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Improved high-performance liquid chromatographic method for simultaneous determination of 12 cytotoxic caged xanthones in gamboges, a potential anticancer resin fromGarcinia hanburyi
Abstract
The potential anti-tumor activity of gamboges, a herbal medicine derived from Garcinia hanburyi, has increasingly gained the interest of scientist worldwide. The major components of gamboges are cytotoxic caged xanthones. In the present study, an improved HPLC method was developed to simultaneously quantify 12 caged xanthones, including three pairs of epimers and four pairs of trans-cis isomers, i.e. forbesione, isomorellic acid, morellic acid, R-30-hydroxygambogic acid, S-30-hydroxygambogic acid, isogambogenic acid, gambogenic acid, gambogellic acid, R-isogambogic acid, S-isogambogic acid, R-gambogic acid and S-gambogic acid. This method was validated to be sensitive, precise and accurate with limits of detection of 0.03-0.08 microg/mL, overall intra-day and inter-day variations less than 7.9% and overall recovery over 93.2%. The correlation coefficients (r(2)) of the calibration curves were higher than 0.995 for all analytes. The newly established method was successfully applied to reveal the difference in the chemical profiles and contents of these analytes in gamboges from different origins. It can be concluded that this method was not only an effective quality control method to ensure the safety and efficacy consistency of gamboges, but also a useful tool for screening and determining more potent cytotoxic xanthones with potential anticancer activity.
... Morellic acid C33H36O8 G. morella, G. gaudichaudii, G. hanburyi [9, 65, 69, 70, 76, 78, [145] 5 Isomorellic acid C33H36O8 G. morella, G. gaudichaudii [76,145] 6 Morellinol C33H38O7 G. morella [49] 7 Moreollin C35H42O8 G. morella [50] 8 Isomoreollin C35H42O8 G. morella, G. gaudichaudii [50,76] 9 Ethoxydihydroisomorellin C35H42O8 G. morella [9] 10 Dihydroisomorellin C33H38O7 G. morella, G. hanburyi [9,65] 11 Gambogic acid C35H44O8 G. hanburyi [9,64,65,67,69,84] 12 Epigambogic acid C35H44O8 G. hanburyi [56,57,64] 13 Isogambogic acid C35H44O8 G. hanburyi [56,57,59] 14 Epiisogambogic acid C35H44O8 G. hanburyi [56,57] 15 30 [11,86] ...
... Morellic acid C33H36O8 G. morella, G. gaudichaudii, G. hanburyi [9, 65, 69, 70, 76, 78, [145] 5 Isomorellic acid C33H36O8 G. morella, G. gaudichaudii [76,145] 6 Morellinol C33H38O7 G. morella [49] 7 Moreollin C35H42O8 G. morella [50] 8 Isomoreollin C35H42O8 G. morella, G. gaudichaudii [50,76] 9 Ethoxydihydroisomorellin C35H42O8 G. morella [9] 10 Dihydroisomorellin C33H38O7 G. morella, G. hanburyi [9,65] 11 Gambogic acid C35H44O8 G. hanburyi [9,64,65,67,69,84] 12 Epigambogic acid C35H44O8 G. hanburyi [56,57,64] 13 Isogambogic acid C35H44O8 G. hanburyi [56,57,59] 14 Epiisogambogic acid C35H44O8 G. hanburyi [56,57] 15 30 [11,86] ...
... Morellic acid C33H36O8 G. morella, G. gaudichaudii, G. hanburyi [9, 65, 69, 70, 76, 78, [145] 5 Isomorellic acid C33H36O8 G. morella, G. gaudichaudii [76,145] 6 Morellinol C33H38O7 G. morella [49] 7 Moreollin C35H42O8 G. morella [50] 8 Isomoreollin C35H42O8 G. morella, G. gaudichaudii [50,76] 9 Ethoxydihydroisomorellin C35H42O8 G. morella [9] 10 Dihydroisomorellin C33H38O7 G. morella, G. hanburyi [9,65] 11 Gambogic acid C35H44O8 G. hanburyi [9,64,65,67,69,84] 12 Epigambogic acid C35H44O8 G. hanburyi [56,57,64] 13 Isogambogic acid C35H44O8 G. hanburyi [56,57,59] 14 Epiisogambogic acid C35H44O8 G. hanburyi [56,57] 15 30 [11,86] ...
Caged xanthones, characterized by a unique 4-oxa-tricyclo[4.3.1.0(3,7)]dec-2-one scaffold, are a special class of bioactive components mainly derived from the Garcinia genus (Guttiferae family). Around 100 compounds from this family have been reported to date and most of them have potent antitumor activity, with gambogic acid being the best representative. During the past decades, inspired by the unusual caged skeleton and remarkable bioactivity, scientists from various fields have shown increasing interest on these promising natural products. In this review, the plant resources, structural characteristics, total synthesis, biological activity and mechanisms of action, structure activity relationship, and anticancer drug development of these caged xanthones are described.
... Therefore, it is important to simultaneously monitor the bioactive constituents for their quality control and also explore the best suited species in terms of active constituents. In recent years, numerous research groups reported analytical methods, using various chromatographic conditions and spectophotometric technologies, to develop quick and accurate analytical approaches for the identification, structural characterization and determination of chemical constituents of Garcinia species ( Acuna et al., 2012;Aisha et al., 2012;Bharate et al., 2014;Kumar, 2006, 2007;Jayaprakasha and Sakariah, 2000;Jena et al., 2002;Ji et al., 2007;Kumar et al., 2013Kumar et al., , 2009Li et al., 2008;Wittenauer et al., 2012;Zhou et al., 2010;Zhou et al., 2009;Zhou et al., 2008aZhou et al., , 2008bZadernowski et al., 2009). Quantitative analysis of the major bioactive constituents of Garcinia is essential for quality control. ...
... Several analytical methods, including high-performance liquid chromatography coupled to photodiode array detection/diode array detection (HPLC-PDA/DAD) and gas chromatography coupled to mass spectrometry (GC-MS) were used to evaluate the quality of Garcinia species ( Acuna et al., 2012;Aisha et al., 2012;Jayaprakasha and Sakariah, 2000;Jena et al., 2002;Ji et al., 2007;Kumar et al., 2013;Li et al., 2008;Zadernowski et al., 2009). Most of the previous researchers have developed HPLC-PDA/DAD methods focusing on the simultaneous determination of only few classes of compounds in one or two Garcinia species except the work by Acuna et al. (2012). ...
... Garcinia hanburyi has a long history of medicinal use in Southeast Asia, and it is used as a folk medicine and coloring agent (1). An improved separation method for the determination of twelve xanthones in gamboges from Garcinia hanburyi enabled researchers to purify GA from Garcinia hanburyi (2). Previous studies have reported that GA has potent antitumor activity via induction of reactive oxygen species accumulation which consequently led to apoptosis of SMMC-7721 cells (3). ...
... Hep3B, Huh7 and WRL68 were purchased from ATCC and cultured according to their protocols. GA was a generous gift from the Chinese Medicine Laboratory, Hong Kong Jockey Club Institute of Chinese Medicine, and was isolated by the established method (2). GA was prepared by dissolving 4 mg of dry GA into 1 ml of DMSO. ...
The anticancer activities of gambogic acid (GA) on two hepatocellular carcinoma cells with either p53 deletion (Hep3B) or p53 mutation (Huh7) were investigated in the present study. GA inhibited the growth of Hep3B and Huh7 through similar apoptotic pathways. After treatment of Hep3B and Huh7 with GA for 24 h, the IC50 was determined for both cell lines at 1.8 and 2.2 µM, respectively. The results showed that both cancer cells underwent morphological changes and DNA fragmentation. GA induced apoptosis in the two cell lines through caspases-3/7, -8 and -9 in the mitochondrial pathway. The results suggest that both the caspases in the extrinsic death receptor pathway and the mitochondrial-dependent pathway are involved in the GA-induced cell apoptosis. The inhibitory effects of GA on Hep3B and Huh7 are independent of p53-associated pathway.
... It is noteworthy that a cross-talk effect was found in the channel B with the DFI at m/z 393, because the type D compounds can also be fragmentized to give the same ion (Fig. 3D). By comparing the MS data in both positive and negative ionization modes and the retention times with those of the reference standards and/or the data in the literatures [3,[19][20][21], the 14 known compounds were readily identified as forbesione (A1), desoxygaudichaudione A (B1), morellic acid (C41), isomorellic acid (C42), isogambogenin (D32), gambogenic acid (D41), isogambogenic acid (D42), S/R-gambogic acid (E41/E42), S/R-isogambogic acid (E43/E44), S-30-hydroxygambogic acid (E51) and R-30-hydroxyisogambogic acid (E52) and a minor known caged xanthone, hanburin (F1), respectively. The trans-and cis-isomers were differentiated by the McLafferty rearrangement fragment ions in positive ionization mode according to the protocol proposed by Zhou et al. [3]. ...
... The trans-and cis-isomers were differentiated by the McLafferty rearrangement fragment ions in positive ionization mode according to the protocol proposed by Zhou et al. [3]. Meanwhile, the elution order of the geometric isomers of the caged xanthone in the reverse phase HPLC experiments is generally the trans before the cis [3,21]. Among the left 17 likely unknown components, 14 ones hit the predicted compounds set (Fig. 2 isogambogeninol (D22), gambogenin (D31), respectively (Table 1). ...
Although the anticancer activities of the resin of Garcinia hanburyi have been well demonstrated, the chemical composition of this medicinal plant is still not fully understood. In this study, a highly effective qualitative method was developed for rapidly profiling the target and non-target caged xanthones in the resin of G. hanburyi. This method mainly involves three steps as follows: (1) prediction of the possible unknown caged xanthones in the resin of G. hanburyi according to the structure characters of the known ones and some well established biosynthetic knowledge; (2) structure classification according to the diagnostic fragment ions (DFIs) of the known caged Garcinia xanthones; (3) detection and characterization of the target and non-target caged xanthones in the resin of G. hanburyi using multiple mass spectrometric (MS) scanning modes. By use of such procedures, mass spectrometric data can be used for confirming the rationally predicted chemical structure rather than sophisticated and time-consumed de novo structure elucidation of a completely unknown component. Finally, a total of 34 caged xanthones including 18 likely new ones from the resin of G. hanburyi were rapidly detected and characterized within one working day.
... The components responsible for the therapeutic effects may be investigated as lead compounds for new drug discovery . Gambogic acid (Figure 2), one of the major caged xanthones of gamboges, is used as a chemical marker for quality control and safety evaluation of gamboges120121122123. As its cytotoxicity is attributed to cell apoptosis induction124125126127128, gambogic acid is a potential lead compound for new anti-cancer drugs124125126 and has recently been approved by the State Food and Drug Administration of China for clinical trials of cancer treat- ment [129]. ...
... The two epimers should be used as separate chemical markers [77]. The two epimers were eluted as a fine peak on a C 18 column; they can be separated on a C 8 column under optimized conditions [122,123]. Gambogic acid (1) and gambogoic acid (2) and the possible derivative schemesFigure 3 Gambogic acid (1) and gambogoic acid (2) and the possible derivative schemes. ...
Selection of chemical markers is crucial for the quality control of herbal medicines, including authentication of genuine species, harvesting the best quality raw materials, evaluation of post-harvesting handling, assessment of intermediates and finished products, and detection of harmful or toxic ingredients. Ideal chemical markers should be the therapeutic components of herbal medicines. However, for most herbal medicines, the therapeutic components have not been fully elucidated or easily monitored. Bioactive, characteristic, main, synergistic, correlative, toxic and general components may be selected. This article reviews the effective use of chemical markers in the quality control of herbal medicines including the selection criteria considering the roles and physicochemical factors which may affect the effective use of chemical markers.
... 9,17) Natural product compounds play a pivotal role in the discovery and development of anticancer drugs. 18,19) Gambogenic acid (GNA), the major bioactive ingredient of Gamboge, 20) was found in the dry resin of Garcinia hanburyi HOOK. f. (Guttiferae). ...
Cutaneous melanoma is an aggressive cancer, which is the most common type of melanoma. In our previous studies, gambogenic acid (GNA) inhibited the proliferation and migration of melanoma cells. Maternally expressed gene 3 (MEG3) is a long noncoding RNA (lncRNA) that has been shown to have inhibitory effects in a variety of cancers. However, the mechanisms in melanoma progression need to be further investigated. In the current study, we investigated the inhibitory effect of GNA on melanoma and its molecular mechanism through a series of cell and animal experiments. We found that GNA could improve epithelial mesenchymal transition by up-regulating the expression of the lncRNA MEG3 gene, thereby inhibiting melanoma metastasis in vitro and in vivo.
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... R-Isogambogic acid, S-isogambogic acid, R-gambogic acid, S-gambogic acid, forbesione, isomorellic acid, morellic acid, R-30-hydroxygambogic acid, S-30-hydroxygambogic acid, isogambogenic acid, gambogenic acid, and gambogellic acid were separated with an improved HPLC method validated to be precise, sensitive, and accurate (8). ...
Garcinia hanburyi, a tropical plant found in south Asia, has a special long history in the development of both medicine and art. This review mainly focuses on the pharmacy research of the bioactive compounds from the plant in recent years. Preparative and analysis separation methods were introduced. Moreover, the chemical structure of the isolated compounds was included. The studies of biological activities of the caged xanthones from the plant, including antitumor, anti-HIV-1, antibacterial, and neurotrophic activities, were reviewed in detail. Furthermore, the mechanisms of its antitumor activity were also reviewed. As mentioned above, some of the xanthones from G. hanburyi can be promising drug candidates, which is worth studying. However, we still need much evidence to prove their efficacy and safety. So, further research is critical for the future application of xanthones from G. hanburyi.
... To date, several methods for the separation and analysis of morellic acid in raw herbs have been reported using high-performance liquid chromatography with photodiode array detector (HPLC-PAD) (14,15), and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS) (16,17). However, no method was reported for quantification of morellic acid in rat plasma. ...
A selective and rapid high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS) was developed for the quantification of morellic acid in rat plasma. HPLC was performed using a Capcell MG C18 (50 × 4.6 mm, i.d., 5 µm) column, and isocratic elution with water-acetonitrile (20:80, v/v) at a flow rate of 0.5 mL/min. Sample preparation of analyte and internal standard (gambogic acid) involved liquid-liquid extraction using ethyl acetate-isopropanol (1:1, v/v) from 50 µL plasma. The precursor → production transitions for analyte and IS were m/z 559.4 → 471.3, and m/z 627.3 → 583.3, respectively, and were monitored on a triple-quadrupole mass spectrometer, operating in negative ion scan mode. The method was validated across the dynamic concentration range of 20-7,500 ng/mL for morellic acid, with a fast run time of 6.0 min. The analytical method measured concentrations of morellic acid with accuracy (% bias) of ≤6.4% and precision (% RSD) of ≤14.0%. Morellic acid was stable during the battery of stability studies. Finally, the applicability of this assay has been successfully demonstrated in vivo pharmacokinetic studies in Sprague-Dawley rats. This method will therefore be useful for further preclinical and clinical pharmacokinetic studies of morellic acid.
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... Semi-synthetic analogues have been prepared in some cases to improve the efficacy and decrease the side effects of the parent compound. To improve clinical (Shi and Li 2007;Tada et al., 1999;Tang et al., 2004;Youn et al., 2007;Zhang et al., 2005) 2. Mitochondriadependent apoptosis 3. Cell differentiation The chronic toxicity study revealed that the targets of Gambogic acid in rats are the kidney and liver (Batova et al., 2010;Chen et al., 2008;Guo et al., 2006;Hahnvajanawong et al., 2010;Han et al., 2006aHan et al., , 2006bHan et al., , 2006cHe et al., 2009;Li et al., 2008Li et al., , 2009Li et al., , 2010Lu et al., 2007b;Nie et al., 2009;Pandey et al., 2007;Qiang et al., 2008;Qin et al., 2007;Reutrakul et al., 2007;Tao et al., 2009;Wang et al., 2009b;Xie et al., 2009;Yang et al., 2007;Yi et al., 2008;Yu et al., 2007;Zhang et al., 2004aZhang et al., , 2007Zhao et al., 2008) 2. Depolymerized microtubules; 3. Antiangiogenesis; 4. Telomerase inhibitor; 5. Multidrug resistance; 6. Mitochondrial and ER stress apoptosis; 7. Inhibit a(4) integrin; ...
Many anticancer drugs are obtained from natural sources. Nature produces a variety of toxic compounds, which are often used as anticancer drugs. Up to now, there are at least 120 species of poisonous botanicals, animals and minerals, of which more than half have been found to possess significant anticancer properties. In spite of their clinical toxicity, they exhibit pharmacological effects and have been used as important traditional Chinese medicines for the different stages of cancer. The article reviews many structures such as alkaloids of Camptotheca acuminata, Catharanthus roseus and Cephalotaxus fortunei, lignans of Dysosma versipellis and Podophyllum emodi, ketones of Garcinia hanburyi, terpenoids of Mylabris and Ginkgo biloba, diterpenoids of Tripterygium wilfordii, Euphorbia fischeriana, Euphorbia lathyris, Euphorbia kansui, Daphne genkwa, Pseudolarix kaempferi and Brucea javanica, triterpenoids of Melia toosendan, steroids of Periploca sepium, Paris polyphylla and Venenum Bufonis, and arsenic compounds including Arsenicum and Realgar. By comparing their related phytochemistry, toxic effects and the recent advances in understanding the mechanisms of action, this review puts forward some ideals and examples about how to increase antitumour activity and/or reduce the side effects experienced with Chinese medicine. Copyright © 2012 John Wiley & Sons, Ltd.
... Due to species, geographic, climatic, and environmental factors, several types of xanthones have been isolated from Swertia spp. [9,10] Several methods including high performance liquid chromatography (HPLC), [11][12][13][14] thin layer chromatography (TLC), [15] and capillary electrophoresis, [16] have been reported for the analysis of different classes of xanthones, but few quantitative analytical methods have been reported for determining xanthones in the Swertia species. In accordance with our previous studies on phytochemistry of Swertia longifolia Boiss. ...
Swertia spp. (Gentianaceae) grow widely in the eastern and southern Asian countries and are used as traditional medicine for gastrointestinal disorders. Swerchirin, one of the xanthones in Swertia spp., has many pharmacological properties, such as, antimalarial, antihepatotoxic, and hypoglycemic effects. Because of the pharmacological importance of Swerchirin in this investigation, it was purified from Swertia longifolia Boiss. as one of the main components and quantified by means of a validated high performance liquid chromatography (HPLC) technique. Aerial parts of the plant were extracted with acetone 80%. Phenolic and non-phenolic constituents of the extract were separated from each other during several processes. The phenolic fraction was injected into the semi-preparative HPLC system, which consisted of a C(18) column and a gradient methanol: 0.1% formic acid mode. Using this method, we were able to purify six xanthones from the plant, in order to use them as standard materials. The analytical method was validated for Swerchirin as one of the most important components of the plant, with more pharmacological activities according to the validation parameters, such as, selectivity, linearity (r(2) > 0.9998), precision (</=3.3), and accuracy, which were measured by the determination of recovery (98-107%). The limits of detection and quantization were found to be 2.1 and 6.3 mug/mL, respectively. On account of the speed and accuracy, the UV-HPLC method may be used for quantitative analysis of Swerchirin.
The previous phytochemical analyses of Garcinia hanburyi revealed that the main structural characteristic associated with its biological activity is the caged polyprenylated xanthones with a unique 4-oxatricyclo [4.3.1.03,7] dec-2-one scaffold, which contains a highly substituted tetrahydrofuran ring with three quaternary carbons. Based on the progress in research of the chemical constituents, pharmacological effects and modification methods of the caged polyprenylated xanthones, this paper presents a preliminary predictive analysis of their drug-like properties based on the absorption, distribution, metabolism, excretion and toxicity (ADME/T) properties. It was found out that these compounds have very similar pharmacokinetic properties because they possess the same caged xanthone structure, the 9,10-double bond in a,b-unsaturated ketones are critical for the antitumor activity. The author believes that there is an urgent need to seek new breakthroughs in the study of these caged polyprenylated xanthones. Thus, the research on the route of administration, therapeutic effect, structural modification and development of such active ingredients is of great interest. It is hoped that this paper will provide ideas for researchers to develop and utilize the active ingredients derived from natural products.
Gamboge, a dried resin secreted by Garcinia hanburyi Hook. f. (Guttiferae), possesses remarkable anticancer activity. However, due to toxicity, it must be processed before use in clinics. Xanthones are the main bioactive ingredients in gamboge. In order to elucidate the influence of processing technology on pharmacological properties of gamboge, an efficient, sensitive, and selective ultra-performance liquid chromatography coupled with triple quadruple mass spectrometry (UPLC-MS/MS) method of five critical xanthones, including β-morellic acid (β-MA), isogambogenic acid (IGNA), gambogenic acid (GNA), R-gambogic acid (GA), and S-GA in rat plasma was established for a comparative pharmacokinetics study of these xanthones after oral administration of crude and processed G. hanburyi extracts. The chromatographic separation of these five xanthones along with an internal standard (I.S.) was carried out on a Waters Acquity UPLC BEH C8 column with a gradient elution method using acetonitrile/0.1% formic acid-water as mobile phases. The eluate was detected by multiple-reaction monitoring (MRM) scanning with an electrospray ionization source operating in the positive ionization mode. Sample preparation involved a liquid-liquid extraction of the five analytes with ethyl acetate. Deoxyschizandrin was employed as an internal standard. This assay method was validated for selectivity, linearity, intra-day and inter-day precision, accuracy, recovery, matrix effect, and stability. The results revealed that the calibration curves displayed good linear regression (r > 0.995), and the lower limit of quantification (LLOQ) was <5.52 ng/mL for each analyte. The intra-day and inter-day precision (RSD) of the five xanthones at low, medium, and high levels was <10.58%, and the bias of the accuracy ranged from -8.54 to 10.2%. All other parameters fulfilled the FDA criteria for bioanalytical validation. In addition, the assay was successfully applied to the determination and pharmacokinetic study of these five xanthones after oral administration of crude and processed gamboge. Furthermore, Cmax of GNA and AUC0-t of IGNA were increased significantly (P < 0.05) after processing, while AUC0-t of β-MA, R-GA, and S-GA decreased remarkably (P < 0.05), which suggested that processing exerted different effects on the absorption of xanthones. The results might be valuable for the clinical reasonable application and understanding the processing mechanism of gamboge.
The Garcinia genus of plants has yielded an intriguing family of caged xanthone-derived natural products that have a documented value in traditional Eastern medicine. Collectively referred to as caged Garcinia xanthones (CGXs), these compounds are defined by an unusual motif in which the C-ring of an allylated xanthone has been converted into a tricyclic cage. This motif is further decorated via A-ring substitutions and peripheral oxidations to produce a variety of related subfamilies. Gambogic acid, the archetype of this family, has shown efficacy in several human tumor xenograft models and has entered clinical trials in China for the treatment of cancer. Its promising therapeutic potential has fueled the evolution of synthetic strategies, aiming to synthesize various natural products of this family and optimize its bioactive scaffold. This review will present the general chemical concepts that have been explored toward the synthesis of CGX natural products and will discuss how they have been applied for the study of the CGX pharmacophore.
Objective
To establish a quality control method for simultaneous determination of multiple components in gamboge.
Methods
A single reference standard for the determination of multiple components (SSDMC) with HPLC was proposed. Seven major components of gamboge including gambogenic acid (S), β-morellic acid (C1), 2R-30-hydroxygambogic acid (C2), isogambogenic acid (C3), gambogellic acid (C4), 2R-gambogic acid (C5), and 2S-gambogic acid (C6) were simultaneously analyzed using gambogenic acid as reference standard. The credibility and feasibility of SSDMC method were validated with respect to linearity, limits of detection and quantification, precision, stability, repeatability, accuracy, ruggedness, and robustness. The relative conversion factors (RCFs) of S and C1-6 were calculated. Twelve batches of gamboge including crude and processed products were successfully analyzed by applying the SSDMC and traditional external standard (ES) methods.
Results
The SSDMC method was credible and feasible. The RCFs of S and C1-6 were 1.000, 0.913, 0.864, 1.064, 0.777, 0.921, and 0.919, respectively. No significant difference was observed in the contents of the seven components between SSDMC and ES methods. The heat-processing technique caused a reduction in the seven components.
Conclusion
SSDMC is a simple, reliable, and effective method for the analysis of the complex multiple components in gamboge, and it is also a practical and economical approach.
A high performance liquid chromatography photo diode array UV detection electrospray ionization tandem mass spectrometry (HPLC-PDA-ESI/MS n) method was developed and validated for the quality evaluation of gamboge (dryed resin exuded from the stems of Garcinia hanburyi). The contents of the three bioactive constituents (gambogenic acid, R-gambogic acid and S-gambogic) were determined by using HPLC-PDA, and their chemical structures were identified by HPLC-ESI-MS n. The limits of detection and quantitation were between 0.039-0.048 μg/mL and 0.13-0.16 μg/mL. The intra- and inter- assay precisions, in terms of percent relative standard deviation, are less than 3.7 and 4.8%, respectively. The accuracy, in terms of recovery percentage, ranged from 96.86 to 101.70%. Good linearity (correlation coefficient > 0.9996) for each calibration curve of standards. HPLC-PDA-ESI-MS n was use to analyze caged xanthones in gamboge. A total of 16 peaks were identified or tentatively characterized. The results indicated that the method could be considered to be a simple, rapid and reliable method for the quality evaluation of gamboge.
The present paper proposes a method that includes optimization of extraction process and high-performance thin-layer chromatographic (HPTLC) method for the rapid and simple quantification of two isomeric anticancer compounds (isogarcinol and garcinol) in Garcinia indica fruit and its validation according to the International Conference on Harmonization (ICH) guidelines. HPTLC of isogarcinol and garcinol was performed on precoated silica 60 F-254S HPTLC plates. For achieving good separation, the mobile phase of n-pentane-ethyl acetate-formic acid (7:3:0.5, v/v/v) was used. Densitometric determinations of the investigated compounds were carried out at 327 nm in reflection-absorption mode. The calibration curves were linear in the range of 990 to 6900 ng for isogarcinol and 200 to 1400 ng for garcinol. The method was found to be reproducible and convenient for quantitative analysis of garcinol and isogarcinol in different solvent extracts of G. indica fruit. Our work demonstrated the optimization of the extraction procedure - rapid and sensitive quantification of two isomeric anticancer compounds -and its validation in the fruit extract of G. indica.
A sensitive ultrahigh performance liquid chromatography tandem mass spectrometry (UHPLC-MS) method was developed for determination of gambogic acid (GA) in rat plasma, urine, bile and main tissues. GA was separated on an Agilent Zorbax XDB-C18 column (50 × 2.1 mm, 1.8 µm) with gradient mobile phase at the flow rate of 0.2 mL/min. The detection was performed by negative electrospray ionization with multiple reaction monitoring mode. The calibration curves of GA were linear between 1.0 and 1000 ng/mL in rat plasma and bile and between 1.0 and 500 ng/mL in urine and tissues. The lowest limit of quantification for all matrices was 1.0 ng/mL. Both accuracy and precision of the assay were satisfactory. This validated method was firstly applied to bioavailability (BA), pharmacokinetics, excretion and tissue distribution in rats. The BAs of GA (40 and 80 mg/kg) in rats were 0.25 and 0.32%, respectively. GA was distributed extensively in rats after oral administration and exhibited the highest level in liver. GA reached the cumulative excretion amount of 25.3 ± 1.7 µg in bile and 0.275 ± 0.08 µg in urine after i.g. 80 mg/kg to rats at 24 h. The present results would be helpful for further clinical use of GA as a potential anticancer drug. Copyright © 2015 John Wiley & Sons, Ltd.
Copyright © 2015 John Wiley & Sons, Ltd.
The present paper proposes a method that includes optimization of extraction process and identification of two anticancer compounds (garcinol and isogarcinol) by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS) system and their quantification through multi-mode multiple reaction monitoring (MRM). The study aimed to develop a fast, accurate and sensitive method for the quantification of two anticancer compounds garcinol and isogarcinol in different extracts of Garcinia indica fruits. Components were detected using multimode LC electrospray ionization (ESI) in positive-ion mode and quantified by multiple-reaction monitoring (MRM) mode using transition mass of m/z 603.3/411, taken as quantifier, and 603.3/343.2 as qualifier for garcinol and isogarcinol. Five point calibration curves were linear in the range of 2 to 10 ng/ml for garcinol and 0.5 ng/ml to 6 ng/ml for isogarcinol, having correlation coefficient of ≥ 0.990 for both molecules. Limit of quantification for garcinol and isogarcinol was 0.06 ng/ml and 0.05 ng/ml, while limit of detection was 0.02 ng/ml and 0.017 ng/ml respectively. Our work demonstrated optimization of extraction procedure, fast and highly sensitive quantification, and validation of the developed method (quantification limit in pico-gram) for the investigated compounds in the fruit extract of G. indica.
Multidrug resistance remains a serious clinical problem in the successful therapy of malignant diseases. It occurs in cultured tumor cell lines, as well as in human cancers. Therefore, it is critical to develop novel anticancer drugs with multidrug-resistance modulating potential to increase the survival rate of leukemia patients. Plant-derived natural products have been used for the treatment of various diseases for thousands of years. This review summarizes the anticancer and multidrug-resistance reversing properties of the extracts and bioactive compounds from traditional medicinal plants in different leukemia cell lines. Further mechanistic studies will pave the road to establish the anticancer potential of plant-derived natural compounds.
This paper describes a method that includes an optimized extraction process and identification and quantification of two anticancer compounds (garcinol and isogarcinol) by LC/electrospray ionization (ESI)-MS/MS in the multiple reaction monitoring (MRM) mode. The study aimed to develop
a fast, accurate, and sensitive method for the quantification of garcinol and isogarcinol in different extracts of Garcinia indica fruits. The compounds were detected using LC/ESI-MS/MS in the positive-ion mode and quantified in the MRM mode using a transition mass of m/z 603.3/411
taken as the quantifier and 603.3/343.2 as the qualifier for garcinol and isogarcinol. Five point calibration curves were linear in the range of 2 to 10 ng/mL for garcinol and 0.5 to 6 ng/mL for isogarcinol, with a correlation coefficient of ≥0.990 for both. LOQ for garcinol and isogarcinol
was 0.06 and 0.05 ng/mL, respectively, while LOD was 0.021 and 0.017 ng/mL respectively. Our work demonstrated optimization of extraction procedure, fast and highly sensitive quantification (pg level LOQ), and validation of the developed method for the investigated compounds in fruit extracts of G. indica.
Gambogic acid and gambogenic acid are two major bioactive components of Garcinia hanburyi, and play a pivotal role in biologic activity. In this study, a specific and sensitive liquid chromatography-tandem mass spectrometry was developed and validated for simultaneous determination of gambogic acid and gambogenic acid in rat plasma. Chromatographic separation was achieved on a C18 column using an isocratic elution with methanol-10 mm ammonium acetate buffer-acetic acid (90:10:0.1, v/v/v) as the mobile phase. The detection was performed on a triple-quadrupole tandem mass spectrometer equipped with electrospray positive ionization using multiple reaction monitoring modes. The transitions monitored were m/z 629.3 [M + H](+) → 573.2 for gambogic acid, m/z 631.2 [M + H](+) → 507.2 for gambogenic acid and m/z 444.2 [M + NH4 ](+) → 83.1 for IS. Linear calibration curves were obtained in the concentration range of 2.00-1000 ng/mL for gambogic acid and 0.500-250 ng/mL for gambogenic acid. The lower limits of quantification of gambogic acid and gambogenic acid in rat plasma were 2.00 and 0.500 ng/mL, respectively. The intra- and inter-day precision (RSD) values were <11.7% and accuracy (RE) was -10.6-12.4% at three QC levels for both analytes. The assay was successfully applied to evaluate pharmacokinetics behavior in rats after oral administration of Garcinia hanburyi extracts. Copyright © 2014 John Wiley & Sons, Ltd.
Safety, efficacy and quality control are the three key elements in the modernization and internationalization of traditional Chinese medicines (TCM). In this paper, we summarize and discuss the issues on the quality control and the research and development of evidence-based TCM. Establishing analytical methodologies and expanding the application of chemical markers are crucial for the quality control of TCM. Apart from the well-known organonepic technique for TCM authentication, various advanced analytical methodologies including DNA sequencing, and hyphenated chromatographic methods have been used for the quantitative and qualitative analysis of the raw materials and products. As for new drug development, we focused on those TCMs that show promising effects on specific diseases or syndromes, including cardiovascular diseases, diabetes, irritable bowel syndrome, depression and menopausal syndrome. The strategies used for new drug development were either extensive evidence-based investigation of traditional formulas that are based on traditional Chinese medical theory, or lead compound discovery through high throughput screening. Both clinical trials of randomized, double-blind and placebo-controlled studies, and pharmacological and toxicological studies with molecular, celluar, organ and animal models were conducted. Factors which hinder the progress of quality control and new drug development are also addressed and discussed. In this review, 74 references published by the authors and collaborators over the past three years, were cited.
Natural products have been a great source of many small molecule drugs for various diseases. In spite of recent advances in biochemical engineering and fermentation technologies that allow us to explore microorganisms and the marine environment as alternative sources of drugs, more than 70 % of the current small molecule therapeutics derive their structures from plants used in traditional medicine. Natural-product-based drug discovery relies heavily on advances made in the sciences of biology and chemistry. Whereas biology aims to investigate the mode of action of a natural product, chemistry aims to overcome challenges related to its supply, bioactivity, and target selectivity. This review summarizes the explorations of the caged Garcinia xanthones, a family of plant metabolites that possess a unique chemical structure, potent bioactivities, and a promising pharmacology for drug design and development.
Bioassay and ultraperformance liquid chromatography/photodiode array/mass spectrometry (UPLC/PDA/MS) guided isolation of the apoptosis-inducing active metabolites on HeLa-C3 cells from the pericarp of Garcinia yunnanensis (Guttiferae) yielded five active compounds, including the new garciyunnanins A (1) and B (2). The structures of the compounds were elucidated by comprehensive nuclear magnetic resonance and mass spectrometry analysis. Garciyunnanin B (2), featured with a natural tetracyclic xanthone skeleton derived from a polyisoprenylated benzophenone, is structurally interesting since it can be seen as an evidence of the previously described cyclization of garcinol by 2,2-diphenyl-1-picrylhydrazyl (DPPH). Garciyunnanin A (1) contains a 3-monohydroxy benzophenone skeleton, which is rarely found in Garcinia species. Both new compounds induce HeLa-C3 cells into apoptosis after 72 h of incubation at 15 microM. It is noteworthy that oblongifolin C (4), the major constituent of this plant, has proved to be the most active one among the isolates for inducing apoptotic cell death in cervical cancer derived HeLa-C3 sensor cells.
On-line ultra high-performance liquid chromatography (UHPLC) coupled with electrospray quadrupole time-of-flight tandem mass spectrometry (ESI-QTOF-MS/MS/MS) has been developed for the analysis of a series of caged xanthones in the resin of Garcinia hanburyi. The fragmentation of protonated molecular ions for 12 known cadged xanthones was carried out using low-energy collision-induced electrospray ionization tandem mass spectrometry. It was found that Retro-Diels-Alder rearrangement occurred in the CID processes and produced the characteristic fragment ions, which are especially valuable for the identification of this class of xanthones. The fragmentation differential between some cis-, trans-isomers was uncovered. Computation methods were utilized to rationalize the observed MS behaviors. On-line UHPLC-ESI-MS/MS/MS method has proved to be rapid and efficient in that within 6min, 15 caged scaffold xanthones, including three pairs of epimers and four pairs of isomers in gamboges, were effectively separated and identified. Among them, two known, namely isogambogenin (13) and isomorellinol (14) and one likely new caged Garcinia xanthones from the Garcinia hanburyi were tentatively characterized based on the tandem mass spectra of known ones.
Despite the presence of lymphocyte infiltration, human hepatocellular carcinoma (HCC) is typically a rapidly progressive disease. The mechanism of regulation of lymphocyte migration is poorly understood. In this study, we investigated various factors regulating T cell migration in HCC patients. We examined serum CXC chemokine levels in HCC patients and demonstrated the production of CXC chemokines by HCC cell lines. We determined the effect of both HCC patient serum and tumor cell conditioned supernatant upon lymphocyte expression of chemokine receptor CXCR3 as well as lymphocyte migration. Lastly, we examined the chemotactic responses of lymphocytes derived from HCC patients.
The serum chemokines IP-10 (CXCL10) and Mig (CXCL9) levels were measured by cytometric bead array (CBA) and the tumor tissue IP-10 concentration was measured by ELISA. The surface expression of CXCR3 on lymphocytes was determined by flow cytometry. The migratory function of lymphocytes to the corresponding chemokines was assessed using an in vitro chemotactic assay. Phosphorylation of extracellular signal-regulated kinase (ERK) was determined by Western blot analysis.
Increased levels of IP-10 and Mig were detected in HCC patient serum and culture supernatants of HCC cell lines. The IP-10 concentration in the tumor was significantly higher than that in the non-involved adjacent liver tissues. HCC cell lines secreted functional chemokines that induced a CXCR3-specific chemotactic response of lymphocytes. Furthermore, tumor-cell-derived chemokines induced initial rapid phosphorylation of lymphocyte ERK followed by later inhibition of ERK phosphorylation. The culture of normal lymphocytes with HCC cell line supernatants or medium containing serum from HCC patients resulted in a significant reduction in the proportion of lymphocytes exhibiting surface expression of CXCR3. The reduction in T cell expression of CXCR3 resulted in reduced migration toward the ligand IP-10, and both CD4+ and CD8+ T cells from HCC patients exhibited diminished chemotactic responses to IP-10 in vitro compared to T cells from healthy control subjects.
This study demonstrates functional desensitization of the chemokine receptor CXCR3 in lymphocytes from HCC patients by CXCR3 ligands secreted by tumor cells. This may cause lymphocyte dysfunction and subsequently impaired immune defense against the tumor.
To investigate the anticancer effect of a traditional Chinese medicine gambogic acid (GA) in human gastric cancer line BGC-823 and further study the mechanism of apoptosis induction of GA.
Low differential human gastric cancer line BGC-823 were treated with GA at different doses and different times, the inhibitory rates were detected by MTT assay. Apoptosis induced by GA in BGC-823 cells was observed by Annexin-V/PI doubling staining flow cytometry assay. And T/C (%) was chosen to detect the inhibition of GA on human gastric adenocarcinoma BGC-823 nude mice xenografts. Apoptosis on nude mice xenografts was observed by Annexin-V/PI doubling staining flow cytometry assay and DNA fragmentation assay. To further determine the molecular mechanism of apoptosis induced by GA, the changes on the expression of bcl-2 and bax genes were detected by RT-PCR.
After incubation with GA, low differential human gastric cancer line BGC-823 was dramatically inhibited in a dose-dependent manner. After these cells was exposed to GA for 24, 48 and 72 h, the IC(50) value were 1.02+/-0.05, 1.41+/-0.20 and 1.14+/-0.19 micromol/L, respectively. Apoptosis in BGC-823 cells induced by GA was observed by Annexin-V/PI doubling staining flow cytometry assay. The apoptotic population of BGC-823 cells was about 12.96% and 24.58%, respectively, when cells were incubated with 1.2 micromol/L GA for 48 and 72 h. T/C (%) of human gastric carcinoma adenocarcinoma BGC-823 nude mice xenografts was 44.3, when the nude mice were treated with GA (8 mg/kg). Meanwhile, apoptosis induced by GA was observed in human gastric carcinoma adenocarcinoma BGC-823 nude mice xenografts. The increase of bax gene and the decrease of bc1-2 gene expressions were found by RT-PCR.
The inhibition of GA on human gastric cancer line BGC-823 was confirmed. This effect connects with the inducing apoptosis in BGC-823 cells and the molecular mechanism might be related to the reduction of expression of apoptosis-regulated gene bcl-2, and the improvement of the expression of apoptosis-regulated gene bax. The result was also confirmed in vivo.
Transferrin receptor (TfR) has been shown to be significantly overexpressed in different types of cancers. We discovered TfR as a target for gambogic acid (GA), used in traditional Chinese medicine and a previously undiscovered link between TfR and the rapid activation of apoptosis. The binding site of GA on TfR is independent of the transferrin binding site, and it appears that GA potentially inhibits TfR internalization. Down-regulation of TfR by RNA interference decreases sensitivity to GA-induced apoptosis, further supporting TfR as the primary GA receptor. In summary, GA binding to TfR induces a unique signal leading to rapid apoptosis of tumor cells. These results suggest that GA may provide an additional approach for targeting the TfR and its use in cancer therapy.
• rapid apoptosis
• caspases
• target identification
Gambogic acid, usually isolated as an inseparable stereomeric mixture of C-2 epimers, was newly separated into two epimers (1 and 2) from the gamboges of Garcinia hanburyi. The stereochemistry at C-2 was clearly defined by extensive spectroscopic analysis and direct comparison of NMR and HPLC data with those of the known R-epimer. Both epimers were examined for their cytotoxicities against human leukemia K562 (K562/S) and doxorubicin-resistant K562 (K562/R) cell lines. Different from doxorubicin (IC (50) = 10.78 microM for K562/R and 0.66 microM for K562/S), epimers 1 and 2 exhibited similar activities against both cell lines (IC(50) = 1.32 and 0.89 microM for 1, IC(50) = 1.11 and 0.86 microM for 2). These results suggested that both epimers were not multidrug resistance (MDR) substrates. Furthermore, epimers 1 and 2 were tested for their inhibitory effects against six human cytochrome P-450 enzymes. Epimers 1 and 2 showed little inhibitory effects toward five of the enzymes except CYP2C9. Interestingly, when tested against CYP2C9, S-epimer 2 had an inhibitory effect 20-fold stronger than that of R-epimer 1.
Aim: To characterize and quantitatively analyze the xanthones in gamboges. Method: A simple and rapid HPLC method was used. Results: Seven major xanthones of gamboges were characterized to be isomorellic acid (1), morellic acid (2), isogambogenic acid (3), gambogenic acid (5), isogambogic acid (6), gambogic acid (7), and 30-hydroxygambogic acid (4) by LC/ESI-MS, respectively. The validation of the quantitative method was performed accordingly. Seven samples from different regions of China and India were examined. They showed similar chromatographic patterns in the HPLC fingerprints, containing the above-mentioned seven major constituents as the characteristic markers. The relative retention time and relative peak area of these characteristic peaks were calculated and set as important parameters for the fingerprinting identification of gamboges. Conclusion: This is a simple, rapid, and efficient quality-control method for the gamboges.
A rapid ion-pair HPLC method was developed and validated for the determination of eight polyprenylated xanthones including three pairs of epimers, namely morellic acid (MA), 30-hydroxygambogic acid (HGA), 30-hydroxyepigambogic acid (HEGA), isogambogic acid (IGA), epiisogambogic acid (EIGA), gambogenic acid (GNA), gambogic acid (GA), and epigambogic acid (EGA), in gamboge resin of Garcinia hanburyi. The separation was performed on a narrow bore C8 column with isocratic elution using a mixture of methanol-ACN-40 mM KH2PO4 buffer (37.5:37.5:25 v/v/v, containing 0.1% tetradecyltrimethylammonium bromide). The newly developed method was used to determine the contents of the eight compounds present in the gamboge. Results showed that GA and EGA are the dominant components of gamboge. The content ratio of each epimer pair remained constant, indicating that the content ratio of epimers can be used as a specific characteristic for the quality control of gamboge.
To investigate the effects and potential mechanisms of gambogic acid (GA), a naturally occurring anticancer agent, on the expression and regulation of telomerase in human gastric carcinoma cells.
GA-induced inhibition of cell proliferation was evaluated by the commonly employed MTT assay on two human gastric carcinoma cell lines, MGC-803 and SGC-7901. Telomerase activity and hTERT mRNA expression were determined by telomeric repeat amplication protocol-polymerase chain reaction and reverse transcription-polymerase chain reaction, respectively. The hTERT promoter activity was measured by luciferase assay. The expression of c-MYC, an apoptotic gene that modulates the expression of hTERT promoter, was quantified by Western blotting.
The proliferation of human gastric carcinoma cell lines, MGC-803 and SGC-7901, was significantly inhibited with GA treatment. Both telomerase activity and hTERT mRNA expression were notably decreased in cells treated with GA. The activity of hTERT promoter and the expression of c-MYC were also remarkably decreased in GA-treated cells.
This study demonstrated that GA treatment of human gastric carcinoma cell lines, MGC-803 and SGC-7901, significantly reduced the expression of c-MYC in a time- and concentration-dependent manner accompanied with the down-regulation of the hTERT transcription and the ultimate reduction in telomerase activity. Our results indicate that the hTERT is a target of c-MYC activity and identify a feasible mechanism of GA's potent anticancer activity.
Eleven novel cytotoxic xanthones, gambogin, morellin dimethyl acetal, isomoreollin B, moreollic acid, gambogenic acid, gambogenin, isogambogenin, desoxygambogenin, gambogenin dimethyl acetal, gambogellic acid and hanburin were isolated together with four known xanthones, gambogic acid, isomorellin, morellic acid and desoxymorellin, from the dry latex of Garcinia hanburyi. The structures were elucidated by a detailed spectroscopic analysis.
To study the inhibitory effect of general gambogic acids (GGA) on transplantation tumor SMMC-7721 in experimental animal model and SMMC-7721 cells in vitro.
Anti-tumor activity of GGA in the experimental transplantation tumor SMMC-7721 was evaluated by relative tumor growth ratio. Cell morphology was observed with inverted microscope and electron microscope. Cell proliferation was measured by MTT assay and the telomerase activity was determined by PCR.
In vivo study indicated that GGA (2, 4, and 8 mg/kg, iv, 3 times per week for 3 weeks) displayed an inhibitory effect on the growth of transplantation tumor SMMC-7721 in nude mice compared with the normal saline group (P<0.01). At the concentrations of 0.625-5.0 mg/L, GGA remarkably inhibited the proliferation of SMMC-7721 cells in vitro. GGA 2 mg/L dramatically changed morphology of SMMC-7721 cells and inhibited the telomerase activity in SMMC-7721 cells.
GGA had inhibitory effect on the growth of SMMC-7721, which might be related to its inhibition of telomerase activity.
The selective induction of apoptosis of gambogic acid (GA) on MGC-803 cells and its probable molecular mechanism were studied. GA greatly inhibited (24, 48, 72 h) the growth of MGC-803 cells (by MTT); the IC(50) value was 0.96 microg/ml at 48 h. Meanwhile, no influence was observed on body weight, number of WBC (white blood cells) in blood or karyote in marrow of rats after GA was injected intravenously. We conclude that GA does not affect normal cells, but that it can induce apoptosis in tumor cells selectively and there were marked morphological changes. A great quantity of apoptotic cells and increasing G(2)/M phase cells were observed by flow cytometry, and a significant percentage of early apoptotic cells were observed by Annexin-V/PI double staining assay. The increase of bax gene and the decrease of bc1-2 gene expressions were detected by immunohistochemistry. Activation of bax and suppression of bc1-2 may contribute to the apoptosis mechanism.
We determined the in vivo and in vitro antitumor activities of gambogic acid (GA) and one of the possible mechanisms for its inhibitory activities. In vivo antitumor activity of GA was evaluated by the relative tumor growth ratio (T/C) in nude mice, and in vitro inhibition of SPC-A1 cells was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and trypan blue exclusion assay. Telomere repeats amplification protocol (TRAP)-polymerase chain reaction (PCR)-enzyme-linked immunosorbent assay (ELISA) and RT-PCR were used to quantitatively detect telomerase activity and the expression of human telomerase reverse transcriptase (hTERT) mRNA, respectively. Results from our in vivo study showed that transplantable tumor growth remained suppressed for up to 21 d with minimal animal weight loss in nude mice treated with gambogic acid (i.v.). Proliferation of SPC-A1 cells cultured in vitro was significantly inhibited (p<0.01), showing time-dependent and dose-dependent inhibition. Telomerase activity and hTERT mRNA expression were both decreased significantly, when cells were exposed to gambogic acid for 24, 48 and 72 h (for 24 h p<0.05, and for 48, 72 h, p<0.01). These results suggeste that gambogic acid could inhibit the growth of SPC-A1 cells and its tumor xenografts, and when treated with gambogic acid for a period of time, telomerase activity and expression of hTERT mRNA in the tumor cells were both inhibited significantly. It is safe, at least in part, to conclude that the down-regulating telomerase activity of GA by modifying partly the expression of hTERT mRNA in SPC-A1 cells may be one possible mechanism for the inhibitory activity of GA in the cells.
The activation of human telomerase, a process regulated by the human telomerase reverse transcriptase (hTERT), is a crucial step during cellular immortalization and malignant transformation. We have reported that gambogic acid (GA), a natural product isolated from the gamboge resin of Garcinia hanburyi tree, is an effective telomerase inhibitor and thus displays potent anticancer activity both in vitro and in vivo. Here we present the direct interaction of GA with oncogene c-MYC, a ubiquitous transcription factor involved in the control of cell proliferation and differentiation, as the molecular mechanism of GA's inhibitory effect on telomerase activity. Consistent with the recently reported association between c-MYC overexpression and induction of telomerase activity, we find here that GA treatment of a human hepatoma cell line SMMC-7721 significantly reduced the expression of c-MYC in a time- and concentration-dependent manner accompanied with the down-regulation of the hTERT transcription and the ultimate reduction in telomerase activity. Our results indicate that the hTERT is a target of c-MYC activity and identify a feasible mechanism of GA's potent anticancer activity.
In this study, the stability of gambogic acid (GA), a polyprenylated xanthone with potent cytotoxicities against various cancer cell lines, was evaluated under several experimental conditions including addition of acids, alkalis and organic solvents. GA was stable when dissolved in acetone, acetonitrile, and chloroform, even when acids were added. However, a new derivative was produced after GA was stored in the methanol solution for a week at room temperature. The addition of alkalis could increase the rate of this chemical transformation. This derivative was determined to be gambogoic acid (GOA) by the HPLC-MS comparison with the known compound. GOA was proposed to be the product of neuclophilic addition of methanol to the olefinic bond at C-10 of GA. Furthermore, when these two compounds were tested for their cytotoxicity, GOA showed significantly weaker inhibitory effects than GA. It was therefore deduced that the alpha,beta-unsaturated carbonyl moiety at C-10 contributed to the cytotoxicity of gambogic acid.
Thirteen xanthones (1-13) were isolated from the resin of Garcinia hanburyi. Among them, two new compounds (namely gaudichaudic acid, and isogambogenic acid, 1, 2), and one new natural product (deoxygaudichaudione A, 3) were identified on the basis of extensive spectral evidence including detailed 2D NMR data. Ten of these xanthones were tested for their cytotoxicities against human leukemia K562 (K562/S) and doxorubicin-resistant K562 (K562/R) cell lines, and showed similar inhibitory effects on both cell lines, suggesting that this group of polyprenylated xanthones might not be multidrug resistance (MDR) substrates.
A simple and rapid liquid chromatographic with diode-array UV-vis spectrophotometric detection (HPLC-DAD) method for identification of natural dyes has been developed. Chromatographic retention of carminic acid, indigotin, crocetin, gambogic acid, alizarin and purpurin has been studied. The mobile phase consisted of 40 mM SDS-10 mM phosphate buffer solution (pH 2.3)-0.1% TFA (eluent A) and acetonitrile (eluent B) using a programmed gradient (5% B to 95% B). Analyses were carried out on a Phenomenex, Luna 5u NH2 100(a) column (250 mm x 4.60 mm i.d., 5 microm particle) and the operating conditions were: 0.6 ml min(-1) flow rate, 20 microl volume injection and 35 degrees C column temperature. Extracts of samples of natural dyes taken from historical maps belonging to The Royal Chancellery Archives in Granada were successfully analyzed using the proposed method including a new technique for sampling.
A recycling counter-current chromatographic system was first set up with a high-speed counter-current chromatography instrument coupled with a column switching valve. This system was first successfully applied to the preparative separation of epimers, gambogic acid and epigambogic acid from Garcinia hanburyi using n-hexane-methanol-water (5:4:1, v/v/v) as the two-phase solvent system. As a result, 28.2 mg gambogic acid and 18.4 mg epigambogic acid were separated from 50 mg of mixture. Their purities were both above 97% as determined by HPLC. The chemical structures were then identified by their (1)H NMR and (13)C NMR spectra.
A high-performance liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (HPLC-APCI-MS) method was established for the determination of gambogic acid (GA) in human plasma using ursolic acid as the internal standard (I.S.). Plasma samples were extracted with ethyl acetate and separated on a Hanbon Lichrospher 5-C18 column with a mobile phase of acetonitrile-tetrahydrofuran-water (70:23:7, v/v). Gambogic acid was determined by using atmospheric pressure chemical ionization (APCI) in a single quadrupole mass spectrometer. HPLC-APCI-MS was performed in the selected ion monitoring (SIM) mode using target ions at [M-H](-)m/z 627.4 for gambogic acid and [M-H](-)m/z 455.4 for the I.S. Calibration curve was linear over the range of 3.108-4144 microg/L. The lower limit of quantification was 3.108 microg/L. The intra- and inter-run precisions were less than 12.3 and 14.1%, respectively. The method has been successfully applied to study the pharmacokinetics of gambogic acid in patients with malignant tumour.
Molecular mechanisms of cell-cycle arrest caused by gambogic acid (GA), a natural product isolated from the gamboge resin
of Garcinia hanburryi tree, have been investigated using BGC-823 human gastric carcinoma cells as a model. Based on our 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazoliumbromide (MTT) assay and flow cytometric analysis, treatment of BGC-823 cells with growth suppressive
concentrations of GA caused an irreversible arrest in the G2/M phase of the cell cycle. Western blot analysis demonstrated that GA-induced cell-cycle arrest in BGC-823 cells was associated
with a significant decrease in CDC2/p34 synthesis, which led to the accumulation of phosphorylated-Tyr15 (inactive) form of CDC2/p34. Real-time PCR, western blot and kinase activity assays revealed that GA-induced reduction of
CDC2/p34 expression was mediated through the inhibition of cyclin-dependent kinase (CDK)-activating kinase (CDK7/cyclin H)
activity. In addition, GA-treated cells were shown to have a low level of CDK7 kinase-phosphorylated-Thr161 CDC2/p34 (active). Taken together, our results suggested that the inhibited proliferation of GA-treated BGC-823 cells was
associated with the decreased production of CDK7 mRNA and protein, which in turn, resulted in the reduction of CDK7 kinase
activity. The reduced CDK7 kinase activity is responsible for the inactivation of CDC2/p34 kinase and the irreversible G2/M phase cell-cycle arrest of human gastric carcinoma BGC-823 cells.
Three new caged xanthones, 7-methoxydesoxymorellin (1), 2-isoprenylforbesione (2) and 8,8a-epoxymorellic acid (3), together with nine known caged xanthones were isolated from the EtOAc extracts of resin and fruits of Garcinia hanburyi. The structures were determined by spectroscopic methods. Most of the isolated compounds showed significant cytotoxicities against a panel of mammalian cancer cell lines. Compound 3, together with the known compounds desoxymorellin, morellic acid, gambogic acid, hanburin, forbesione and dihydroisomorellin, exhibited anti-HIV-1 activity in the reverse transcriptase (RT) assay while the known compounds desoxygambogenin and dihydroisomorellin were found moderately active in the syncytium assay. This work represents the first report on the anti-HIV-1 activities of caged xanthones.
Two new polyprenylated xanthone epimers were isolated from gamboges of Garcinia hanburyi, and identified by detailed spectroscopic analysis as 30-hydroxygambogic acid (2a) and its (2S)-epimer 30-hydroxyepigambogic acid (2b). Both compounds exhibited significant cytotoxicities against the human leukemia K562/S and the corresponding doxorubicin-resistant K562/R cell lines (Table 2).
A rapid, simple and sensitive high-performance liquid chromatography method for the quantification of gambogic acid in dog plasma was developed and validated. After acidification with hydrochloric acid, dog plasma was extracted with ethyl acetate and determined by HPLC. The analysis was carried out on a reversed-phase C(18) analytical column. The mobile phase consisted of a mixture of methanol-0.05% phosphoric acid (94:6, v/v), and the column temperature was maintained at 35 degrees C. A constant mobile phase flow rate of 1.0 mL/min was employed throughout the analyses. The ultraviolet detector was set at 360 nm. Chromatographic separation was achieved in less than 10 min and the calibration curve was linear over a concentration range of 0.156-20 microg/mL. The intra-assay and inter-assay variability values were less than 10.0%. The accuracy ranged from 93.0 to 104.2%. The established method has been successfully applied to a pharmacokinetic study of gambogic acid in dogs.
In Thai folklore medicine, gamboge, the yellow gum-resin secreted from Garcinia hanburyi, is used for infected wound, pain and edema The ethyl acetate extract from Garcinia hanburyi (GH5763) was assessed for anti-inflammatory, analgesic and antipyretic activities using experimental animal models. It was found that GH5763 possessed inhibitory activity on acute phase of inflammation as seen in ethyl phenylpropiolate-induced ear edema and carrageenin-induced hind paw edema in rats. However, GH5763 did not elicit any inhibitory effect on arachidonic acid-induced hind paw edema. In subchronic inflammatory model, GH5763 provoked a significant reduction of both transudative and proliferative phase when tested on cotton pellet-induced granuloma model. GH5763 also reduced the alkaline phosphatase activity in serum of rats in this animal model. In the analgesic test, GH5763 elicited inhibitory activity on acetic acid-induced writhing response and on both the early and the late phase of formalin test. Moreover, GH5763 also possessed an excellent antipyretic effect when tested in yeast-induced hyperthermic rats. It is postulated that the anti-inflammatory, analgesic and antipyretic activities of GH5763 are caused by the inhibition of the prostaglandin biosynthesis.
A simple and rapid capillary electrophoretic method with UV detection (CE-UV) has been developed for the identification of five natural dyes namely, carmine, indigo, saffron, gamboge and Rubia tinctoria root. The separation was performed in a fused-silica capillary of 64.5 cm length and 50 microm id. The running buffer was 40 mM sodium tetraborate buffer solution (pH 9.25). The applied potential was 30 kV, the temperature was 25 degrees C and detections were performed at 196, 232, 252, 300 and 356 nm. The injections were under pressure of 50 mbar during 13 s. The method was applied to the identification of carminic acid, gambogic acid, crocetin, indigotin, alizarin and purpurin in the collection of drawings and maps at the Royal Chancellery Archives in Granada (Spain). The method was validated by using HPLC as a reference method.
Gambogic acid (GA), a xanthone derived from the resin of the Garcinia hanburyi, has been recently demonstrated to bind transferrin receptor and exhibit potential anticancer effects through a signaling mechanism that is not fully understood. Because of the critical role of NF-kappaB signaling pathway, we investigated the effects of GA on NF-kappaB-mediated cellular responses and NF-kappaB-regulated gene products in human leukemia cancer cells. Treatment of cells with GA enhanced apoptosis induced by tumor necrosis factor (TNF) and chemotherapeutic agents, inhibited the expression of gene products involved in antiapoptosis (IAP1 and IAP2, Bcl-2, Bcl-x(L), and TRAF1), proliferation (cyclin D1 and c-Myc), invasion (COX-2 and MMP-9), and angiogenesis (VEGF), all of which are known to be regulated by NF-kappaB. GA suppressed NF-kappaB activation induced by various inflammatory agents and carcinogens and this, accompanied by the inhibition of TAK1/TAB1-mediated IKK activation, inhibited IkappaBalpha phosphorylation and degradation, suppressed p65 phosphorylation and nuclear translocation, and finally abrogated NF-kappaB-dependent reporter gene expression. The NF-kappaB activation induced by TNFR1, TRADD, TRAF2, NIK, TAK1/TAB1, and IKKbeta was also inhibited. The effect of GA mediated through transferrin receptor as down-regulation of the receptor by RNA interference reversed its effects on NF-kappaB and apoptosis. Overall our results demonstrate that GA inhibits NF-kappaB signaling pathway and potentiates apoptosis through its interaction with the transferrin receptor.
Gambogic acid (GA) is the major active ingredient of gamboge, a brownish resin exuded from Garcinia hanburryi tree in Southeast Asia. In this study, we compared the different apoptotic induction of GA on human normal embryonic hepatic L02 cells and human hepatoma SMMC-7721 cells by detecting growth inhibition, observing morphological changes, and the expressions of the relative apoptotic proteins (Bax, Bcl-2 and caspase-3). The results indicated that GA could selectively induce apoptosis of SMMC-7721 cells, while had relatively less effect on L02 cells. To illustrate the distinct selective antitumor mechanism of GA, we further study its distribution in cultured cells and in tumor-bearing mice. The results indicated that SMMC-7721 cells have higher GA binding activity than L02 cells. The retention time of GA in grafted tumor was longer than in liver, renal and other organs. Collectively, the selective anticancer activity of GA could be due to its significant apoptotic inducing effects as well as its higher distribution and longer retention time in tumor cells compared to the normal cells. So GA might be a kind of highly effective anticancer drug candidate with low toxicity to normal tissue.
The anticancer activity of gambogic acid in vitro
- Jin Ty Lv
- Dong Yc Fd
- Liu Jm
- Lei
- Chen Qm
- Br
Dong C, Jin TY, Lv FD, Dong YC, Liu JM, Lei QM and Chen BR. The anticancer activity of gambogic acid in vitro. Bulletin of Chinese Pharmacy 1988; 23(2): 89–90.
Determination of gambogic acid in gamboges by RP-HPLC
- Liu Wy
- F Feng
- You
- Zhang
Liu WY, Feng F, You QD and Zhang ZX. Determination of gambogic acid in gamboges by RP-HPLC. Chinese Traditional and Herbal Drugs 2003; 34: 706 –707.
DOI: 10.1002/bmc 644 S Isolation and structure of neo-gambogic acid from gamboges (Garcinia hanburryi)
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