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Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 185476 18 pageshttpdxdoiorg1011552013185476

Research ArticleProstaglandin Analogous and Antioxidant Activity MediatedGastroprotective Action of Tabernaemontana divaricata (L) RBr Flower Methanolic Extract against Chemically InducedGastric Ulcers in Rats

Mohammed Safwan Ali Khan12 Abdul Manan Mat Jais1 and Adiba Afreen2

1 Department of Biomedical Sciences Faculty of Medicine and Health Sciences Universiti Putra Malaysia43400 Serdang Selangor Malaysia

2 Natural Products Research Laboratory Anwarul Uloom College of Pharmacy New Mallepally HyderabadAndhra Pradesh 500001 India

Correspondence should be addressed to Mohammed Safwan Ali Khan safwanpharmagmailcom

Received 31 March 2013 Revised 23 May 2013 Accepted 12 June 2013

Academic Editor Richard Gomer

Copyright copy 2013 Mohammed Safwan Ali Khan et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

The present study was conducted to evaluate the antiulcerogenic effect and recognize the basic mechanism of action ofTabernaemontana divaricata (L) R Br flowers T divaricata flower methanolic extract (TDFME) was screened for antiulceractivity versus aspirin and ethanol induced gastric ulcers at three dosesmdash125 250 and 500mgkgmdashorally using misoprostol asa standard Besides histopathological examination seven parameters that is ulcer index total protein nonprotein sulphhydrylsmucin catalase malondialdehyde and superoxide dismutase levels were estimated In addition toHPLCprofiling GC-MS analysisand electrospray ionizationmdashhigh resolution mass spectral (ESI-HRMS) analysis of crude TDFME were carried out in an attemptto identify known phytochemicals present in the extract on the basis of mz value The results revealed a significant increase inthe levels of catalase superoxide dismutase mucin and nonprotein sulphhydryls while they revealed a reduction in ulcer indexthe levels of total protein and malondialdehyde Histopathological observations also demonstrated the protective effect Thoughall the doses of TDFME exhibited gastroprotective function higher doses were found to be more effective Mass spectral analysisgave a few characteristicmz values suggesting the presence of a few known indole alkaloids while HPLC profiling highlighted thecomplexity of the extract TDFMEwas found to exhibit its gastroprotective effect through antioxidantmechanism and by enhancingthe production of gastric mucous

1 Introduction

Tabernaemontana divaricata (L) R Br ex Roemer andSchultes (Apocynaceae) has high medicinal value and itis used in the ethnic systems of medicine all over theworld Traditionally crepe jasmine flowers are used to treatcorneal inflammation [1] ophthalmia [2 3] and in Unanimedicine for the management of pain [4] Also the flowerssoaked water is sprinkled on smallpox patients [5] Recentinvestigations on T divaricata flowers reported hippocraticscreening [6] antianxiety [7] anticonvulsant [8 9] antidi-abetic and cytotoxic [10] antifertility [11] antiinflammatory

[12 13] antioxidant [13] and gastroprotective effects [14] Incontinuation to our research on pharmacological assessmentof the gastroprotective potential of T divaricata flowers thepresent study was conducted to understand its mechanism ofaction in chemically induced gastric ulcers

2 Material and Methods

21 Plant Material and the Preparation of Extract The pin-wheel flowers were collected from the local areas of Ameerpetand Mallepally of Hyderabad district Flowers of Tabernae-montana divaricata (L) R Br ex Roemer and JA Schultes

2 BioMed Research International

belonging to the Apocynaceae family were authenticated bya plant taxonomist Dr P V Prasanna (ScientistmdashE) at theBotanical Survey of India Deccan Regional Centre Hyder-abad (establishment under the Ministry of Environment ampForests Government of India) The specimen deposited inthe herbariumwas assigned a voucher number BSID 887Theextract was prepared as per the procedure described earlier[14] Briefly after collection flowers were shade dried andcoarsely powdered Approximately 500 gm of the powderedflowers was extracted using methanol 99 pure (SD Fine-Chem) in a soxhlet apparatus The extract was concentratedunder reduced pressure and stored in an airtight containerin a refrigerator at the temperature below 10∘C The driedmass of crudeTabernaemontana divaricataflowermethanolicextract (TDFME) was found to be 34 ww with respect tothe dried flowersThe solution of TDFMEwas prepared usingdistilled water for the evaluation of the antiulcer activity

22 Drugs and Chemicals Chloroform AR diethyl ethermethanol AR phenolphthalein pH indicator solution andsodium hydroxide pellets were procured from SD Fine-Chem Limited Mumbai while pure aspirin was obtainedfrom Divis Laboratories Hyderabad Absolute ethanol waspurchased from Changshu Yangyuan Chem China andmisoprostol (as misoprost-200) was purchased from CiplaLtd Goa while surgical spirit was obtained from KakatiyaPharma Hyderabad Topferrsquos Reagent and distilled waterwere obtained fromNice Chemicals China and Stangen FineChemicals Hyderabad respectively All chemicals were usedwithout further purification

23 Animals Adult male Wistar rats weighing 150ndash200 gmwere used for the evaluation of antiulcer activityThe animalswere maintained under standard laboratory conditions inpolypropylene cages under 12 hr lightdark cycle controlledtemperature (24 plusmn 2∘C) fed with commercial pellet dietand water ad libitum in an animal house approved by theCommittee for the Purpose and Supervision on Experimentson Animals (Reg no 1534PO-a11CPCSEA) All the ani-mals were acclimatized to the laboratory environment for 10days before the initiation of the experiments The protocol(IAECAUCP2011-1202) was approved by the InstitutionalAnimal Ethical Committee before the commencement ofanimal experimentation All measures were taken to ensurethat the experiments were conducted in accordance with theinstructions of IAEC Anwarul Uloom College of PharmacyNew Mallepally Hyderabad Andhra Pradesh India

24 Acute Toxicity Test and Selection of Test Doses Three testdoses (125 250 and 500mgkg) were selected in range of 116to 14 of the maximum oral safe doses reported in earlierarticles [7 9 11]

25 Evaluation of Antiulcer Activity by In Vivo Assays

251 Acute Ethanol Induced Gastric Ulcers All the animals(119899 = 6) in each group were fasted for 36 hours before theadministration of ethanol The standard drug (misoprostol

100 120583gkg po) or the test extract was administered onehour before ethanol administration Ethanol (90) wasadministered to all animals at a dose of 1mL200 gm Afterone hour all animals were sacrificed and stomachs wereisolated [15] Lesion severity was determined by measuringulcer index Later stomachs were subjected to the estimationof catalase superoxide dismutase malondialdehyde mucintotal protein and nonprotein sulphhydryl contents followingstandard procedures

(1) Ulcer Index (UI) Mean ulcer score for each animal isexpressed as ulcer index The stomachs were washed withrunning water to see the ulcers in the glandular portion of thestomach The number of the ulcers per stomach were notedand the severity of the ulcers was scoredmicroscopically withthe help of hand lens (10x) and scoring was done as perKulkarni (1999) [16] as following

0 = normal stomach

05 = red colouration

1 = spot ulcers

15 = hemorrhagic streaks

2 = ulcers gt 3mm but lt5mm

3 = ulcers gt 5mm

(2) Assessment of the Oxidative Damage in Gastric Tis-sue After measuring the ulcer index the stomachs werewashed with 09 (wv) NaCl cut into small pieces andhomogenized with a glass homogenizer in ice-cold 015MKCl solution to produce a 20 (wv) homogenate Thehomogenate was used for the determination of variousbiochemical parameters

(a) Estimation of Catalase (CAT)Catalase activity in stomachtissue was determined according to the method of Leyck andParnham (1990) [17] The stomach tissue was scraped andhomogenized in ice cold saline medium with the help of ahomogenizer The solution was centrifuged for 10 minutesat 3000 g-force and collected for the experiment 100 120583L ofthe supernatant was added to a solution of 3 120583L of H

2O2

phosphate buffer mixture (50mM phosphate buffer pH 70and 30 H

2O2) The change in optical density at 240 nm per

unit time was measured

(b) Estimation of Superoxide Dismutase (SOD) Superoxidedismutase activity in stomach tissue was determined accord-ing to the method of Fridovich (1995) [18] The stomachtissue was scrapped and homogenized in ice cold normalsaline medium with the help of a homogenizer Then thetissue homogenate was centrifuged for 10 minutes at 3000 g-force and the supernatant was collected and used for theestimation of SOD activity 10mL of the solution was taken ina test tube andmixed with 05mL of 50mMphosphate buffer(pH 78) 01mM of EDTA 005mM xanthine and 001mMcytochrome c and then 100mL of 25mM of xanthine

BioMed Research International 3

oxidase was added to start the reaction and the absorbancewas measured at 550 nm

(c) Determination of Malondialdehyde (MDA) The methodreported by Utley et al (1967) [19] was followed The animalswere killed 1 h after ethanol administration The stomachswere removed and each was homogenized in 015MolL KCl(at 4∘C) in a homogenizer to give a 10 wv homogenateAliquots of homogenate 1mL in volume were incubatedat 37∘C for 3 h in a metabolic shaker Then 1mL of 10aqueous trichloroacetic acid (TCA) was added and mixedThe mixture was then centrifuged at 800 g-force for 10minOnemLof the supernatantwas removed andmixedwith 1mLof 067 2-thiobarbituric acid inwater and placed in a boilingwater bath for 10min The mixture was cooled and dilutedwith 1mL distilled water The absorbance of the solution wasthen read at 535 nmThe content ofmalondialdehyde (nmolgwet tissue) and index of the magnitude of lipid peroxidationwere then calculated by referring to a standard curve of themalondialdehyde solution

(d) Determination of Gastric Wall Mucin Content (GWM)Mucin content was estimated by the method describedby Corne et al (1974) [20] with some modifications Theglandular segments of the stomachs of rats subjected to theethanol induced ulcers model were isolated and weighedEach glandular segment was immediately immersed in 10mLof 01 alcian blue solution (016M sucrose005M sodiumacetate pH 58) After immersion for 2 h excess dye wasremoved by two successive rinses with 10mL of 025Msucrose first for 15min and then for 45min The stomachswere all sequentially transferred to a 05M magnesiumchloride and shaken for 2 h FourmL of the blue extract wasthen shaken vigorously with an equal volume of ether Theresulting emulsion was centrifuged at 3600 g-force and theabsorbance of the aqueous layer was read at 580 nm Theamount of alcian blue extracted per gram of wet glandulartissue was then calculated

(e) Estimation of Nonprotein Sulphhydryls (NPSH) Gastricmucosal nonprotein sulphhydryls were measured accordingto the method of Sedlak and Lindsay (1968) [21] Theglandular part of the stomach was homogenized in ice-cold 002 mMolL ethylenediaminetetraacetic acid (EDTA)Aliquots of 5mL of the homogenates was mixed in 15mLtest tubes with 4mL of distilled water and 1mL of 50wv trichloroacetic acid (TCA) The tubes were shakenintermittently for 10min and centrifuged at 3000 g-forceTwomL of supernatant were mixed with 4mL of 04molLTris buffer at pH 89 Then 01mL of 001M 551015840-dithiobis(2-nitrobenzoic acid) (DTNB) was added and the volumewas made up to 10mL with 39mL of absolute methanol Thesample was shaken and the absorbance was measured within5min of DTNB addition at 412 nm against a reagent blank

(f) Estimation of Total Protein Content (TP) Total proteincontent was estimated by the method of Lowry et al (1951)[22] The dissolved proteins in gastric juice were estimatedin the alcoholic precipitate obtained by adding 90 alcohol

with gastric juice in a 9 1 ratio respectively Then 01mL ofalcoholic precipitate of gastric juice was dissolved in 1mL of01 N NaOH From this 005mL was taken in another testtube and 4mL of alkaline mixture was added and allowedto stand After 10min 04mL of phenol reagent was addedand again allowed to stand for 10min for the developmentof colour Reading was taken against a blank prepared withdistilled water at 610 nm The protein content was calculatedfrom the standard curve prepared with bovine albumin andhas been expressed in terms of 120583gmL of gastric juice

252 Aspirin (NSAID) Induced Gastric Mucosal DamageThe gastric ulcers were induced by administering aspirin(200mgkg po) once daily for five days [23] The animalswere divided into five groups (119899 = 6) Group-I served asnegative control and received only vehicle Group-II servedas standard and was treated with standard drug (misoprostol100 120583gkg po) [24] Groups-III -IV and -V received threedifferent doses (125 250 and 500mgkg) of test extract andserved as test groups All the treatments were made 30minbefore administering aspirin once daily for five days Therats were sacrificed on the fifth day and the ulcer index wasdetermined as mentioned earlier

(1) Histopathological Studies The isolated stomachs werepreserved in 15 formalin solution and were sent to thepathologist for histopathological examination by stainingwith haematoxylin and eosin The morphological changeswere observed and recorded with 100x lenses [25]

26 Analytical Profile of TDFME

261 High Performance Liquid Chromatographic AnalysisHPLC analysis was carried out at the analytical developmentlaboratory Mylan Laboratories Limited Bolarum Hyder-abad HPLC analysis of TDFME was performed by gradientsystem using Waters 2996 photodiode array HPLC systemwith Kromasil C

18- (250 times 45mm 5120583m) column Two

solvents A (water with 01 trifluoroacetic acid) and B(acetonitrile with 01 trifluoroacetic acid) were used forelution of constituents The column was equilibrated in85 A15 B prior to the injection of the sample andupon injection this composition was then changed to 60A40 B over 30min utilizing a linear gradient followed bychanging to 50 A50 B over the next 10min and thenthe concentration was returned to 85 A15 B over thefinal 10min The flow rate was set to 1mLmin injectionvolume was 10 120583L and column temperature was maintainedat 30∘CThe system was run for 60 minutesThe eluents weremonitored at 255 and 350 nm The peak numbers retentiontimes areas and percentage areas were recorded

262 Gas ChromatographymdashMass Spectral AnalysisTDFME was subjected to GC-MS analysis by Agilent 6890GC with 5973N MSD CP-Sil 8 CB column of 30m length times025mm id times 025 120583 and hydrogen gas 12mLmin was usedas mobile phase The sample was electro-ionized by a 70 eVsource and quadruple temperatures were maintained at


temperature 230∘C and 150∘C respectively The transferlineand injection temperature were 270∘CThe oven temperatureprogramwas set as 50∘C for 2min initially then asgt10∘Cminramp with final temperature as 280∘C held for 5min

263 Electrospray IonizationmdashHigh Resolution Mass SpectralAnalysis Crude TDFME was subjected to high resolutionmass spectral analysis by Thermo Exactive having a quadru-ple detector mass analyzer at the National Centre for MassSpectrometry the Indian Institute of Chemical TechnologyHyderabad by Dr U V R Vijayasarthi The sample wasionized by heated electro-spray ionization (HESI) methodin positive mode A mixture of methanol (80) and formicacid (01) was used as a mobile phase and the sample flowrate was set to 300120583Lmin The capillary voltage was 40 kVand the source and transferline temperatures were 350∘C and300∘C respectively Nitrogen was used as an auxiliary andsheath gas the supply was set to 35 and 10 units for respectivepurposes The nitrogen was supplied through Peak ScientificNM30L nitrogen generatorThe119898119911 values were recorded inthe range of 0ndash1000

264 Ehrlichrsquos Confirmatory Chemical Test for Indole Alka-loids For the detection of indole alkaloids Ehrlichrsquos reagent(025 g of p-dimethylaminobenzaldehyde dissolved in 130mLsulphuric acid and 70mL distilled water allowed to standfor 24 hours) was used [26] Test extract gave a blue colourindicating the presence of indole alkaloids

27 Statistical Analysis Data obtained was analyzed by one-way ANOVA followed by Dunnettrsquos multiple comparisonsposthoc test using Graphpad Prism version 40 32 bit forwindows Graphpad software San Diego California USA(httpwwwgraphpadcom) The values are expressed asMean plusmn standard error of mean (SEM) 119875 lt 005 wasconsidered statistically significant

3 Results and Discussion

31 Results of Ethanol Induced Gastric Ulcers Model Ethanolis a common potential ulcerogenic agent that induces gastrichemorrhagic erosion on intragastric administration (Shettyet al 2000) [27] Ulcers caused by chemical inducers likeethanol are due to a number of contributing factors whichinclude effects on mucosal blood flow platelet thrombidamage to capillary endothelium and release of arachidonatemetabolites leukotriene C

4D4(LTC4D4) and platelet acti-

vating factor (PAF) (Goel and Bhattacharya 1991) [28] Ratgastric mucosal damage induced by high concentration ofethanol is widely used to investigate gastroprotective effectof medicinal plants (Zhu et al 1997) [29] Ethanol inducedlesion formation is due to different factors like stasis of gastricblood flow contributing significantly to the development ofhemorrhagic as well as necrotic aspects of tissue injuryMillerat al (1985) stated that the free radicals produced duringlipid peroxidation of cells and the cell membranes producemucosal mast cell degranulation leading to the release ofvasoactive mediators including histamine [30] Histamine

is responsible for the activation of adenyl cyclase whichproduces cyclic AMP which in turn activates the gastricproton pumpand releases hydrogen ions (Tripathi 2003) [31]The amount of hydrogen ions generated is proportional tothe gastric acid formed When there is an increase in thegastric acid production this leads to erosion or self-digestionof the gastric mucosa that protects the stomach Thus thismay render that gastric mucosa more vulnerable to furtherdamage or infections

The products of 5-lipoxygenase pathway may also playa key role in the development of such ulcer (Lange et al1985) [32] Mizui and Doteuchi (1986) and Terano et al(1989) reported the involvement of free radicals in ethanolinduced gastric ulceration and antioxidant action and lipidperoxidation respectively [33 34] Alcohol causes ulcerationdue to the excessive production of gastric mucosal LTC-4 and LTD-4 (Nielsen et al 1987) [35] Glavin and Szabo(1992) further explained the mechanism of ethanol inducedgastric ulcers They mentioned that ethanol induces ulcersby reducing the gastric mucosal blood flow and mucusproduction in the gastric lumen decreasing endogenous glu-tathione and prostaglandin levels and increasing ischemiagastric vascular permeability acid ldquoback diffusionrdquo histaminerelease efflux of sodium and potassium influx of calciumgeneration of free radicals and the production of leukotrienes[36] According to Al-Harbi et al (1997) the genesis ofethanol induced gastric lesions is multifactorial with thedepletion of gastric wall mucous and leukotriene release [37]

Oral administration of absolute alcohol causes severedamage to the gastric mucosaThe hemorrhagic and necroticeffects of ethanol can be reversed by prostaglandins as theyeffectively protect the mucosa against chemically inducedgastric injuries (Robert et al 1979) [38] Therefore treatmentwith analogous prostaglandins could be effective in reducingthis type of damage Considering the above fact misoprostol(methyl-PGE

1ester) was used as the standard drug in the

present study The mechanisms involved in gastric mucosalprotection of misoprostol are stimulation of gastric andduodenal mucosal production and bicarbonate secretion bythe gastric mucosa the increase in mucosal blood flow whichmaintains mucosal integrity the efficient removal of toxicsubstances the inhibition of basal gastric acid secretion andin response to food histamine pentagastrin and coffee bydirect action on parietal cells [39ndash42] A comparative studywas conducted in rats to assess mucosal protection offeredby misoprostol cimetidine and sucralfate against gastriculcers induced by aspirin indomethacin stress sodiumtaurocholate and ethanol Misoprostol offered better gastro-protection by reducing the mean number of gastric lesionsby 58 and 70 of the control group at 100 and 200120583gkgrespectively Further it is stated that misoprostol providesmucosal protection by a mechanism that does not requirea reduction of gastric acid secretion for its protective effect[43] The results of ethanol induced gastric ulcers model arepresented in Table 1

311 Ulcer Index (UI) The standard drug misoprostol100 120583gkg and test extract (TDFME) at all the test doses used


Table 1 Results of ethanol induced gastric ulcers model

Parameters Negative controlStandard

misoprostol100120583gkg

Test-ITDFME125mgkg

Test-IITDFME250mgkg

Test-IIITDFME500mgkg

Catalase 289 plusmn 011 (027) 409 plusmn 018 (044)b 386 plusmn 012 (049)a 394 plusmn 020 (049)a 423 plusmn 038 (094)b

Malondialdehyde 711 plusmn 030 (074) 269 plusmn 023 (057)c 564 plusmn 029 (073)b 494 plusmn 016 (040)c 358 plusmn 024 (060)c

Mucin content 052 plusmn 006 (015) 107 plusmn 011 (027)b 095 plusmn 015 (037)a 115 plusmn 010 (024)b 189 plusmn 011 (029)c

Nonprotein sulphhydrylconcentration 231 plusmn 018 (045) 455 plusmn 017 (042)c 300 plusmn 019 (047)b 307 plusmn 009 (024)b 498 plusmn 000 (023)c

Superoxide dismutase 275 plusmn 035 (086) 599 plusmn 031 (078)c 244 plusmn 030 (074)ns 405 plusmn 023 (057)a 407 plusmn 024 (059)a

Total protein content 3425 plusmn 409 (1003) 3132 plusmn 322 (791)c 3217 plusmn 468 (1147)b 3165 plusmn 550 (1349)b 2517 plusmn 415 (1017)c

Ulcer index 10 plusmn 028 (070) 450 plusmn 042 (104)c 708 plusmn 058 (142)c 400 plusmn 036 (089)c 150 plusmn 046 (114)c

Note sample size (119899) = 6 rats per group Data is expressed as Mean plusmn Standard Error of Mean and Standard Deviation in parenthesis a119875 lt 005 b119875 lt 001c119875 lt 0001 and nsnonsignificant versus negative control (on statistical analysis with ANOVA followed by Dunnettrsquos multiple comparison post-hoc test)

in the study (125ndash500mgkg) showed significant reduction(119875 lt 0001) in ulcer index

(1) Catalase (CAT) and Superoxide Dismutase (SOD) Thestandard drug and the high dose of test extract significantly(119875 lt 001) raised the level of catalase Low and moderatedoses of TDFME also produced a similar effect but the levelof significance found was 119875 lt 005 Misoprostol and testextract at moderate and high doses (250 and 500mgkg)used in the study produced significant increase in level ofsuperoxide dismutase with 119875 lt 0001 and 119875 lt 005respectively while there was no significant effect observedwith the low dose of test extract (125mgkg) Das et al (1997)reported that in stress-induced gastric ulcerationMDA levelssignificantly increase with the concomitant decrease in CATand SOD concentrations [44] Test extract at various doses inthe present study significantly decreasedMDA and increasedCAT and SOD concentrations suggesting possibility ofantioxidant activity mediated gastroprotection

(2) Malondialdehyde (MDA) Misoprostol and TDFME atmoderate and high doses produced a significant decrease(119875 lt 0001) in the level of malondialdehyde Low testdose also limited MDA concentration with 119875 lt 001 Stressinactivates mucosal prostaglandin synthesis by accumulatinghydrogen peroxide (prostaglandin biosynthesis inhibitor)and by generating reactive oxygen species (ROS) (Bandy-opadhyay et al 1999) [45]The increase inMDA levels resultsin an increase in free radicals like superoxide anion hydrogenperoxide andhydroxyl radicalsThese radicals are responsiblefor cell degranulation by increasing the peroxidation of cellmembrane lipids causing a loss of structural and functionalintegrity of cell membranes Accumulation of hydrogenperoxide occurs in the mitochondria and cytosol which leadsto an increase in lipid peroxidation if not scavenged by CAT(Michiels et al 1994) [46] Further a strong relationshipbetween the levels of gastric mucosal lipid peroxidation endproducts (marker of oxidative stress) and gastric ulceration instress induced ulcers has been reported (Tandon et al 2004)[47] The decline in malondialdehyde (MDA) concentrationindicates a decrease in lipid peroxidation since it is the endproduct of lipid peroxidation (Buege and Aust 1978) [48]

(3) Gastric Wall Mucus (GWM) Dose dependent increasein mucin content was observed in test groups (with 119875 valueranging from lt005 to lt0001) The effect of the moderatetest dose was comparable with that of the standard groupThe maximum increase in mucin content was found to be inanimals receiving high test dose 500mgkg with (119875 lt 0001)Gastric mucous is an important protective factor for thegastric mucosa and consists of a viscous elastic adherent andtransparent gel formed by 95 water and 5 glycoproteinsthat covers the entire gastrointestinal mucosa Moreovergastric mucous is capable of acting as an antioxidant andthus can reduce mucosal damage mediated by oxygen freeradicals (Repetto and Llesuy 2002) [49] The mucous gellayer is a complex secretion containing inorganic materialssecretory IgA lactoferin high molecular weight proteinscontaining serine threonine proline and alanine and carbo-hydrates mainly galactose galactosamine glucosamine andfructose Approximately 600 carbohydrate side chains permolecule of glycoprotein are present (Schrager 1969) [50]The physicochemical properties make it a relatively resistantacid barrier (Flemstrom and Garner 1982) [51] Mucin is themajor part of gastric mucous an important pre-epithelialfactor that acts as a first line of defense against ulcerogens(Zalewsky and Moody 1979) [52] Prostaglandins E

2and I2

are predominantly synthesized by the gastric mucosa and areknown to inhibit the secretion of gastric acid and stimulatethe secretion of mucous and bicarbonate Hydrophobic sur-factants like phospholipid secretion in the gastric epithelialcells are also stimulated by the prostaglandins (Aly 1987) [53]Decrease in mucosal secretion is thus considered importantin gastric ulceration (Goel and Bhattacharya 1991) [28] Thetest extract and the standard drug showed similar effectsthat is increase in the production of mucin The degree ofenhancement observed in standard and test groups was alsocomparable It would be reasonable to comment that TDFMEexhibited prostaglandin analogous effect on gastric mucosa

(4) Nonprotein Sulphhydryls (NPSH) Treatment with thestandard drug and TDFME 500mgkg produced significantrestorative effect on nonprotein sulphhydryl concentration(with 119875 lt 0001) TDFME low and medium dose (125and 250mgkg) also elevated NPSH content Sulphhydryl


(a) (b) (c) (d)

Figure 1 Photos of ratrsquos stomachs subjected to aspirin induced gastric ulcers Note (a) standard (misoprostol 100 120583gkg) (b) test-I (TDFME125mgkg) (c) test-II (TDFME 250mgkg) (d) test-III (TDFME 500mgkg)

(a) (b) (c) (d) (e)

Figure 2 Histopathological slides of ratrsquos stomachs subjected to aspirin induced gastric ulcers Note (a) negative control (b) standard(misoprostol 100 120583gkg) (c) test-I (TDFME 125mgkg) (d) test-II (TDFME 250mgkg) and (e) test-III (TDFME 500mgkg)

Table 2 Results of Aspirin induced gastric ulcers model


Misoprostol100120583gkg

Test-ITDFME125mgkg

Test-IITDFME250mgkg


Ulcer index 95 plusmn 042 400 plusmn 036c

716 plusmn 040b

75 plusmn 056b

150 plusmn 028c

b119875 lt 001 c119875 lt 0001

compounds in living organisms play a central role in themaintenance of gastric integrity particularly when ROS areinvolved in the pathogenesis of tissue damage (Kimura etal 2001) [54] It is reported that a significant decrease ingastric non-protein sulph-hydryls occurs following ethanoladministration by generating enormous amount of freeradicals (AlMofleh et al 2007) [55]Hiraishi et al (1994) andSener-Muratoglu et al (2001) demonstrated that the increasein mucosal nonprotein sulphhydryl exerts a gastroprotectiveeffect [56 57]

(5) Total Protein (TP) TDFME pretreatment at all the testdoses significantly decreased protein content in gastric juice500mgkg test dosewasmore effective than the standard doseindicating strengthening potential onmucosal barrier and anincrease in resistance against aggressive factors Leakage ofplasma protein into the gastric juice can cause weakening ofthe gastric mucosal barrier (Mizushima and Kobayashi 1968Grossman 1978) [58 59]

32 Results of Aspirin Induced Gastric Ulcers Model Aspirininduced gastric ulceration model showed dose dependent

increase in ulcer protection Test-I (TDFME 125mgkg)and Test-II (TDFME 250mgkg) significantly (119875 lt 001)decreased the ulcer index while the standard and Test-III (TDFME 500mgkg) more significantly (119875 lt 0001)decreased the mean ulcer score (ulcer index) Thereby theulcer protective effect of high test dose (TDFME 500mgkg)was found to be more prominent than the standard (miso-prostol) Results are shown in Figure 1 and Table 2

321 Histopathological Findings

(A) Negative Control Stomachs of the negative con-trol group showed aggregates of inflammatory cellsmucosal ulceration degenerated epithelial cells andnecrosis (thick arrow in Figure 2(a)) Severe oedemainflammatory infiltration (macrophages and neu-trophils) congestion in vascular spaceswere observedin submucosa (Short arrow in Figure 2(a)) Results areshown in Figure 2

(B) Standard Mucosa of rats treated with the standarddrug (misoprostol 100 120583gkg) was found to be intact


010

005

000

000 500 1000 1500 2000 2500(Minutes)

Chromatogram

(AU

)

3000 3500 4000 4500 5000 5500 6000

Injection 1 Vial 18 Injection volume 1000 Run time 6000 Sample set name TCL 080612Instrument method name TCL INST trial6 Processed channel descr PDA 2550 nm

Processing method TCL process21Flow rate 05 mLmin

Figure 3 HPLC chromatogram of TDFME

13

24 5

6

7 8

9

10

11

(a)

12

13 14

15

16

(b)

Figure 4 GC-MS spectra of TDFME

while few scattered lymphocytes were present indicat-ing inflammation (Thick arrow in Figure 2(b))

(C) TDFME (125mgkg) TDFME (125mgkg) treatedgroup revealed gastric ulceration and haemorrhage(haemorrhagic streaks) in mucosa (Thick arrow inFigure 2(c)) Submucosa showed moderate oedemaand few scattered inflammatory cells (Short arrow inFigure 2(c))

(D) TDFME (250mgkg) Mucosa of rats treated withTDFME (250mgkg) was found to be intact with fewscattered lymphocytes (thick arrow in Figure 2(d))Submucosa revealed mild oedema congestion invascular spaces (short arrow in Figure 2(d))

(E) TDFME (500mgkg) Mucosa of rats treated withTDFME (500mgkg) was found to be normal(Thick arrow in Figure 2(e)) Submucosa showedmild oedema and scattered inflammatory infiltration(Short arrow in Figure 2(e))

33 Results of HPLC Analysis 40 peaks and their respectiveretention times (RT) were recorded on HPLC analysis ofTDFME as per the method described earlier Peaks 5 8 1214 15 16 20 29 and 37 had a noteworthy area and percentagearea The results are shown in Figure 3 and Table 3

34 Results of GC-MS Analysis The GC-MS analysis ofTDFME reveals the presence of 120572-tocopherol (Vit E)cycloartenol docosane eicosane ergost-5-en-ol ibogamine-18-carboxylic acid 12-methoxy methyl ester 9-Octadecenoicacid 11-Octadecenoic acid methyl ester 912-octadecedienoicacid palmitic acid squalene stigmasterol tetracosane andurs-12-en-24-oic acid 3-oxo methyl ester The retentiontimes structures molecular formulae and weights of com-pounds quality match with Wiley 7N Database and the per-centage of the total composition is summarized in Table 4 AGC-MS spectrum showing corresponding peaks is presentedin Figure 4

35 Results of Electrospray IonizationmdashHigh ResolutionMass Spectral Analysis A few characteristic 119898119911 peakswere observed on ESI-HRMS analysis of crude TDFMEThe recorded molecular weights and formulae werefound to match with the reported indole alkaloids Apartfrom corynanthean and ibogan indole alkaloids like 11-methoxy-N-methyldihydropericyclivine 19-epivoacangineisovoacangine and isovoacristine which have been reportedearlier in the flowers (Arambewela and Ranatunge 1991)[60] the observed [M + H]+ peaks in positive mode ESI-HRMS analysis reveal the probable presence of few moreindole alkaloids belonging to subclasses aspidospermatan


Table 3 Results of the HPLC analysis of TDFME

Peak number Retention time Percentage area1 480 3952 575 0983 645 2064 673 0695 770 7156 842 0207 887 0478 1046 6139 1106 18310 1207 17711 1350 03612 1577 60313 1613 10914 1747 199115 1807 76816 1831 40717 1922 02218 1968 06819 2028 22220 2088 65621 2121 03222 2167 31923 2253 21724 2334 20125 2378 21426 2487 01827 2702 02828 2733 01829 2805 45630 2868 01931 2938 02032 3033 03233 3115 03434 3181 02435 3220 05536 3304 07937 3346 58038 3542 15539 3589 02540 3861 069

corynanthean ibogan plumeran and miscellaneous sub-classes for the first time in the flowers The compoundscould be Apparicine Voafinine Vobasine Voacangine 3-Oxocoronaridine 5-Oxocoronaridine 6-Oxocoronaridine5-Hydroxyvoaphylline Lochnericine Pachysiphine and314419-tetrahydro-Olivacine or structural analogues withsimilar molecular formulae and weights The observed[M + H]+ peaks and the list of probable compounds withtheir exact mz values and molecular formulae as per theliterature are summarized in Table 5 The mz peaks and

molecular formulae are shown in ESI-HR mass spectra inFigure 5 The members of Apocynaceae are rich in indolealkaloids and in particular T divaricata contains at least66 characterized indole alkaloids (Pratchayasakul et al2008) [61] The present study highlights the presence ofcertain uncharacterized indole alkaloids or their analoguesin flowers that need to be elucidated with further exhaustivephytochemical studies and may possess medicinal propertiesand find application in modern medicine

Falcao et al (2008) documented an extensive literature onantiulcer and antisecretory activities of various alkaloids [62]Tabersonine is reported to be present in T divaricata flowers(Gomez-Gonzalez et al 1981) [63] Tan et al (2002) reportedthe antiulcer activity of Tabersonine isolated from Enantiachlorantha and Voacanga africana at a dose of 50mgkgorally in rats [64] In addition to this an indole alkaloidTabernaemontanine isolated from flowers leaves stem barkand roots of T divaricata improved the blood supply tovarious organs and vascular tissue by vasodilation in dogs(Elkeiy et al 1966 Van Beek et al 1984) [5 65] Thisalkaloid may exhibit therapeutic effect in gastric injuriesresulting from decreased blood flow to the gastric mucosaFurther the crude extracts of T divaricata are reported topossess antiinflammatory activity (Henriques et al 1996Satyanarayana et al 2004 Thambi et al 2006) [12 13 66]According to the recent review report of Awaad et al (2013)the prophylactic antiulcerogenic activity of natural productsinvolves the antioxidant and antiinflammatory mechanismswhile the therapeutic effects are due to the antisecretory andhealing properties [67]

The present day treatment approach towards manage-ment of peptic ulcer disease employs antihistaminics (H

2-

receptor antagonists like cimetidine famotidine ranitidineroxatidine) and anticholinergics (selective M

1muscarinic

receptor antagonists like pirenzepine) that produce antise-cretory and antispasmodic effects respectively (Miller 1985)[30] T divaricata contains isovoacristine an indole alkaloidthat exhibited antihistaminic anticholinergic and skeletalmuscle relaxant properties in a study on isolated guineapig ileum and rabbit respectively (Rao and Singri 1979)[68] Hence the protective effect could be a consequence ofantihistaminic and anticholinergic activities demonstrated bysuch phytochemicals Besides these facts the hippocratic invivo screening of T divaricata leaves and flowers conductedin rats by Taesotikul et al (1989) suggests depressive effectson both peripheral and central nervous systems [6] Thedepressant effect can limit the release of histamine animportant local hormone and chemical mediator that evokesgastric acid secretion with other stimulating factors likegastrin vagal stimulation and food by activating protonpump (H+K+ATPase) through H

2receptors (Tripathi 2003)

[31] Therefore the gastroprotective effect exhibited by Tdivaricata flowers can be attributed mostly to the indolealkaloids present in it

Furthermore a few indole alkaloids from T divari-cata have shown antimicrobial activity in in vitro studiesApparicine isolated by cell suspension culture of T divaricatainhibited Polio III virus at a concentration of 250120583gmL(Pawelka and Stockigt 1983 Andrade et al 2005 Farnsworth


Table 4 Results of the GC-MS analysis of TDFME

Peak no Retentiontime in min Compounds with structures molecular formulae and molecular weights

Qualitymatch with

Wileydatabase

Percentage oftotal

composition

1 1762O

O

Hexadecanoic acid methyl ester (methyl palmitate)

C17H34O2 [27045]

98 103

2 1812 O

OH

Hexadecanoic acid (Palmitic acid)

C16H32O2 [25642]

99 080

3 1925 O

O

912-Octadecadienoic acid methyl ester

C19H34O2 [29447]

99 073

4 1931O

O

11-Octadecenoic acid methyl ester

C19H36O2 [29648]

99 092

5 1978

9-Octadecenoic acid (Oleic acid)

HO

O

C18H34O2 [28246]

96 096

6 2016

Docosane

C22H46 [31060]

93 018

7 2103

Eicosane

C20H42 [28255]

97 050


Table 4 Continued


Qualitymatch with

Wileydatabase

Percentage oftotal

composition

8 2185

Tetracosane

C24H50 [33865]

99 016

9 2515

C30H50 [41072]

Squalene

87 1141

10 2633 NH

N

HO

MeO

Et

MeO

H

Voacangine (Ibogamine-18-carboxylic acid 12-methoxy methyl ester)

C22H28N2O3 [36847]

94 137

11 2726O

HO

C29H50O2 [43071]

Vitamin E (120572-Tocopherol)

97 030

12 2803

H3C

H3C

CH3

CH3

CH3

CH3

HO

(Campestanol)Ergost-5-en-3120573-ol

C28H48O [40068]

99 190


Table 4 Continued


Qualitymatch with

Wileydatabase

Percentage oftotal

composition

13 2829

OH

HH

H

H

Stigma-522-dien-3120573-ol

C29H48O [41269]

99 420

14 2880

H H

HH

HO

2223-dihydro-stigmasterol (120573-Sitosterol)

C29H50O [41471]

97 292

15 2956

H

H

HO

C30H50O [42672]

919-Cycloanost-24-en-3120573-ol (Cycloarternol)

98 1301

16 3074

H

H

H

O

OO

H

H

C31H48O3 [46871]

Urs-12-en-24-oic acid 3-oxo- methyl ester

95 4103


Table 5 Mass spectral data of Crude TDFME

Observed [M+H]+ peaks and correspondingmzvalues in +ESI-MS

Molecular formulae [mz values] and structures ofreported compounds [5]

C17

H19

N2

[25115]

NH

R1 = H and R2 = CH3

C17H18N2 [25014]

2

7 20

15

143

16

314419-tetrahydro-Olivacine (Miscellaneous)

R1 R2

N4

17CH3

C18

H21

N2

[26516]N

N

MeH

H CH2

Apparicine (Aspidospermatan)C18H20N2 [26416]

C19

H25

N2

O2

[31319]N

N

N

H

H

N O

O

HO

OH Et

5-Hydroxyvoaphylline (Plumeran)

OrVoafinine (Aspidospermatan)

C19H24N2O2 [31218]

C21

H25

N2

O3

[35318]

Or

O

O

NH

HN

OMe

Et

Lochnericine (Plumeran)C21H24N2O3 [35217]


Table 5 Continued



Or

NH

H NO

HO

MeO

Et

3-Oxocoronaridine (Ibogan)

C21

H25

N2

O3

[35318]

Or

H

H

N

N

HO

MeO

Et

O5-Oxocoronaridine (Ibogan)

Or


NH

N

H

H

O

O

MeO

Et

Or

NH

N O

Et

H

Pachysiphine (Plumeran)

CO2Me

NN

H

H

H

O

O

Me

Me

Vobasine (Corynanthean)

MeO


Table 5 Continued



C22

H29

N2

O3

[36921]

NH

N

HOOr

MeO

MeOEt

H

Voacangine (Ibogan)C22H28N2O3 [36821]

NH

N

HO

MeOEt

MeO

H

Or

Isovoacangine (Ibogan)

11-methoxy-N-methyldihydropericyclivine(Corynanthean)

N

H

N

H

O

Me Et

MeO

MeO

H

Or

NH

N

HO

MeOEt

MeO

H

19-Epivoacangine (Ibogan)

et al 1968) [69ndash71] It exhibited antibacterial activityagainst Corynebacterium Escherichia Proteus PseudomonasSalmonella Shigella and Staphylococcus in an in vitro studyat a concentration of 12 (Rojas Hernandez and Dıaz Perez1977) [72] Voacamine found in bark leaves stem and rootsof T divaricata by Karawya and Aboutabl (1979) demon-strated strong antimicrobial activity against gram positivebacteria such as Bacillus subtilis and Staphylococcus aureusand moderate activity against gram negative bacteria likeEscherichia coli and Pseudomonas aeruginosa (Van Beek et al1984 Pratchayasakul et al 2008) [5 61 73] As mentioned inour previous article T divaricata extracts or its componentscan be effective against Helicobacter pylori The biological

activity of T divaricata extracts against H pylori is yet to beestablished [14]

Earlier T divaricata was also studied for antioxidanteffects by various investigators Mandal and Mukherji (2001)demonstrated that T divaricata is a good scavenger of airpollutants [74] They also established that this plant has highlevel of antioxidative agents action like ascorbate peroxidasecatalase glutathione phenolic peroxidase and superoxidedismutase Further Gupta et al (2004) studied antioxidantand hepatoprotective effects of T divaricata methanolicextract of leaves in carbon tetrachloride induced hepatotoxi-city model The extract produced significant dose dependentantioxidant and hepatoprotective effects by diminishing lipid


TDFME-2 20ndash99 RT 009ndash036 AV 80 NL 149E7T FTMS 1 1 + p ESI full ms [10000ndash200000]

26516930

z = 1

C18H21N2 = 26516993

minus235716ppm

25115374

z

mz

= 1

C17H19N2 = 25115428

minus214970ppm

27711333

z = 1C8H14O2N8Na = 27711319

051243ppm

30014344

z = 1

114054ppmC13H19O2N5Na = 30014

95100

230 240

Rela

tive a

bund

ance

250 260 270 280 290 300

9085807570656055504540353025201510

50

(a)

mz

Rela

tive a

bund

ance

z = 1

z = 1

36921680

38521154

35318547

z = 1

110836ppm

minus127746ppm

minus140455 ppm

95100105

9085807570656055504540353025201510

50

310 320 330 340 350 370360 380 390 400

C22H29O3N2 = 36921727

C21H25O3N2 = 35318597z = 1

31319035

minus226396 ppmC19H25O2N2 = 31319105

C21H26N6Na = 38521112


(b)

Figure 5 Electrospray ionizationmdashhigh resolution mass spectra of crude TDFME

peroxidation and elevating levels of antioxidant agents likeCatalaseGlutathione and Superoxide dismutase [75] Amongthe chief non-alkaloidal bioactive molecules is a flavonoidthe kaempferol reported by Farooq et al (1959) [76] It exertsgastroprotective effect by scavenging stress generated oxygenmetabolites and suppressing neutrophils infiltration in theinflammation that occurs during gastric ulceration (Borrelliand Izzo 2000 Cheng andKoo 2000) [77 78]Thepossibilityof synergismbetween indole alkaloids and kaempferol cannotbe ruled out

4 Conclusion

It is concluded that T divaricata (L) R Br flower methanolicextract exhibits gastroprotective effect by enhancing theproduction of the gastric mucosa or preventing its depletionby aggressive factors while exhibiting antioxidant activityin chemically induced gastric ulceration in rats The massspectral analysis signifies the probable presence of few aspi-dospermatan corynanthean ibogan and plumeran sub-typesof indole alkaloids in the flowers for the first time The


study also highlights the possible role of indole alkaloids ingastroprotective function of T divaricata

Acknowledgments

The authors would like to acknowledge the support of DrA Gopala Reddy Dr D Madhuri and Dr Shiva Kumarfrom the College of Veterinary Science Acharya NG RangaAgricultural University and Mr Hari Narayana DirectormdashNishka Labs Uppal Hyderabad for providing necessaryfacilities and technical assistance in conducting the biochem-ical assays The authors also express their gratitude to Profes-sor Dr Mohib Khan Oriental College of Pharmacy and MrMohammed Azeemuddin Mukhram Research AssociateHimalaya Drugs Company for their valuable suggestions

References

[1] W Dymock C J HWarden and D Hooper PharmacographiaIndica vol 2 Kegan Paul Trench Trubner London UKEducation Societyrsquos Press Byculla Bombay India Thacker ampSpink Calcutta India 1891

[2] W Ainslie Materia Medica of Hindoostan Government PressMadras 1813

[3] M Parinitha G U Harish N C Vivek T Mahesh and M BShivanna ldquoEthno-botanical wealth of Bhadra wild life sanctu-ary in Karnatakardquo Indian Journal of Traditional Knowledge vol3 no 1 pp 37ndash50 2004

[4] S A Warsi and B Ahmed Pakistan Journal of Science vol 1 p128 1949

[5] T A Van Beek R Verpoorte and A B Svendsen ldquoTaber-naemontana L (Apocynaceae) a review of its taxonomyphytochemistry ethnobotany and pharmacologyrdquo Journal ofEthnopharmacology vol 10 no 1 pp 1ndash156 1984

[6] T Taesotikul A Panthong D Kanjanapothi R Verpoorte andJ J C Scheffer ldquoHippocratic screening of ethanolic extractsfrom two Tabernaemontana speciesrdquo Journal of Ethnopharma-cology vol 27 no 1-2 pp 99ndash106 1989

[7] P Basavaraj B Shivakumar and H Shivakumar ldquoAnxiolyticactivity of Tabernaemontana divaricata (Linn) R Br flowersextract in micerdquo International Journal of Pharma and BioSciences vol 2 no 3 pp 65ndash72 2011

[8] M S A Khan and M A Mukhram ldquoAnti-Seizure activity ofTabernaemontana divaricata (L) rbr flowermethanolic extractagainst maximal electroshock and pentylene tetrazole inducedconvulsions in experimental animalsrdquo Pharmacologyonline vol1 pp 784ndash798 2011

[9] P Basavaraj B S Shivakumar H S Shivakumar and V JManjunath ldquoEvaluation of anticonvulsant activity of Tabernae-montana divaricata (Linn) r br flower extractrdquo InternationalJournal of Pharmacy and Pharmaceutical Sciences vol 3 no 3pp 310ndash315 2011

[10] M Rahman M S Islam M S Ali M R Islam and M ZHossain ldquoAntidiabetic and cytotoxic activities of methanolicextract of Tabernaemontana divaricata (L) flowersrdquo Interna-tional Journal of Drug Development and Research vol 3 no 3pp 270ndash276 2011

[11] M A Mukhram H Shivakumar G L Viswanatha and SRajesh ldquoAnti-fertility effect of flower extracts of Tabernaemon-tana divaricata in ratsrdquo Chinese Journal of Natural Medicinesvol 10 no 1 pp 58ndash62 2012

[12] D Satyanarayana A B Joshi K S Chandrashekhar and KVijayanarayana ldquoAnti inflammatory activity of the flowers ofTabernaemontana divaricata (L) RBRrdquo Indian Drugs vol 41no 7 pp 405ndash407 2004

[13] PThambi B Kuzhivelil M Sabu and C Jolly ldquoAntioxidant andantiinflammatory activities of the flowers of Tabernaemontanacoronaria (L) RBrrdquo Indian Journal of Pharmaceutical Sciencesvol 68 no 3 pp 352ndash355 2006

[14] M S A Khan A M M Jais M Khan Z A Zakariaand M Ranjbar ldquoGastroprotective effect of Tabernaemontanadivaricata (L) RBr flower methanolic extract in ratsrdquo Pharma-cologyonline vol 2 pp 24ndash35 2011

[15] A S Salim ldquoRemoving oxygen-derived free radicals stimulateshealing of ethanol-induced erosive gastritis in the ratrdquo Diges-tion vol 47 no 1 pp 24ndash28 1990

[16] S K Kulkarni Handbook of Experimental Pharmacology Val-labh Prakashan New Delhi India 3rd edition 1999

[17] S Leyck and M J Parnham ldquoAcute antiinflammatory andgastric effects of the seleno-organic compound ebselenrdquo Agentsand Actions vol 30 no 3-4 pp 426ndash431 1990

[18] I Fridovich ldquoSuperoxide radical and superoxide dismutasesrdquoAnnual Review of Biochemistry vol 64 pp 97ndash112 1995

[19] H G Utley F Bernheim and P Hochstein ldquoEffect of sulfhydrylreagents on peroxidation in microsomesrdquo Archives of Biochem-istry and Biophysics vol 118 no 1 pp 29ndash32 1967

[20] S J Corne S M Morrissey and R J Woods ldquoA method forthe quantitative estimation of gastric barrier mucusrdquo Journal ofPhysiology vol 242 no 2 pp 116ndash117 1974

[21] J Sedlak and R H Lindsay ldquoEstimation of total protein-bound andnonprotein sulfhydryl groups in tissuewith Ellmanrsquosreagentrdquo Analytical Biochemistry vol 25 pp 192ndash205 1968

[22] O H Lowry N J Rosebrough A L Farr and R J RANDALLldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[23] N Kannappan S Jaikumar R Manavalan and A K MuthuldquoAntiulcer activity of methanolic extract of Jatropha curcas(Linn) on aspirin-induced gastric lesions in Wistar ratsrdquoPharmacologyonline vol 1 pp 279ndash293 2008

[24] S Khare M Asad S Dhamanigi and V Prasad ldquoAntiulceractivity of cod liver oil in ratsrdquo Indian Journal of Pharmacologyvol 40 no 5 pp 209ndash214 2008

[25] N Ahmed M S A Khan A M Mat Jais et al ldquoAnti-ulcer activity of sandalwood (Santalum album L) stem hydro-alcoholic extract in three gastric-ulceration models of wistarratsrdquo Boletın Latinoamericano y del Caribe de Plantas Medici-nales y Aromaticas vol 12 no 1 pp 81ndash91 2013

[26] C K Kokate Practical Pharmacognosy Vallabh PrakashanNew Delhi India 19th edition 1994

[27] R Shetty K V Kumar M U R Naidu and K S RatnakarldquoEffect of Gingko biloba extract on ethanol-induced gastricmucosal lesions in ratsrdquo Indian Journal of Pharmacology vol32 no 5 pp 313ndash317 2000

[28] R K Goel and S K Bhattacharya ldquoGastroduodenal mucosaldefence and mucosal protective agentsrdquo Indian Journal ofExperimental Biology vol 29 no 8 pp 701ndash714 1991

[29] M Zhu T H Lew and C T Luk ldquoGastric protective effectof Lentinus edodes against ethanol-induced ulcerationrdquo Fitoter-apia vol 68 no 6 pp 537ndash542 1997

[30] T A Miller D Li Y J Kuo K L Schmidt and L LShanbour ldquoNonprotein sulfhydryl compounds in canine gastricmucosa effects of PGE

2

and ethanolrdquo The American journal ofphysiology vol 249 no 1 pp G137ndashG144 1985


[31] K D Tripathi Essentials of Medical Pharmacology JaypeeBrothers Medical Publishers New Delhi India 5th edition2003

[32] K Lange B A Peskar and B M Peskar ldquoStimulation of ratgastric mucosal leukotriene formation by ethanolrdquo NaunynSchmiedbergrsquos Archives of Pharmacology supplement 330 p R271985

[33] T Mizui and M Doteuchi ldquoLipid peroxidation a possible rolein gastric damage induced by ethanol in ratsrdquo Life Sciences vol38 no 23 pp 2163ndash2167 1986

[34] A Terano H Hiraishi S-I Ota J Shiga and T SugimotoldquoRole of superoxide and hydroxyl radicals in rat gastricmucosalinjury induced by ethanolrdquo Gastroenterologia Japonica vol 24no 5 pp 488ndash493 1989

[35] S T Nielsen L Beninati and J Chang ldquoREV 5901 and Ly171883 protect rat gastric mucosa against ethanol-induceddamagerdquo Agents and Actions vol 21 no 3-4 pp 320ndash322 1987

[36] G B Glavin and S Szabo ldquoExperimental gastric mucosalinjury laboratory models reveal mechanisms of pathogenesisand new therapeutic strategiesrdquo FASEB Journal vol 6 no 3 pp825ndash831 1992

[37] M M Al-Harbi S Qureshi M Raza M M Ahmed M Afzaland A H Shah ldquoGastric antiulcer and cytoprotective effect ofCommiphora molmol in ratsrdquo Journal of Ethnopharmacologyvol 55 no 2 pp 141ndash150 1997

[38] A Robert J E Nezamis C Lancaster and A J HancharldquoCytoprotection by prostaglandins in rats Prevention of gastricnecrosis produced by alcohol HCl NaOH hypertonic NaCland thermal injuryrdquoGastroenterology vol 77 no 3 pp 433ndash4431979

[39] N Al Somal K E Coley P C Molan and B M HancockldquoSusceptibility ofHelicobacter pylori to the antibacterial activityof manuka honeyrdquo Journal of the Royal Society of Medicine vol87 no 1 pp 9ndash12 1994

[40] M Asante H Ahmed P Patel et al ldquoGastric mucosalhydrophobicity in duodenal ulceration role of Helicobacterpylori and infection density and mucus lipidsrdquo Gastroenterol-ogy vol 113 no 2 pp 449ndash454 1997

[41] A Ballinger ldquoCytoprotection with misoprostol use in thetreatment and prevention of ulcersrdquo Digestive Diseases vol 12no 1 pp 37ndash45 1994

[42] R F Bauer R G Bianchi J Casler and B Goldstin ldquoCompar-ative mucosal protective properties of misoprostol cimetidineand sucralfaterdquoDigestive Diseases and Sciences vol 31 no 2 pp81Sndash85S 1986

[43] K Berstad R Sjodahl and A Berstad ldquoPhospholipase A2

activity in gastric juice from patients with active and H pylori-eradicated healed duodenal ulcerrdquo Alimentary PharmacologyandTherapeutics vol 8 no 2 pp 175ndash180 1994

[44] D Das D Bandyopadhyay M Bhattacharjee and R K Baner-jee ldquoHydroxyl radical is the major causative factor in stress-induced gastric ulcerationrdquo Free Radical Biology and Medicinevol 23 no 1 pp 8ndash18 1997

[45] U Bandyopadhyay D Das D Bandyopadhyay M Bhattachar-jee and R K Banerjee ldquoRole of reactive oxygen species inmercapto-methylimidazole-induced gastric acid secretion andstress-induced gastric ulcerationrdquo Current Science vol 76 no1 pp 55ndash63 1999

[46] CMichielsM Raes O Toussaint and J Remacle ldquoImportanceof se-glutathione peroxidase catalase and CuZn-SOD forcell survival against oxidative stressrdquo Free Radical Biology andMedicine vol 17 no 3 pp 235ndash248 1994

[47] R Tandon H D Khanna M Dorababu and R K GoelldquoOxidative stress and antioxidants status in peptic ulcer and gas-tric carcinomardquo Indian Journal of Physiology and Pharmacologyvol 48 no 1 pp 115ndash118 2004

[48] J A Buege and S D Aust ldquoMicrosomal lipid peroxidationrdquoMethods in Enzymology vol 52 no C pp 302ndash310 1978

[49] M G Repetto and S F Llesuy ldquoAntioxidant properties ofnatural compounds used in popularmedicine for gastric ulcersrdquoBrazilian Journal of Medical and Biological Research vol 35 no5 pp 523ndash534 2002

[50] J Schrager ldquoThe composition and some structural features ofthe principal gastric glycoproteinrdquo Digestion vol 2 no 2 pp73ndash89 1969

[51] G Flemstrom and A Garner ldquoGastroduodenal HCO3

trans-port characteristics and proposed role in acidity regulation andmucosal protectionrdquo American Journal of Physiology vol 5 no3 pp G183ndashG193 1982

[52] C A Zalewsky and F GMoody ldquoMechanisms ofmucus releasein exposed canine gastric mucosardquoGastroenterology vol 77 no4 I pp 719ndash729 1979

[53] A Aly ldquoProstaglandins in clinical treatment of gastroduodenalmucosal lesions a reviewrdquo Scandinavian Journal of Gastroen-terology Supplement vol 22 no 137 pp 43ndash49 1987

[54] M Kimura S Goto Y Ihara et al ldquoImpairment of glu-tathione metabolism in human gastric epithelial cells treatedwith vacuolating cytotoxin from Helicobacter pylorirdquoMicrobialPathogenesis vol 31 no 1 pp 29ndash36 2001

[55] I A AlMofleh AAAlhalder J SMossaMOAl-Soohalbaniand S Rafatullah ldquoAqueous suspension of anise ldquoPimpinellaanisumrdquo protects rats against chemically induced gastric ulcersrdquoWorld Journal of Gastroenterology vol 13 no 7 pp 1112ndash11182007

[56] H Hiraishi A Terano S Ota et al ldquoProtection of culturedrat gastric cells against oxidant-induced damage by exogenousglutathionerdquo Gastroenterology vol 106 no 5 pp 1199ndash12071994

[57] G Sener-Muratoglu K Paskaloglu S Arbak CHHurdag andG Ayanoglu-Dulger ldquoProtective effect of famotidine omepra-zole and melatonin against acetylsalicylic acid-induced gastricdamage in ratsrdquo Digestive Diseases and Sciences vol 46 no 2pp 318ndash330 2001

[58] Y Mizushima and M Kobayashi ldquoInteraction of anti-inflammatory drugs with serum proteins especially withsome biologically active proteinsrdquo Journal of Pharmacy andPharmacology vol 20 no 3 pp 169ndash173 1968

[59] M I Grossman ldquoControl of gastric secretionrdquo in GastroIntestI-nal Disease Pathophysiology Diagnosis andManagement M HSleisenzer and J S Fordtran Eds pp 640ndash659 WB SaundersPhiladelphia Pa USA 2nd edition 1978

[60] L S R Arambewela and T Ranatunge ldquoIndole alkaloids fromTabernaemontana divaricatardquo Phytochemistry vol 30 no 5 pp1740ndash1741 1991

[61] W Pratchayasakul A Pongchaidecha N Chattipakorn and SChattipakorn ldquoEthnobotany and ethnopharmacology of Taber-naemontana divaricatardquo Indian Journal ofMedical Research vol127 no 4 pp 317ndash335 2008

[62] HD S Falcao J A Leite JM Barbosa-Filho et al ldquoGastric andduodenal antiulcer activity of alkaloids a reviewrdquoMolecules vol13 no 12 pp 3198ndash3223 2008

[63] C Gomez-Gonzalez C Navajas Polo S Corzo Rodriguez andA L Padilla Mendez ldquoFitoquimica de Ervatamia coronaria


Stapf (IV) Fraccionamiento de las bases totals presents en lasflores en un gradient de acidezrdquo Revista Cubana De Farmaciavol 15 no 3 pp 192ndash199 1981

[64] P V Tan B Nyasse T Dimo P Wafo and B T AkahkuhldquoSynergistic and potentiating effects of ranitidine and two newanti-ulcer compounds from Enantia chlorantha and Voacangaafricana in experimental animalmodelsrdquoPharmazie vol 57 no6 pp 409ndash412 2002

[65] M A Elkeiy S M Abd Elwahab and A Y Zaki JournalofPharmaceutical Sciences of United Arab Republic vol 7 p 971966

[66] A T Henriques A A Melo P R H Moreno L L Ene JA P Henriques and E E S Schapoval ldquoErvatamia coronariachemical constituents and some pharmacological activitiesrdquoJournal of Ethnopharmacology vol 50 no 1 pp 19ndash25 1996

[67] A S Awaad R M El-Meligy and G A Soliman ldquoNaturalproducts in treatment of ulcerative colitis and peptic ulcerrdquoJournal of Saudi Chemical Society vol 17 pp 101ndash124 2013

[68] P G Rao and B P Singri Indian Journal ofChemistry vol 17 p414 1979

[69] K-H Pawelka and J Stockigt ldquoIndole alkaloids from cellsuspension cultures of Tabernaemontana divaricata and Taber-nanthe ibogardquo Plant Cell Reports vol 2 no 2 pp 105ndash107 1983

[70] M T Andrade J A Lima A C Pinto C M RezendeM P Carvalho and R A Epifanio ldquoIndole alkaloids fromTabernaemontana australis (Muell Arg) Miers that inhibitacetylcholinesterase enzymerdquo Bioorganic and Medicinal Chem-istry vol 13 no 12 pp 4092ndash4095 2005

[71] N R Farnsworth G H Svoboda and R N Blomster ldquoAntiviralactivity of selected Catharanthus alkaloidsrdquo Journal of Pharma-ceutical Sciences vol 57 no 12 pp 2174ndash2175 1968

[72] N M Rojas Hernandez and C Dıaz Perez ldquoFungal activity ofvarious alkaloids isolated from Catharanthus roseus G DonrdquoRevista cubana de medicina tropical vol 29 no 3 pp 147ndash1521977

[73] M S Karawya and E A Aboutabl ldquoPhytochemical studiesof Abrus precatorius alkaloidsrdquo Journal of Pharmaceutical Sci-ences vol 20 p 241 1979

[74] M Mandal and S Mukherji ldquoA study on the activities of afew free radicals scavenging enzymes present in five roadsideplantsrdquo Journal of Environmental Biology vol 22 no 4 pp 301ndash305 2001

[75] M Gupta U K Mazumder R S Kumar T Sivakumar andP Gomathi ldquoAntioxidant and protective effects of Ervatamiacoronaria stapf leaves against carbon tetrachloride-inducedliver injuryrdquo European Bulletin of Drug Research vol 12 pp 13ndash22 2004

[76] M O Farooq W Rahman and M Ilyas ldquoKaempferol fromflowers of Ervatamia coronaria stapfrdquo Die Naturwissenschaftenvol 46 no 12 pp 401ndash402 1959

[77] F Borrelli and A A Izzo ldquoThe plant kingdom as a source ofanti-ulcer remediesrdquo Phytotherapy Research vol 14 pp 581ndash5912000

[78] C L Cheng and M W L Koo ldquoEffects of Centella asiatica onethanol induced gastric mucosal lesions in ratsrdquo Life Sciencesvol 67 no 21 pp 2647ndash2653 2000

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


belonging to the Apocynaceae family were authenticated bya plant taxonomist Dr P V Prasanna (ScientistmdashE) at theBotanical Survey of India Deccan Regional Centre Hyder-abad (establishment under the Ministry of Environment ampForests Government of India) The specimen deposited inthe herbariumwas assigned a voucher number BSID 887Theextract was prepared as per the procedure described earlier[14] Briefly after collection flowers were shade dried andcoarsely powdered Approximately 500 gm of the powderedflowers was extracted using methanol 99 pure (SD Fine-Chem) in a soxhlet apparatus The extract was concentratedunder reduced pressure and stored in an airtight containerin a refrigerator at the temperature below 10∘C The driedmass of crudeTabernaemontana divaricataflowermethanolicextract (TDFME) was found to be 34 ww with respect tothe dried flowersThe solution of TDFMEwas prepared usingdistilled water for the evaluation of the antiulcer activity

22 Drugs and Chemicals Chloroform AR diethyl ethermethanol AR phenolphthalein pH indicator solution andsodium hydroxide pellets were procured from SD Fine-Chem Limited Mumbai while pure aspirin was obtainedfrom Divis Laboratories Hyderabad Absolute ethanol waspurchased from Changshu Yangyuan Chem China andmisoprostol (as misoprost-200) was purchased from CiplaLtd Goa while surgical spirit was obtained from KakatiyaPharma Hyderabad Topferrsquos Reagent and distilled waterwere obtained fromNice Chemicals China and Stangen FineChemicals Hyderabad respectively All chemicals were usedwithout further purification

23 Animals Adult male Wistar rats weighing 150ndash200 gmwere used for the evaluation of antiulcer activityThe animalswere maintained under standard laboratory conditions inpolypropylene cages under 12 hr lightdark cycle controlledtemperature (24 plusmn 2∘C) fed with commercial pellet dietand water ad libitum in an animal house approved by theCommittee for the Purpose and Supervision on Experimentson Animals (Reg no 1534PO-a11CPCSEA) All the ani-mals were acclimatized to the laboratory environment for 10days before the initiation of the experiments The protocol(IAECAUCP2011-1202) was approved by the InstitutionalAnimal Ethical Committee before the commencement ofanimal experimentation All measures were taken to ensurethat the experiments were conducted in accordance with theinstructions of IAEC Anwarul Uloom College of PharmacyNew Mallepally Hyderabad Andhra Pradesh India

24 Acute Toxicity Test and Selection of Test Doses Three testdoses (125 250 and 500mgkg) were selected in range of 116to 14 of the maximum oral safe doses reported in earlierarticles [7 9 11]

25 Evaluation of Antiulcer Activity by In Vivo Assays

251 Acute Ethanol Induced Gastric Ulcers All the animals(119899 = 6) in each group were fasted for 36 hours before theadministration of ethanol The standard drug (misoprostol

100 120583gkg po) or the test extract was administered onehour before ethanol administration Ethanol (90) wasadministered to all animals at a dose of 1mL200 gm Afterone hour all animals were sacrificed and stomachs wereisolated [15] Lesion severity was determined by measuringulcer index Later stomachs were subjected to the estimationof catalase superoxide dismutase malondialdehyde mucintotal protein and nonprotein sulphhydryl contents followingstandard procedures

(1) Ulcer Index (UI) Mean ulcer score for each animal isexpressed as ulcer index The stomachs were washed withrunning water to see the ulcers in the glandular portion of thestomach The number of the ulcers per stomach were notedand the severity of the ulcers was scoredmicroscopically withthe help of hand lens (10x) and scoring was done as perKulkarni (1999) [16] as following

0 = normal stomach

05 = red colouration

1 = spot ulcers

15 = hemorrhagic streaks

2 = ulcers gt 3mm but lt5mm

3 = ulcers gt 5mm

(2) Assessment of the Oxidative Damage in Gastric Tis-sue After measuring the ulcer index the stomachs werewashed with 09 (wv) NaCl cut into small pieces andhomogenized with a glass homogenizer in ice-cold 015MKCl solution to produce a 20 (wv) homogenate Thehomogenate was used for the determination of variousbiochemical parameters

(a) Estimation of Catalase (CAT)Catalase activity in stomachtissue was determined according to the method of Leyck andParnham (1990) [17] The stomach tissue was scraped andhomogenized in ice cold saline medium with the help of ahomogenizer The solution was centrifuged for 10 minutesat 3000 g-force and collected for the experiment 100 120583L ofthe supernatant was added to a solution of 3 120583L of H

2O2

phosphate buffer mixture (50mM phosphate buffer pH 70and 30 H

2O2) The change in optical density at 240 nm per

unit time was measured

(b) Estimation of Superoxide Dismutase (SOD) Superoxidedismutase activity in stomach tissue was determined accord-ing to the method of Fridovich (1995) [18] The stomachtissue was scrapped and homogenized in ice cold normalsaline medium with the help of a homogenizer Then thetissue homogenate was centrifuged for 10 minutes at 3000 g-force and the supernatant was collected and used for theestimation of SOD activity 10mL of the solution was taken ina test tube andmixed with 05mL of 50mMphosphate buffer(pH 78) 01mM of EDTA 005mM xanthine and 001mMcytochrome c and then 100mL of 25mM of xanthine


































Test-ITDFME125mgkg

Test-IITDFME250mgkg














2and I2




(a) (b) (c) (d)


(a) (b) (c) (d) (e)





Test-ITDFME125mgkg

Test-IITDFME250mgkg



716 plusmn 040b

75 plusmn 056b

150 plusmn 028c

b119875 lt 001 c119875 lt 0001









010

005

000

000 500 1000 1500 2000 2500(Minutes)

Chromatogram

(AU

)

3000 3500 4000 4500 5000 5500 6000




13

24 5

6

7 8

9

10

11

(a)

12

13 14

15

16

(b)
















2-


1muscarinic








Qualitymatch with

Wileydatabase

Percentage oftotal

composition

1 1762O

O


C17H34O2 [27045]

98 103

2 1812 O

OH


C16H32O2 [25642]

99 080

3 1925 O

O


C19H34O2 [29447]

99 073

4 1931O

O


C19H36O2 [29648]

99 092

5 1978


HO

O

C18H34O2 [28246]

96 096

6 2016

Docosane

C22H46 [31060]

93 018

7 2103

Eicosane

C20H42 [28255]

97 050


Table 4 Continued


Qualitymatch with

Wileydatabase

Percentage oftotal

composition

8 2185

Tetracosane

C24H50 [33865]

99 016

9 2515

C30H50 [41072]

Squalene

87 1141

10 2633 NH

N

HO

MeO

Et

MeO

H


C22H28N2O3 [36847]

94 137

11 2726O

HO

C29H50O2 [43071]


97 030

12 2803

H3C

H3C

CH3

CH3

CH3

CH3

HO


C28H48O [40068]

99 190


Table 4 Continued


Qualitymatch with

Wileydatabase

Percentage oftotal

composition

13 2829

OH

HH

H

H


C29H48O [41269]

99 420

14 2880

H H

HH

HO


C29H50O [41471]

97 292

15 2956

H

H

HO

C30H50O [42672]


98 1301

16 3074

H

H

H

O

OO

H

H

C31H48O3 [46871]


95 4103





C17

H19

N2

[25115]

NH

R1 = H and R2 = CH3

C17H18N2 [25014]

2

7 20

15

143

16


R1 R2

N4

17CH3

C18

H21

N2

[26516]N

N

MeH

H CH2


C19

H25

N2

O2

[31319]N

N

N

H

H

N O

O

HO

OH Et



C19H24N2O2 [31218]

C21

H25

N2

O3

[35318]

Or

O

O

NH

HN

OMe

Et



Table 5 Continued



Or

NH

H NO

HO

MeO

Et


C21

H25

N2

O3

[35318]

Or

H

H

N

N

HO

MeO

Et


Or


NH

N

H

H

O

O

MeO

Et

Or

NH

N O

Et

H


CO2Me

NN

H

H

H

O

O

Me

Me


MeO


Table 5 Continued



C22

H29

N2

O3

[36921]

NH

N

HOOr

MeO

MeOEt

H


NH

N

HO

MeOEt

MeO

H

Or



N

H

N

H

O

Me Et

MeO

MeO

H

Or

NH

N

HO

MeOEt

MeO

H







26516930

z = 1

C18H21N2 = 26516993

minus235716ppm

25115374

z

mz

= 1

C17H19N2 = 25115428

minus214970ppm

27711333

z = 1C8H14O2N8Na = 27711319

051243ppm

30014344

z = 1

114054ppmC13H19O2N5Na = 30014

95100

230 240

Rela

tive a

bund

ance

250 260 270 280 290 300

9085807570656055504540353025201510

50

(a)

mz

Rela

tive a

bund

ance

z = 1

z = 1

36921680

38521154

35318547

z = 1

110836ppm

minus127746ppm

minus140455 ppm

95100105

9085807570656055504540353025201510

50

310 320 330 340 350 370360 380 390 400

C22H29O3N2 = 36921727

C21H25O3N2 = 35318597z = 1

31319035

minus226396 ppmC19H25O2N2 = 31319105

C21H26N6Na = 38521112


(b)



4 Conclusion




Acknowledgments


References































2



























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


































Test-ITDFME125mgkg

Test-IITDFME250mgkg














2and I2




(a) (b) (c) (d)


(a) (b) (c) (d) (e)





Test-ITDFME125mgkg

Test-IITDFME250mgkg



716 plusmn 040b

75 plusmn 056b

150 plusmn 028c

b119875 lt 001 c119875 lt 0001









010

005

000

000 500 1000 1500 2000 2500(Minutes)

Chromatogram

(AU

)

3000 3500 4000 4500 5000 5500 6000




13

24 5

6

7 8

9

10

11

(a)

12

13 14

15

16

(b)
















2-


1muscarinic








Qualitymatch with

Wileydatabase

Percentage oftotal

composition

1 1762O

O


C17H34O2 [27045]

98 103

2 1812 O

OH


C16H32O2 [25642]

99 080

3 1925 O

O


C19H34O2 [29447]

99 073

4 1931O

O


C19H36O2 [29648]

99 092

5 1978


HO

O

C18H34O2 [28246]

96 096

6 2016

Docosane

C22H46 [31060]

93 018

7 2103

Eicosane

C20H42 [28255]

97 050


Table 4 Continued


Qualitymatch with

Wileydatabase

Percentage oftotal

composition

8 2185

Tetracosane

C24H50 [33865]

99 016

9 2515

C30H50 [41072]

Squalene

87 1141

10 2633 NH

N

HO

MeO

Et

MeO

H


C22H28N2O3 [36847]

94 137

11 2726O

HO

C29H50O2 [43071]


97 030

12 2803

H3C

H3C

CH3

CH3

CH3

CH3

HO


C28H48O [40068]

99 190


Table 4 Continued


Qualitymatch with

Wileydatabase

Percentage oftotal

composition

13 2829

OH

HH

H

H


C29H48O [41269]

99 420

14 2880

H H

HH

HO


C29H50O [41471]

97 292

15 2956

H

H

HO

C30H50O [42672]


98 1301

16 3074

H

H

H

O

OO

H

H

C31H48O3 [46871]


95 4103





C17

H19

N2

[25115]

NH

R1 = H and R2 = CH3

C17H18N2 [25014]

2

7 20

15

143

16


R1 R2

N4

17CH3

C18

H21

N2

[26516]N

N

MeH

H CH2


C19

H25

N2

O2

[31319]N

N

N

H

H

N O

O

HO

OH Et



C19H24N2O2 [31218]

C21

H25

N2

O3

[35318]

Or

O

O

NH

HN

OMe

Et



Table 5 Continued



Or

NH

H NO

HO

MeO

Et


C21

H25

N2

O3

[35318]

Or

H

H

N

N

HO

MeO

Et


Or


NH

N

H

H

O

O

MeO

Et

Or

NH

N O

Et

H


CO2Me

NN

H

H

H

O

O

Me

Me


MeO


Table 5 Continued



C22

H29

N2

O3

[36921]

NH

N

HOOr

MeO

MeOEt

H


NH

N

HO

MeOEt

MeO

H

Or



N

H

N

H

O

Me Et

MeO

MeO

H

Or

NH

N

HO

MeOEt

MeO

H







26516930

z = 1

C18H21N2 = 26516993

minus235716ppm

25115374

z

mz

= 1

C17H19N2 = 25115428

minus214970ppm

27711333

z = 1C8H14O2N8Na = 27711319

051243ppm

30014344

z = 1

114054ppmC13H19O2N5Na = 30014

95100

230 240

Rela

tive a

bund

ance

250 260 270 280 290 300

9085807570656055504540353025201510

50

(a)

mz

Rela

tive a

bund

ance

z = 1

z = 1

36921680

38521154

35318547

z = 1

110836ppm

minus127746ppm

minus140455 ppm

95100105

9085807570656055504540353025201510

50

310 320 330 340 350 370360 380 390 400

C22H29O3N2 = 36921727

C21H25O3N2 = 35318597z = 1

31319035

minus226396 ppmC19H25O2N2 = 31319105

C21H26N6Na = 38521112


(b)



4 Conclusion




Acknowledgments


References































2



























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


(a) (b) (c) (d)


(a) (b) (c) (d) (e)





Test-ITDFME125mgkg

Test-IITDFME250mgkg



716 plusmn 040b

75 plusmn 056b

150 plusmn 028c

b119875 lt 001 c119875 lt 0001









010

005

000

000 500 1000 1500 2000 2500(Minutes)

Chromatogram

(AU

)

3000 3500 4000 4500 5000 5500 6000




13

24 5

6

7 8

9

10

11

(a)

12

13 14

15

16

(b)
















2-


1muscarinic








Qualitymatch with

Wileydatabase

Percentage oftotal

composition

1 1762O

O


C17H34O2 [27045]

98 103

2 1812 O

OH


C16H32O2 [25642]

99 080

3 1925 O

O


C19H34O2 [29447]

99 073

4 1931O

O


C19H36O2 [29648]

99 092

5 1978


HO

O

C18H34O2 [28246]

96 096

6 2016

Docosane

C22H46 [31060]

93 018

7 2103

Eicosane

C20H42 [28255]

97 050


Table 4 Continued


Qualitymatch with

Wileydatabase

Percentage oftotal

composition

8 2185

Tetracosane

C24H50 [33865]

99 016

9 2515

C30H50 [41072]

Squalene

87 1141

10 2633 NH

N

HO

MeO

Et

MeO

H


C22H28N2O3 [36847]

94 137

11 2726O

HO

C29H50O2 [43071]


97 030

12 2803

H3C

H3C

CH3

CH3

CH3

CH3

HO


C28H48O [40068]

99 190


Table 4 Continued


Qualitymatch with

Wileydatabase

Percentage oftotal

composition

13 2829

OH

HH

H

H


C29H48O [41269]

99 420

14 2880

H H

HH

HO


C29H50O [41471]

97 292

15 2956

H

H

HO

C30H50O [42672]


98 1301

16 3074

H

H

H

O

OO

H

H

C31H48O3 [46871]


95 4103





C17

H19

N2

[25115]

NH

R1 = H and R2 = CH3

C17H18N2 [25014]

2

7 20

15

143

16


R1 R2

N4

17CH3

C18

H21

N2

[26516]N

N

MeH

H CH2


C19

H25

N2

O2

[31319]N

N

N

H

H

N O

O

HO

OH Et



C19H24N2O2 [31218]

C21

H25

N2

O3

[35318]

Or

O

O

NH

HN

OMe

Et



Table 5 Continued



Or

NH

H NO

HO

MeO

Et


C21

H25

N2

O3

[35318]

Or

H

H

N

N

HO

MeO

Et


Or


NH

N

H

H

O

O

MeO

Et

Or

NH

N O

Et

H


CO2Me

NN

H

H

H

O

O

Me

Me


MeO


Table 5 Continued



C22

H29

N2

O3

[36921]

NH

N

HOOr

MeO

MeOEt

H


NH

N

HO

MeOEt

MeO

H

Or



N

H

N

H

O

Me Et

MeO

MeO

H

Or

NH

N

HO

MeOEt

MeO

H







26516930

z = 1

C18H21N2 = 26516993

minus235716ppm

25115374

z

mz

= 1

C17H19N2 = 25115428

minus214970ppm

27711333

z = 1C8H14O2N8Na = 27711319

051243ppm

30014344

z = 1

114054ppmC13H19O2N5Na = 30014

95100

230 240

Rela

tive a

bund

ance

250 260 270 280 290 300

9085807570656055504540353025201510

50

(a)

mz

Rela

tive a

bund

ance

z = 1

z = 1

36921680

38521154

35318547

z = 1

110836ppm

minus127746ppm

minus140455 ppm

95100105

9085807570656055504540353025201510

50

310 320 330 340 350 370360 380 390 400

C22H29O3N2 = 36921727

C21H25O3N2 = 35318597z = 1

31319035

minus226396 ppmC19H25O2N2 = 31319105

C21H26N6Na = 38521112


(b)



4 Conclusion




Acknowledgments


References































2



























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


010

005

000

000 500 1000 1500 2000 2500(Minutes)

Chromatogram

(AU

)

3000 3500 4000 4500 5000 5500 6000




13

24 5

6

7 8

9

10

11

(a)

12

13 14

15

16

(b)
















2-


1muscarinic








Qualitymatch with

Wileydatabase

Percentage oftotal

composition

1 1762O

O


C17H34O2 [27045]

98 103

2 1812 O

OH


C16H32O2 [25642]

99 080

3 1925 O

O


C19H34O2 [29447]

99 073

4 1931O

O


C19H36O2 [29648]

99 092

5 1978


HO

O

C18H34O2 [28246]

96 096

6 2016

Docosane

C22H46 [31060]

93 018

7 2103

Eicosane

C20H42 [28255]

97 050


Table 4 Continued


Qualitymatch with

Wileydatabase

Percentage oftotal

composition

8 2185

Tetracosane

C24H50 [33865]

99 016

9 2515

C30H50 [41072]

Squalene

87 1141

10 2633 NH

N

HO

MeO

Et

MeO

H


C22H28N2O3 [36847]

94 137

11 2726O

HO

C29H50O2 [43071]


97 030

12 2803

H3C

H3C

CH3

CH3

CH3

CH3

HO


C28H48O [40068]

99 190


Table 4 Continued


Qualitymatch with

Wileydatabase

Percentage oftotal

composition

13 2829

OH

HH

H

H


C29H48O [41269]

99 420

14 2880

H H

HH

HO


C29H50O [41471]

97 292

15 2956

H

H

HO

C30H50O [42672]


98 1301

16 3074

H

H

H

O

OO

H

H

C31H48O3 [46871]


95 4103





C17

H19

N2

[25115]

NH

R1 = H and R2 = CH3

C17H18N2 [25014]

2

7 20

15

143

16


R1 R2

N4

17CH3

C18

H21

N2

[26516]N

N

MeH

H CH2


C19

H25

N2

O2

[31319]N

N

N

H

H

N O

O

HO

OH Et



C19H24N2O2 [31218]

C21

H25

N2

O3

[35318]

Or

O

O

NH

HN

OMe

Et



Table 5 Continued



Or

NH

H NO

HO

MeO

Et


C21

H25

N2

O3

[35318]

Or

H

H

N

N

HO

MeO

Et


Or


NH

N

H

H

O

O

MeO

Et

Or

NH

N O

Et

H


CO2Me

NN

H

H

H

O

O

Me

Me


MeO


Table 5 Continued



C22

H29

N2

O3

[36921]

NH

N

HOOr

MeO

MeOEt

H


NH

N

HO

MeOEt

MeO

H

Or



N

H

N

H

O

Me Et

MeO

MeO

H

Or

NH

N

HO

MeOEt

MeO

H







26516930

z = 1

C18H21N2 = 26516993

minus235716ppm

25115374

z

mz

= 1

C17H19N2 = 25115428

minus214970ppm

27711333

z = 1C8H14O2N8Na = 27711319

051243ppm

30014344

z = 1

114054ppmC13H19O2N5Na = 30014

95100

230 240

Rela

tive a

bund

ance

250 260 270 280 290 300

9085807570656055504540353025201510

50

(a)

mz

Rela

tive a

bund

ance

z = 1

z = 1

36921680

38521154

35318547

z = 1

110836ppm

minus127746ppm

minus140455 ppm

95100105

9085807570656055504540353025201510

50

310 320 330 340 350 370360 380 390 400

C22H29O3N2 = 36921727

C21H25O3N2 = 35318597z = 1

31319035

minus226396 ppmC19H25O2N2 = 31319105

C21H26N6Na = 38521112


(b)



4 Conclusion




Acknowledgments


References































2



























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of








2-


1muscarinic








Qualitymatch with

Wileydatabase

Percentage oftotal

composition

1 1762O

O


C17H34O2 [27045]

98 103

2 1812 O

OH


C16H32O2 [25642]

99 080

3 1925 O

O


C19H34O2 [29447]

99 073

4 1931O

O


C19H36O2 [29648]

99 092

5 1978


HO

O

C18H34O2 [28246]

96 096

6 2016

Docosane

C22H46 [31060]

93 018

7 2103

Eicosane

C20H42 [28255]

97 050


Table 4 Continued


Qualitymatch with

Wileydatabase

Percentage oftotal

composition

8 2185

Tetracosane

C24H50 [33865]

99 016

9 2515

C30H50 [41072]

Squalene

87 1141

10 2633 NH

N

HO

MeO

Et

MeO

H


C22H28N2O3 [36847]

94 137

11 2726O

HO

C29H50O2 [43071]


97 030

12 2803

H3C

H3C

CH3

CH3

CH3

CH3

HO


C28H48O [40068]

99 190


Table 4 Continued


Qualitymatch with

Wileydatabase

Percentage oftotal

composition

13 2829

OH

HH

H

H


C29H48O [41269]

99 420

14 2880

H H

HH

HO


C29H50O [41471]

97 292

15 2956

H

H

HO

C30H50O [42672]


98 1301

16 3074

H

H

H

O

OO

H

H

C31H48O3 [46871]


95 4103





C17

H19

N2

[25115]

NH

R1 = H and R2 = CH3

C17H18N2 [25014]

2

7 20

15

143

16


R1 R2

N4

17CH3

C18

H21

N2

[26516]N

N

MeH

H CH2


C19

H25

N2

O2

[31319]N

N

N

H

H

N O

O

HO

OH Et



C19H24N2O2 [31218]

C21

H25

N2

O3

[35318]

Or

O

O

NH

HN

OMe

Et



Table 5 Continued



Or

NH

H NO

HO

MeO

Et


C21

H25

N2

O3

[35318]

Or

H

H

N

N

HO

MeO

Et


Or


NH

N

H

H

O

O

MeO

Et

Or

NH

N O

Et

H


CO2Me

NN

H

H

H

O

O

Me

Me


MeO


Table 5 Continued



C22

H29

N2

O3

[36921]

NH

N

HOOr

MeO

MeOEt

H


NH

N

HO

MeOEt

MeO

H

Or



N

H

N

H

O

Me Et

MeO

MeO

H

Or

NH

N

HO

MeOEt

MeO

H







26516930

z = 1

C18H21N2 = 26516993

minus235716ppm

25115374

z

mz

= 1

C17H19N2 = 25115428

minus214970ppm

27711333

z = 1C8H14O2N8Na = 27711319

051243ppm

30014344

z = 1

114054ppmC13H19O2N5Na = 30014

95100

230 240

Rela

tive a

bund

ance

250 260 270 280 290 300

9085807570656055504540353025201510

50

(a)

mz

Rela

tive a

bund

ance

z = 1

z = 1

36921680

38521154

35318547

z = 1

110836ppm

minus127746ppm

minus140455 ppm

95100105

9085807570656055504540353025201510

50

310 320 330 340 350 370360 380 390 400

C22H29O3N2 = 36921727

C21H25O3N2 = 35318597z = 1

31319035

minus226396 ppmC19H25O2N2 = 31319105

C21H26N6Na = 38521112


(b)



4 Conclusion




Acknowledgments


References































2



























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




Qualitymatch with

Wileydatabase

Percentage oftotal

composition

1 1762O

O


C17H34O2 [27045]

98 103

2 1812 O

OH


C16H32O2 [25642]

99 080

3 1925 O

O


C19H34O2 [29447]

99 073

4 1931O

O


C19H36O2 [29648]

99 092

5 1978


HO

O

C18H34O2 [28246]

96 096

6 2016

Docosane

C22H46 [31060]

93 018

7 2103

Eicosane

C20H42 [28255]

97 050


Table 4 Continued


Qualitymatch with

Wileydatabase

Percentage oftotal

composition

8 2185

Tetracosane

C24H50 [33865]

99 016

9 2515

C30H50 [41072]

Squalene

87 1141

10 2633 NH

N

HO

MeO

Et

MeO

H


C22H28N2O3 [36847]

94 137

11 2726O

HO

C29H50O2 [43071]


97 030

12 2803

H3C

H3C

CH3

CH3

CH3

CH3

HO


C28H48O [40068]

99 190


Table 4 Continued


Qualitymatch with

Wileydatabase

Percentage oftotal

composition

13 2829

OH

HH

H

H


C29H48O [41269]

99 420

14 2880

H H

HH

HO


C29H50O [41471]

97 292

15 2956

H

H

HO

C30H50O [42672]


98 1301

16 3074

H

H

H

O

OO

H

H

C31H48O3 [46871]


95 4103





C17

H19

N2

[25115]

NH

R1 = H and R2 = CH3

C17H18N2 [25014]

2

7 20

15

143

16


R1 R2

N4

17CH3

C18

H21

N2

[26516]N

N

MeH

H CH2


C19

H25

N2

O2

[31319]N

N

N

H

H

N O

O

HO

OH Et



C19H24N2O2 [31218]

C21

H25

N2

O3

[35318]

Or

O

O

NH

HN

OMe

Et



Table 5 Continued



Or

NH

H NO

HO

MeO

Et


C21

H25

N2

O3

[35318]

Or

H

H

N

N

HO

MeO

Et


Or


NH

N

H

H

O

O

MeO

Et

Or

NH

N O

Et

H


CO2Me

NN

H

H

H

O

O

Me

Me


MeO


Table 5 Continued



C22

H29

N2

O3

[36921]

NH

N

HOOr

MeO

MeOEt

H


NH

N

HO

MeOEt

MeO

H

Or



N

H

N

H

O

Me Et

MeO

MeO

H

Or

NH

N

HO

MeOEt

MeO

H







26516930

z = 1

C18H21N2 = 26516993

minus235716ppm

25115374

z

mz

= 1

C17H19N2 = 25115428

minus214970ppm

27711333

z = 1C8H14O2N8Na = 27711319

051243ppm

30014344

z = 1

114054ppmC13H19O2N5Na = 30014

95100

230 240

Rela

tive a

bund

ance

250 260 270 280 290 300

9085807570656055504540353025201510

50

(a)

mz

Rela

tive a

bund

ance

z = 1

z = 1

36921680

38521154

35318547

z = 1

110836ppm

minus127746ppm

minus140455 ppm

95100105

9085807570656055504540353025201510

50

310 320 330 340 350 370360 380 390 400

C22H29O3N2 = 36921727

C21H25O3N2 = 35318597z = 1

31319035

minus226396 ppmC19H25O2N2 = 31319105

C21H26N6Na = 38521112


(b)



4 Conclusion




Acknowledgments


References































2



























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


Table 4 Continued


Qualitymatch with

Wileydatabase

Percentage oftotal

composition

8 2185

Tetracosane

C24H50 [33865]

99 016

9 2515

C30H50 [41072]

Squalene

87 1141

10 2633 NH

N

HO

MeO

Et

MeO

H


C22H28N2O3 [36847]

94 137

11 2726O

HO

C29H50O2 [43071]


97 030

12 2803

H3C

H3C

CH3

CH3

CH3

CH3

HO


C28H48O [40068]

99 190


Table 4 Continued


Qualitymatch with

Wileydatabase

Percentage oftotal

composition

13 2829

OH

HH

H

H


C29H48O [41269]

99 420

14 2880

H H

HH

HO


C29H50O [41471]

97 292

15 2956

H

H

HO

C30H50O [42672]


98 1301

16 3074

H

H

H

O

OO

H

H

C31H48O3 [46871]


95 4103





C17

H19

N2

[25115]

NH

R1 = H and R2 = CH3

C17H18N2 [25014]

2

7 20

15

143

16


R1 R2

N4

17CH3

C18

H21

N2

[26516]N

N

MeH

H CH2


C19

H25

N2

O2

[31319]N

N

N

H

H

N O

O

HO

OH Et



C19H24N2O2 [31218]

C21

H25

N2

O3

[35318]

Or

O

O

NH

HN

OMe

Et



Table 5 Continued



Or

NH

H NO

HO

MeO

Et


C21

H25

N2

O3

[35318]

Or

H

H

N

N

HO

MeO

Et


Or


NH

N

H

H

O

O

MeO

Et

Or

NH

N O

Et

H


CO2Me

NN

H

H

H

O

O

Me

Me


MeO


Table 5 Continued



C22

H29

N2

O3

[36921]

NH

N

HOOr

MeO

MeOEt

H


NH

N

HO

MeOEt

MeO

H

Or



N

H

N

H

O

Me Et

MeO

MeO

H

Or

NH

N

HO

MeOEt

MeO

H







26516930

z = 1

C18H21N2 = 26516993

minus235716ppm

25115374

z

mz

= 1

C17H19N2 = 25115428

minus214970ppm

27711333

z = 1C8H14O2N8Na = 27711319

051243ppm

30014344

z = 1

114054ppmC13H19O2N5Na = 30014

95100

230 240

Rela

tive a

bund

ance

250 260 270 280 290 300

9085807570656055504540353025201510

50

(a)

mz

Rela

tive a

bund

ance

z = 1

z = 1

36921680

38521154

35318547

z = 1

110836ppm

minus127746ppm

minus140455 ppm

95100105

9085807570656055504540353025201510

50

310 320 330 340 350 370360 380 390 400

C22H29O3N2 = 36921727

C21H25O3N2 = 35318597z = 1

31319035

minus226396 ppmC19H25O2N2 = 31319105

C21H26N6Na = 38521112


(b)



4 Conclusion




Acknowledgments


References































2



























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


Table 4 Continued


Qualitymatch with

Wileydatabase

Percentage oftotal

composition

13 2829

OH

HH

H

H


C29H48O [41269]

99 420

14 2880

H H

HH

HO


C29H50O [41471]

97 292

15 2956

H

H

HO

C30H50O [42672]


98 1301

16 3074

H

H

H

O

OO

H

H

C31H48O3 [46871]


95 4103





C17

H19

N2

[25115]

NH

R1 = H and R2 = CH3

C17H18N2 [25014]

2

7 20

15

143

16


R1 R2

N4

17CH3

C18

H21

N2

[26516]N

N

MeH

H CH2


C19

H25

N2

O2

[31319]N

N

N

H

H

N O

O

HO

OH Et



C19H24N2O2 [31218]

C21

H25

N2

O3

[35318]

Or

O

O

NH

HN

OMe

Et



Table 5 Continued



Or

NH

H NO

HO

MeO

Et


C21

H25

N2

O3

[35318]

Or

H

H

N

N

HO

MeO

Et


Or


NH

N

H

H

O

O

MeO

Et

Or

NH

N O

Et

H


CO2Me

NN

H

H

H

O

O

Me

Me


MeO


Table 5 Continued



C22

H29

N2

O3

[36921]

NH

N

HOOr

MeO

MeOEt

H


NH

N

HO

MeOEt

MeO

H

Or



N

H

N

H

O

Me Et

MeO

MeO

H

Or

NH

N

HO

MeOEt

MeO

H







26516930

z = 1

C18H21N2 = 26516993

minus235716ppm

25115374

z

mz

= 1

C17H19N2 = 25115428

minus214970ppm

27711333

z = 1C8H14O2N8Na = 27711319

051243ppm

30014344

z = 1

114054ppmC13H19O2N5Na = 30014

95100

230 240

Rela

tive a

bund

ance

250 260 270 280 290 300

9085807570656055504540353025201510

50

(a)

mz

Rela

tive a

bund

ance

z = 1

z = 1

36921680

38521154

35318547

z = 1

110836ppm

minus127746ppm

minus140455 ppm

95100105

9085807570656055504540353025201510

50

310 320 330 340 350 370360 380 390 400

C22H29O3N2 = 36921727

C21H25O3N2 = 35318597z = 1

31319035

minus226396 ppmC19H25O2N2 = 31319105

C21H26N6Na = 38521112


(b)



4 Conclusion




Acknowledgments


References































2



























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





C17

H19

N2

[25115]

NH

R1 = H and R2 = CH3

C17H18N2 [25014]

2

7 20

15

143

16


R1 R2

N4

17CH3

C18

H21

N2

[26516]N

N

MeH

H CH2


C19

H25

N2

O2

[31319]N

N

N

H

H

N O

O

HO

OH Et



C19H24N2O2 [31218]

C21

H25

N2

O3

[35318]

Or

O

O

NH

HN

OMe

Et



Table 5 Continued



Or

NH

H NO

HO

MeO

Et


C21

H25

N2

O3

[35318]

Or

H

H

N

N

HO

MeO

Et


Or


NH

N

H

H

O

O

MeO

Et

Or

NH

N O

Et

H


CO2Me

NN

H

H

H

O

O

Me

Me


MeO


Table 5 Continued



C22

H29

N2

O3

[36921]

NH

N

HOOr

MeO

MeOEt

H


NH

N

HO

MeOEt

MeO

H

Or



N

H

N

H

O

Me Et

MeO

MeO

H

Or

NH

N

HO

MeOEt

MeO

H







26516930

z = 1

C18H21N2 = 26516993

minus235716ppm

25115374

z

mz

= 1

C17H19N2 = 25115428

minus214970ppm

27711333

z = 1C8H14O2N8Na = 27711319

051243ppm

30014344

z = 1

114054ppmC13H19O2N5Na = 30014

95100

230 240

Rela

tive a

bund

ance

250 260 270 280 290 300

9085807570656055504540353025201510

50

(a)

mz

Rela

tive a

bund

ance

z = 1

z = 1

36921680

38521154

35318547

z = 1

110836ppm

minus127746ppm

minus140455 ppm

95100105

9085807570656055504540353025201510

50

310 320 330 340 350 370360 380 390 400

C22H29O3N2 = 36921727

C21H25O3N2 = 35318597z = 1

31319035

minus226396 ppmC19H25O2N2 = 31319105

C21H26N6Na = 38521112


(b)



4 Conclusion




Acknowledgments


References































2



























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


Table 5 Continued



Or

NH

H NO

HO

MeO

Et


C21

H25

N2

O3

[35318]

Or

H

H

N

N

HO

MeO

Et


Or


NH

N

H

H

O

O

MeO

Et

Or

NH

N O

Et

H


CO2Me

NN

H

H

H

O

O

Me

Me


MeO


Table 5 Continued



C22

H29

N2

O3

[36921]

NH

N

HOOr

MeO

MeOEt

H


NH

N

HO

MeOEt

MeO

H

Or



N

H

N

H

O

Me Et

MeO

MeO

H

Or

NH

N

HO

MeOEt

MeO

H







26516930

z = 1

C18H21N2 = 26516993

minus235716ppm

25115374

z

mz

= 1

C17H19N2 = 25115428

minus214970ppm

27711333

z = 1C8H14O2N8Na = 27711319

051243ppm

30014344

z = 1

114054ppmC13H19O2N5Na = 30014

95100

230 240

Rela

tive a

bund

ance

250 260 270 280 290 300

9085807570656055504540353025201510

50

(a)

mz

Rela

tive a

bund

ance

z = 1

z = 1

36921680

38521154

35318547

z = 1

110836ppm

minus127746ppm

minus140455 ppm

95100105

9085807570656055504540353025201510

50

310 320 330 340 350 370360 380 390 400

C22H29O3N2 = 36921727

C21H25O3N2 = 35318597z = 1

31319035

minus226396 ppmC19H25O2N2 = 31319105

C21H26N6Na = 38521112


(b)



4 Conclusion




Acknowledgments


References































2



























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


Table 5 Continued



C22

H29

N2

O3

[36921]

NH

N

HOOr

MeO

MeOEt

H


NH

N

HO

MeOEt

MeO

H

Or



N

H

N

H

O

Me Et

MeO

MeO

H

Or

NH

N

HO

MeOEt

MeO

H







26516930

z = 1

C18H21N2 = 26516993

minus235716ppm

25115374

z

mz

= 1

C17H19N2 = 25115428

minus214970ppm

27711333

z = 1C8H14O2N8Na = 27711319

051243ppm

30014344

z = 1

114054ppmC13H19O2N5Na = 30014

95100

230 240

Rela

tive a

bund

ance

250 260 270 280 290 300

9085807570656055504540353025201510

50

(a)

mz

Rela

tive a

bund

ance

z = 1

z = 1

36921680

38521154

35318547

z = 1

110836ppm

minus127746ppm

minus140455 ppm

95100105

9085807570656055504540353025201510

50

310 320 330 340 350 370360 380 390 400

C22H29O3N2 = 36921727

C21H25O3N2 = 35318597z = 1

31319035

minus226396 ppmC19H25O2N2 = 31319105

C21H26N6Na = 38521112


(b)



4 Conclusion




Acknowledgments


References































2



























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



26516930

z = 1

C18H21N2 = 26516993

minus235716ppm

25115374

z

mz

= 1

C17H19N2 = 25115428

minus214970ppm

27711333

z = 1C8H14O2N8Na = 27711319

051243ppm

30014344

z = 1

114054ppmC13H19O2N5Na = 30014

95100

230 240

Rela

tive a

bund

ance

250 260 270 280 290 300

9085807570656055504540353025201510

50

(a)

mz

Rela

tive a

bund

ance

z = 1

z = 1

36921680

38521154

35318547

z = 1

110836ppm

minus127746ppm

minus140455 ppm

95100105

9085807570656055504540353025201510

50

310 320 330 340 350 370360 380 390 400

C22H29O3N2 = 36921727

C21H25O3N2 = 35318597z = 1

31319035

minus226396 ppmC19H25O2N2 = 31319105

C21H26N6Na = 38521112


(b)



4 Conclusion




Acknowledgments


References































2



























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



Acknowledgments


References































2



























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

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