improved hplc method for determination of amaryllidaceae alkaloids

IMPROVED HPLC METHOD FOR DETERMINATION OF AMARYLLIDACEAE ALKALOIDS

I. Ivanov1, S. Berkov2 and A. Pavlov1

1Department of Microbial Biosynthesis and Biotechnologies – Laboratory in Plovdiv, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Plovdiv, Bulgaria, 2 Departament de Productes Naturals, Biologia Vegetal i Edafologia, Facultat de Farmàcia, Universitat de Barcelona, Barcelona, Catalonia, Spain Correspondence to: Ivan Ivanov E-mail: [email protected]

ABSTRACT An improved HPLC method for quantitative determination of galanthamine, lycorine and norgalanthamine was developed. HPLC separation was achieved using a reversed phase C18 column Symmetry® and a gradient system with acetonitrile as an organic phase and 1% (w/v) ammonium acetate buffer adjusted to pH 6.6 with acetic acid (flow rate 0.3 – 0.5 ml/min.). The calibration curves were linear from 5 to 150 μg/ml (r2>0.99). The reliability of the proposed system was proved through reproducibility test with alkaloids extracts from in vitro shoot-clumps culture obtained from Leucojum aestivum L. and Pancratium maritimum L. Keywords: Amaryllidaceae alkaloids, galanthamine, HPLC, lycorine

Introduction Amaryllidaceae are known as ornamental plants, furthermore some species of this family contain alkaloids that are know to exhibit a wide range of biological activities (6). Galanthamine (1), an isoquinoline alkaloid, is long acting, selective, reversible, and competitive acetylcholinesterase inhibitor used for symptomatic treatment of senile dementia of the Alzheimer’s type (8), poliomyelitis and other neurological diseases (17). Lycorine (3), a pyrrolophenanthridine alkaloid, displays a strong antiviral effect (9) against poliovirus, measles, and Herpes simplex type 1 viruses as well as high antiretroviral (19), strong antimitotic (10), and cytotoxic activities (20). Different analytical techniques have been described for the qualitative and quantitative determination of Amaryllidaceae alkaloids in various parts of different Amaryllidaceae plants including CGC-MS (4), spectrophotometric (13), HPTLC (1, 2) and enzyme immunoassay (15). HPLC methods were used for determination of some Amaryllidaceae alkaloids in plant and tissue-culture extracts of Narcissus ssp. (11, 12, 18) and Leucojum aestivum L. (11, 14, 16). These methods differ mainly in the column type, pH range of the mobile phase, and

analysed plants. However reported methods do not meet our requirements for determination of galanthamine (3), lycorine (2) and norgalanthamine (1) in extracts from in vitro L. aestivum shoots. In the presented manuscript we describe an improved HPLC method for determination of Amaryllidaceae alkaloids mixtures from in vitro cultures.

Materials and methods

Plant material In vitro shoot-clumps cultures were obtained from calli of Leucojum aestovum L. and Pancratium maritimum L. (14). The shoot-clumps were cultivated on sold MS medium supplemented with 3% sucrose, 5.5 g/l “Plant agar” (Duchefa Biochemie, Netherlands), 2 mg/l Benzylamino purine and 1.15 mg/l α - Naphthaleneacetic acid at 26°C under illumination 16h light/8h darkness photoperiod.

Alkaloid extraction 0.2-0.3 g dry biomass was extracted three times with 5 ml of methanol in an ultrasonic bath for 15 min. combined extracts were concentrated under vacuum and dissolved in 2 ml of 3% sulfuric acid. The neutral compounds were removed by extraction (tree times) with diethyl ether. The alkaloids were fractionated after basification of the extracts with 1 ml of 25% ammonia and extraction (3 X 3 ml) with chloroform.

BIOTECHNOL. & BIOTECHNOL. EQ. 23/2009/SE XI ANNIVERSARY SCIENTIFIC CONFERENCE SPECIAL EDITION/ON-LINE 120 YEARS OF ACADEMIC EDUCATION IN BIOLOGY 45 YEARS FACULTY OF BIOLOGY

809

The chloroform extracts were dried over anhydrous sodium sulphate and evaporated to dryness.

Equipment HPLC analysis system Waters 2487 Dual λ Absorbanc Detector and Waters 1525 Binary HPLC pump (Waters, Milford, USA). The chromatographic assay was performed on a Symmetry® C18 column (150 x 4.6 mm) reversed phase matrix (5 μm) (Waters) and elution was carried out in a gradient system with acetonitrile as the organic phase (solvent A) and 1% (w/v) ammonium acetate buffer adjusted to pH 6,6 with acetic acid (solvent B). UV detector was set at 287 nm and the volume of injection was 20 μl. The gradient system and flow rate are shown in Table 1.

Results and Discussion One of the most used HPLC method for the quantitative determination of galanthamine and some other Amaryllidaceae alkaloids was developed by Sellés et al. (18). This method was successfully applied for galanthamine quantification in plants and tissue shoot-clump cultures from Narcissus confusus Pugsley. Unfortunately, it was not adapted to plants producing lycorine (3) and norgalanthamine (1) (3) because it was unable to separate efficiently a combination of these compounds and galanthamine (Fig. 1A) therefore it was inapplicable for analyses of extracts from L.aestivum and P.maritimum shoot in vitro cultures, which contain all these compounds in their alkaloids mixtures (7, 14). For this reason, an adaptation of the analytical method was performed, based on optimization of HPLC chromatographic conditions.

For good separation of the alkaloids, especially lycorine

(3) and galanthamine (2), the gradient profiles and mobile phases were optimized in order to obtain better peak resolution. Оptimizаtion was accomplished by a change of the pH of inorganic mobile phase (solvent B) - adjusted to pH 6.6. As compared to the original method, the gradient starts from 10% of solvent A instead 31% as in base system, and reaches up to 90% (18 min) instead of 70% (10 min) Table 1. The identification and quantitative determination of the compounds were accomplished both by a comparison of retention time, peak heights and areas with standard methanolic solutions of the alkaloids and by internal standards (Fig. 1B). The linearity of the HPLC method was checked by injecting six mixtures of solutions containing 10, 25, 50, 75, 100 and 150 μg/ml of norgalanthamine (1), lycorine (2) and galanthamine (3). The relationships between peak height ratio (Y) and the concentration injected (X; μg/ml) is listed in Table 2. Calibration curves showed linearity between 1 – 150 μg/ml with correlation coefficients higher than 0.999.

The optimized method allowed successful identification and quantification of target compounds galanthamine (3), lycorine (2) and norgalanthamine (1) in the samples from shoot-clumps cultures of L. aestivum and P. maritimum (Fig 2). Priority of the method was separation of these tree alkaloids from the mixtures of other the accompaning Amaryllydaceae alkaloids. These samples were used to determine the reproducibility of results. Mean values, standard deviations and coefficient of variation of alkaloids concentration were presented in Table 3.

TABLE 1 Gradient elution scheme employed for the HPLC analysis of alkaloids

Gradient profile of origin method Gradient profile of improved method Time, min Flow rate,

ml/min %A %B Time, min Flow rate,

ml/min %A %B

1. 0,50 31,0 69,0 0,40 10,0 90,0 2. 6,00 0,50 31,0 69,0 11,00 0,30 31,0 69,0 3. 9,00 0,50 70,0 30,0 15,00 0,50 70,0 30,0 4. 10,00 0,50 70,0 30,0 16,00 0,50 90,0 10,0 5. 12,00 0,50 31,0 69,0 18,00 0,50 90,0 10,0 6. 20,00 0,50 31,0 69,0 20,00 0,40 31,0 69,0 7. - - - - 22,00 0,40 10,0 90,0 8. - - - - 31,00 0,40 10,0 90,0

XI ANNIVERSARY SCIENTIFIC CONFERENCE BIOTECHNOL. & BIOTECHNOL. EQ. 23/2009/SE 120 YEARS OF ACADEMIC EDUCATION IN BIOLOGY SPECIAL EDITION/ON-LINE 45 YEARS FACULTY OF BIOLOGY

810

Fig 1. HPLC chromatogram of standarts (A) gradient system described from Sellés et al. (18) (B) optimized gradient system 1.- norgalanthamine, 2. – licorine and 3. – galanthamine.

Fig 2. HPLC chromatograms: (A) Shoot-clumps culture from L.aestivum, (B) Shoot-clumps culture from P. maritimum 1.- norgalanthamine, 2. - lycorine and 3. - galanthamine.


811

TABLE 2 Parameters of retention time and calibration curves for separated alkaloids Alkaloid Retention time (min) Equation r2

Norgalanthamine (1) 10,600 Y= 2.41x104X + 8.17x103 0,9997 Lycorine (2) 14,430 Y= 3.62x104X + 4.35x104 0,9980 Galanthamine (3) 15,130 Y= 4,55x104X – 7.14x104 0,9998

TABLE 3 Reproducibility test: determination of norgalanthamine [1], lycorine [2] and galanthamine [3] in shoot-clumps culture samples of L. aestivum and P. maritimum.

L.aestivum culture P. maritimum culture Nor -galanthamine (μg/ml)

Lycorine (μg/ml)

Galanthamine (μg/ml)

Nor -galanthamine (μg/ml)

Lycorine (μg/ml)

Galanthamine (μg/ml)

1 51.784 9.106 48.195 12.433 82.291 28.566 2 54.650 8.327 49.824 11.539 75.697 26.780 3 53.735 9.276 50.943 11.455 82.065 29.080 4 52.528 8.497 52.331 13.482 83.976 29.521 5 58.266 8,994 49.753 12.846 84.266 29.170 Mean ± SD

54.190 ± 2.530

8.840 ± 0.408

50.209 ± 1.537

12.351 ± 0.865

81.660 ± 3.470

28.623 ± 1.086

CV% 4.670 4.610 3.060 7.000 4.250 3.790

Conclusion The improved HPLC method is suitable for quantitative determination of norgalanthamine (1), lycorine (2) and galanthamine (3) from complex alkaloid mixtures in plants extracts and shoot-clumps cultures obtained from Amaryllidaceae species. Moreover this method may be used by biotechnology industry for quality control of these compounds.

Acknowledgment The work was supported by the Bulgarian Science Foundation, Bulgarian Ministry of Education and Science (project TK – B – 1605/2006).

REFERENCES 1. Abou-Donia А, Toaima S., Hammoda H. and Shawky

E. (2007) Chromatographia, 65, 497 – 500. 2. Abou-Donia А, Toaima S., Hammoda H. and Shawky

E. (2008) Phytochem. Anal, 19, 353 – 358. 3. Bergoñón S., Codina C., Bastida J., Viladomat F. and

Melé E. (1996) Plant Cell, Tissue and Organ Culture, 45, 191 – 199.

4. Berkov S., Pavlov A., Ilieva M., Burrus M., Popov S. and Stanilova M. (2005) Phytochem Anal, 16, 98 – 103.

5. Diop M., Hehn A., Ptac A., Chretien F., Doerper S., Gontier E., Bourgaud F., Henry M., Chapleur Y. And Laurain-Mattar D. (2007) Phytochem. Rev, 6, 137 – 141.

6. Gabrielsen B., Monath T., Huggins J., Kefauver D., Petit G., Groszek G., Hollingshead M., Kirsi J., Shannon W., Schubert E., Dare J., Ugarkar B. And Ussery M., Phelan M. (1992) J. Nat. Prod., 55, 1569 – 1581.

7. Georgieva L., Berkov s., Kondakova V., Bastida J., Viladomat F., Atanassov A. and Codina C. (2007) Z. Naturforsch, 62, 627 – 635.

8. Harvey A.L. (1995) Pharm. And Therap., 68, 113 – 128. 9. Li S.Y., Chen C., Zhang H.Q., Guo HY., Wang H.,

Wang L., Zhang X., Hua S.N., Yu J., Xiao P.G., Li R.S. and Tan X. (2005) Antiviral Res., 67(1), 18 – 23.

10. Liu J., Hu W.X., He L.F., Ye M. and Li Y. (2004) FEBS Letters, 578(3), 245 – 250.

11. Lόpez S., Bastida J., Viladomat F. and Codina C. (2002) Phytochem. Anal., 13, 311-315.

12. Mustafa NR., Rhee IK. and Verpoorte R. (2003) J. Liq. Chromatogr. & Relat. Technol., 26(19), 3217-3233.

13. Novikova Y. and Tulaganov A.A. (2002) Pharm. Chem. J., 36(11), 623-627.

14. Pavlov A., Berkov S., Courot E., Gocheva T., Tuneva D., Pandova B., Georgiev M., Georgiev V., Yanev S.,

XI ANNIVERSARY SCIENTIFIC CONFERENCE BIOTECHNOL. & BIOTECHNOL. EQ. 23/2009/SE 120 YEARS OF ACADEMIC EDUCATION IN BIOLOGY SPECIAL EDITION/ON-LINE 45 YEARS FACULTY OF BIOLOGY

812

Burrus M. and Ilieva M. (2007) Process Biochemistry, 42, 734-739.

15. Poulev A., Neumann B. and Zenk M. (1993) Planta Med., 59, 442 – 446.

16. Ptak A., Tahchy AE., Dupire F., Boisbrun M., Henry M., Chapleur Y., Moś M. and Laurain-Matter D. (2009) J. Nat. Prod., 72, 142-147.

17. Radicheva N., Vydevska M. and Mileva K. (1996) Methods Find Exp. Clin. Pharm., 18, 301-308.

18. Sellés M., Viladomat F., Bastida J. and Codina C. (1999) Plant Cell Reports, 18, 646-651.

19. Szlávik L., Gyuris A., Minarovits J., Forgo P., Molnar J. and Hohmann J. (2004) Planta Med., 70(9), 871 – 873.

20. Weniger B., Italiano L., Beck J., Bastida J., Bergόnon S., Codina C., Lobstein A. and Anton R. (1995) Planta Med., 61, 77 – 79.


813

improved hplc method for determination of amaryllidaceae alkaloids

Documents