This article was downloaded by: [Dicle University] On: 06 November 2014, At: 06:01 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Screening for novel plant sources of prenyloxyanthraquinones: Senna alexandrina Mill. and Aloe vera (L.) Burm. F. a
a
a
Francesco Epifano , Serena Fiorito , Marcello Locatelli , Vito a
a
Alessandro Taddeo & Salvatore Genovese a
Department of Pharmacy, University “G. D'Annunzio” of ChietiPescara, Via dei Vestini 31, 66100 Chieti Scalo (CH), Italy Published online: 24 Oct 2014.
To cite this article: Francesco Epifano, Serena Fiorito, Marcello Locatelli, Vito Alessandro Taddeo & Salvatore Genovese (2015) Screening for novel plant sources of prenyloxyanthraquinones: Senna alexandrina Mill. and Aloe vera (L.) Burm. F., Natural Product Research: Formerly Natural Product Letters, 29:2, 180-184, DOI: 10.1080/14786419.2014.971792 To link to this article: http://dx.doi.org/10.1080/14786419.2014.971792
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
Downloaded by [Dicle University] at 06:01 06 November 2014
Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2015 Vol. 29, No. 2, 180–184, http://dx.doi.org/10.1080/14786419.2014.971792
Screening for novel plant sources of prenyloxyanthraquinones: Senna alexandrina Mill. and Aloe vera (L.) Burm. F. Francesco Epifano*, Serena Fiorito, Marcello Locatelli, Vito Alessandro Taddeo and Salvatore Genovese Department of Pharmacy, University “G. D’Annunzio” of Chieti-Pescara, Via dei Vestini 31, 66100 Chieti Scalo (CH), Italy
Downloaded by [Dicle University] at 06:01 06 November 2014
(Received 7 July 2014; final version received 21 September 2014) As a continuation of our ongoing studies aimed to reveal the presence of oxyprenylated anthraquinones in plants claimed to have a laxative effect, in this article, we describe the extraction and HPLC separation of madagascin (3-isopentenyloxyemodin) and 3geranyloxyemodine from dried leaves and fruits of Senna alexandrina Mill. (Leguminosae) and leaves and gel of Aloe vera (L.) Burm. F. (Xanthorrhoeaceae). Both compounds are described herein for the first time as components of extracts of the title plants. Keywords: Aloe vera; anthraquinones; 3-geranyloxyemodin; madagascin; oxyprenylated secondary metabolites; polyketides; Senna alexandrina
1. Introduction Oxyprenylated anthraquinones have been reported in the literature for the first time in 1981 when Amonkar et al. (1981) described the isolation of 6-geranyloxy-3-methyl-1,8dihydroxyanthrone from the root extracts of Psorospermum febrifugum Spach (Guttiferae). In subsequent years, such secondary metabolites have been described also in the genera Psorospermum, Vismia, Harungana and Rhamnus (Epifano et al. 2012; 2013; Genovese et al. 2012). As a continuation of our ongoing studies aimed at the quantification of anthraquinones in plants having laxative effects, we decided to investigate the presence of two oxyprenylated anthraquinones, namely madagascine (1) and 3-geranyloxyemodine (2) (Figure 1), in extracts obtained from dried leaves and fruits of Senna alexandrina Mill. (syn. Cassia angustifolia M. Vahl, Cassia senna L.) (Leguminosae), and dried leaves and gel of Aloe vera (L.) Burm. F. (syn. Aloe barbadensis Mill.) (Xanthorrhoeaceae).
2. Results and discussion 2.1. Chemical synthesis As reported previously (Bruyere et al. 2011), the title compounds were obtained in 54% and 68% yield and purity $ 98% using commercially available emodin as the starting material by DBUpromoted alkylation with 3,3-dimethylallyl or geranyl bromide, respectively, followed by crystallisation with n-hexane.
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
Natural Product Research OH
O
181
OH
O
R
O 1R=H 2 R = isopentenyl
Downloaded by [Dicle University] at 06:01 06 November 2014
Figure 1. Chemical structures of madagascine (1) and 3-geranyloxyemodine (2).
2.2. Plant material and preparation of extracts Dried leaves and fruit samples of S. alexandrina were purchased commercially and carefully checked for the presence of adulteration and/or falsifications by microscopic characterisation. The samples were correctly identified by Dr Francesco Epifano and Dr Salvatore Genovese. Voucher specimens (No. CA-001) have been deposited in the Herbarium of the Department of Pharmacy of the University of ‘Gabriele d’Annunzio’ Chieti-Pescara, Italy. Leaves of A. vera were collected in Termoli (Molise, Italy) in October 2012. The samples were correctly identified by Dr Francesco Epifano and Dr Salvatore Genovese. Voucher specimens (No. AL-001) have been deposited in the Herbarium of the Department of Pharmacy of the University of ‘Gabriele d’Annunzio’ Chieti-Pescara, Italy. Aloe gel was obtained by incision of fresh leaves followed by drop-by-drop collection of the resulting exudate. The extraction of all the vegetable materials consisted of a multi-step process: trituration followed by an overnight maceration in n-hexane (10 mL/g of vegetable material), separation and evaporation of the solution, filtration of the residual vegetable material and its overnight maceration using methanol and finally a second filtration of the latter. The n-hexane extract, once concentrated to complete dryness under vacuum and re-dissolved in methanol, was combined to the methanol extract. The methanol solution was subsequently subjected to reflux for 30 min with a 6N HCl aqueous solution in order to hydrolyse glycosides, followed by the extraction of hydroalcoholic mixture with ethyl acetate to analyse the total content of anthraquinones. The dark red syrupy samples so obtained were stored at 2 208C in amber glass tubes before HPLC analysis. 2.3. Limits of detection, limits of quantitation and linearity Limits of detection were calculated by measuring S/N values obtained in the mobile phase spiked at 1 mg/mL level and extrapolation of the corresponding values to S/N ¼ 3. The limits of quantitation of the method was defined according to the Guidance for Industry on the validation of bioanalytical methods as the concentration of the lowest standard on the calibration curve for which (a) the analyte peak is identifiable and discrete, (b) the analyte response is at least ten times the response of the blank sample and (c) the analyte response is reproducible with a precision less than 20% and trueness better of 80 –120%. Under these experimental conditions the retention time and other key parameters related to the analysis were as follows: (a) for madagascin (1) retention time of 33.3 (^ 0.4) min, linearity range of 0.5– 100 mM, determination coefficients (r 2) of 0.9850, (b) for 3-geranyloxyemodin (2) retention time of 44.8 (^ 1.0) min, linearity range of 5.0 –125 mM, determination coefficients (r 2) of 0.9925. The dead retention time, calculated with uracil, was 1.83 min. The within-assay precision (repeatability) of the method was in the range 0.6– 12.4, while between-assay precision (intermediate precision) was in the range 2 0.3 to 12.4. The trueness of the method was evaluated at the same analyte concentration levels by comparing the measured anthraquinone concentrations of the QC samples with their nominal values. The within-assay trueness of the method was in the range
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2 12.0 to 11.5 range, while between-assay trueness was in the range 2 12.2 to 12.7 range. Calibration curves, obtained at 435 nm, were plotted using weighted (1/x 2) linear least-squares regression analysis. The weighting factor was chosen to minimise deviation of back-calculated values from theoretical concentrations, especially for the lowest concentration levels, as permitted by method validation guidelines, that report that ‘standard curve fitting is determined by applying the simplest model that adequately describes the concentration – response relationship using appropriate weighting’.
Downloaded by [Dicle University] at 06:01 06 November 2014
2.4. Quantification of madagascine (1) and 3-geranyloxyemodin (2) Results about the concentration of madagascine and 3-geranyloxyemodine in S. alexandrina and A. vera extracts are reported in Table 1. For comparison, values of the concentration of ‘classic’ unprenylated anthraquinones, such as aloe-emodin, rhein, emodin, physcioine and chrysophanol, estimated using the same extractive and analytical procedures are also reported. Structural assignments of peaks corresponding to compounds have been made by means of MS techniques. MS fragmentations of peaks corresponding to compounds 1 and 2 were in full agreement with those already reported for the same compounds (Genovese et al. 2012). The content of madagascine (1) and 3-geranyloxyemodin (2) is in some instances by far higher to the individual content of the main unprenylated anthranoids that are commonly found both in S. alexandrina and A. vera. This is particularly true in the leaf extract of A. vera, where the concentration of madagascine is very close or even higher than that of aloe-emodin, chrysophanol and physcioine, being also about 100-fold higher than its biosynthetic precursor emodin and 11-fold higher than its geranylated counterpart. On the contrary, low values for all anthraquinones under study have been recorded in the gel obtained as an exudate from A. vera leaves. In the extracts from the leaves and fruits of S. alexandrina, similar concentrations of 1 and 2 were found together with high content of aloe-emodin and rhein. The presence of oxyprenylated anthraquinone derivatives in extracts from Senna and Aloe plants is reported in this study for the first time in the literature. The high concentration of the two oxyprenylated anthraquinones under investigation recorded herein suggests that these secondary metabolites of mixed biosynthetic origin may be taken into account in re-defining the anthraquinone profile of reported and unreported Senna and Aloe plants, and to be part of the chemotaxonomic markers of both genera. These compounds in fact were known so far to naturally occur in few species belonging to the genera Vismia, Psorospermum, Rhamnus and Harungana. By means of slight modifications of both extraction methodology and HPLC experimental conditions with respect to the already reported processes, we were able to simultaneously detect molecules with different polarities: the five ‘classic’ polar anthraquinones and the more lipophilic oxyprenylated madagascin and 3-geranyloxyemodin. In this respect, the overall method we set up could be of great help in analysing the total content of anthraquinones (both unprenylated and prenylated) of Table 1. Madagascine and 3-geranyloxyemodin concentrations in S. alexandrina and A. vera extracts. Madagascine Aloe gel Aloe leaves Senna leaves and fruits
3-Geranyloxyemodin
Aloeemodin
Rhein
Emodin
Chrysophanol
Physcione
20.1 ^ 0.3 41.5 ^ 1.1 19.2 ^ 0.4 1.3 ^ 0.1 2.4 ^ 0.1 35.0 ^ 1.0 50.6 ^ 1.0 659.5 ^ 7.6 55.2 ^ 1.0 344.0 ^ 6.6 13.5 ^ 0.8 6.4 ^ 0.4 949.1 ^ 12.3 478.7 ^ 11.1 290.9 ^ 8.7 402.5 ^ 16.2 197.5 ^ 3.4 1042.7 ^ 20.4 27.6 ^ 1.0 19.5 ^ 0.5 ND
Notes: Values expressed as mg/g of vegetable material. ND, not detected. Data are reported as mean ^ standard deviation (n ¼ 10).
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Senna and Aloe plants of pharmacological importance. It is in fact nowadays well known that extracts from several species belonging to these two genera containing a high content of differently substituted anthraquinones are traditionally used for therapeutic purposes as laxative and purgative agents (Boudreau & Beland 2006). Recently, anthraquinones and anthraquinone based-phytopreparations from Senna and Aloe spp. have been intensively investigated for alternative targets, such as the use as anti-ulcer (Paguigan et al. 2014), anti-diabetic (Boaduo et al. 2014; Yimam et al. 2014), anti-viral (Li et al. 2014) and anti-cancer (Chen et al. 2014) agents, and for several others effects. The presence of oxyprenylated anthraquinones in Senna and Aloe extracts disclosed herein lead us to hypothesise that these secondary metabolites may also contribute to the until now observed biological activities, and the pharmacognosy of Senna and Aloe genera could be partially revised in light of our findings in future. Thus, this investigation may be a stimulus to re-investigate the laxative effects of Senna- and Aloe-based phytopreparations in the light of the high content of madagascine and 3-geranyloxyemodine revealed herein and also to study in more details the effects of these last two O-prenyl secondary metabolites on smooth muscles of the large bowel.
3. Conclusions In this article, we reported an extractive and HPLC procedure for the qualitative and quantitative analysis in S. alexandrina and A. vera of two naturally occurring biologically active anthraquinones: madagascine (1) and 3-geranyloxyemodin (2). By means of effective chemical and analytical methodologies it was possible to detect in a single chromatographic run typical anthranoids components such as emodin, physcione, aloe-emodin, rhein and chrysophanol, and additional lipophilic compounds such as compounds 1 and 2. The findings described herein open the possibility to re-investigate the anthraquinone profile of all plants containing this class of natural products, comprising those used for medicinal purposes. The analytical experimental conditions we adopted herein will be certainly of help in future for the simultaneous detection of unprenylated and prenylated anthraquinones in these plants thus contributing to the definition of chemical fingerprints and to take into account oxyprenylated metabolites also for the biological activity. To this aim, using the synthetic chemical, extractive and analytical methodologies we described, we will be able to analyse several other anthraquinones-containing plant species (e.g. rhubarb, butternut, yellow dock and others) and these studies are currently ongoing in our laboratories.
Supplementary material Supplementary material relating to this paper is available online, alongside Figure S1.
Funding Financial support to this study from University “G. D’Annunzio” of Chieti-Pescara is gratefully acknowledged.
References Amonkar A, Chang CJ, Cassady JM. 1981. 6-Geranyloxy-3-methyl- 1,8-dihydroxyanthrone, a novel antileukemic agent from Psorospermum febrifugum Sprach var. ferrugineum (Hook. fil). Experientia. 37:1138–1139. Boaduo NK, Katerere D, Eloff JN, Naidoo V. 2014. Evaluation of six plant species used traditionally in the treatment and control of diabetes mellitus in South Africa using in vitro methods. Pharm Biol. 52:756–761. Boudreau MD, Beland FA. 2006. An evaluation of the biological and toxicological properties of Aloe barbadensis (Miller) Aloe vera. J Environ Sci Health C Environ Carcinog Ecotoxicol Rev. 24:103–154.
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Bruyere C, Genovese S, Lallemand B, Ionescu-Motatu A, Curini M, Kiss R, Epifano F. 2011. Growth inhibitory activities of oxyprenylated and non-prenylated naturally ccurring phenylpropanoids in cancer cell lines. Bioorg Med Chem Lett. 21:4173–4178. Chen R, Zhang J, Hu Y, Wang S, Chen M, Wang Y. 2014. Potential antineoplastic effects of Aloe-emodin: a comprehensive review. Am J Chin Med. 42:275–288. Epifano F, Fiorito S, Carlucci G, Locatelli M, Genovese S. 2013. Phytochemistry and pharmacognosy of naturally occurring prenyloxyanthraquinones. Curr Drug Targets. 14:959–963. Epifano F, Genovese S, Kremer D, Randic M, Carlucci G, Locatelli M. 2012. Re-investigation of the anthraquinone pool of Rhamnus spp.: madagascine from the fruits of Rhamnus cathartica and R. intermedia. Nat Prod Commun. 7:1029–1032. Genovese S, Epifano F, Curini M, Kremer D, Carlucci G, Locatelli M. 2012. Screening for oxyprenylated anthraquinones in Mediterranean Rhamnus species. Biochem Syst Ecol. 43:125–127. Li SW, Yang TC, Lai CC, Huang SH, Liao JM, Wan L, Lin YJ, Lin CW. 2014. Antiviral activity of aloe-emodin against influenza a virus via galectin-3 up-regulation. Eur J Pharmacol. doi:10.1016/j.ejphar.2014.05.028. Paguigan ND, Castillho DH, Chichioco-Hernandez CL. 2014. Anti-ulcer activity of leguminosae plants. Arch Gastroenterol. 51:64–67. Yimam M, Zhao J, Corneliusen B, Pantier M, Brownell L, Jia Q. 2014. Blood glucose lowering activity of aloe based composition, UP780, in alloxan induced insulin dependent mouse diabetes model. Diabetol Metab Syndr. 6:61–63.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Screening for novel plant sources of prenyloxyanthraquinones: Senna alexandrina Mill. and Aloe vera (L.) Burm. F. a
a
a
Francesco Epifano , Serena Fiorito , Marcello Locatelli , Vito a
a
Alessandro Taddeo & Salvatore Genovese a
Department of Pharmacy, University “G. D'Annunzio” of ChietiPescara, Via dei Vestini 31, 66100 Chieti Scalo (CH), Italy Published online: 24 Oct 2014.
To cite this article: Francesco Epifano, Serena Fiorito, Marcello Locatelli, Vito Alessandro Taddeo & Salvatore Genovese (2015) Screening for novel plant sources of prenyloxyanthraquinones: Senna alexandrina Mill. and Aloe vera (L.) Burm. F., Natural Product Research: Formerly Natural Product Letters, 29:2, 180-184, DOI: 10.1080/14786419.2014.971792 To link to this article: http://dx.doi.org/10.1080/14786419.2014.971792
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
Downloaded by [Dicle University] at 06:01 06 November 2014
Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2015 Vol. 29, No. 2, 180–184, http://dx.doi.org/10.1080/14786419.2014.971792
Screening for novel plant sources of prenyloxyanthraquinones: Senna alexandrina Mill. and Aloe vera (L.) Burm. F. Francesco Epifano*, Serena Fiorito, Marcello Locatelli, Vito Alessandro Taddeo and Salvatore Genovese Department of Pharmacy, University “G. D’Annunzio” of Chieti-Pescara, Via dei Vestini 31, 66100 Chieti Scalo (CH), Italy
Downloaded by [Dicle University] at 06:01 06 November 2014
(Received 7 July 2014; final version received 21 September 2014) As a continuation of our ongoing studies aimed to reveal the presence of oxyprenylated anthraquinones in plants claimed to have a laxative effect, in this article, we describe the extraction and HPLC separation of madagascin (3-isopentenyloxyemodin) and 3geranyloxyemodine from dried leaves and fruits of Senna alexandrina Mill. (Leguminosae) and leaves and gel of Aloe vera (L.) Burm. F. (Xanthorrhoeaceae). Both compounds are described herein for the first time as components of extracts of the title plants. Keywords: Aloe vera; anthraquinones; 3-geranyloxyemodin; madagascin; oxyprenylated secondary metabolites; polyketides; Senna alexandrina
1. Introduction Oxyprenylated anthraquinones have been reported in the literature for the first time in 1981 when Amonkar et al. (1981) described the isolation of 6-geranyloxy-3-methyl-1,8dihydroxyanthrone from the root extracts of Psorospermum febrifugum Spach (Guttiferae). In subsequent years, such secondary metabolites have been described also in the genera Psorospermum, Vismia, Harungana and Rhamnus (Epifano et al. 2012; 2013; Genovese et al. 2012). As a continuation of our ongoing studies aimed at the quantification of anthraquinones in plants having laxative effects, we decided to investigate the presence of two oxyprenylated anthraquinones, namely madagascine (1) and 3-geranyloxyemodine (2) (Figure 1), in extracts obtained from dried leaves and fruits of Senna alexandrina Mill. (syn. Cassia angustifolia M. Vahl, Cassia senna L.) (Leguminosae), and dried leaves and gel of Aloe vera (L.) Burm. F. (syn. Aloe barbadensis Mill.) (Xanthorrhoeaceae).
2. Results and discussion 2.1. Chemical synthesis As reported previously (Bruyere et al. 2011), the title compounds were obtained in 54% and 68% yield and purity $ 98% using commercially available emodin as the starting material by DBUpromoted alkylation with 3,3-dimethylallyl or geranyl bromide, respectively, followed by crystallisation with n-hexane.
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
Natural Product Research OH
O
181
OH
O
R
O 1R=H 2 R = isopentenyl
Downloaded by [Dicle University] at 06:01 06 November 2014
Figure 1. Chemical structures of madagascine (1) and 3-geranyloxyemodine (2).
2.2. Plant material and preparation of extracts Dried leaves and fruit samples of S. alexandrina were purchased commercially and carefully checked for the presence of adulteration and/or falsifications by microscopic characterisation. The samples were correctly identified by Dr Francesco Epifano and Dr Salvatore Genovese. Voucher specimens (No. CA-001) have been deposited in the Herbarium of the Department of Pharmacy of the University of ‘Gabriele d’Annunzio’ Chieti-Pescara, Italy. Leaves of A. vera were collected in Termoli (Molise, Italy) in October 2012. The samples were correctly identified by Dr Francesco Epifano and Dr Salvatore Genovese. Voucher specimens (No. AL-001) have been deposited in the Herbarium of the Department of Pharmacy of the University of ‘Gabriele d’Annunzio’ Chieti-Pescara, Italy. Aloe gel was obtained by incision of fresh leaves followed by drop-by-drop collection of the resulting exudate. The extraction of all the vegetable materials consisted of a multi-step process: trituration followed by an overnight maceration in n-hexane (10 mL/g of vegetable material), separation and evaporation of the solution, filtration of the residual vegetable material and its overnight maceration using methanol and finally a second filtration of the latter. The n-hexane extract, once concentrated to complete dryness under vacuum and re-dissolved in methanol, was combined to the methanol extract. The methanol solution was subsequently subjected to reflux for 30 min with a 6N HCl aqueous solution in order to hydrolyse glycosides, followed by the extraction of hydroalcoholic mixture with ethyl acetate to analyse the total content of anthraquinones. The dark red syrupy samples so obtained were stored at 2 208C in amber glass tubes before HPLC analysis. 2.3. Limits of detection, limits of quantitation and linearity Limits of detection were calculated by measuring S/N values obtained in the mobile phase spiked at 1 mg/mL level and extrapolation of the corresponding values to S/N ¼ 3. The limits of quantitation of the method was defined according to the Guidance for Industry on the validation of bioanalytical methods as the concentration of the lowest standard on the calibration curve for which (a) the analyte peak is identifiable and discrete, (b) the analyte response is at least ten times the response of the blank sample and (c) the analyte response is reproducible with a precision less than 20% and trueness better of 80 –120%. Under these experimental conditions the retention time and other key parameters related to the analysis were as follows: (a) for madagascin (1) retention time of 33.3 (^ 0.4) min, linearity range of 0.5– 100 mM, determination coefficients (r 2) of 0.9850, (b) for 3-geranyloxyemodin (2) retention time of 44.8 (^ 1.0) min, linearity range of 5.0 –125 mM, determination coefficients (r 2) of 0.9925. The dead retention time, calculated with uracil, was 1.83 min. The within-assay precision (repeatability) of the method was in the range 0.6– 12.4, while between-assay precision (intermediate precision) was in the range 2 0.3 to 12.4. The trueness of the method was evaluated at the same analyte concentration levels by comparing the measured anthraquinone concentrations of the QC samples with their nominal values. The within-assay trueness of the method was in the range
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2 12.0 to 11.5 range, while between-assay trueness was in the range 2 12.2 to 12.7 range. Calibration curves, obtained at 435 nm, were plotted using weighted (1/x 2) linear least-squares regression analysis. The weighting factor was chosen to minimise deviation of back-calculated values from theoretical concentrations, especially for the lowest concentration levels, as permitted by method validation guidelines, that report that ‘standard curve fitting is determined by applying the simplest model that adequately describes the concentration – response relationship using appropriate weighting’.
Downloaded by [Dicle University] at 06:01 06 November 2014
2.4. Quantification of madagascine (1) and 3-geranyloxyemodin (2) Results about the concentration of madagascine and 3-geranyloxyemodine in S. alexandrina and A. vera extracts are reported in Table 1. For comparison, values of the concentration of ‘classic’ unprenylated anthraquinones, such as aloe-emodin, rhein, emodin, physcioine and chrysophanol, estimated using the same extractive and analytical procedures are also reported. Structural assignments of peaks corresponding to compounds have been made by means of MS techniques. MS fragmentations of peaks corresponding to compounds 1 and 2 were in full agreement with those already reported for the same compounds (Genovese et al. 2012). The content of madagascine (1) and 3-geranyloxyemodin (2) is in some instances by far higher to the individual content of the main unprenylated anthranoids that are commonly found both in S. alexandrina and A. vera. This is particularly true in the leaf extract of A. vera, where the concentration of madagascine is very close or even higher than that of aloe-emodin, chrysophanol and physcioine, being also about 100-fold higher than its biosynthetic precursor emodin and 11-fold higher than its geranylated counterpart. On the contrary, low values for all anthraquinones under study have been recorded in the gel obtained as an exudate from A. vera leaves. In the extracts from the leaves and fruits of S. alexandrina, similar concentrations of 1 and 2 were found together with high content of aloe-emodin and rhein. The presence of oxyprenylated anthraquinone derivatives in extracts from Senna and Aloe plants is reported in this study for the first time in the literature. The high concentration of the two oxyprenylated anthraquinones under investigation recorded herein suggests that these secondary metabolites of mixed biosynthetic origin may be taken into account in re-defining the anthraquinone profile of reported and unreported Senna and Aloe plants, and to be part of the chemotaxonomic markers of both genera. These compounds in fact were known so far to naturally occur in few species belonging to the genera Vismia, Psorospermum, Rhamnus and Harungana. By means of slight modifications of both extraction methodology and HPLC experimental conditions with respect to the already reported processes, we were able to simultaneously detect molecules with different polarities: the five ‘classic’ polar anthraquinones and the more lipophilic oxyprenylated madagascin and 3-geranyloxyemodin. In this respect, the overall method we set up could be of great help in analysing the total content of anthraquinones (both unprenylated and prenylated) of Table 1. Madagascine and 3-geranyloxyemodin concentrations in S. alexandrina and A. vera extracts. Madagascine Aloe gel Aloe leaves Senna leaves and fruits
3-Geranyloxyemodin
Aloeemodin
Rhein
Emodin
Chrysophanol
Physcione
20.1 ^ 0.3 41.5 ^ 1.1 19.2 ^ 0.4 1.3 ^ 0.1 2.4 ^ 0.1 35.0 ^ 1.0 50.6 ^ 1.0 659.5 ^ 7.6 55.2 ^ 1.0 344.0 ^ 6.6 13.5 ^ 0.8 6.4 ^ 0.4 949.1 ^ 12.3 478.7 ^ 11.1 290.9 ^ 8.7 402.5 ^ 16.2 197.5 ^ 3.4 1042.7 ^ 20.4 27.6 ^ 1.0 19.5 ^ 0.5 ND
Notes: Values expressed as mg/g of vegetable material. ND, not detected. Data are reported as mean ^ standard deviation (n ¼ 10).
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Senna and Aloe plants of pharmacological importance. It is in fact nowadays well known that extracts from several species belonging to these two genera containing a high content of differently substituted anthraquinones are traditionally used for therapeutic purposes as laxative and purgative agents (Boudreau & Beland 2006). Recently, anthraquinones and anthraquinone based-phytopreparations from Senna and Aloe spp. have been intensively investigated for alternative targets, such as the use as anti-ulcer (Paguigan et al. 2014), anti-diabetic (Boaduo et al. 2014; Yimam et al. 2014), anti-viral (Li et al. 2014) and anti-cancer (Chen et al. 2014) agents, and for several others effects. The presence of oxyprenylated anthraquinones in Senna and Aloe extracts disclosed herein lead us to hypothesise that these secondary metabolites may also contribute to the until now observed biological activities, and the pharmacognosy of Senna and Aloe genera could be partially revised in light of our findings in future. Thus, this investigation may be a stimulus to re-investigate the laxative effects of Senna- and Aloe-based phytopreparations in the light of the high content of madagascine and 3-geranyloxyemodine revealed herein and also to study in more details the effects of these last two O-prenyl secondary metabolites on smooth muscles of the large bowel.
3. Conclusions In this article, we reported an extractive and HPLC procedure for the qualitative and quantitative analysis in S. alexandrina and A. vera of two naturally occurring biologically active anthraquinones: madagascine (1) and 3-geranyloxyemodin (2). By means of effective chemical and analytical methodologies it was possible to detect in a single chromatographic run typical anthranoids components such as emodin, physcione, aloe-emodin, rhein and chrysophanol, and additional lipophilic compounds such as compounds 1 and 2. The findings described herein open the possibility to re-investigate the anthraquinone profile of all plants containing this class of natural products, comprising those used for medicinal purposes. The analytical experimental conditions we adopted herein will be certainly of help in future for the simultaneous detection of unprenylated and prenylated anthraquinones in these plants thus contributing to the definition of chemical fingerprints and to take into account oxyprenylated metabolites also for the biological activity. To this aim, using the synthetic chemical, extractive and analytical methodologies we described, we will be able to analyse several other anthraquinones-containing plant species (e.g. rhubarb, butternut, yellow dock and others) and these studies are currently ongoing in our laboratories.
Supplementary material Supplementary material relating to this paper is available online, alongside Figure S1.
Funding Financial support to this study from University “G. D’Annunzio” of Chieti-Pescara is gratefully acknowledged.
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