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Antiproliferative activity of chloroformic fractions from leaves and inflorescences of Ageratina gracilis
Abstract
To obtain a scientific basis and justification of plant domestication in the use of Ageratina gracilis, we did an in vitro study of the anticancer potential of extracts and fractions from its leaves and inflorescences. Firstly, cytotoxicity was evaluated against five human tumorigenic cell lines by MTT assay. Subsequently, the chloroformic fractions, considered the most cytotoxic were tested for genotoxicity by comet assay, morphological effects were analyzed by fluorescent microscopy, cell cycle arrest by flow cytometry and early apoptosis induction through fluorescein-5-isothiocyanate (FITC) labeled Annexin-V assay. Non-polar extracts with IC50 values of <53μg/ml showed a high cytotoxicity. The highest cytotoxicity was achieved by chloroformic fraction from petroleum ether extract of leaves and inflorescences and chloroformic fraction from ethanolic extract of leaves, displaying a significant inhibition of cell viability particularly on A549 cells with an IC50 value of 25.9 μg/mL. Chloroformic fractions caused a high percent of DNA damage above 60 percent on A549 and MDAMB-231.The fractions also induced G1/S phase arrest of the cell cycle in A549 cells, furthermore it was confirmed the apoptotic activity chloroformic fraction from petroleum ether extract of inflorescences and chloroformic fraction from ethanolic extract of leaves on those cells by Annexin-V assay. These preliminary results indicate that A. gracilis has an antiproliferative activity against cancer cells, being a starting point for forthcoming studies about the antineoplastic activity and its domestication conditions.
... Formazan crystals were dissolved with 100 µL of DMSO. The results were determined by the optical density determined by the absorbance at 570 nm [5,39]. Estimation of the halfmaximal inhibitory concentration (IC 50 ) was performed using non-linear regression from plotting cell survival (%) versus treatment concentration (µg/mL). ...
... DNA staining was performed with 1.0 mg/mL of DAPI (Invitrogen, Waltham, MA, USA). Fluorescence was monitored using an epifluorescence microscope (Motic AE31, Schertz, TX, USA), and the images were captured with MoticCamPro 282A and analyzed using Motic Image plus 2.0 software [5,23,39]. ...
Cancer treatment frequently carries side effects, therefore, the search for new selective and effective molecules is indispensable. Hymenaea courbaril L. has been used in traditional medicine in South America to treat several diseases, including prostate cancer. Leaves’ extracts from different polarities were evaluated using the 3-(4,5-methyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) cell viability assay to determine the cytotoxicity in prostate p53-null cells, followed by bio-guided fractionations to obtain the most cytotoxic fraction considering the selectivity index. The most cytotoxic fraction was analyzed by GC/MS to identify the active compounds. The majority compound, caryophyllene oxide, induced early and late apoptosis, depolarized the mitochondrial membrane, leading to several morphological changes and shifts in apoptotic proteins, and caspases were evidenced. Depolarization of the mitochondrial membrane releases the pro-apoptotic protein Bax from Bcl-xL. The apoptosis process is caspase-7 activation-dependent. Caryophyllene oxide is a safe anti-proliferative agent against PC-3 cells, inducing apoptosis with low toxicity towards normal cells.
Background:
Microtubule-targeting agents (MTAs) disrupt microtubule dynamics, thereby inducing apoptosis via mitochondrial pathway activation through the modulation in the expression of the Bcl-2 family.
Methods:
To describe topological features of the MTAs networks associated to intrinsic apoptosis induction in p53-null prostate cancer cells, we predicted and compared the interactomes and topological properties of Paclitaxel and Vincristine, and thus, the essential nodes corresponding with the pro- and anti-apoptotic proteins and their kinetics were subjected to experimental analysis in PC-3 cell line.
Results:
The essential nodes of the apoptotic pathways, TP53, and CASP3, were identified in both, Paclitaxel and Vincristine networks, but the intrinsic pathway markers BCL2, BAX, and BCL2L1 were identified as hub nodes only in the Paclitaxel network. An in vitro analysis demonstrated an increase in BimEL and the cleaved-caspase-3 proteins in PC-3 cells exposed to both treatments. Immunoprecipitation analysis showed that treatments induced the releasing of Bax from the anti-apoptotic complex with Bcl-2 protein and the role of BimEL as a de-repressor from sequestering complexes, in addition, new protein complexes were identified between BimEL or Bcl-2 and cleaved-caspase-3, contributing data to the Vincristine network for p53-null cells in response to MTAs.
Conclusion:
The differences in sensitivities, protein profiles, and protein complex kinetics observed between the drugs confirmed that the selectivity and stimulation of the apoptotic system vary depending on the cell's genotype, the drug used and its exposure period.
This contribution is a completely updated and expanded version of the four prior analogous reviews that were published in this journal in 1997, 2003, 2007, and 2012. In the case of all approved therapeutic agents, the time frame has been extended to cover the 34 years from January 1, 1981, to December 31, 2014, for all diseases worldwide, and from 1950 (earliest so far identified) to December 2014 for all approved antitumor drugs worldwide. As mentioned in the 2012 review, we have continued to utilize our secondary subdivision of a "natural product mimic", or "NM", to join the original primary divisions and the designation "natural product botanical", or "NB", to cover those botanical "defined mixtures" now recognized as drug entities by the U.S. FDA (and similar organizations). From the data presented in this review, the utilization of natural products and/or their novel structures, in order to discover and develop the final drug entity, is still alive and well. For example, in the area of cancer, over the time frame from around the 1940s to the end of 2014, of the 175 small molecules approved, 131, or 75%, are other than "S" (synthetic), with 85, or 49%, actually being either natural products or directly derived therefrom. In other areas, the influence of natural product structures is quite marked, with, as expected from prior information, the anti-infective area being dependent on natural products and their structures. We wish to draw the attention of readers to the rapidly evolving recognition that a significant number of natural product drugs/leads are actually produced by microbes and/or microbial interactions with the "host from whence it was isolated", and therefore it is considered that this area of natural product research should be expanded significantly.
The following study is designed to determine the anticancer and cytotoxic potential of hydroalcoholic extract from rhizomes of Pa ris polyphylla to produce any cytotoxic effect on human A549 lung cancer cell lines. The hydroalcoholic analysed for spectral analysis through NMR, HPLC - DAD and LC - ESI - MS/MS methods and found to have Diosgenyl and pennogenyl saponins. The test conducted us ing MTT method using hum an lung cancer A549 cell lines as part of the in vitro preclinical characterization of compound and compared against Doxorubicin . More than 97 % increment in cell k illing at a concentration of 500 µ g/ml recorded in the cell line . Th e EC50 for the extract was calculated to be 52.34 ug/ml and at the concentration of 1.8559 ug/ml, doxorubicin exhibited approximately 98 % killing of the cells. The EC50 for the Doxorubicin was calculated to be 0.579 ug/ml . From the study it is concluded t he hydroalcoholic rhizome extracts Paris polyphylla has potential to exhibit anticancer activity.
9-Oxo-10, 11-dehydroageraphorone (euptox A), a cadenine sesquiterpene, is the main toxin from Eupatorium adenophorum. The aim of the present study was to examine the induction and mechanism of HeLa cell apoptosis by euptox A. The apoptosis‑inducing effect of the euptox A on HeLa cells was examined by MTT assay. The underlying mechanism was analyzed by flow cytometry and quantitative PCR. Flow cytometry results suggested that euptox A effectively inhibited the proliferation of HeLa cells, arrested the cell cycle transition from S to G2/M phase, did not continue to complete the cell cycle activity (mainly from 4 times and mitosis), and induced cell proliferation. The RT-qPCR detection results showed that euptox A induced apoptosis by improving the gene expression level of apoptotic proteases such as caspase-10 in HeLa cells. Its mechanism of action was associated with the upregulation of apoptotic gene expression and arresting of the cell cycle.
There is an increasing interest to identify plant-derived natural products with antitumor activities. In this work, we have studied the effects of aqueous leaf extracts from Amazonian Vismia and Piper species on human hepatocarcinoma cell toxicity. Results showed that, depending on the cell type, the plants displayed differential effects; thus, Vismia baccifera induced the selective killing of HepG2, while increasing cell growth of PLC-PRF and SK-HEP-1. In contrast, these two last cell lines were sensitive to the toxicity by Piper krukoffii and Piper putumayoense, while the Piperaceae did not affect HepG2 growth. All the extracts induced cytotoxicity to rat hepatoma McA-RH7777, but were innocuous (V. baccifera at concentrations < 75 µg/mL) or even protected cells from basal death (P. putumayoense) in primary cultures of rat hepatocytes. In every case, cytotoxicity was accompanied by an intracellular accumulation of reactive oxygen species (ROS). These results provide evidence for the anticancer activities of the studied plants on specific cell lines and suggest that cell killing could be mediated by ROS, thus involving mechanisms independent of the plants free radical scavenging activities. Results also support the use of these extracts of the Vismia and Piper genera with opposite effects as a model system to study the mechanisms of the antitumoral activity against different types of hepatocarcinoma.
Although apoptosis and necrosis have distinct features, the identification and discrimination of apoptotic and necrotic cell death in vitro is challenging. Immunocytological and biochemical assays represent the current gold standard for monitoring cell death pathways; however, these standard assays are invasive, render large numbers of cells and impede continuous monitoring experiments. In this study, both room temperature (RT)-induced apoptosis and heat-triggered necrosis were analyzed in individual Saos-2 and SW-1353 cells by utilizing Raman microspectroscopy. A targeted analysis of defined cell death modalities, including early and late apoptosis as well as necrosis, was facilitated based on the combination of Raman spectroscopy with fluorescence microscopy. Spectral shifts were identified in the two cell lines that reflect biochemical changes specific for either RT-induced apoptosis or heat-mediated necrosis. A supervised classification model specified apoptotic and necrotic cell death based on single cell Raman spectra. To conclude, Raman spectroscopy allows a non-invasive, continuous monitoring of cell death, which may help shedding new light on complex pathophysiological or drug-induced cell death processes.
Combretastatin A-4 (CA-4) is one of the most effective agents used in chemotherapy. Nevertheless, the contribution of p53 and Bim proteins in the CA-4-induced apoptosis in non-small lung cancer cells (NSCLC) remains unresolved, specifically on involving of p53 in the mitochondrial pathway activation by a transcription-independent mechanism. In this context, the p53-null H1299 and wt-p53 H460 NSCLC cells, in the absence and presence of pifithrin-µ (PFTµ), an inhibitor of p53 mitochondrial-translocation, were treated with CA-4 and different cellular endpoints were analyzed. In contrast to previous observations in H460 cells, CA-4 failed in the activation of an apoptotic response in H1299 cells, thus indicating an involvement of p53 in the cell death induced by the drug. We found that CA-4 led to p53 cellular re-localization in H460 cells; in particular, p53 was released from the microtubular network and accumulated at mitochondria where in it interact with Bim protein and other proteins of the Bcl-2 (B-cell leukaemia-2) family, leading to cytochrome c release, alteration in the mitochondrial membrane polarization, cell cycle arrest at the G2/M-phase, and cell death. Interestingly, the cytosolic and the mitochondrial accumulation of protein Bim was strictly dependent on p53 status. The extent of cell death was not reduced in H460 after combined treatment of PFTµ with CA-4. Overall, the data support a model of CA-4-induced apoptosis in NSCLC, for which the expression of p53 protein is essential, but its mitochondrial function, linked to p53-transcription independent apoptosis pathway, is negligible.
Anticancer drug therapy activates both molecular cell death and autophagy pathways. Here we show that even sublethal concentrations of DNA-damaging drugs, such as etoposide and cisplatin, induce the expression of autophagy-related protein 5 (ATG5), which is both necessary and sufficient for the subsequent induction of mitotic catastrophe. We demonstrate that ATG5 translocates to the nucleus, where it physically interacts with survivin in response to DNA-damaging agents both in vitro and in carcinoma tissues obtained from patients who had undergone radiotherapy and/or chemotherapy. As a consequence, elements of the chromosomal passenger complex are displaced during mitosis, resulting in chromosome misalignment and segregation defects. Pharmacological inhibition of autophagy does not prevent ATG5-dependent mitotic catastrophe, but shifts the balance to an early caspase-dependent cell death. Our data suggest a dual role for ATG5 in response to drug-induced DNA damage, where it acts in two signalling pathways in two distinct cellular compartments, the cytosol and the nucleus.
Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely used in conjunction with Annexin V to determine if cells are viable, apoptotic, or necrotic through differences in plasma membrane integrity and permeability. The Annexin V/PI protocol is a commonly used approach for studying apoptotic cells. PI is used more often than other nuclear stains because it is economical, stable and a good indicator of cell viability, based on its capacity to exclude dye in living cells. The ability of PI to enter a cell is dependent upon the permeability of the membrane; PI does not stain live or early apoptotic cells due to the presence of an intact plasma membrane. In late apoptotic and necrotic cells, the integrity of the plasma and nuclear membranes decreases, allowing PI to pass through the membranes, intercalate into nucleic acids, and display red fluorescence. Unfortunately, we find that conventional Annexin V/ PI protocols lead to a significant number of false positive events (up to 40%), which are associated with PI staining of RNA within the cytoplasmic compartment. Primary cells and cell lines in a broad range of animal models are affected, with large cells (nuclear: cytoplasmic ratios <0.5) showing the highest occurrence. Herein, we demonstrate a modified Annexin V/ PI method that provides a significant improvement for assessment of cell death compared to conventional methods. This protocol takes advantage of changes in cellular permeability during cell fixing to promote entry of RNase A into cells following staining. Both the timing and concentration of RNase A have been optimized for removal of cytoplasmic RNA. The result is a significant improvement over conventional Annexin V/ PI protocols (< 5% events with cytoplasmic PI staining).
The species Ageratina pichinchensis (Asteraceae) has been used for a long time in Mexican traditional medicine for the treatment of different skin conditions and injuries. In this study, the healing capacity of the plant extracts obtained was evaluated and, in order to understand the mechanism of healing, we also analyzed its effect on cell proliferation IN VITRO, cytotoxicity, and skin irritation. Different extracts obtained from the aerial parts of A. pichinchensis, topically administrated, were evaluated in a healing model by scalpel-blade incision on the rat. The extracts, at 10 % concentrations, were administrated daily during an eight-day period. A control group, to which the vehicle was administered, was used; while fibrinolysin (Fibrase SA®) was administered for positive control purposes. Reduction in wound size and the histological characteristics of the skin at the end of the treatment were evaluated. Cytotoxicity was evaluated in cell lines KB (nasopharyngeal carcinoma), UISO (squamous cell carcinoma of the cervix), OVCAR (ovarian carcinoma), and HCT-15 (colon carcinoma). In addition, the effect on cell proliferation of cell line MRC-5 (normal cells from human fetal lung) was measured, and skin irritation was evaluated. The results showed an important healing capacity of A. pichinchensis extract in noninfected wounds; the aqueous extract was found to be the most efficient. The extracts exhibited no cytotoxic effect; however, there was an effect that promoted cell proliferation in cell line MRC-5. The products tested demonstrated no skin irritant effects.
The in vivo micronucleus (MN) assay has proven to be an effective measure of genotoxicity potential. However, sampling a single tissue (bone marrow) for a single indicator of genetic damage using the MN assay provides a limited genotoxicity profile. The in vivo alkaline (pH >13) Comet assay, which detects a broad spectrum of DNA damage, can be applied to a variety of rodent tissues following administration of test agents. To determine if the Comet assay is a useful supplement to the in vivo MN assay, a combined test protocol (MN/Comet assay) was conducted in male B6C3F1 mice and F344/N rats using four model genotoxicants: ethyl methanesulfonate (EMS), acrylamide (ACM), cyclophosphamide (CP), and vincristine sulfate (VS). Test compounds were administered on 4 consecutive days at 24-hr intervals (VS was administered to rats for 3 days); animals were euthanized 4 hr after the last administration. All compounds induced significant increases in micronucleated reticulocytes (MN-RET) in the peripheral blood of mice, and all but ACM induced MN-RET in rats. EMS and ACM induced significant increases in DNA damage, measured by the Comet assay, in multiple tissues of mice and rats. CP-induced DNA damage was detected in leukocytes and duodenum cells. VS, a spindle fiber disrupting agent, was negative in the Comet assay. Based on these results, the MN/Comet assay holds promise for providing more comprehensive assessments of potential genotoxicants, and the National Toxicology Program (NTP) is presently using this combined protocol in its overall evaluation of the genotoxicity of substances of public health concern.
Although reactive oxygen species (ROS)-related cell damage has been implicated in pathogenesis of fibrogenetic pulmonary disorders, features of ROS-mediated cell death in human lung fibroblasts are not completely understood. We therefore examined the effects of hydrogen peroxide (H2O2) on cell growth kinetics in human lung fibroblasts (HFL-1 cells) and tested the roles of antioxidants on the H2O2-induced cell death (i.e., necrosis and apoptosis) in HFL-1 cells. We found that the relatively low concentrations of H2O2 ranging from 10 microM to 100 microM induced predominantly apoptosis, whereas higher concentration of H2O2 ranging 1 mM-10 mM induced predominantly necrosis in HFL-1 cells. Extracellular supplementation of glutathione (GSH) in culture media significantly abolished the H2O2-induced cell death, whereas GSH-depleted cells by pretreatment with buthionine sulfoxime (BSO) were likely to undergo cell death caused by a lower concentration of H2O2 than normal HFL-1 cells without BSO treatment. These results indicate that H2O2 induces both necrosis and apoptosis of human lung fibroblasts at least in part through the action of ROS and that modulation of the ROS production inside and outside of cells may influence the cell survival during oxidative insults.
Cell death is discriminated into two main forms: apoptosis and necrosis. In contrast to necrosis, apoptosis is a regulated, energy-dependent form of cell death leading to phagocytosis of cellular remnants by neighboring cells. Characteristic morphological features of these two forms of cell death will be discussed and correlated to underlying molecular mechanisms.
Significance
Drugs targeting microtubules are among the most active anticancer agents. In vitro and in preclinical models, these agents are said to interfere with mitosis. However human tumors divide too slowly for this paradigm to apply, evidenced by the failure of over a dozen well-designed antimitotic agents targeting the aurora kinases and kinesin spindle protein that had minimal antitumor activity but caused severe bone marrow suppression. We have proposed that microtubule-targeting agents interfere with the trafficking of critical proteins in interphase microtubules. If true, then one must identify critical proteins whose traffic on microtubules is impacted. We identify nine DNA repair proteins that traffic on microtubules, explaining why combinations of a microtubule-targeting agent and a DNA-damaging agent are frequently used in cancer therapy.
The cardiotoxicity induced by the anti-cancer Doxorubicin (DOX) involves increased oxidative stress, disruption of calcium homeostasis and activation of cardiomyocyte death. Nevertheless, antioxidants and caspase inhibitors often show little efficacy in preventing cell death. We hypothesize that a caspase-independent cell death mechanism with the release of the Apoptosis Inducing Factor (AIF) from mitochondria is involved in DOX toxicity. To test the hypothesis, H9c2 cardiomyoblasts were used as model for cardiac cells. Our results demonstrate that z-VAD-fmk, a pan-caspase inhibitor, does not prevent DOX toxicity in this cell line. DOX treatment results in AIF translocation to the nuclei, as confirmed by Western Blotting of cell fractions and confocal microscopy. Also, DOX treatment of H9c2 cardiomyoblasts resulted in the appearance of 50 kbp DNA fragments, a hallmark of AIF nuclear effects. AIF knockdown using a siRNA approach in H9c2 cells resulted in a reduction of DOX toxicity, including decreased p53 activation and poly-ADP-ribose-polymerase (PARP) cleavage. Among the proteases that could be responsible for AIF cleavage, DOX decreased calpain activity but increased cathepsin B activation, with inhibition of the latter partly decreasing DOX toxicity. Altogether, the results support that AIF release is involved in DOX-induced H9c2 cell death, which explains the limited ability of caspase inhibitors to prevent toxicity.
Chromatin has been successfully used as a tool for the study of genome function in cancers. Vincristine as a vinca alkaloid anticancer drug exerts its action by binding to tubulins. In this study the effect of vincristine on DNA and chromatin was investigated employing various spectroscopy techniques as well as thermal denaturation, equilibrium dialysis and DNA-cellulose affinity. The results showed that the binding of vincristine to DNA and chromatin reduced absorbance at both 260 and 210 nm with different extent. Chromopheres of chromatin quenched with the drug and fluorescence emission intensity decreased in a dose-dependent manner. Chromatin exhibited higher emission intensity changes compared to DNA. Upon addition of vincristine, Tm of DNA and chromatin exhibited hypochromicity without any shift in Tm. The binding of the drug induced structural changes in both positive and negative extremes of circular dichroism spectra and exhibited a cooperative binding pattern as illustrated by a positive slope observed in low r values of the binding isotherm. Vincristine showed higher binding affinity to double stranded DNA compared to single stranded one. The results suggest that vincristine binds with higher affinity to chromatin compared to DNA. The interaction is through intercalation along with binding to phosphate sugar backbone and histone proteins play fundamental role in this process. The binding of the drug to chromatin opens a new insight into vincristine action in the cell nucleus.
Microtubules are dynamic polymers that play crucial roles in a large number of cellular functions. Their pivotal role in mitosis makes them a target for the development of anticancer drugs. Microtubule-damaging agents suppress microtubule dynamics, leading to disruption of the mitotic spindle in dividing cells, cell cycle arrest at M phase, and late apoptosis. A better understanding of the processes coupling microtubule damage to the onset of apoptosis will reveal sites of potential intervention in cancer chemotherapy. Inhibition of microtubule dynamics induces persistent modification of biological processes (M arrest) and signaling pathways (mitotic spindle assembly checkpoint activation, Bcl-2 phosphorylation, c-Jun NH(2)-terminal kinase activation), which ultimately lead to apoptosis through the accumulation of signals that finally reach the threshold for the onset of apoptosis or through diminishing the threshold for engagement of cell death. Microtubules serve also as scaffolds for signaling molecules that regulate apoptosis, such as Bim and survivin, and their release from microtubules affect the activities of these apoptosis regulators. Thus, sustained modification of signaling routes and changes in the scaffolding properties of microtubules seem to constitute two major processes in the apoptotic response induced by microtubule-interfering agents.
According to a widespread model, anti-cancer chemotherapy involves the triggering of tumor cells to undergo apoptosis, so apoptosis-resistant cells would be recalcitrant to such therapy. However, in addition to apoptosis, which is mainly dependent on the activity of the tumor suppressor protein p53, cells can be eliminated following DNA damage by other mechanisms. Mitotic catastrophe, a form of cell death that results from abnormal mitosis, is one such mechanism. While the term mitotic catastrophe has been used to describe a type of cell death that occurs during mitosis, there is still no broadly accepted definition. Occasionally, mitotic catastrophe is used restrictively for abnormal mitosis leading to cell death, which can occur through necrosis or apoptosis, rather than cell death itself. Although different classes of cytotoxic agents induce mitotic catastrophe, the pathways of abnormal mitosis differ depending on the nature of the inducer and the status of cell-cycle checkpoints. Moreover, mitotic catastrophe can also develop because of aberrant re-entry of tumor cells into the cell cycle after prolonged growth arrest. Elucidation of the factors that regulate different aspects of treatment-induced mitotic catastrophe should assist in improving the efficacy of anti-cancer therapy, providing opportunities for the development of new drugs.
Four new cadinane sesquiterpenes (1-4), including a dimeric cadinane derivative (2) and a peroxide cadinane analogue (3), have been isolated from the leaves of Eupatorium adenophorum. Their structures including absolute configurations were determined on the basis of spectroscopic data interpretation and single-crystal X-ray crystallography. Compound 4 showed in vitro cytotoxicity against the HCT-8, Bel-7402, and A2780 cancer cell lines. © 2008 American Chemical Society and American Society of Pharmacognosy.
GCC: Global compositae checklist
- Y Roskov
- L Abucay
- T Orrell
- D Nicolson
- T Kunze
- C Flann
- N Bailly
- P Kirk
- T Bourgoin
- E Dewalt
- W Decock
- A Dewever
Roskov, Y., L. Abucay, T. Orrell, D. Nicolson, T. Kunze, C. Flann, N.
Bailly, P. Kirk, T. Bourgoin, R., E. DeWalt, W. Decock and A.
DeWever. 2016. GCC: Global compositae checklist. In: Species
2000 and ITIS Catalogue of Life. Digital Resource. Available
from: http://www.catalogueoflife.org/col/details/species/id/4eee6
fe3fbf2731f796f81ea2faae5ad. [Last accessed on 2016 Sep 19].
Flavonoides de Eupatorium gracile
- R Torrenegra
- J Pedrozo
- S Escarria
Torrenegra, R., J. Pedrozo and S. Escarria. 1984. Flavonoides de
Eupatorium gracile. Rev. Latinoamer. Quim. 15: 129-130.