA and 6-BA occurs due to the expression of key genes involved in jasmonic acid signal transduction. This study is the first to analyze the production and hormonal regulation of medicinal A. bidentata metabolites at the molecular level. The results herein contribute to a better understanding of the regulation of oleanane-type triterpenoid saponins accumulation and define strategies to improve the yield of these useful metabolites.

Dual-targeting liposomes for enhanced anticancer effect in somatostatin receptor II-positive tumor model.

We developed octreotide-modified magnetic liposomes (OMlips) as dual-targeting drug carriers to enhance the drug accumulation in tumor site. Octreotide acts as a modified ligand for receptor-mediated targeting and the coated Fe3O4 nanoparticles offer the magnetic targeting property. SSTR2 overexpressed A549 cells and S180 cells were chosen to explore the targeting ability and antitumor effect of the oleanolic acid (OA)-loaded OMlips in vitro and in vivo. The OMlips platform significantly improves the targeting, penetrating and accumulation of OA at the SSTR2 overexpressed cells and SSTR2-positive tumor-bearing mice. The OA-loaded OMlips have better antitumor effect and lower systemic toxicity. Such a receptor-mediated and magnetically-orienting dual-targeting drug nanocarriers may have great potentials in clinical practice.

Use of ion pair amphiphile as an alternative of natural phospholipids in enhancing the stability and anticancer activity of oleanolic acid loaded nanostructured lipid carriers

Nanostructured lipid carriers (NLC) comprising soy lecithin (SLC), tristearin (TS) and palmitic acid (PA) having molar ratio 2:2:1 was modified by partially replacing SLC with ion pair amphiphile (NLCIPA), prepared by mixing equimolar aqueous solution of sodium dodecyl sulphate and hexadecyltrimethylammonium bromide. Hot homogenization followed by ultrasonication method was employed for the preparation of NLC and NLCIPA. Desirable SLC / IPA ratio for NLCIPA formulations were obtained by analyzing the interfacial behavior of the lipidic components in the presence of IPA at air-water interface. Considering the interfacial behavior and mutual miscibility, lipid compositions having SLC/IPA ratio equal to 2:3, 3:7 and 1:4 were selected for the preparation of NLCIPA. Analytical techniques like dynamic light scattering, differential scanning calorimetry, transmission electron microscopy, fridge fractured electron microscopy and atomic force electron microscopy assured higher stability of NLCIPA formulations compared to the conventional NLC and the NLCIPA with 30 mol% SLC was found to be optimum. Experimental evaluation of interfacial, solution phase and thermal characteristics of oleanolic acid (OA) loaded NLC and NLCIPA formulations indicated the accumulation of OA on the palisade layer and the drug acquisition was higher in NLCIPA than the conventional NLC. NLCIPA formulations were found even more promising to sustain the release of OA to a desirable extent. Higher cytotoxic activity for the OA loaded NLCIPA than the NLC was noted during the cytotoxicity studies on hepatocellular carcinoma, hepatocyte-derived carcinoma and colorectal carcinoma cancer cell lines. The NLCIPA formulations, therefore, can be considered as promising alternate to conventional NLC in improving the therapeutic efficacy of OA as the anticancer drug.

Enhanced biosynthesis of saponins by coronatine in cell suspension culture of Kalopanax septemlobus.

Kalopanax septemlobus is a medicinal woody species of the family Araliaceae, and the pharmaceutical properties of saponins obtained from K. septemlobus suggest that K. septemlobus has the potential to be a crude drug and dietary health supplement. In this study, we established cell suspension culture of K. septemlobus to develop a sustainable source of natura-ceuticals. Friable calli were used for establishing cell suspension culture. The maximum amount of total saponins (1.56mg/60ml suspension) was obtained during the 15th day of incubation, whereas the maximum capacity of saponin production was reached after day 6 (0.42μg/mg of fresh weight). The total saponin production in the cell suspension of K. septemlobus was significantly increased by coronatine (COR) at 160% at a dose of 1μM compared with the mock-treated control, whereas methyl jasmonate treated cells exhibited less increase in total saponin level as compared to the COR-treated cells. In addition, the elicitation of COR strongly induced the expression of beta-amyrin synthase, thus resulting in the accumulation of oleanolic acid (2.369±0.98μg/mg of extract), a precursor for oleanane-type triterpene saponins. These results indicate that COR is an efficient elicitor for inducing phytochemicals in cell suspension culture and that it provides the possibility for producing saponins of K. septemlobus using cell suspension culture.

Methyl Jasmonate and Salicylic Acid Enhanced the Production of Ursolic and Oleanolic Acid in Callus Cultures of Lepechinia Caulescens.

Background:The production of triterpenes from plants for pharmacological purposes varies in concentration, due to genetic and environmental factors. In vitro culture enables the control and increase of these bioactive molecules.Objective:To evaluate the effect of plant growth regulators and elicitors in the induction of calli and the production of ursolic acid (UA) and oleanolic acid (OA) in Lepechinia caulescens.Materials and Methods:Leaf explants were exposed for the induction of calli at different concentrations and combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BAP). Methyl jasmonate (MJ) and salicylic acid were used as elicitors. High-performance liquid chromatography method was used to quantify UA and OA content in each treatment.Results:Treatment with 3.0 mg/L of 2,4-D and 0.1 mg/L of BAP produced the best results for calli induction and production of UA (1.57 mg/g dry weight [DW]) and OA (1.13 mg/g DW). Both elicitors facilitated the accumulation of triterpenes.Conclusion:The combination of auxins and cytokinins showed favorable results for the induction of calli. Variation concerning the accumulation of UA and OA was observed between treatments. MJ increased the production of triterpenes five times after 8 h of exposure, compared to control treatment. There is a greater accumulation of UA (16.58 mg/g DW) and OA (1.94 mg/g DW) in leaves of wild plants.SUMMARY Callus cultures of Lepechinia caulescens were obtained from leaf explants treated with 2,4-dichlorophenoxyacetic acid and 6-bencylaminopurineResulting cultures were elicited with methyl jasmonate (MJ) and salicylic acid to increase the production of the triterpenes, ursolic acid (UA), and oleanolic acid (OA)The cultures elicited with MJ increased the production of UA and OA five times, as compared to the control. Abbreviations used: 2,4-D: 2,4-dichlorophenoxyacetic acid, BAP: 6-benzylaminopurine, DW: Dry weight, MJ: Methyl jasmonate, OA: Oleanolic acid, PGRs: Plant growth regulators, UA: Ursolic acid, SA: Salicylic acid.

An Intronless β-amyrin Synthase Gene is More Efficient in Oleanolic Acid Accumulation than its Paralog in Gentiana straminea.

Paralogous members of the oxidosqualene cyclase (OSC) family encode a diversity of enzymes that are important in triterpenoid biosynthesis. This report describes the isolation of the Gentiana straminea gene GsAS2 that encodes a β-amyrin synthase (βAS) enzyme. Unlike its previously isolated paralog GsAS1, GsAS2 lacks introns. Its predicted protein product was is a 759 residue polypeptide that shares high homology with other known β-amyrin synthases (βASs). Heterologously expressed GsAS2 generates more β-amyrin in yeast than does GsAS1. Constitutive over-expression of GsAS2 resulted in a 5.7 fold increase in oleanolic acid accumulation, while over-expression of GsAS1 led to a 3 fold increase. Additionally, RNAi-directed suppression of GsAS2 and GsAS1 in G. straminea decreased oleonolic acid levels by 65.9% and 21% respectively, indicating that GsAS2 plays a more important role than GsAS1 in oleanolic acid biosynthesis in G. straminea. We uses a docking model to explore the catalytic mechanism of GsAS1/2 and predicted that GsAS2, with its Y560, have higher efficiency than GsAS1 and mutated versions of GsAS2 in β-amyrin produce. When the key residue in GsAS2 was mutagenized, it produced about 41.29% and 71.15% less β-amyrin than native, while the key residue in GsAS1 was mutagenized to that in GsAS2, the mutant produced 38.02% more β-amyrin than native GsAS1.

Effect of Drought and Nitrogen on Betulin and Oleanolic Acid Accumulation and OSC Gene Expression in White Birch Saplings

Betulin and oleanolic acid are pentacyclic triterpene compounds with a broad range of biological and medicinal properties. Herein, we demonstrated that 2-year-old white birch saplings were capable of synthesizing betulin and oleanolic acid, and the accumulation of these compounds was significantly affected by seasonal and environmental factors. Indeed, the triterpenes accumulated mainly in the stem bark, with little in the root bark and leaves. From May to October, betulin and oleanolic acid stem bark accumulation displayed an inverted “V” curve, peaking in late July to late August (warmest summer months in Haerbin, China). Interestingly, low nitrogen levels promoted root bark and leaf accumulation of oleanolic acid, while high nitrogen amounts were more favorable to oleanolic acid and betulin accumulation in the stem bark. Moderate and mild drought during the accumulation peak period promoted oleanolic acid and betulin accumulation in the stem bark. Serious shortage of soil moisture inhibited the accumulation of both triterpenes. From July to August, high nitrogen (N3) treatment increased lupeol synthase (BPW) and beta-amyrin synthase (BPY) gene expression in the stem bark. Water stress, especially mild and moderate, induced BPW and BPY expression and downregulated cycloartenol synthase (BPX) in the stem bark. This study provides a strong basis for understanding triterpenoid synthesis in birch at the molecular level.

Optimisation of potassium chloride nutrition for proper growth, physiological development and bioactive component production in Prunella vulgaris L.

Prunella vulgaris L. is an important medicinal plant with a variety of pharmacological activities, but limited information is available about its response to potassium chloride (KCl) supplementation. P. vulgaris seedlings were cultured in media with four different KCl levels (0, 1.00, 6.00 and 40.00 mM). Characteristics relating to the growth, foliar potassium, water and chlorophyll content, photosynthesis, transpiration, nitrogen metabolism, bioactive constituent concentrations and yield were determined after three months. The appropriate KCl concentration was 6.00 mM to result in the highest values for dry weight, shoot height, spica and root weight, spica length and number in P. vulgaris. The optimum KCl concentration resulted in a maximum net photosynthetic rate (Pn) that could be associated with the highest chlorophyll content and fully open stomata conductance. A supply of surplus KCl resulted in a higher concentration of foliar potassium and negatively correlated with the biomass. Plants that were treated with the appropriate KCl level showed a greater capacity for nitrate assimilation. The Pn was significantly and positively correlated with nitrate reductase (NR) and glutamine synthetase (GS) activities and was positively correlated with leaf-soluble protein and free amino acid (FAA) contents. Both KCl starvation (0 mM) and high KCl (40.00 mM) led to water loss through a high transpiration rate and low water absorption, respectively, and resulted in increased concentrations of ursolic acid (UA), oleanolic acid (OA) and flavonoids, with the exception of rosmarinic acid (RA). Moreover, the optimum concentration of KCl significantly increased the yields of RA, UA, OA and flavonoids. Our findings suggested that significantly higher plant biomass; chlorophyll content; Pn; stronger nitrogen anabolism; lower RA, UA, OA and flavonoid accumulation; and greater RA, UA, OA and flavonoid yields in P. vulgaris could be expected in the presence of the appropriate KCl concentration (6.00 mM).

The effect of elicitors on oleanolic acid accumulation and expression of triterpenoid synthesis genes in Gentiana straminea

Gentiana straminea is native to the Qinghai-Tibet plateau, where it is exposed to extremes of cold and strong UV-B radiation. Here we showed that low temperature, but not UV-B radiation, affected the accumulation of the triterpenoid oleanolic acid. Neither of these stresses altered the expression of known triterpenoid synthesis genes. However, the application of methyl jasmonate (MeJA), but not salicylic acid (SA), significantly enhanced the accumulation of oleanolic acid, and up-regulated the triterpenoid synthesis genes, especially the expression of βAS, the gene encoding β-amyrin.

Distribution and expression characteristics of triterpenoids and OSC genes in white birch (Betula platyphylla suk.)

Betulin and oleanolic acids (pentacyclic triterpenoid secondary metabolites) have broad pharmacological activities and can be potentially used for the development of anti-cancer and anti-AIDS drugs. In this study, we detected the accumulation and the distribution characteristics of betulin and oleanolic acid in various organs of white birch at different ages. We also determined the expression of 4 OSC genes (LUS, β-AS, CAS1 and CAS2) involved in the triterpenoid synthesis pathways by real time RT-PCR. The result showed that the 1-year old birch can synthesize betulin and oleanolic acid. In addition, betulin and oleanolic acids were mainly distributed in the bark, while the content in the root skin and leaf was very low. The content of betulin and oleanolic acid in birch varied in different seasons. The content of betulin and oleanolic acid and their corresponding LUS and β-AS gene expression were very low in 1-year old birch. With increasing age of birch, betulin content was increased, while oleanolic acid was decreased. Similar changes were also observed for their corresponding synthesis genes LUS and β-AS. In the leaf of 1-year old plant, the highest expression of CAS1 and CAS2 occurred at end of September, while expression of LUS and the β-AS was low from June to October. In the stem skin,high expression of β-AS and the LUS genes occurred from the end of July to September. In the root, high expression of the β-AS gene was observed at the end of October. These results indicated that triterpenoid gene expression was similar to the triterpene accumulation. Expression of LUS gene and β-AS gene in birch with different ages were corresponding to the betulinic and oleanolic acid accumulation. Expression of CAS1 and CAS2 genes were elevated with increasing age of birch. This study provides molecular mechanisms of triterpenes synthesis in birch plants.

Herbaceous Peony (Paeonia lactiflora Pall.) as an Alternative Source of Oleanolic and Ursolic Acids

Oleanolic acid (OA) and ursolic acid (UA) have been proven to possess many biological activities, and much attention is focused on the search for plants which are rich in OA and UA. In this report, the OA and UA accumulation characteristics were investigated in 47 cultivars of Chinese herbaceous peony (Paeonia lactiflora Pall.) and were followed in three cultivars over different developmental stages as measured by high performance liquid chromatography (HPLC). OA and UA levels in leaves and stems demonstrated an overall upward trend from May 1 to September 15 except for UA in the leaves of “Hong Feng”. The maximum values of OA and UA in leaves of “Yangfei Chu Yu”, “Fen Zhu Pan” and “Hong Feng” were 852.98, 575.60, 290.48 μg/g FW and 924.94, 827.36, 432.67 μg/g FW, respectively. The maximum values of OA and UA in stems of “Yangfei Chu Yu”, “Fen Zhu Pan” and “Hong Feng” were 359.28, 90.49, 43.90 μg/g FW and 326.86, 82.25, 56.63 μg/g FW, respectively. OA and UA contents in leaves of 47 different herbaceous peony cultivars ranged from 66.73–618.12 and 36.23–665.14 μg/g FW, respectively, with average values of 171.62 and 227.57 μg/g FW, respectively. The results suggested that the aboveground parts of herbaceous peony may be used as an alternative source of OA and UA for medicinal purposes in addition to its ornamental purposes.

Significant enhancement of oleanolic acid accumulation by biotic elicitors in cell suspension cultures of Calendula officinalis L.

The effects of elicitors on cell growth and oleanolic acid (OA) accumulation in shaken cell suspension cultures of Calendula officinalis were investigated. Elicitors were added individually at various concentrations to 5-day-old cell cultures and their effects monitored at 24 h intervals for 4 days. Different effects on OA accumulation were observed depending on the day of treatment. Jasmonic acid was the most efficient elicitor. After 72 h of treatment with 100 μM JA, the intracellular content of OA reached its maximum value (0.84 mg g −1 DW), which was 9.4-fold greater than that recorded in an untreated control cultures. The addition of chitosan at 50 mg l −1 produced a 5-fold enhancement of OA accumulation (0.37 mg g −1 DW) after 48 h of treatment. Treatment with yeast extract at 200 mg l −1 for 96 h or with pectin at 2 mg l −1 for 48 h produced identical cellular levels of OA (0.22 mg g −1 DW). Lastly, 48 h elicitation with homogenate of the fungus Trichoderma viride produced a 1.8-fold increase in oleanolic acid content (0.12 mg g −1 DW). In addition to significantly stimulating OA accumulation and its secretion into the culture medium, the elicitors also caused slight inhibition of cell growth.

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.