Qualitative and Quantitative Determination of Main Alkaloids of Chelidonium Majus L

Original Acta Chromatographica 29(2017)3, 385–397 Research Paper DOI: 10.1556/1326.2017.29.3.09

Qualitative and Quantitative Determination of Main Alkaloids of Chelidonium majus L. Using Thin-Layer Chromatographic–Densitometric Method

ANNA BOGUCKA-KOCKA* AND DANIEL ZALEWSKI

Chair and Department of Biology with Genetics, Medical University in Lublin, Chodźki 4, 200-93 Lublin, Poland *E-mail: [email protected]

Summary. We present a new simple thin-layer chromatographic method designed for determination of the main alkaloids of Chelidonium majus L. In this study, we used roots and herb of the plant collected in spring and autumn. The alkaloid fractions were prepared according to modified pharmacopeial procedure [1]. In our method, we performed two-step elution onto silica gel plates. The first eluent consisted of chloroform, methanol, and water mixed with 70:30:4 proportion. The second eluent comprised of toluene, ethyl acetate, and methanol with 83:15:2 proportion. The described thin-layer chromatography (TLC) system allows qualitative and quantitative determination of the following alkaloids: sanguinarine, chelerythrine, chelidonine, coptisine, and berberine. For determination of protopine, eluent with n-buthanol, acetic acid, and water in 15:1.5:4 proportion was investigated. The dominant alkaloids observed in studied fractions were coptisine (1027.096 ± 13.367–287.474 ± 3.069 mg/100 g dry matter ± sdv) and chelidonine (1780.667 ± 263.522– 115.929 ± 14.694 mg/100 g dry matter ± sdv). The alkaloid detected in the least amount was chelerythrine (30.74 ± 7.526–1.143 ± 0.0651 mg/100 g dry matter ± sdv). The highest total amount of all alkaloids was determined in the fractions obtained from herbs in spring, and the lowest amount was detected in herbs autumn. Additionally, we compared amounts of studied alkaloids in different parts of plants (aerial parts and roots). The plants were collected in spring and autumn. Authors concluded that the presented method can be used as a valuable tool for screening studies on C. majus L.

Key Words: Chelidonium, alkaloids, thin-layer chromatography, densitometry

Introduction

Chelidonium majus L. (greater celandine, the Papaveraceae family) is a peren- nial plant growing in Europe, North America, and Asia [2]. Characteristic

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (https://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted use, distribution, and reproduction in any medium for non-commercial purposes, provided the original author and source are credited, a link to the CC License is provided, and changes - if any - are indicated. First published online: Novermber 29, 2016 ISSN 2083-5736 © The Author(s)

Unauthenticated | Downloaded 09/24/21 05:04 AM UTC 386 Bogucka-Kocka and Zalewski feature of this plant is orange latex containing 0.2–3% of alkaloids exuded when the plant is injured. This complex group comprises benzophe- nanthridine, berberine, and protopine alkaloids. The major alkaloids are chelidonine, coptisine, berberine, chelerythrine, and sanguinarine [3]. Preparations containing extracts of C. majus L. have been traditionally used for treatment of liver disease, oral infections, gallstones, gastric ulcer, and skin diseases [3]. They are known and used in folk medicine due to their anti-inflammatory, antitumor, choleretic, antimicrobial, and spasmo- lytic properties. Antitumor activity of C. majus L. alkaloids is the most in- tensively researched area [4–10]. Thiophosphoric acid, derivative of chelidonine, has been found to have potential anticancer properties [11, 12, 13]. There have not been many qualitative and quantitative comparative thin-layer chromatography (TLC) analysis of the alkaloid content in C. majus L. harvested in various periods published in the literature. In 1995, Tome and Colombo presented a study in which they compared a distribution of five alkaloids in chloroformic extracts of latex, leaves, and roots in winter and summer during a day using high-performance liquid chromatography [14]. Thin-layer chromatography is a simple method, used to research secondary plant metabolites, including alkaloids. There had been several TLC systems used to study the composition of the C. majus L. alkaloids presented to date [1, 15–21]. Among them, only the method investigated by Sarkozi et al. was developed for quantitative analysis of separated alkaloids in analytical range [1]. They utilized two different mobile phases: one for chelidonine, chelerythrine, and sanguinarine separation and the other for coptisine and berberine analysis which were applied on different silica gel plates. Our goal was to develop a TLC system which allows for simultaneous separation and determination of these alkaloids, as well as protopine, on a one single plate. In this paper, we report TLC method, which enables qualitative and quantitative simultaneous determination of chelidonine, coptisine, berberine, chelerythrine, and sanguinarine in alkaloid fractions obtained from aerial parts and roots of C. majus L. The compounds used in the study were obtained from plants collected in spring and autumn. To the best of our knowledge, in the current literature, no papers were published comparing alkaloids compositions in different parts of specimens of C. majus L. collected in varied growing seasons.

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Experimental

Chemicals

Reference substances: sanguinarine, chelerythrine, chelidonine, protopine, and berberine were purchased from Sigma-Aldrich (USA), and coptisine, from ChromaDex (USA). Water was distilled using DirectQ3UV apparatus (Millipore, France). All remaining reagents used in the study were purchased from POCH Gliwice (Gliwice, Poland).

Plant Materials

The plant material included aerial parts and roots of C. majus L. Specimens were harvested in their natural state in Rozwadów, Poland in May and Oc- tober. Collected plants were cleaned, dried in air at 40 °C without light, and authenticated in the Chair and Department of Pharmaceutical Botany, Medical University in Lublin, Poland. The samples dedicated to the research were marked as root spring (roots harvested in May), herb spring (aerial parts harvested in May), root autumn (roots harvested in October), and herb autumn (aerial parts harvested in October).

Preparation of Alkaloid Fractions

Alkaloid fractions designed to TLC investigations were prepared according to modified pharmacopeial procedure [1]. The powdered samples of plant (100 g) were extracted with 1000 mL aqueous acetic acid (12% v/v) in a water bath for 30 min. Subsequently, extracts were filtered and the process of extraction was repeated. Aliquots of the filtrates were combined, basified with 25% ammonium solution (to pH = 8–9), and extracted with n-butyl al- cohol (15 × 150 mL). The combined organic phases were dried over anhy- drous sodium sulfate and, after evaporation to dry residue, were weighed and dissolved in 40 mL of methanol. The total content of alkaloids was studied by spectrophotometry according to Polish Pharmacopeia VIII [22]. The results of study samples were expressed in chelidonine and presented in Fig. 1.

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10 9 8 ) 7 6 5 4 3 Alkaloid content (% content Alkaloid 2 1 0 Root spring Herb spring Root autumn Herb autumn

Fig. 1. Content of alkaloids in plant samples according to Polish Pharmacopeia VIII [22] (n = 3)

Thin-Layer Chromatography

TLC study was carried on 0.25-cm-thick precoated silica gel plates Si 60 F254, 20 cm × 10 cm and 10 cm × 10 cm (Merck, Darmstadt, Germany). The reference stock solutions of standard alkaloids was prepared in methanol (berberine, 17 μg mL−1; sanguinarine, 2.87 μg mL−1; chelerythrine, 10 μg mL−1; chelidonine, 481 μg mL−1; protopine, 115.5 μg mL−1; coptisine, 0.982 μg mL−1) (Fig. 2). The stock solutions were spotted on plates in the form of bands of 6 mm width using Camag ATS-4 Autosampler (Camag, Switzer- land). The extracted samples were spotted in the same way and cochromatographed with reference samples of alkaloid solutions. On each plate, samples of fractions and standard solutions in five different volumes (calibration spots) were spotted at a distance of 10 mm (Fig. 3). All chromatograms were developed in DS type chambers (Chromdes, Poland). For chelidonine, coptisine, berberine, chelerythrine, and sanguinarine analysis, a unidimensional two-fold elution on 10 × 20 cm plates was performed. The plates were developed by using mobile phase containing chloroform– methanol–water (70:30:4) with 50 mm distance. After air-drying the plate (15 min), the second mobile phase composed of toluene–ethyl acetate– methanol (83:15:2) was applied on 160 mm distance. This eluent was previously reported by Gadzikowska and Gołkiewicz in 1998 [16]. Before devel-

Unauthenticated | Downloaded 09/24/21 05:04 AM UTC TLC–Densitometric Method 389 oping the second mobile phase, air in used chamber had been previously saturated by this eluent for 30 min. Before first mobile phase developing, the saturation step was not necessary.

Fig. 2. Densitogram of root autumn (1), herb autumn (2), root spring (3) fractions cochromatographed with stock solutions of reference alkaloids: chelidonine (4), protopine (5), chelerythrine (6), sanguinarine (7), and berberine (8). λ = 366 nm

Investigated chromatographic system allows performing satisfactory separation of five studied alkaloids: sanguinarine, chelerythrine, chelidonine, coptisine, and berberine. The separation of protopine was not sufficient; therefore, we optimized a different mobile phase containing n-butyl alco- hol–acetic acid–water (15:1.5:4). In this case, a unidimensional one-step elution mode was applied on a distance of 90 mm on 10 cm × 10 cm silica gel plates.

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Fig. 3. Densitogram of herb spring (1), root spring (2), herb autumn (3), and root autumn (4) diluted fractions cochromatographed with successive concentrations of coptisine stock solution (tracks 5–9), λ = 360 nm

Qualitative and quantitative densitometric analyses were performed with TLC Scanner 3 (Camag, Switzerland). All developed plates were thor- oughly dried in a fume cupboard before the experiment. To optimize scan- ning wavelengths, densitometric measurement of the spots between 100 nm and 400 nm in 5 nm steps was performed. In the case of sanguinarine, chelerythrine, coptisine, and berberine, the maximum wavelength of fluorescence was 330 nm, 320 nm, 360 nm, and 340 nm, respectively. Chelidonine and protopine showed poor fluorescence effect; therefore, maximum wavelength of absorbance (287 nm and 290 nm, respectively) was used. The beam of ultraviolet (UV) light was set at optimal size of 4 mm × 0.3 mm. Densitometric investigations of the standard spots were used for drawing linear-type calibration plot with compared peak areas. Identification of alkaloids in studied samples was based on analysis and comparison of Rf values, color, and UV spectrum of standard spots. Data obtained from the

Unauthenticated | Downloaded 09/24/21 05:04 AM UTC TLC–Densitometric Method 391 analysis (calibration curve, correlation coefficient, equation of the calibration plot, standard deviation, Rf value, wavelength corresponding to the maximum of the fluorescence or absorption, content of individual alkaloids, and purity of alkaloid standard) were obtained using a WinCATS version 1.4.4. software (Table I). The first value of calibration graphs is a limit of de- tection, and RSD values show a satisfactory repeatability. The recovery values were determined by the standard addition procedure (Table 2).

Table I. Densitometric data of quantitative investigations

Linear range Linear regression Correlation Purityb R Compound f (ng) equaliationa y = ax + b coefficient (%) (mm)

Sanguinarine 5–100 y = 67.52x + 149.9 0.99990 95 0.45

Chelerythrine 10–100 y = 51.85x + 127.3 0.99996 67 0.35

Berberine 100–400 y = 33.51x + 1747 0.99649 68 0.24

Coptisine 200–800 y = 17.54x + 3931 0.98861 71 0.16

Chelidonine 1000–10 000 y = 0.5365x + 373.2 0.99563 66 0.40

Protopine 1000–4000 y = 2.391x + 1878 0.99098 82 0.20

ay = area, x = amount (ng) bPurity of standard alkaloids calculated by WinCATS version 1.4.4. software

Table II. Statistical parameters of quantification study alkaloids

Alkaloid RSD (%) LOD (ng) LOQ (ng) Recovery (%)

Sanguinarine 17.14 2 5 96

Chelerythrine 5.7 5 10 98

Berberine 4.35 10 50 93

Coptisine 1.30 5 20 95

Chelidonine 12.675 300 1000 93

Protopine 0.319 300 1000 94

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Results and Discussion

Total Content of Alkaloids

The total content of alkaloids in different parts of C. majus collected in May and October was performed according to Polish Pharmacopeia VIII [22]. The results were expressed in chelidonine and presented on Fig. 1. The plant material used for our study was in accordance with pharmacopeial requirements because the total alkaloid content was greater than 0.6% and amounted 1.18% in the autumn herb, 8.45% in the spring herb, 3.77% in the autumn root, and 7.99% in the root collected in the spring (Fig. 1). The larg- est amount of the alkaloids was obtained in the herb collected in the spring (8.45%). In spring root, we determined slightly lower content of alkaloids compounds (7.99%). Moreover, the total content of alkaloids in the roots collected in autumn (3.77%) was over two times lower than the content of alkaloids in the spring root (7.99%). Autumn herb (1.18%) contained over seven times less alkaloids than herb spring (8.45%) and over three times fewer alkaloids than autumn root (3.77%) (Fig. 1). These results correlate with research by Táborská et al. who found that C. majus L. collected in autumn contains much more alkaloid compounds in roots than in aerial parts [24]. The highest content of alkaloid compounds was included in specimens collected in the spring. This observation is in contrary to the general under- standing that autumn is high time for an accumulation of alkaloids in plants.

Optimization of TLC Mobile Phase Composition

Our aim was to establish an optimized composition of TLC mobile phase, which would allow simultaneous separation of sanguinarine, chelerythrine, chelidonine, protopine, berberine, and coptisine. We performed numerous TLC analyses with many different combinations and modifications of chromatographic mobile phases. Based on the obtained chromatograms, it was found that the simultaneous separation of tested alkaloids was not sufficient using only one eluent mixture. For berberine and coptisine separation, we modified a mobile phase described by Sarkozi et al. in 2006 (chloroform–methanol, 60:30), and finally, we found that chloroform–methanol– water (70:30:4) with distance of 50 mm is the most optimal. On developed plates, sanguinarine, chelerythrine, and chelidonine were located at the

Unauthenticated | Downloaded 09/24/21 05:04 AM UTC TLC–Densitometric Method 393 front of mobile phase; therefore, we applied a mobile phase toluene–ethyl acetate–methanol (83:15:2) [15] on distance of 160 mm. Still, we were not able to receive acceptable separation of protopine; therefore, different mobile phase was necessary. Based on the composition investigated by Rout et al. in 2008 (n-buthanol–acetic acid–water, 8:1:1), we found n-buthanol–acetic acid–water mobile phase in the ratio 15:1.5:4 with a distance of 90 mm as a most effective.

Content of Alkaloids in Study Fractions

Amounts of particular alkaloids in study samples are shown in Table III. The dominant alkaloid in the fraction obtained from the spring herb, autumn herb, and autumn root was coptisine, and in the spring root — chelidonine. Spring root fraction contained also the highest amount of chelidonine, berberine, protopine, sanguinarine, and chelerythrine. The highest quantity found in autumn herb fraction was presented by coptisine. Among studied alkaloids, sanguinarine was expressed in the least amount (in the fraction obtained from spring herb, autumn herb, and spring root) and berberine (in fraction from autumn roots). The fractions derived from plant material collected in the spring contained twice (in herb) and five-fold (in roots) greater amounts of protopine than the fractions obtained from the related autumnal plant material (Table III).

Table III. Content of alkaloids in study fractions

Content of alkaloids (mg/100 g dry matter ± sdv) Alkaloid Spring herb Spring root Autumn herb Autumn root

Sanguinarine 4.188 ± 0.718 30.834 ± 1.895 4.418 ± 0.464 15.836 ± 2.704

Chelerythrine 1.143 ± 0.0651 30.74 ± 7.526 3.906 ± 0.322 10.361 ± 1.455

Berberine 30.231 ± 1.315 60.098 ± 9.002 25.545 ± 2.258 8.866 ± 0.274

Coptisine 1027.096 ± 13.367 746.901 ± 9.119 287.474 ± 3.069 819.745 ± 6.752

Chelidonine 115.929 ± 14.694 1780.667 ± 263.522 187.233 ± 18.05 382.439 ± 18.54

Protopine 241.725 ± 0.772 368.95 ± 1.501 125.385 ± 3.304 72.036 ± 0.223

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In the fractions obtained from the roots, we found significantly higher content of sanguinarine, chelerythrine, and chelidonine in comparison to the fractions made from the herb C. majus L. collected in corresponding in- tervals of time. For sanguinarine, it was 7.4-fold and 3.5-fold higher for spring root and autumn root, respectively. For chelerythrine, it was 27-fold and 2.7-fold higher in spring root and autumn root, respectively. In the case of chelidonine, it was 15.4-fold and twice higher in spring root and autumn root (Table III). The fractions obtained from plant material harvested in the spring characterized a stable content of berberine, which is 2.13% and 1.99% of all determined alkaloids in the spring herb and the spring root. The percentage of protopine observed also showed certain degree of stability resulting in spring herb 17.02%, and the spring root 12.22% of the total alkaloid compounds quantified (Table III). In the case of fractions made from specimens obtained in autumn, the most stable alkaloid is chelidonine. In the fractions obtained from the herb and the roots, this alkaloid was presented in 29.53% and 29.21% of determined alkaloids (Table III). Our findings are in accordance with the study by Sarkozi et al. They indicate that the dominant alkaloid presenting in the aerial parts of C. majus L. is coptisine with chelidonine expressed in the roots and berberine occurring in roots in lower amount [1]. Similar results were published by Tábor- ská et al., using reversed-phase high-performance liquid chromatography (RP-HPLC) method to accomplish the identification of 21 alkaloids in the herb and roots of C. majus L. Among those alkaloids, coptisine was a compound which was present in the greatest amount [24]. Similar results were obtained by Gu et al.; using the ultra-performance liquid chromatography (UPLC) method, they determined coptisine as a dominant alkaloid in C. majus L. and berberine to be present in the smallest amount [25]. Suchomelova et al. in 2007, using a gradient HPLC method, determined the content of sanguinarine, chelerythrine, berberine, and protopine in the roots of C. majus L. Interestingly, the authors did not detect the presence of coptisine and chelidonine, which, in our research, were the dominant compounds [26]. The differences between the results obtained in our work and the literature reports can be caused by several factors. The plant material is derived from a variety of habitats and plants grow in different climatic conditions, which have a significant impact on the amount of secondary metabolites. The season in which the plants were harvested also plays an important role, as well as drying, storage, and preparation of samples. The content of alkaloids in the extracts used for the tests depends on the techniques of extrac-

Unauthenticated | Downloaded 09/24/21 05:04 AM UTC TLC–Densitometric Method 395 tion as well. Scientists reporting the number of alkaloids or individual alkaloid content use variety of methods with different parameters such as accu- racy, sensitivity, and reproducibility of the results. Remarkably, obtained chromatograms allowed to determine parts of C. majus L. plant which the fraction was made of. We indicated two spots of plausible alkaloid compounds clearly visible in 366 nm, with Rf = 0.29 and Rf = 0.75, presented only on chromatograms of the fractions from the aerial parts. We observed one spot at Rf = 0.05 visible also at 366 nm, occurring only in fractions obtained from the roots (Fig. 4, spots marked by circles). The presence of these compounds could serve as a marker which would en- able identification of plant material. It could also allow to detect contamina- tions with foreign plant material in preparations with C. majus L. According to the literature, the alkaloids occurring only in the herb of C. majus L. are chelamine and chelamidine with magnoflorine identified only in the roots [24]. To confirm this observation and to use these compounds as the marker spots require further study.

Fig. 4. The chromatogram of root autumn (track 1), herb autumn (track 2), root spring (track 3), and herb spring (track 4) fractions with spots (marked by circles) occurring only in the aerial parts (Rf = 0.29 and 0.75) or root (Rf = 0.05) fractions (λ = 366 nm)

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C. majus L. is a rich source of alkaloid compound, which is being used in contemporary medicine. There is therefore a continuing need for developing new methods that will help quickly and easily define the content of active pharmacological substances in plant material. We believe that the presented method can be our contribution to achieve this goal.

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