Tigliane-Type Diterpene Esters from the Fruits of Shirakiopsis indica and Their Anti-HIV Activity.

Four new diterpene esters, shirakindicans A-D (1-4), along with eight related known diterpene esters (5-12), were isolated from the fruits of the Bangladeshi medicinal plant Shirakiopsis indica. The structures of 1-4 were elucidated by spectroscopic data analysis and electronic circular dichroism (ECD) calculations. Shirakindican A (1) was assigned as a tigliane-type diterpene ester possessing an unusual 6ß-hydroxy-1,7-dien-3-one structure, while shirakindican B (2) exhibits a tiglia-1,5-dien-3,7-dione structure. The anti-HIV activities of the isolated diterpene esters were evaluated and showed significant activities for sapintoxins A (5) and D (11), with EC50 values of 0.0074 and 0.044 µM, respectively, and TI values of 1 100 and 5 290. Sapatoxin A (12) also exhibited anti-HIV activity with an EC50 value of 0.13 µM and a TI value of 161.

DP from Euphorbia fischeriana S. mediated apoptosis in leukemia cells via the PI3k/Akt signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Euphorbia fischeriana S. (E. fischeriana) is a classic Chinese herb with toxicity that is mainly used for cancer treatment and in insect repellent, anti-inflammatory and anti-edema applications (Liu et al., 2001). 12-Deoxyphorbol13-palmitate (DP), a tetracyclic diterpene monomer compound, was extracted from the roots of E. fischeriana by our research groups. AIM: Previous studies found that DP could inhibit the proliferation of leukemia cells in vitro. However, the underlying mechanism of DP in leukemia is unknown. Hence, DP's pharmacological effect on leukemia cells was investigated in this study. MATERIALS AND METHODS: DP was obtained from the Natural Medicine Chemistry Laboratory of Qiqihaer Medical University. In vitro, K562 cells and HL60 cells were incubated with DP or DP combined with LY294002 at different concentrations. Cell proliferation and apoptosis were detected by the relevant experimental methods. In vivo, nude mouse xenograft models were established by injecting K562 cells. DP was intraperitoneally administered to observe the influence on the growth of transplanted tumors. Gene detection and immunoblot analysis were performed to validate the mechanisms. RESULTS: The cell counting kit-8 (CCK-8) assay proved that DP inhibited the growth of K562 and HL60 cells in a time- or dose-dependent manner. At 12 h, DP could induce apoptosis by Annexin V-FITC/propidium iodide (PI) dual labeling, loss of mitochondrial membrane potential (MMP), intracellular reactive oxygen species (ROS), acridine orange/ethidium bromide (AO/EB) staining and transmission electron microscopy (TEM) observation in K562 or HL60 cells. Furthermore, in an assay of gene and protein expression, we found that DP could downregulate the gene and protein expression levels of Bcl-2, upregulate the gene and protein expression levels of Bax and Bim, and downregulate the protein expression levels of PI3k, p-Akt, and p-FoxO3a. Moreover, the effects of DP on proliferation and apoptosis in K562 cells were enhanced by LY294002. Then, we tested the antitumor effects of DP in vivo. Nude mouse xenograft models were established by subcutaneously injecting K562 cells. We found that tumor volume was significantly decreased in DP-treated xenograft nude mice. Morphologic changes, apoptosis degree, and related gene and protein expression levels in transplanted tumor tissue of DP-treated nude mice were assessed by different experimental methods. CONCLUSIONS: The in vivo and in vitro experimental results indicated that DP might inhibit the proliferation and induce the apoptosis of leukemia cells, which might be a result of suppressing the PI3k/Akt signaling pathways.

Lab-made solid phase microextraction phases for off line extraction and direct mass spectrometry analysis: Evaluating the extraction parameters.

The analyses of drugs and metabolites in complex matrices have been widely studied in recent years. However, due to high levels endogenous compounds and matrix complexity, these analyses require a sample pre-treatment step. To this aim, two lab-made extractive phases were integrated to probe electrospray ionization mass spectrometry (PESI-MS) technique for direct analysis of illicit drugs in biological fluids and phorbol esters in Jatropha curcas extract. The polypyrrole (PPy) phase was electropolymerized onto a platinum wire surface by cyclic voltammetry. The molecularly imprinted polymer (MIP) was synthesized and adhered onto a stainless-steel needle with epoxy resin. The PPy-PESI-MS method showed to be linear in a concentration range from 1 to 500 µg L-1, with accuracy values between -2.1 and 14%, and precision values between 0.8 and 10.8%. The MIP-PESI-MS method showed to be linear in a concentration range from 0.9 to 30 mg L-1, with accuracy values between -1.6 and -15.3%, and precision values between 4.1 and 13.5%.

Current Strategies for the Detoxification of Jatropha curcas Seed Cake: A Review.

Jatropha curcas is an important oilseed plant, with considerable potential in the development of biodiesel. Although Jatropha seed cake, the byproduct of oil extraction, is a residue rich in nitrogen, phosphorus, potassium, and carbon, with high protein content suitable for application in animal feed, the presence of toxic phorbol esters limits its application in feed supplements and fertilizers. This review summarizes the current methods available for detoxification of this residue, based upon chemical, physical, biological, or combined processes. The advantages and disadvantages of each process are discussed, and future directions involving genomic and proteomic approaches for advancing our understanding of biodegradation processes involving microorganisms are highlighted.

Anti-HIV Activities and Mechanism of 12-O-Tricosanoylphorbol-20-acetate, a Novel Phorbol Ester from Ostodes katharinae.

APOBEC3G is a member of the human cytidine deaminase family that restricts Vif-deficient viruses by being packaged with progeny virions and inducing the G to A mutation during the synthesis of HIV-1 viral DNA when the progeny virus infects new cells. HIV-1 Vif protein resists the activity of A3G by mediating A3G degradation. Phorbol esters are plant-derived organic compounds belonging to the tigliane family of diterpenes and could activate the PKC pathway. In this study, we identified an inhibitor 12-O-tricosanoylphorbol-20-acetate (hop-8), a novel ester of phorbol which was isolated from Ostodes katharinae of the family Euphorbiaceae, that inhibited the replication of wild-type HIV-1 and HIV-2 strains and drug-resistant strains broadly both in C8166 cells and PBMCs with low cytotoxicity and the EC50 values ranged from 0.106 µM to 7.987 µM. One of the main mechanisms of hop-8 is to stimulate A3G expressing in HIV-1 producing cells and upregulate the A3G level in progeny virions, which results in reducing the infectivity of the progeny virus. This novel mechanism of hop-8 inhibition of HIV replication might represents a promising approach for developing new therapeutics for HIV infection.

Isolation and Structure Characterization of Cytotoxic Phorbol Esters from the Seeds of Croton tiglium.

Nine new and eleven known phorbol esters were isolated from an acetone extract of the seeds of Croton tiglium. Their structures were determined by extensive analysis of spectroscopic data. Eleven of these compounds were evaluated for their inhibition activity on human tumor cell lines HL-60 and lung carcinoma A549. 12-O-Tiglylphorbol-13-acetate (11), 12-O-(2-methyl)-butyrylphorbol-13-aetate (12), and 12-O-tiglylphorbol-13-isobutyrate (13) exhibited strong inhibition activity against both HL-60 and A549 cell lines with IC50 values ≤ 0.02 and ≤ 0.1 µg/mL, respectively. Compound 18 showed strong inhibition activity against the HL-60 cell line with an IC50 value of 0.02 µg/mL.

Inflammation Modulatory Phorbol Esters from the Seeds of Aquilaria malaccensis.

The tree Aquilaria malaccensis is a valuable source of agarwood, which is used in herbal medicinal preparations. Phytochemical research on A. malaccensis seeds has led to the isolation of four new phorbol esters (1-4), two known phorbol esters (5, isolated from Nature for the first time, and 6), and two known glycerides (7 and 8). The structures of these isolates were elucidated by means of spectroscopic data interpretation. The inflammation-modulatory activities of the isolates on elastase release and superoxide anion generation in human neutrophils were evaluated. Interestingly, phorbol esters 1, 5, and 6 showed potent inhibitory activity on elastase release in human neutrophils, with IC50 values of 2.7, 0.8, and 2.1 µM, respectively. All isolated phorbol esters exerted enhancing activity on superoxide anion generation. The results indicated that phorbol esters may play a bilateral modulatory role in the processes of inflammation. In addition, the compounds were evaluated for their cytotoxic properties against HepG2 (hepatoma), MDA-MB-231 (breast), and A549 (lung) cancer cells, but all compounds were inactive for all cell lines used (IC50 > 10 µM).

Phorbol ester-type diterpenoids from the twigs and leaves of Croton tiglium.

Phytochemical investigation of the ethanol extract from the twigs and leaves of Croton tiglium led to the isolation of two new phorbol esters (1-2) and seven known ones (3-9). Their structures were elucidated by the analyses of extensive spectroscopic data (IR, MS, and 1D and 2D NMR) and comparing with related compounds. Meanwhile, compounds 1-9 were determined for their cytotoxic activities on human lung cancer cell line A549. Among them, 1-2 were inactive against the cell line A549 (IC50 > 100 µM), but compounds 3 and 7 showed weak activities.

Nineteen-step total synthesis of (+)-phorbol.

Phorbol, the flagship member of the tigliane diterpene family, has been known for over 80 years and has attracted attention from many chemists and biologists owing to its intriguing chemical structure and the medicinal potential of phorbol esters. Access to useful quantities of phorbol and related analogues has relied on isolation from natural sources and semisynthesis. Despite efforts spanning 40 years, chemical synthesis has been unable to compete with these strategies, owing to its complexity and unusual placement of oxygen atoms. Purely synthetic enantiopure phorbol has remained elusive, and biological synthesis has not led to even the simplest members of this terpene family. Recently, the chemical syntheses of eudesmanes, germacrenes, taxanes and ingenanes have all benefited from a strategy inspired by the logic of two-phase terpene biosynthesis in which powerful C-C bond constructions and C-H bond oxidations go hand in hand. Here we implement a two-phase terpene synthesis strategy to achieve enantiospecific total synthesis of (+)-phorbol in only 19 steps from the abundant monoterpene (+)-3-carene. The purpose of this synthesis route is not to displace isolation or semisynthesis as a means of generating the natural product per se, but rather to enable access to analogues containing unique placements of oxygen atoms that are otherwise inaccessible.

Five new phorbol esters with cytotoxic and selective anti-inflammatory activities from Croton tiglium.

Five new phorbol esters, (four phorbol diesters, 1-4, and one 4-deoxy-4α-phorbol diester, 5), as well as four known phorbol esters analogues (6-9) were isolated and identified from the branches and leaves of Croton tiglium. Their structures were elucidated mainly by extensive NMR spectroscopic, and mass spectrometric analysis. Among them, compound (1) was the first example of a naturally occurring phorbol ester with the 20-aldehyde group. Compounds 2-5, and 7-9 showed potent cytotoxicity against the K562, A549, DU145, H1975, MCF-7, U937, SGC-7901, HL60, Hela, and MOLT-4 cell lines, with IC50 values ranging from 1.0 to 43 µM, while none of the compounds exhibited cytotoxic effects on normal human cell lines 293T and LX-2, respectively. In addition, compound 3 exhibited moderate COX-1 and COX-2 inhibition, with IC50 values of 0.14 and 8.5 µM, respectively.

Rapid isolation and purification of phorbol esters from Jatropha curcas by high-speed countercurrent chromatography.

In this work, a high-speed countercurrent chromatography (HSCCC) method was established for the preparation of phorbol esters (PEs) from Jatropha curcas. n-Hexane-ethyl acetate-methanol-water (1.5:1.5:1.2:0.5, v/v) was selected as the optimum two-phase solvent system to separate and purify jatropha factor C1 (JC1) with a purity of 85.2%, as determined by HPLC, and to obtain a mixture containing four or five PEs. Subsequently, continuous semipreparative HPLC was applied to further purify JC1 (99.8% as determined by HPLC). In addition, UPLC-PDA and UPLC-MS were established and successfully used to evaluate the isolated JC1 and PE-rich crude extract. The purity of JC1 was only 87.8% by UPLC-UV. A peak (a compound highly similar to JC1) was indentified as the isomer of JC1 by comparing the characteristic UV absorption and MS spectra. Meanwhile, this strategy was also applied to analyze the PE-rich crude extract from J. curcas. It is interesting that there may be more than 15 PEs according to the same quasi-molecular ion peaks, highly similar sequence-specific fragment ions, and similar UV absorption spectrum.

Tigliane diterpenes from Croton mauritianus as inhibitors of chikungunya virus replication.

A bioassay-guided purification of an EtOAc extract of the leaves of Croton mauritianus using a chikungunya virus-cell-based assay led to the isolation of 12-O-decanoylphorbol-13-acetate (1) and the new 12-O-decanoyl-7-hydroperoxy-phorbol-5-ene-13-acetate (2), along with loliolide, vomifoliol, dehydrovomifoliol, annuionone D and bluemol C. The planar structure and the relative configuration of compound 2 were elucidated based on spectroscopic analysis, including 1D- and 2D-NMR experiments, mass spectrometry, and comparison with literature data. Compounds 1 and 2 inhibited chikungunya virus-induced cell death in cell culture with EC50s of 2.4±0.3 and 4.0±0.8 µM, respectively.

Alienusolin, a new 4α-deoxyphorbol ester derivative, and crotonimide C, a new glutarimide alkaloid from the Kenyan Croton alienus.

Two novel compounds, alienusolin, a 4α-deoxyphorbol ester (1), crotonimide C, a glutarimide alkaloid derivative (2), and ten known compounds, julocrotine (3), crotepoxide (4), monodeacetyl crotepoxide (5), dideacetylcrotepoxide, (6), ß-senepoxide (7), α-senepoxide (8), (+)-(2S,3R-diacetoxy-1-benzoyloxymethylenecyclohex-4,6-diene (9), benzyl benzoate (10), acetyl aleuritolic (11), and 24-ethylcholesta-4,22-dien-3-one (12) were isolated from the Kenyan Croton alienus. The structures of the compounds were determined using NMR, GCMS, and HRESIMS studies.

Cytotoxic phorbol esters of Croton tiglium.

Chemical investigation of the seeds of Croton tiglium afforded eight new phorbol diesters (three phorbol diesters, 1-3, and five 4-deoxy-4α-phorbol diesters, 4-8), together with 11 known phorbol diesters (nine phorbol diesters, 9-17, and two 4-deoxy-4α-phorbol diesters, 18 and 19). The structures of compounds 1-8 were determined by spectroscopic data information and chemical degradation experiments. The cytotoxic activities of the phorbol diesters were evaluated against the SNU387 hepatic tumor cell line, and compound 3 exhibited the most potent activity (IC50 1.2 µM).

12-Deoxyphorbol 13-palmitate inhibit VEGF-induced angiogenesis via suppression of VEGFR-2-signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: 12-Deoxyphorbol 13-palmitate (G) is one toxic compound isolated from Euphorbia fischeriana, an Asian spice used for cancer treatment as a folk remedy. However, whether 12-deoxyphorbol 13-palmitate affects angiogenesis remains unclear. AIM OF THE STUDY: To explore the in vitro and in vivo antiangiogenic effects of 12-deoxyphorbol 13-palmitate and its underlying mechanisms. MATERIALS AND METHODS: We explored antigenic functions in human umbilical vein endothelial cells (HUVEC) by 12-deoxyphorbol 13-palmitate, including proliferation, migration and metastasis through matrigel plug assay, chorioallantoic membrane assay, in vitro migration assay, tube formation assay, motility assay. Antibody chip was applied to screen differentially expressed proteins modulated by 12-deoxyphorbol 13-palmitate, and was further confirmed by RT-PCR and western blot analysis. Tumor xenograft mice were applied to investigate whether 12-deoxyphorbol 13-palmitate could inhibit microvessel density in vivo. RESULTS: 12-Deoxyphorbol 13-palmitate inhibited vascular endothelial growth factor (VEGF)-induced angiogenic processes in vitro, such as proliferation, in vitro migration, and tube formation of HUVEC. In chorioallantoic membrane assay, 12-deoxyphorbol 13-palmitate significantly inhibited neovessel formation. Antibody chip technology demonstrated decreased expression of TIMP-1, TIMP-2, VEGF, basic fibroblast growth factor (bFGF), matrix metalloproteinases (MMP)-2, VEGFR-2 and VEGFR-3 proteins in HUVEC after 24h. In addition, 12-deoyphorbol 13-palmitate inhibited the in vivo growth of MCF-7 cells in grafted mouse model. Immunohistochemistry staining showed decreased microvessel density (CD31) and attenuated VEGFR-2 signaling pathways by 12-deoxyphorbol 13-palmitate. CONCLUSION: 12-Deoxyphorbol 13-palmitate may be utilized to target active angiogenesis through VEGF/VEGFR2 signal pathway for cancer.

Phorbol esters isolated from Jatropha meal induced apoptosis-mediated inhibition in proliferation of chang and Vero cell lines.

The direct feeding of Jatropha meal containing phorbol esters (PEs) indicated mild to severe toxicity symptoms in various organs of different animals. However, limited information is available on cellular and molecular mechanism of toxicity caused by PEs present in Jatropha meal. Thus, the present study was conducted to determine the cytotoxic and mode of action of PEs isolated from Jatropha meal using human hepatocyte (Chang) and African green monkey kidney (Vero) cell lines. The results showed that isolated PEs inhibited cell proliferation in a dose-dependent manner in both cell lines with the CC(50) of 125.9 and 110.3 µg/mL, respectively. These values were compatible to that of phorbol 12-myristate 13-acetate (PMA) values as positive control i.e., 124.5 and 106.3 µg/mL respectively. Microscopic examination, flow cytometry and DNA fragmentation results confirmed cell death due to apoptosis upon treatment with PEs and PMA at CC(50) concentration for 24 h in both cell lines. The Western blot analysis revealed the overexpression of PKC-δ and activation of caspase-3 proteins which could be involved in the mechanism of action of PEs and PMA. Consequently, the PEs isolated form Jatropha meal caused toxicity and induced apoptosis-mediated proliferation inhibition toward Chang and Vero cell lines involving over-expression of PKC-δ and caspase-3 as their mode of actions.

Phorbol esters from Jatropha meal triggered apoptosis, activated PKC-δ, caspase-3 proteins and down-regulated the proto-oncogenes in MCF-7 and HeLa cancer cell lines.

Jatropha meal produced from the kernel of Jatropha curcas Linn. grown in Malaysia contains phorbol esters (PEs). The potential benefits of PEs present in the meal as anticancer agent are still not well understood. Hence, this study was conducted to evaluate the cytotoxic effects and mode of actions of PEs isolated from Jatropha meal against breast (MCF-7) and cervical (HeLa) cancer cell lines. Isolated PEs inhibited cells proliferation in a dose-dependent manner of both MCF-7 and HeLa cell lines with the IC50 of 128.6 ± 2.51 and 133.0 ± 1.96 µg PMA equivalents/mL respectively, while the values for the phorbol 12-myristate 13-acetate (PMA) as positive control were 114.7 ± 1.73 and 119.6 ± 3.73 µg/mL, respectively. Microscopic examination showed significant morphological changes that resemble apoptosis in both cell lines when treated with PEs and PMA at IC50 concentration after 24 h. Flow cytometry analysis and DNA fragmentation results confirmed the apoptosis induction of PEs and PMA in both cell lines. The PEs isolated from Jatropha meal activated the PKC-δ and down-regulated the proto-oncogenes (c-Myc, c-Fos and c-Jun). These changes probably led to the activation of Caspase-3 protein and apoptosis cell death occurred in MCF-7 and HeLa cell lines upon 24 h treatment with PEs and PMA. Phorbol esters of Jatropha meal were found to be promising as an alternative to replace the chemotherapeutic drugs for cancer therapy.

Phorbol esters with wnt signal-augmenting effects isolated from Excoecaria indica.

A screening of natural products using a luciferase assay targeting the Wnt signaling pathway was carried out, and the bioassay-guided fractionation of Excoecaria indica (Euphorbiaceae) collected from Bangladesh afforded three phorbol esters (1 - 3). These compounds exhibited Wnt signal-augmenting effects with 1 causing a 25-fold increase in TCF/beta-catenin (TOP) transcriptional activity at 95 nM.

Chemical constituents of Euphorbia tangutica.

Sixteen known compounds isolated from the whole plants of Euphorbia tangutica, including phorbol-13-actate (1) previously synthesised and obtained from a natural source for the first time, were evaluated in vitro against a panel of human cancer cell lines using the MTT method. Among them, ergosterol (6) exhibited significant cytotoxic activity against HL-60 cell line with an IC(50) value of 3.3 µM, and 3ß,5α-dihydroxy-15ß-cinnamoyloxy-14-oxolathyra-6 Z,12 E-diene (7) also displayed moderate activity.

Isolation, stability and bioactivity of Jatropha curcas phorbol esters.

Jatropha curcas seed oil, which can be utilized for biodiesel production upon transesterification, is also rich in phorbol esters (PEs). In this study, PEs from J. curcas oil (Jatropha factors C1 and C2 (purified to homogeneity), Jatropha factors C3 and (C4+C5) (obtained as mixtures) and PE-rich extract (containing all the above stated Jatropha factors) were investigated. The concentrations of Jatropha PEs were expressed equivalent to Jatropha factor C1. In the snail (Physa fontinalis) bioassay, the order of potency (EC50, µg/L) was: PE-rich extract