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Application of HPLC-DAD for the quantification of Lycorine in Galanthus elwesii Hook

ABSTRACT

In the present study, a reversed-phase high-performance liquid chromatographic method has been used for the quantitative determination of lycorine in the aerial parts and bulbs of G. elwesii Hook. A simple method for the extraction of lycorine in low mass plant samples was employed utilizing pre-packed columns with diatomaceous earth (Extrelut(r)). The chromatographic separation was performed using an isocratic system with a mobile phase of trifluoroacetic acid-water-acetonitrile (0.01:92.5:7.5, v/v/v) applied at a flow rate 1 mL min-1 using diode array detector. The content of lycorine in the bulbs and aerial parts of G. elwesii collected from Demirci (Manisa) was found as 0.130 and 0.162 %, respectively. Additionally, in the bulbs of the specimens collected from Sogucak (Balikesir), lycorine was quantified as 0.055 %, whereas in the aerial parts, it was determined as 0.006 %. The method was validated partially with respect to system specificity, linearity, accuracy, precision, limits of detection (LOD) and quantitation (LOQ). Validation procedures displayed that the method was specific, accurate and precise.

Uniterms:
Alkaloids; Amaryllidaceae; Plant extracts; High Performance Liquid Chromatography/method validation; High Perfomance Liquid Chromatography/quantitative analysis.

INTRODUCTION

Galanthus elwesii Hook. (Amaryllidaceae) is an easily recognized species with broad glaucous leaves, large flowers and bold markings on the inner segments. It has a relatively wide distribution and can be found in eastern parts of the former Yugoslavia, northern Greece, the eastern Aegean Islands, southern Ukraine, Bulgaria and Turkey. Within Turkey, this species has the widest distribution among others and naturally grows in northwestern, western and southern Anatolia (Bıshop, Davıs, Grımshaw, 2006; Yuzbasioglu, 2012YUZBASIOGLU, S. Morphological variations of Galanthus elwesii in Turkey and difficulties on identification. Bocconea, v.24, p.335-339, 2012.).

Plants of the Amaryllidaceae are known to produce alkaloids with a wide range of pharmacological activities (Unver, 2007UNVER, N. New skeletons and new concepts in Amaryllidaceae alkaloids. Phytochem. Rev., v.6, p.125-135, 2007.; Hoshino, 1998HOSHINO, O. The Amaryllidaceae alkaloids. In: CORDELL, G.A. (ed). The alkaloids: chemistry and physiology. San Diego: Academic Press, 1998. p.323-424. ). Among the Amaryllidaceae alkaloids, galanthamine, is used for the treatment of mild and moderate cases of Alzheimer's disease (AD) (Howes, Perry, 2011HOWES, M.J.; PERRY, E. The role of phytochemicals in the treatment and prevention of dementia. Drug Aging, v.28, p.439-468, 2011.). Lycorine, a major alkaloid found in many Amaryllidaceae species, has been shown to have a wide variety of biological activities such as antitumoral (Wang et al., 2014WANG, P.; YUAN, H.; ZHANG, X.; LI, Y.; SHANG, L.; YIN, Z.; Novel lycorine derivatives as anticancer agents: synthesis and in vitro biological evaluation. Molecules, v.19, p.2469-2480, 2014.), antimalarial (Cedron et al., 2010CEDRON, J.C.; GUTIERREZ, D.; FLORES, N.; RAVELO, A.G.; ESTEVEZ-BRAUN, A. Synthesis and antiplasmodial activity of lycorine derivatives. Bioorg. Med. Chem., v.18, p.4694-701, 2010.), hepatoprotective (Ilavenil, Kaleeswaran, Ravikumar, 2012ILAVENIL, S.; KALEESWARAN, B.; RAVIKUMAR, S. Protective effects of lycorine against carbon tetrachloride induced hepatotoxicity in Swiss albino mice. Fundam. Clin. Pharmacol., v.26, n.3, p.393-401, 2012.), antiviral (He et al., 2013HE, J.; QI, W.; WANG, L.; TIAN, J.; JIAO, P.; LIU, G.; YE, W.; LIAO, M. Amaryllidaceae alkaloids inhibit nuclear-to-cytoplasmic export of ribonucleoprotein (RNP) complex of highly pathogenic avian influenza virus H5N1. Influenza Other Respir. Viruses, v.7, n.6, p.922-931, 2013.), antifungal (Shen et al., 2014SHEN, J.; RUAN, Y.; REN, W.; MA, B.; WANG, X.; ZHENG, C. Lycorine: a potential broad-spectrum agent against crop pathogenic fungi. J. Microbiol. Biotechnol., v.24, p.354-358, 2014.) and antiparasitic (Giordani et al., 2012GIORDANI, R.B.; JUNIOR, C.O.R.; ANDRADE, J.P.; BASTIDA, J.; ZUANAZZI, J.A.S.; TASCA, T.; ALMEIDA, M.V. Lycorine derivatives against Trichomonas vaginalis. Chem. Biol. Drug. Des., v.80, p.129-133, 2012.) activities. Due to its diverse biological properties, it has been to the interest of phytochemists to determine the content of this alkaloid using various analytical techniques in Amaryllidaceous plants (Abou-Donia et al., 2007; Petruczynik et al., 2016PETRUCZYNIK, A.; MISIUREK, J.; TUZIMSKI, T.; USZYŃSKI, R.; SZYMCZAK, G.; CHERNETSKYY, M.; WAKSMUNDZKA-HAJNOS, M. Comparison of different HPLC systems for analysis of galanthamine and lycorine in various species of Amaryllidaceae family. J. Liq. Chromatogr. Relat. Technol., v.39, p.574-579, 2016.).

In the present study, during the course of our ongoing phytochemical studies on Turkish Galanthus species, aerial parts and bulbs of G. elwesii, collected from two different localities in Western Turkey, were quantitatively analyzed for their lycorine content by using high performance liquid chromatography (HPLC) coupled with a diode array detector (DAD). In the context of validation procedures, linearity, precision, limits of detection and quantification, accuracy, and specificity of the method were also displayed. In addition, the specimens were analyzed for their galanthamine content.

MATERIAL AND METHODS

Material

Standard sample of lycorine was previously isolated in our laboratory and authenticated by detailed spectral analysis (UV, IR, NMR, MS) (Kaya et al., 2004aKAYA, G.I.; UNVER, N.; GOZLER, B.; BASTIDA, J. (−)-Capnoidine and (+)-bulbocapnine from an Amaryllidaceae species, Galanthus nivalis subsp. cilicicus. Biochem. Syst. Ecol., v.32, p.1059-1062, 2004a.). The chemical structure of lycorine was given in Figure 1. TFA (trifluoroacetic acid) (Merck), HPLC grade acetonitrile (Lab-Scan Analytical Sciences) and chromatographic grade double-distilled water were used for the HPLC analysis. Other chemicals were of analytical grade.

FIGURE 1
Chemical structure of lycorine.

Apparatus

Both the analysis of the samples and the validation studies were performed on a liquid chromatographic system (Agilent 1100 series), equipped with a quaternary pump, a vacuum degasser, a thermostatted column compartment, a manual injector with 20 µL loop (Rheodyne 7725i), a diode array detector (DAD) (Agilent 1200 series) and a Hichrom C18 column (250 x 4.6 mm, i.d., particle size 5 µm).

Procedure

Samples

Specimens of G. elwesii were collected from Sogucak (Balikesir) and Demirci (Manisa) in Western Turkey, during flowering time. The plants were identified by Prof. M. Ali Onur from the Department of Pharmacognosy, Faculty of Pharmacy, Ege University, Izmir, Turkey. Voucher samples of G. elwesii (No's 1363 and 1401) are deposited in the Herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Ege University.

Preparation of samples and standard solutions

The extraction procedure of lycorine from the plant material and preparation of standard lycorine solutions were performed according to a previously used method (Kaya et al., 2010KAYA, G.I.; CICEK, D.; SARIKAYA, B.; ONUR, M.A.; UNVER-SOMER, N. HPLC - DAD analysis of lycorine in Amaryllidaceae species. Nat. Prod. Commun., v.5, n.6, p.873-876, 2010.).

HPLC determination

The extract was dissolved in 1 mL 0.1 % TFA. The injection volume was 20 mL and the flow rate was 1 mL/min. The chromatographic run time was 50 min. Column temperature was set at 25 °C. The chromatographic separation was achieved isocratically using a mobile phase consisted of TFA-water-acetonitrile (0.01:92.5:7.5, v/v/v) on a Hichrom C18 column and detection was carried out at 290 nm (Kaya et al., 2010KAYA, G.I.; CICEK, D.; SARIKAYA, B.; ONUR, M.A.; UNVER-SOMER, N. HPLC - DAD analysis of lycorine in Amaryllidaceae species. Nat. Prod. Commun., v.5, n.6, p.873-876, 2010.; Mustafa, Rhee, Verpoorte, 2003MUSTAFA, N.R.; RHEE, I.K.; VERPOORTE, R. Rapid method for determination of galanthamine in Amaryllidaceae plants using HPLC. J. Liq. Chromatogr. Relat. Technol., v.26, n.19, p.3217-3233, 2003.). Quantitative determination was carried out by an external standard method based on peak areas.

RESULTS AND DISCUSSION

The previously proposed method (Kaya et al., 2010KAYA, G.I.; CICEK, D.; SARIKAYA, B.; ONUR, M.A.; UNVER-SOMER, N. HPLC - DAD analysis of lycorine in Amaryllidaceae species. Nat. Prod. Commun., v.5, n.6, p.873-876, 2010.; Mustafa, Rhee, Verpoorte, 2003MUSTAFA, N.R.; RHEE, I.K.; VERPOORTE, R. Rapid method for determination of galanthamine in Amaryllidaceae plants using HPLC. J. Liq. Chromatogr. Relat. Technol., v.26, n.19, p.3217-3233, 2003.) was applied for the detection of lycorine and galanthamine plus for the quantification of lycorine in G. elwesii specimens. Under the above-stated chromatographic conditions, lycorine was resolved within approximately 9 min. (Figure 2 and 3). Quantitative determination was carried out by the external standard method based on peak areas. The results of the mean values of three replicate injections of lycorine were reported in Table I. Significant variation in the amounts of lycorine was found in the analyzed samples. The lycorine content ranged between 0.006-0.162%. The highest amount was found in the aerial parts of G. elwesii collected from Demirci (Manisa). Linearity, accuracy and precision studies were carried out according to the ICH validation guidelines on the validation of analytical procedures (ICH, 2005).

FIGURE 2
Chromatogram of lycorine.

FIGURE 3
Chromatogram of an extract of G. elwesii.

TABLE I
Statistical data showing recovery studies of lycorine (n=3)

Specificity

Specificity is described as the ability to measure the analyte response in the presence of components such as impurities, degradants, matrix, etc. (ICH, 2005). The specificity of the method for the analysis of lycorine was evaluated in the presence of other components of the extracts. Peak purities of lycorine was evaluated by the acquisition of UV spectra with the DAD detector.

Linearity

Stock standard solution of lycorine was prepared by dissolving 2 mg in 10 mL 0.1 % TFA. The linearity of the method was shown by injecting seven known concentrations of the standard lycorine in the range of 2.5-200 µg/mL. Each standard solution (20 mL) was injected into the column in triplicate and then the calibration curve of the analyte was obtained by plotting the peak area versus the concentration. The regression equation for lycorine was found as y= 16.17864x -3.12657. Excellent linearity was obtained (r2=0.99997) exhibiting a good correlation between the alkaloid concentration and the peak area.

Accuracy

Accuracy may be defined as the agreement between the found value and true value of the reference material and can be presented as a percent recovery. Standard addition analysis was performed for the recovery of the method. Three known amounts of the individual standard were added to sample solutions and the mixtures were analyzed by the same method used in the analysis of lycorine in the plant samples. Recovery assay was carried out by spiking three different known concentrations of lycorine into the sample solutions prior to extraction. The mean extraction recovery of lycorine was found as in the range of 94.24 - 100.60 %. The results of the experiments are given in Table I.

Precision

The precision of the method was evaluated by studying intra-day and inter-day variations. Intra-day precision was determined by injecting solutions of seven different concentrations of standard lycorine (2.5, 5, 10, 25, 50, 100, 200 µg/mL) in triplicate on the same day. Inter-day precision was calculated by performing the same procedure on two different days. The results of the precision analysis of lycorine are summarized in Table II. RSD was found to be always less than 2.0 % when the analysis was performed at seven different concentrations.

TABLE II
Intra-day and inter-day precision of the method

Limit of detection and limit of quantification

Limit of detection (LOD) and limit of quantification were evaluated based on the signal-to-noise ratio (S/N) of 3 and 10, respectively. LOD and LOQ were experimentally determined by 10 injections of lycorine. The LOD and the LOQ of the analyte were calculated as 0.0423µg/mL and 0.14921 µg/mL respectively.

CONCLUSION

Comprising a part of our phytochemical studies on Galanthus species of Turkish origin, the present study was undertaken to evaluate the lycorine and galanthamine contents of two Galanthus elwesii specimens naturally growing in Western Turkey. As a result, lycorine was determined both in the aerial parts and bulbs of these specimens, however galanthamine was not detected in any of the plant samples. The employed method has several advantages including simple and rapid sample preparation, requirement of a small amount of plant material, and a simple mobile phase. The method was also validated with respect to linearity, intra and inter-day precision, recovery, limits of detection and quantification. Validation studies showed that the method could be successfully used to determine lycorine in G. elwesii samples, which provided an efficient and reliable method for the quality control of G. elwesii. G. elwesii specimens collected from different localities in Turkey have been investigated for their content of lycorine and galanthamine in previous studies (Kaya et al., 2004bKAYA, G.I.; FILLIK, A.; HISIL, Y.; UNVER, N. High pressure liquid chromatographic analysis of lycorine in four Galanthus species growing in Turkey. Turk J. Pharm. Sci., v.1, p.105-114, 2004b.; Celik-Sarier, 2002; Kaya, Gozler, 2003; Muhtar, Sener, 1996MUHTAR, F.; SENER, B. Evaluation of Amaryllidaceae plants exported from Turkey regarding lycorine. In: MEETING ON PLANT CRUDE DRUGS, 11, Ankara, 1996. Proceedings. Ankara, 1996. p.384-388.; Kaya et al., 2014). The results of these studies together with the results of the present study are summarized in Table III.

TABLE III
Lycorine and galanthamine content of Galanthus elwesii from different localities

The results obtained in the present study differ significantly from the results of previous studies on Galanthus elwesii (Table III). This could be caused by endogenous and exogenous factors. Karyological investigations on Turkish Galanthus populations have been made by several researchers (Şenel, Ozkan, Kandemir, 2002; Zeybek, 1988ZEYBEK, N. Taxonomical investigations on Turkish snowdrops (Galanthus L.). Turk J Botany., v.12, p.89-103, 1988.; Zeybek, Sauer, 1994) and these studies have revealed a polymorphism among different karyotypes and SAT -chromosomes which were reported to be important in the variation (Kandemir, 2010). Earlier reports showed that the concentration of alkaloids vary in great amount in different Amaryllidaceae plants with the seasonal growth (Katoch et al., 2012KATOCH, D.; KUMAR, S.; KUMAR, N.; SINGH, B. Simultaneous quantification of Amaryllidaceae alkaloids from Zephyranthes grandiflora by UPLC-DAD/ESI-MS/MS. J. Pharm. Biomed. Anal., v.71, p.187-192, 2012.; Tram et al., 2002TRAM, N.T.N.; MITOVA, M.; BANKOVA, V.; HANDJIEVA, N.; POPOV, S.S. GC-MS of Crinum latifolium L. alkaloids. Z. Naturforsch., v.57, p.239-242, 2002.). In addition, it has been documented that a relationship existed between the alkaloid content of an Amaryllidaceous species Leucojum aestivum L. and the chemical composition of the soil (Gorinova et al., 1993GORINOVA, N.I.; ATANASSOV, A.I.; STOJANOV, D.V.; TENCHEVA, J. Influence of chemical composition of soils on the galanthamine content in Leucojum aestivum. J. Plant Nutr., v.16, n.9, p.1631-1636, 1993.). Therefore, the variations of the alkaloid content in G. elwesii specimens may be influenced by several factors including polymorphism, collection site and time.

ACKNOWLEDGMENTS

This study was financially supported by the Ege University Research Fund (Project No: 09/ECZ/037) and partially supported by TUBITAK (104T272) and EBILTEM (2007/BIL/007).

REFERENCES

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Publication Dates

  • Publication in this collection
    2017

History

  • Received
    27 Mar 2015
  • Accepted
    25 Oct 2016
Universidade de São Paulo, Faculdade de Ciências Farmacêuticas Av. Prof. Lineu Prestes, n. 580, 05508-000 S. Paulo/SP Brasil, Tel.: (55 11) 3091-3824 - São Paulo - SP - Brazil
E-mail: bjps@usp.br