Isolation and Characterization of Antitumor Alkaloid from Poppy Capsules ( Papaver somniferum )

Noscapine, a phthalideisoquinoline alkaloid derived from opium, has been used in the treatment of various cancer types. Its lowtoxicity pro�le has increased attention to this alkaloid. �ith regard to increasing demand for this compound, we developed a new method for isolation of noscapine from dried capsules of Papaver somniferum. Noscapine was successfully isolated from poppy capsules for the �rst time and the purity of the isolated compound was determined to be over 99.59% by HPLC analysis. e structure of noscapine was con�rmed by H NMR, C NMR, FT-IR, elemental analysis, and HR-ESI-MS methods.


Introduction
Important class of pharmaceutical compounds, they are alkaloids form, which can be found in natural products such as the opium poppy, Papaver somniferum.Alkaloids are normally removed from natural products using solvent extraction.However, the extraction of very small quantities of alkaloids in natural plants required the development of new and improved experimental methods.Noscapine, (3S)-6,7-Dimethoxy-3-[(5R)-4-methoxy-6-methyl-5,6,7,8-tetrahydro-1,3-dioxolo[4,5-g]isoquinolin-5-yl]isobenzofuran-1(3H)one, is a natural phthalideisoquinoline alkaloid obtained from the opium poppy which has been used for many decades as a very safe cough suppressant with no side effects [1,2].Unlike other alkaloids, noscapine is not addictive.Recently, antitumor properties of noscapine have been reported by Joshi�s group for the �rst time [3] and research on the therapeutic effect of noscapine in inhibiting tumor progression in experimental animals is ongoing [4][5][6].Moreover, histological studies have shown that noscapine is not toxic to normal tissues [1,7,8].Generally, alkaloids are obtained from opium poppy, are structurally complex, and most of them cannot be economically synthesized.Noscapine has an excellent pharmacological pro�le, therefore the pure yield of this alkaloid from natural plants is very important.
Isolation of noscapine from opium was studied in literature.Sim showed the separation of alkaloids from opium in a step-wise process and produced noscapine as the last product [9].Also Ramanathan and Chandra report several processes for purifying narcotine which is known as an impure form of "Noscapine" [10].In these methods are used opium as the starting material.
On the other hand, the poppy straw is an alternative source which is chemically used to extract the alkaloids [11].For this method, the capsules of P. somniferum are cut and dried in air in the �eld then harvested and crushed to straw.In many countries this method is present as an alternative route to obtain alkaloids like noscapine due to the fact that the usage of crude opium which contains the controlled substance morphine may be limited by governmental rules.Because of this restriction, noscapine producers have been searching for a new source and poppy straw, which is harvested when it is fully mature and dry, which is suitable for this.Also, it is important to say that poppy straw may contain much higher quantities of noscapine relative to morphine content [12].However, P. somniferum has been genetically modi�ed by gene insertion to either enhance alkaloid production or to "switch off " the genes responsible for production of nonrequired alkaloids.
A simple process for isolating the alkaloids is highly desirable to evaluate product quality.is paper describes a method for the extraction and puri�cation of noscapine that is present in the poppy straw.In this method, noscapine was obtained in purity suitable for use in pharmacopy (99.59%).e chemical structure of this alkaloid is shown in Figure 1.

General. Melting point was determined in open glass
capillary tube by means of a BUCHI Melting Point B-540 apparatus.Infrared (FT-IR) spectra were taken using a PerkinElmer Spectrum One FT-IR spectrometer,  max in cm −1 .e 1 H and 13 C nuclear magnetic resonance (NMR) were taken with a Bruker 500 NMR spectrometer.Chemical shis are reported in ppm ().High resolution electrospray ionization mass spectra (HR-ESI-MS) were obtained with MeOH on a Bruker micrOTOF-Q.Elemental analyses (EA) were performed using a ermo Finnigan FLASH 1112 SERIES EA instrument.e speci�c rotation was measured on an Optical Activity Ltd.A55 polarimeter instrument using 2 cm 3 cell with a 0.5 dm path length and the sample concentration is given in g/100 cm 3 unit.Chemical reagents for isolation were of analytical grade and purchased from Merck (Darmstadt, 90 Germany).All solvents were �ltered through 0.45 m membrane �lters before being injected into the HPLC.

Plant Material.
Poppy capsules were procured from Turkish Grain Board in August 2009.For this method, the capsules of P. somniferum are cut and dried in air in the �eld then harvested and crushed to straw.ese capsules are grown from genetically engineered seeds and contain 1.5% (w/w) noscapine.

Extraction of Crude Noscapine.
A three-neck round bottom �ask was charged with 500 g of air-dried and ground poppy straw, 2000 cm 3 of ethanol, and 250 cm 3 of demineralised water.e contents of the �ask were stirred mechanically.e suspension was heated and re�uxed for two hours at 80-85 ○ C. e suspension was �ltered.e alcohol extract was concentrated under reduced pressure to give 200 cm 3 of brown aqua phase.e pH of the solution was set 3.5 by adding acetic acid.5.0 g of activated charcoal were added into this solution and contents was heated to 60 ○ C and stirred mechanically for �een minutes.is solution was �ltered.e �ltrate was extracted with chloroform (4 times 250 cm 3 ) and the extracts are combined.e chloroform extract was concentrated to give 15 g of dark brown residue.is residue was dissolved in 400 mL of 10% acetic acid solution and heated to 50 ○ C. e pH of this solution was made 9.3 by adding 25% ammonia slowly.Crude noscapine was precipitated.e precipitate was �ltered and dried in an oven at 105 ○ C.

2.�. Puri�cation of Crude Noscapine.
Crude noscapine was suspended in 50 cm 3 of toluene.e suspension was heated to 70-80 ○ C. 5.0 g of activated charcoal was added and stirred mechanically for �een minutes at room temperature.e solution was �ltered.e �ltrate was concentrated under reduced pressure to give white precipitate and stirred mechanically for thirty minutes in an ice bath.e white precipitate was �ltered and dried in an oven at 105 ○ C. e resulting white solid corresponded to a yield of 90% and purity (by HPLC) of 99.59%.M.p.: 174-177 ○ C (decomp);

Determination of Alkaloid Content of Poppy Capsule,
Crude Noscapine and Pure Noscapine by HPLC 2.5.1.Sample Preparation.200 mg of homogenized, powdered, dried poppy capsule was shaken with 50.0 cm 3 0.1 M hydrochloric acid for 2 hours.An aliquot of the extract was �ltrated and the �ltrate was �lled to a vial.50 mg of crude noscapine or pure noscapine was dissolved in 5 cm 3 of 10% acetic acid solution is added approximately and 100 cm 3 of demineralised water, sonicated to dissolve, then diluted to 250 cm 3 with demineralised water.It was mixed well and �lled to a vial.

Chromatographic
System.An Agilent 1200 HPLC with UV-vis detection capabilities and a 0.01 cm 3 injection volume was used.e wavelength used during the analysis was 288 nm.A Eurospher-100 column (KNAUER) measuring 120 × 4 mm, 5 m particle size and with C18 stationary phase was used.e column was heated and maintained at 40 ○ C. e mobile phases consisted of (A) water with 0.67% tri�uoroacetic acid (TFA) and (B) 0.67% TFA in a mixture of methanol, acetonitrile, and water (500/16/129 cm 3 ) using a gradient system.e �ow rate was 1.0 mL/min and the applied elution program is described in Table 1.

Results and Discussion
Noscapine was isolated as a white crystalline solid with negative rotation [] 22  = −176 ○ ( = 0.5 g/100 cm 3 , CHCl 3 ).Several elution systems were tested in HPLC separation.e results indicated that when methanol-acetonitrile-water was used as mobile phase in gradient mode (Table 1), good seperation results could be obtained.HPLC analysis of poppy straw, crude extract, and pure noscapine show that noscapine was clearly seperated and exhibited well-de�ned chromatogram with retention times of 14.586 min in the selected experimental conditions (Figure 2).e content of noscapine in the crude extracts was 49.4%.Aer puri�cation treatment, the purity of the collected fraction peak 1 was found to be 99.59%.
Its molecular formula was determined as C 22 H 23 NO 7 on the basis of HR-ESI-MS (/ 414.1553 [M + H] + , calcd for C 22 H 23 NO 7 414.1532).Figure 3 shows the expected molecular ion peak at / = 414.1553,indicating formation of [M + H] + .e chemical structure of peak fraction in Figure 2(c) was identi�ed according to its FT-IR, 1 H NMR data 13 C NMR. ese were well-matched with the literature [12][13][14][15].Figure 4 shows infrared spectra of pure noscapine product and noscapine standard (purchased from Turkish Grain Board).IR spectra show numerous sharp bands between 700 and 1500 cm −1 which are assigned to deformation and stretching vibrations of the alkaloid ring system.Also, the strong band at 1756 cm −1 is the most characteristic for C=O stretching vibration.e NMR spectrum of noscapine shows two one-proton doublets at 6.96 and 6.08 ppm assigned to the aromatic protons in the phthalidyl ring.e one-proton singlet signal at 6.31 ppm is attributed to aromatic proton in the isoquinoline ring.e three-proton singlets at 3.86 ppm, 4.05, and 4.09 ppm are ascribed to methoxy protons in isoquinoline and phthalidyl rings, respectively.e signal at 5.94 which is given as a two-proton singlet is accounted the methylenedioxy protons.e singlet signal at 2.55 ppm is due to the -N-CH 3 protons.e -CH 2 -CH 2 -protons of isoquinoline ring are appeared as a multiplet at 2.32-2.38 ppm.
e 13 C NMR spectrum of compound exhibited signals for aromatic carbons between 117.2-134.1 ppm, methylenedioxy carbon at 100.7 ppm, and carbonyl carbon at 168.0 ppm.e signals at 56.9, 59.4, and 62.2 ppm are ascribed to methoxy carbon atoms.e carbon atom which bonded nitrogen atom is observed at 46.3 ppm.

Conclusions
Prior to now, noscapine is isolated from opium.Recently, following seed development and gene studies, poppy seed including capsules that are rich in noscapine have been developed.To our knowledge, this is the �rst report on the isolation of noscapine from poppy capsules.e isolation method is very short compared with other production methods from opium.Hence, poppy straw could be used as an accessible source of noscapine product with high yield.Its chemical structure was clearly con�rmed by using various instrumental techniques.e obtained product has pharmacological purity and does not include impurities.

F 3 :F 4 :
HR-ESI-MS data of pure noscapine product.FT-IR spectrums of noscapine product and noscapine standard (purchased from Turkish Grain Board).