Improved Hplc Method For Determination Of Amaryllidaceae Alkaloids

IMPROVED HPLC METHOD FOR DETERMINATION OF AMARYLLIDACEAE ALKALOIDS I. Ivanov1, S. Berkov2 and A. Pavlov1 1 Department 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 BIOTECHNOL. & BIOTECHNOL. EQ. 23/2009/SE SPECIAL EDITION/ON-LINE

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.

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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 Time, min

1. 2. 3. 4. 5. 6. 7. 8.

6,00 9,00 10,00 12,00 20,00 -

Gradient profile of improved method

Flow rate, ml/min

%A

%B

Time, min

0,50 0,50 0,50 0,50 0,50 0,50 -

31,0 31,0 70,0 70,0 31,0 31,0 -

69,0 69,0 30,0 30,0 69,0 69,0 -

11,00 15,00 16,00 18,00 20,00 22,00 31,00

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Flow rate, ml/min

%A

%B

0,40 0,30 0,50 0,50 0,50 0,40 0,40 0,40

10,0 31,0 70,0 90,0 90,0 31,0 10,0 10,0

90,0 69,0 30,0 10,0 10,0 69,0 90,0 90,0

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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.

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TABLE 2 Parameters of retention time and calibration curves for separated alkaloids Alkaloid Norgalanthamine (1) Lycorine (2) Galanthamine (3)

Retention time (min) 10,600 14,430 15,130

r2 0,9997 0,9980 0,9998

Equation Y= 2.41x104X + 8.17x103 Y= 3.62x104X + 4.35x104 Y= 4,55x104X – 7.14x104

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

1 2 3 4 5 Mean ± SD CV%

L.aestivum culture Nor galanthamine (μg/ml) 51.784 54.650 53.735 52.528 58.266 54.190 ± 2.530 4.670

Lycorine (μg/ml)

Galanthamine (μg/ml)

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

Galanthamine (μg/ml)

9.106 8.327 9.276 8.497 8,994 8.840 ± 0.408 4.610

48.195 49.824 50.943 52.331 49.753 50.209 ± 1.537 3.060

12.433 11.539 11.455 13.482 12.846 12.351 ± 0.865 7.000

28.566 26.780 29.080 29.521 29.170 28.623 ± 1.086 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).

82.291 75.697 82.065 83.976 84.266 81.660 ± 3.470 4.250

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