American Journal of Ethnomedicine, 2015, Vol. 2, No. 2 ISSN: 2348-9502 Available online at http://www.ajethno.com © American Journal of Ethnomedicine

Studies on Structure Elucidation of Aconitum Alkaloids Using LC-ESI-MS Technique

Bilal Ahmad Dar*1, Ali Mohamad Lone1, Rafiq Ahmad Khan2 and Mushtaq Ahmad Qurishi1

1University of Kashmir, Hazratbal Srinagar, Kashmir, India 2Department of physics, GDC Bemina, Srinagar, India

*Corresponding author e-mail: [email protected]

ABSTRACT

Development of a rapid, sensitive and specific liquid chromatography-electron spray-mass spectrometry (LC-ESI-MS) method as an effective tool for identification of different types of alkaloids from Aconitum chasmanthum collected from Sopi Kargil that can be used for quality checking of aconitum formulations. Chromatographic separation were achieved on C18 column and peaks determined by mass spectrometry in positive and negative modes. Chromatographic conditions were optimized to obtain high resolution and short run time. Use of the validated method led to tentative identification of seven alkaloids heterophylisine, atidine, atisine, chasm aconitine, 3-bichaconine, neoline and heteroatisine out of the twelve alkaloids picked from total ion chromatogram at the retention time of 1.74- 13.75 m on the basis of their retention times and fragmentation patterns in MS-MS data. Results showed that the positive mode response was much higher than the negative ion mode as most of the characteristic and adduct peaks were obtained in positive mode. Moreover this method has proved to be an effective tool to analyse the components of especially endangered plant species and can be used in quality control.

Keywords- Aconitum chasmanthum, LC-ESI-MS, Sopi, Kargil.

INTRODUCTION tuberous roots. Stem is simple and erect bearing numerous leaves around 50-100 cm Aconitum chasmanthum stapf.ex Holmes. is high. Inflorescence is long, narrow, stiff the botanical name of a plant commonly raceme over 30 cm long. It bears white or called vatsanabha (classical), Aconite, bluish flowers with oblong follicles. The 1 Monk’s hood, Holf’s bane (English), flowering season is from july-september . It bachnak, beesh, atees (urdu), banbalang, has a wide distribution in sub alpine and mohri, mohand (kashmiri) which belongs to alpine regions of western Himalayas from family renunculaceae. It is an erect, Chitral and Hazara to Kashmir and Lahul perennial herb with biennial, paired and between altitudes of 2100-3600 m. It is also

Page 110 www.ajethno.com American Journal of Ethnomedicine ______ISSN: 2348-9502 found in the mountains of Assam and sub gaining significance. Different species of alpine grasslands of Himachal Pradesh. The Aconitum are rich in diterpene alkaloids genus Aconitum has over 250 species2. and flavonoids with wide spectrum of Different species of Aconitum3 with their activities. Alkaloids like mesaconitine, distribution in the Himalayas and medicinal aconitine, hypaconitine, heteratisine, hetero- importance have been given by shah. phyllisine, heterophylline, heterophyllidine, Genus Aconitum is enlisted as endangered atidine, hetidine, hetsinone and medicinal plant in Red Data Book. The benzoylheteratisine have been reported from factors like overgrazing, prolonged seed different species of aconitum6. Although dormancy, seedling mortality and highly toxic, Aconitum tubers are used as unsustainable exploitation of plants in herbal drugs after heat or alkaline treatment conditions other than their natural habitat resulting in reduction in toxicity on account have increased threat to natural population of deacetylation, debenzoylation and of medicinal plants. Research studies on A. oxidation. The processed tubers have shown sinomontanum4 has revealed that chilling is lesser alkaloid content than unprocessed the prerequisite condition for overall ones when separated by modified the HPLC improvement in seed germination. Similarly method using C18 column gradient eluted the low temperature condition was reported with acetonitrile and ammonium bicarbonate to be the requirement for embryo growth in buffer7. Eleven diterpene alkaloids were A. lycoctonum. Besides, the low pollen isolated from Aconitum with oxazolidine8 germinability in alpine conditions is ring containing isoatisine and coryphine responsible for poor seed set. Pollen fertility being the most active ones. Seven C19 can aid in the successful adaptation of plant norditerpenoid and a C20 diterpene alkaloid species. Recovery and conservation of have been reported in Aconitum jaulense9. threatened species is possible through use of A new alkaloid 8-O-Azeloyl-14- reproductive biology5. Conservation and benzoylaconine was reported from the roots commercial cultivation of the endangered of Aconitum karacolicum10. 6- species is possible through effective dehydroacetyl-sepaconitine and 13- breeding programs. Conservation by hydroxylappoconitine, two new cultivation may prove to be lucrative as it is norditerpenoids and Heterophylline A, trouble free. Some standard methods are to Heterophylline B were reported from the be developed by scientists and researchers roots of Aconitum heterophyllum Wall11, 12. for benefit of farmers in particular and Spicatine A and Spicatine B, two new consumer in general. Tissue culture can norditerpenoids were isolated in A. greatly aid in the conservation of threatened Spicatum13. Swatinine, 4[2-(methoxy- Aconitum sp. As small amounts of plant carbonyl) anilino] 4-oxobutanoic acid, material can generate a large number of delphatine, lappaconitine, puberanine and N- disease free propagules which can be acetylsepaconitine have been isolated from retaken to their native habitats. It also helps A. Leave Royle. Two flavonoids Quercitin in broadening the genetic database, and kaempferol derrivatives with two known augmenting the yield and production of flavnol glycisides clovin and robinin have active secondary metabolites. It has the been isolated and identified from roots of A. potential to act as the source of future plant anthora L14. liquiritigenin and liquiritin- based medicines. On account of increased flavonoids were reported from A. demand for herbal products, the concern for carmichaeli15. increasing the secondary metabolite production through genetic engineering is

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MATERIALS AND METHODS resolution and short run time. The use of 0.1% formic acid has been adopted to The root part of Aconitum increase ionization efficiency and improve chasmanthum (15 Kg) were collected from peak shape. sopi-a place 85 km away from Kargil Ladakh (J&K, India) in august 2010. The Liquid chromatography–tandem mass specimen was identified by Akhtar H. spectrometry (LC–ESI-MSMS) Malik, Curator, Centre for Biodiversity & For LC-ESI-MSn experiments, LC- Taxanomy, University of Kashmir MS QqQ-6410B equipment (Agilent (Specimen deposited under accession No. Technologies) comprising a chromato- 341320 and Collection No. 1300- Bilal, graphic system 1260 Infinity (Agilent Kash). Technologies) coupled with an Agilent Triple Quad mass spectrometer fitted with Extraction an ESI source was used. The conditions of The air dried, finely powdered root ESI source were set as follows: sheath and material (5Kg) was extracted for 72 hours auxillary gases flow, 25 and 3 arbitrary with methanol to afford the respective units, respectively; spray voltage, 4KV; extract, which was concentrated under capillary temperature 325oC and tube length reduced pressure and was coded as ACE. voltage: 120 V. The samples were analyzed in both positive and negative modes and full Reagents and chemicals scan mass range was set between m/z 100- Acetonitrile methanol and formic 1200 with the acquisition of centroided-type acid (Sigma Aldrich) HPLC grade were mass spectra. Accurate mass analyses were purchased. HPLC grade deionised water calibrated according to manufacturers (18.2 MΩ) prepared by using the Milli-Q guidelines. In the MSn experiments, data water purification system (USA) was dependent MSn scanning was performed to procured. Other reagents were of analytical minimize total analysis time as it can trigger grade and are available commercially. fragmentation spectra of target ions and prevent repitition by dynamic exclusion LC System settings. The software of the Agilent The analyses were obtained based on technologies was used for data analysis. an Angilent 1260 infinite series, which After the automated removal of noise and consisted of a pump, Autosampler, detector baseline signals, the theoretical extract and a column component. The chromatogram of main alkaloids was shown chromatographic separations were in the plot (fig. 1). performed on chromo lith RP-18e column (4.6mm ID, 50mm length) which was RESULTS AND DISCUSSION maintained at room temperature. Mobile phase consisted of (A) aqueous formic acid For LC-MS experiments different (0.1%) and (B) methanol, with the gradient fragmentation measurements were elution as follows:0-8 min, 12%-25% B; 8- conducted in independent LC-MSn 12 min, isocratic at 25% of B; 12-16 min, experiments. In the first measurement the 25%-40% of B; 16-40 min, 40%-50% of B, survey scan was performed in analyser at 40-50 min, 50%-100% of B. The flow rate (R=30,000) followed by MS2 scan at was 1ml/min. The optimized (R=15,000). The accurate tandem mass chromatographic conditions like selection of spectra can provide strong evidences for mobile phase are used to obtain a good identification of fragments and neutral loss.

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In the separate measurement in which ESI reference to Uttaranchal (India). Med. Plant. MS2 and MS3 experiments using dynode conserv, 2005, 11: 9-15. detection provided more fragment ions and 4. Dosmann MS, Stratification improves and is order of fragmentation could be determined likely required for germination of Aconitum to show evidence of structural identification. sinomontanum. Hort. Technol, 2002, 12: 423-425. Twelve alkaloids were picked from the 5. Kuniyal CP, Rajsekaran C, Prasad P and chromatogram at the retention times from Bhadula SK, Propagation of a threatened 1.74-13.75 min. Among them seven mediinal herb Aconitum atrox (Bruhl) Muk. compounds were identified as through tuber segments. Plant Genet. heterophyllisine, atidine, atisine, Resour. Newsl. 2003, 135: 59-62. chasmoaconitine, 3- bichaconine, neoline 6. Wang Z, Wen J, Xing J and He and heteroatisine by comparing their Y, Quantitative determination alkaloids in retention times and on the basis of their four species of Aconitum by HPLC. J. fragmentation patterns in MS-MS data. The Pharm. Biomed. Anal., 2006, 40: 1031- other compounds were tentatively identified 1034. by comparing their mass data with those 7. Jiang ZH, Xie Y, Zhou H, Wang JR and Liu ZQ et al., Quantification of Aconitum reported in literature. The Retention time alkaloids in aconite roots by a modified RP- values and mass data of deduced compounds HPLC method. Phytochem. Anal., 2005, 16: from the peaks are summarized in table 1. 415-421. The fragmentation patterns of the identified 8. Dzhakhang FN and Bessonova compounds are shown in (fig. 2a to2e). IA, Alkaloids of Aconitum coreanum. X. Curare-like activity structure relationship. CONCLUSION Chem. Nat. Comp, 2002, 38: 74-77. 9. Shim SH, Kim JS, Kang SS, Son KH and In this study a simple and efficient Bae KH, Alkaloidal constituents LC-ESI-MSn method has been developed from Aconitum jaulense. Arch. Pharmacol. for providing a sensitive and applicable tool Res., 2003 9: 725-729. for determination and identification of 10. Chodoeva A, Bosc JJ, Guillon J, Decendit alkaloids from the roots of aconitum A and Petraud M et al., 8-O-Azeloyl-14- chasmanthum which can also be used in benzoylaconine: A new alkaloid from the other Aconitum herbs and their roots of Aconitum karacolicum Rapcs. and its antiproliferative activities. Bioorg. Med. formulations. Moreover the LC-ESI-Mass Chem, 2005 13: 6493-6501. spectrometry has been demonstrated to be an 11. Ahmad M, Ahmad W, Ahmad M, Zeeshan effective tool for analysis of the components M, Obaidullah and Shaheen F, and searching of novel compounds in plant Norditerpenoid alkaloids from the roots extracts of especially endangered plant of Aconitum heterophyllum Wall with species. It also provided the essential data antibacterial activity. J. Enzyme Inhibition for further pharmacological and Med. Chem, 2008 23: 1018-1022. toxicological studies on Aconitum plants. 12. Nisar M, Ahmad M, Wadood N, Lodhi MA, Shaheen F and Choudhary MI, New REFERENCES diterpenoid alkaloids from Aconitum heterophyllum wall: Selective butyryl- 1. Bakshi, Guha DN, A Lexicon of medicinal cholinestrase inhibitors. J. Enzyme plants in india, 1999, vol 2. Inhibition Med. Chem, 2009, 24: 47-51. 2. Lane B, the encyclopedia of forensic 13. Gao LM, Yan LY, He HY and Wei science. Med. History, 2004, 36:53-69. XM, Norditerpenoid alkaloids from 3. Shah NC, Conservation aspects of Aconitum Aconitum spicatum Stapf. J. Integr. Plant species in the Himalayas with special Biol, 2006, 48: 364-369.

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14. Mariani C, Braca A, Vitalini S, De Tommasi 15. Lyu HN, Ho-Young K, Dae-Young L, N, Visioli F and Fico G, Flavonoid Kyong-Tai K, Se-Young K and Nam-In characterization and In vitro antioxidant B, Isolation of flavonoids from processed activity of Aconitum anthora L. aconiti tuber. J. Appl. Biol. Chem., 2008, (Ranunculaceae). Phytochemistry, 2008, 69: 51:165-168. 1220-1226.

Table 1. Compounds identified in Aconitum chasmanthum

TR in Characteristic S. No m/z Adduct peaks Identification (min) fragment ions [M+2]377[M- MS2[376]: 374, 354, 1 7.6-8.10 375.4 [C22H33NO4] Heterophyllisine 1]374 324 2 8.4-8.8 359.9 [C22H33NO3] [M+1]360.9 MS2[361]: 316, 208 Atidine

3 8.8-10.3 343.4[C22H33NO2] [M+33]377 - Atisine

4 [M+2]616.3 MS2[614]:492, 432, 11.2-11.6 614.3[C34H48O9N] Chasmoaconitine [M+Na]637 386, 330 5 [M+1]484.4 11.8-12.3 483.4[C25H41NO8] MS2[483]: 465, 303 3-α Bikhaconine [M+3H]486 6 [M+1]393.3 12.4:12.8 392.3[C22H33NO5] MS2[392]:303,305 Heteratisine [M+42]434] 7 [M+19]456 12.4-12.9 438.5[C24H39NO6] MS2[438]:393, 302 Neoline [M+33]470]

Figure 1. LCMS- Total ion chromatogram of Aconitum chasmanthum

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Figure 2a.

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Figure 2a. Fragmentation p attern of chasmo aconitine

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Figure 2b. Fragmentation of neoline

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Figure 2c. Fragmentation of bichaconine

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Figure 2d. Fragmentation of heterophylisine

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Figure 2d.

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Figure 2e. Fragmentation of atidine

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