Spatial localisation of curcumin and rapid screening of the chemical compositions of turmeric rhizomes (Curcuma longa Linn.) using Direct Analysis in Real Time-Mass Spectrometry (DART-MS)

Food Chemistry 173 (2015) 489–494

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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Spatial localisation of curcumin and rapid screening of the chemical compositions of turmeric rhizomes (Curcuma longa Linn.) using Direct Analysis in Real Time-Mass Spectrometry (DART-MS) A.F.M. Motiur Rahman a,⇑, Rihab F. Angawi b, Adnan A. Kadi a a b

Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia Department of Chemistry, Faculty of Science, King Abdul Aziz University, Jeddah 21589, Saudia Arabia

a r t i c l e

i n f o

Article history: Received 29 April 2014 Received in revised form 20 July 2014 Accepted 13 October 2014 Available online 18 October 2014 Keywords: Curcumin Turmeric Curcuma longa Linn. DART

a b s t r a c t Curcumin is a potent antioxidant agent having versatile biological activities is present in turmeric rhizomes (Curcuma longa Linn.). Powder of turmeric rhizomes is consumes as curry spicy worldwide, especially in Asia. In this study, we demonstrate that, bioactive curcumin and its analog demethoxycurcumin are chiefly concentrated in the pith rather than the other parts of the turmeric rhizomes and it was discovered using modern atmospheric ionisation source ‘Direct Analysis in Real Time’ (DART) connected with an Ion Trap Mass Spectrometry. In addition, all the major components present in turmeric rhizomes were detected in positive and/or in negative ion mode using DART. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction The name turmeric (Curcuma longa Linn.) originated from the Arabic name ‘kurkum’ (Goel, Kunnumakkara, & Aggarwal, 2008). It is a rhizomes of a perennial plant (family Zingiberaceae) having leaves and stems (Chan et al., 2009). Turmeric comes in various forms and peoples consume turmeric rhizomes powder by adding in curry in their daily meals (Thomas-Eapen, 2009). The importance of turmeric rhizomes from the fact that it has been used in folk medicine for the treatment of inflammatory related diseases such as jaundice (liver disease), indigestion, urinary tract infection, rheumatoid arthritis, and insect bites (Ammon & Wahl, 1991). Its phytochemical component curcumin [(1E,6E)-1,7-bis(4-hydroxy3-methoxyphenyl)hepta-1,6-diene-3,5-dione] is a b-diketone having diverse biological activities including antitumor (Choi, Chun, Kim, Kim, & Park, 2006) antioxidant, antiarthritic, antiamyloid and anti-ischaemic (Shukla, Khanna, Ali, Khan, & Srimal, 2008), antimutagenic, antiangiogenesis, antimicrobial, and immunomodulation activities (Chattopadhyay, Biswas, Bandyopadhyay, & Banerjee, 2004; Um et al., 2008). Curcumin also shows growth inhibition and apoptosis induction in vitro as well as inhibition of tumorigenesis in vivo against variety of cancer cell lines (Bisht

⇑ Corresponding author. Tel.: +966 (0)14670237; fax: +966 (0)14676220. E-mail address: [email protected] (A.F.M.M. Rahman). http://dx.doi.org/10.1016/j.foodchem.2014.10.049 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.

et al., 2007; Kawamori et al., 1999). In addition, turmeric decreases the amyloid pathology of Alzheimer’s disease (AD) (Ringman, Frautschy, Cole, Masterman, & Cummings, 2005). On the other hand, synthetic derivatives of curcumin also show potent biological activities (Adams et al., 2004; Amolins, Peterson, & Blagg, 2009; Mishra, Narain, Mishra, & Misra, 2005; Robinson et al., 2005; Selvam, Jachak, Thilagavathi, & Chakraborti, 2005; Shao et al., 2006; Venkateswarlu, Ramachandra, & Subbaraju, 2005). Even though turmeric having versatile biological activities (most probably because of its main bioactive component curcumin), only Asians are consuming it as spicy in their daily meals. Usually, addition of turmeric as spicy to the curry occurs while cooking, it makes the curry yellow in colour and produced strong odour. Most probably due to the yellow colour and strong odour numbers of peoples around the world do not prefer to eat these types of curry. As a result, they are losing this opportunity to intake bioactive components, which is very cheap and obtain naturally. Whereas, it could be helpful for all of us if we intake this bioactive component without any problems. To overcome these problems and make easily accessible the bioactive curcumin, we need to figure out where exactly the location of bioactive curcumin is. Once figure out the location, it will be easier to extract and isolate the bioactive components, hence, people can intake the part where bioactive curcumin are present or add them in curry. If so, curry might have no or less colour, no or less odour, etc. Not only turmeric but also for other natural herbs, if we figure out the presence of bioactive

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components whether it is in the seeds, skin, cortex, pith, leaf, stems or in root, etc., then it will be very much easier to extract, isolation, characterisation, structure elucidation and getting their biological activities. Therefore, fining bioactive molecules in natural herbs will be time saving, more cost-effective, easily accessible and it could be helpful for all of us around the world. Considering the importance of bioactive natural components present in natural herbs, beside traditional method (extraction, purification, characterisation, structure elucidation and then checking biological activities), a number of routine works have been conducting on the identification of bioactive components using various mass spectrometric methods (HPLC–MS, GC–MS etc.). Although spectrometric methods has been applying for the detection, characterisation and structure elucidation of natural raw materials, still there is an important requirements for efficient methods for the detection and spatial localisation of bioactive components in natural raw materials in their real state to find out whether it is present or not. This will open the door for the scientists to choose the natural raw materials for further analysis, hence, saving time, economy as well as energy and so on. It should be noted that, DART is a short and efficient ionisation technique for the identification of the chemical compositions present in raw natural materials. DART can be shoot to synthetic mixture even in a mixture of chemical compositions to decent known/unknown compounds. It does not require sample preparations, solvents, columns, etc. except some cases needed minimal sample preparation. Now a days, application of DART is increasing rapidly and number of studies were reported including, analysis of curcumin in turmeric powder (Kim & Jang, 2009), analysis of printing and writing paper (Adams, 2011), crushed garlic (Block, Dane, Thomas, & Cody, 2010), allium siculum (Kubec et al., 2010), profiling of Piper betle Linn. (Bajpai, Sharma, Kumar, & Madhusudanan, 2010), milk powder (Vaclavik, Rosmus, Popping, & Hajslova, 2010), quality assessment of Radix Aconiti Preparata (Zhu et al., 2012), olive oil quality (Vaclavik, Cajka, Hrbek, & Hajslova, 2009), and very recently chalcone fragmentation by our group (Rahman, Mohamed, Pervez, Mohammad, & Adnan, 2013), etc. DART usually produces the molecular ions [M]+ or [M+H]+ in positive mode, and [M] or [M H] in negative mode. As a part of our interests detecting natural bioactive components present in natural herbs, here, we have chosen turmeric rhizomes for the spatial localisation of bioactive components besides rapid screening using DART ion source. In this study, atmospheric pressure ionisation technique DART ion source coupled to a Ion Trap mass spectrometer was applied for the spatial localisation of bioactive components especially curcumin present in turmeric rhizomes beside rapid screening of the bioactive components. Initially, various forms of samples (raw, dry and commercial) of turmeric rhizomes in their real state and the methanol extracts of them, as well as juice of raw rhizomes were analysed. Finally, DART was shooting to the cortex and pith of raw turmeric rhizomes for the spatial localisation of the bioactive components.

2.2. DART-MS sample Powder: raw turmeric rhizomes powder was prepared using traditional was, where, turmeric rhizomes was boiled for 30–40 min., dried in open air for 2/3 days and crashed into powder. Juice: turmeric rhizomes were cut into pieces, crushed in a mortar and pestle and juice were collected by filtration. Extract: raw rhizomes were cut into pieces/dried rhizomes were crushed into powder, and commercial turmeric powder were separately stirred in absolute methanol (approximately 1 g in 10 mL methanol) at room temperature for 24 h, filtered through Agilent technologies syringe filters and extracted solution were reconstituted using N2. Pith and cortex: a cross-section of raw turmeric rhizomes was separated in pith and cortex. 2.3. Instrumentation and conditions All experiments were performed using an Agilent 6320 Ion Trap LC/MS mass spectrometer fitted with an IonSense (IonSense, Saugus, MA, USA) DART source. The operating conditions of a DART ion source were as follows: positive/negative ion mode; helium flow: 4.0 L min 1; gas heater temperature was set to 350 °C; and the capillary voltage was 4000 V. The distance between the outlet of the DART and the inlet of the orifice of Ion Trap was 1 cm. Sample introduction was accomplished by moving, slowly, the closed end of a glass capillary (for powder and liquids) which was dipped into a powdered analytes and was carried across the helium gas stream between the DART source and the orifice of the Ion Trap. Raw samples were introduced using forceps. 3. Results and discussion In order to detect the bioactive components present in turmeric rhizomes using DART atmospheric pressure ion source connected with an Ion Trap mass spectrometer, various samples of turmeric rhizomes (Fig. 1) including raw (Fig. 1B), dried powder (Fig. 1D), commercially available powder, juice of raw rhizomes (Fig. 1E) and their methanol extracts were analysed.

2. Materials and methods 2.1. Chemicals and samples All reagents were analytical grade. Methanol (MeOH) was obtained from VWR International Ltd. (Hurter Boulevard, Lutterworth, Leicester, LE17 4XN, UK), and ultra pure deionized water was obtained from Milli-Q plus purification system, Millipore, Waters (Millipore, Bedford, MA, USA). Raw and commercial turmeric rhizomes were collected from local market in Riyadh, Saudi Arabia.

Fig. 1. Various forms of turmeric rhizomes: (A) turmeric plant; (B) raw turmeric rhizomes, (C) dry turmeric rhizomes, (D) powder of dried turmeric rhizomes, and (E) juice of raw turmeric rhizomes.

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It should be noted that, traditionally, turmeric rhizomes powder preparing by boiling raw turmeric rhizomes for 30–40 min. then drying in open air for 2/3 days and then crashing into powder. As shown in Fig. 1, here, we followed the traditional way to prepare powder sample of dried turmeric rhizomes (Fig. 1D). Juice sample (Fig. 1D) of raw turmeric rhizomes was prepared by crushing the raw turmeric rhizomes in a mortar and pestle. For methanol extract samples: raw turmeric rhizomes were cut into pieces, dried rhizomes were crushed into powder, and commercial turmeric powder were stirred separately in absolute methanol (approximately 1 g in 10 mL methanol) at room temperature for 24 h, filtered through Agilent technologies syringe filters, reconstituted under nitrogen gas flow and introduced directly into the DART. Screening of the chemical compositions of turmeric rhizomes were accomplished by introducing both the raw and methanol

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extract to the DART in positive and negative modes. From the mass spectra of raw turmeric rhizomes (Fig. 2), thirteen bioactive compounds have been identified including curcumin (1). Chart 1 demonstrates the structure of these compounds (1–9); alphaturmerone (2a, m/z = 219.1 [M+H]+)/dihydro-ar-turmerone (2b, m/z = 219.1 [M+H]+)/(E)-alpha-atlantone (2c, m/z = 219.1 [M+H]+), 1,3-dihydroisobenzofuran (3, m/z = 121.1 [M+H]+), caffeic acid (4, m/z = 179.4 [M H] ), methyl 8,11-octadecadienoate (5, m/z = 293.4 [M H] ), ar-turmerone (6a, m/z = 217.3 [M+H]+)/(E)-3methyl-6-p-tolylhepta-1,4-dien-3-ol (6b, m/z = 217.3 [M+H]+), 6-(3-hydroxy-4-methylcyclohexa-2,4-dienyl)-2-methylhept-2-en4-one (7a, m/z = 235.0 [M+H]+)/7-(cyclohexa-1,3-dienyl)-5hydroxy-2,6-dimethylhept-2-en-4-one (7b, m/z = 235.0 [M+H]+), n-hexadecanoic acid (8, m/z = 255.4 [M H] ), linoleic acid (9, m/z = 279.3 [M H] ) as major peak.

Fig. 2. Mass spectra of turmeric rhizomes: (a) raw samples in positive mode; (b) raw samples in negative mode; (c) methanol extract of raw samples in positive mode; (d) methanol extract of raw samples in negative mode; (e) turmeric rhizomes powder in positive mode; (f) turmeric rhizomes powder in negative mode; (g) methanol extract of turmeric rhizomes powder in positive mode; (h) methanol extract of turmeric rhizomes powder in negative mode.

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O

O

O

O

Curcumin (1) OH Exact Mass: 368.1260 m/z 391.13 [M+Na]+ or m/z 367.2 [M-H]-

HO

O

O

2a

2b Dihydro-ar-turmerone Exact Mass: 218.1671

alpha-turmerone Exact Mass: 218.1671

O

O O

2c (E)-alpha-Atlantone Exact Mass: 218.1671

HO

3 1,3-dihydroisobenzofuran Exact Mass: 120.0575

5

OH 4

HO

O

O methyl 8,11-octadecadienoate Exact Mass: 294.2559

caffeic acid Exact Mass: 180.0423

OH

O

O

O OH OH

6a ar-turmerone Exact Mass: 216.1514

6b (E )-3-methyl-6-ptolylhepta-1,4-dien-3-ol Exact Mass: 216.1514

7b

7a

6-(3-hydroxy-4methylcyclohexa-2,4-dienyl)-2methylhept-2-en-4-one Exact Mass: 234.1620

7-(cyclohexa-1,3-dienyl)-5hydroxy-2,6-dimethylhept-2en-4-one Exact Mass: 234.1620

O 8

OH n-hexadecanoic acid Exact Mass: 256.2402

O OH 9

linoleic acid

Exact Mass: 280.2402

Chart 1. Chemical structures of bioactive compounds detected in turmeric rhizomes using DART.

Fig. 3. Mass spectra of commercial turmeric powder: (a) commercial turmeric powder in positive mode; (b) commercial turmeric powder in negative mode; (c) methanol extract of commercial turmeric powder in positive mode; (d) methanol extract of commercial turmeric powder in negative mode; (e) juice of raw turmeric rhizomes in positive mode; (f) juice of raw turmeric rhizomes in negative mode.

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Table 1 MS scan data of various turmeric rhizomes samples using DART ion source. Entry

Samples

MS scan data in m/z Positive mode

Negative mode

1 2

Raw turmeric rhizomes

Raw Methanol extract

391.2, 279.1, 219.1, 121.2, 83.3 391.1, 251.0, 235.0, 219.3, 217.3, 179.2, 121.2, 83.3

367.2, 279.2, 247.2, 231.2, 193, 151.9, 120.9, 89.0 397.3, 281.3, 255.3, 231.2, 191.1, 136.0, 121.0, 89.0

3 4

Dry turmeric rhizomes powder

Powder Methanol extract

391.4, 235.1, 217.1, 179.3, 121.2 391.4, 375.3, 235.2, 217.3, 121.2, 119.3

325.8, 293.4, 265.5., 256.0, 249.5, 231.8, 217.7 279.3, 255.4, 231.1, 151.9, 121.0, 89.0

5 6

Commercial turmeric rhizomes powder

Powder Methanol extract

235.0, 217.1, 179.1, 119.1, 83.2 278.9, 234.9, 216.9, 118.9, 83.1

367.4, 337.3, 279.4, 255.4, 231.2, 149.0, 121.0, 89.0 293.6, 279.4, 255.5, 231.4, 265.1, 252.1, 136.1, 121.2, 103.2, 89.3

7

Raw turmeric

Juice

391.5, 279.5, 219.5, 121.7, 93.7, 83.7

367.2, 311.5, 283.5, 255.5, 179.2, 89.2

Fig. 4. (a) Cross-section of raw turmeric rhizomes; (b) mass spectra of pith in negative mode; (c) mass spectra of cortex in negative mode.

The mass spectra shows a number of peaks including sodium adduct of the bioactive curcumin (1, m/z = 391 [M + Na]+) is present in positive mode for both raw turmeric rhizomes (Fig. 2a) and the methanol extract of raw turmeric rhizomes (Fig. 2c) samples. But in negative mode, curcumin (1, m/z = 367.2 [M H] ) is present only in raw turmeric rhizomes sample (Fig. 2b). Analysis of the mass spectrum of the dry turmeric rhizomes reveals peaks in the positive mode (1, m/z = 391.4 [M+Na]+; Fig. 2e) and in the negative mode (m/z = 367.2 [M H] ; Fig. 2f) corresponding to the sodium adduct and deprotonated bioactive curcumin, respectively. On the other hand, the mass spectrum of the methanol extract did not show curcumin in both positive/negative modes (Fig. 2g and h). In addition, comparison of the data of turmeric rhizomes powder (Fig. 2e–h) with raw turmeric rhizomes (Fig. 2a–d) shows the disappearance of compounds 4 and 5 in turmeric rhizomes powder samples and was identified ferulic acid (10, m/z = 193.0 [M H] ) with very nice peak (Fig. 2f) as an additional compound. The mass spectra of commercial turmeric powder and their methanol extract did not show curcumin (1) peaks in positive mode (Fig. 3a). However, in negative mode only commercial turmeric powder shows the bioactive curcumin peak (1, m/z = 367.2 [M H] ; Fig. 3b). Compounds 4 and 5 were not found in the commercial powder of turmeric rhizomes. Interestingly, demethoxycurcumin was found in negative mode (11, m/z = 337.3 [M H] ; Fig. 3b) in the powder of commercial of turmeric rhizomes. Fig. 3e shows the mass spectra of raw turmeric rhizomes juice. A peak for sodium adduct of curcumin (1) in the positive mode (1, m/z = 391.5 [M+Na]+; Fig. 3e) and as a deprotonated curcumin in negative mode (1, m/z = 367.2 [M H] ; Fig. 3f) was observed. In

addition, compounds 2, 3, 4, 8 along with additional stearic acid (12, m/z = 283.5 [M H] ; Fig. 3f) were detected. Table 1 shows a summary of the peaks obtained from all samples of turmeric rhizomes. As the table illustrates, a peak for the sodium adduct of curcumin (m/z = 391 [M + Na]+) was obtained in the positive mode except for the commercial turmeric rhizomes powder. On the other hand, curcumin peak (m/z = 367 [M H] ) was obtained in the negative mode from all samples except that for the dry turmeric rhizomes powder. Based on summarised table (Table 1) it can be concluded that, bioactive curcumin is present in turmeric rhizomes and it is easy to identify using DART in negative mode with m/z value of 367 (exact mass is 368.13) rather positive mode. It should be noted that, in the positive mode only sodium adduct of bioactive curcumin was observed (m/z = 391). Finally, spatial localisation of bioactive curcumin presence in turmeric rhizomes was evaluated by shooting DART to the pith and cortex of the cross-section (Fig. 4a) of raw turmeric rhizomes. Surprisingly, bioactive curcumin (1) found in the pith of the cross-section of raw turmeric rhizomes with m/z value of 367.2 in negative mode along with demethoxycurcumin (11, m/z = 337.2 [M H] ; Fig. 4b). Interestingly, neither curcumin nor demethoxycurcumin were observed in the cortex of the cross-section of raw turmeric rhizomes (Fig. 4c) sample.

4. Conclusion Modern atmospheric pressure ionisation source ‘Direct Analysis in Real Time’ (DART) coupled to an Ion Trap mass spectrometer

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was applied for the spatial localisation of bioactive components curcumin present in turmeric rhizomes. It was found that bioactive curcumin and its analog demethoxycurcumin are chiefly concentrated in the pith rather than the other parts of the turmeric rhizomes. In addition, 15 chemical components were detected in turmeric rhizomes. Since, curcumin is chiefly concentrated in the pith of turmeric rhizomes it can be packed in small quantity for industrialise. Therefore, not only Asians but also everyone can intake the industrialised pith of turmeric rhizomes which is having fewer odours, less in colour (here, less in colour means less amount addition in curry so colour of the curry will be less), at the same time getting benefit from it. On the other hand, This DART-MS method is easy, efficient and more cost-effective for the detection of bioactive components present in natural raw material in their real states. Applications of DART for other natural raw materials are in progress and the results remain to be explored. Acknowledgements Partial financial support from ‘Distinguished Scientist Fellowship Program (DSFP)’, King Saud University, Riyadh, Saudi Arabia is gratefully acknowledged. References Adams, J. (2011). Analysis of printing and writing papers by using direct analysis in real time mass spectrometry. International Journal of Mass Spectrometry, 301(1– 3), 109–126. Adams, B. K., Ferstl, E. M., Davis, M. C., Herold, M., Kurtkaya, S., Camalier, R. F., et al. (2004). Synthesis and biological evaluation of novel curcumin analogs as anticancer and anti-angiogenesis agents. Bioorganic & Medicinal Chemistry, 12(14), 3871–3883. Ammon, H. P. T., & Wahl, M. A. (1991). Pharmacology of Curcuma longa. Planta Medica, 57(01), 1–7. Amolins, M. W., Peterson, L. B., & Blagg, B. S. J. (2009). Synthesis and evaluation of electron-rich curcumin analogues. Bioorganic & Medicinal Chemistry, 17(1), 360–367. Bajpai, V., Sharma, D., Kumar, B., & Madhusudanan, K. P. (2010). Profiling of Piper betle Linn. cultivars by direct analysis in real time mass spectrometric technique. Biomedical Chromatography, 24(12), 1283–1286. Bisht, S., Feldmann, G., Soni, S., Ravi, R., Karikar, C., Maitra, A., et al. (2007). Polymeric nanoparticle-encapsulated curcumin (‘‘nanocurcumin’’): A novel strategy for human cancer therapy. Journal of Nanobiotechnology, 5(1), 1–18. Block, E., Dane, A. J., Thomas, S., & Cody, R. B. (2010). Applications of direct analysis in real time mass spectrometry (DART-MS) in allium chemistry. 2Propenesulfenic and 2-propenesulfinic acids, diallyl trisulfane S-oxide, and other reactive sulfur compounds from crushed garlic and other alliums. Journal of Agricultural and Food Chemistry, 58(8), 4617–4625. Chan, E. W. C., Lim, Y. Y., Wong, S. K., Lim, K. K., Tan, S. P., Lianto, F. S., et al. (2009). Effects of different drying methods on the antioxidant properties of leaves and tea of ginger species. Food Chemistry, 113(1), 166–172. Chattopadhyay, I., Biswas, K., Bandyopadhyay, U., & Banerjee, R. K. (2004). Turmeric and curcumin: Biological actions and medicinal applications. Current Science, 87(1), 44–53.

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