Phytochemical study of the alkaloiods of Iraqi Rhazya stricta Decaisne

Alaa mohamad khalil ,Dr Abdul Mutalib Nasser

Abstracts In the first part of the thesis, an introduction to the genus Rhazya is given together with a review on the reported alkaloids known to be present in this genus .Reference is also made to the chemical and pharmacological studies of these alkaloids and the folkloric usage of the plant Rhazya stricta Decaisne.The second part, deals with materials , instruments , general techniques and experiments carried on the Iraqi species of Rhazya stricta .The third part , gives the results obtained together with the detailed discussion of these results and characterization of the isolated alkaloids . The isolated alkaloids are characterized as :1.1,2Dehydroaspidospermidine 2.Didecarbomethoxytetrahydrosecamine 3. 16 R-Decarbomethoxytetrahydrosecamine Two of these alkaloids namely didecarbomethoxytetrahydro- secamine ,16R-decarbomethoxytetrahydrosecamine is considered a new alkaloids isolated from Iraqi Rhazya,While 1,2 dehydroaspidospermidine were isolated from genus Rhazya from the first time. Moreover ,a discussion of the experiments and the interrelationship of the isolated alkaloids relative to the general proposed biosynthetic pathway is given. IntroductionThe genus Rhazya (family Apocynaceae) comprises two species (3,4), namely Rhazya stricta Decaisne and Rhazya orientalis (5) . Rhazya species were called after the name of a Muslim scientist Abu Bakr Mohammed bin Zakariya Ar-Razi , known in Europe mostly under the Latinized name of Rhazes (1) R. stricta is a small glabrous, erect under shrub or shrubabout 90 cm high , with a smooth central stem and dense semi-erect branches ; leaves alternate, 6 to 10 1 to 2 cm, ellipticlanceolate, thick or leathery, sessile, turning yellow with age; flowers white in short branched cymes; fruit pale yellow follicles; seeds shortly winged (9). Vernacular names It is known harmal in Arabic, in Iraq locally name (( Luwiza)) (10) however, one should distinguish between the harmal for Peganum harmala and the harmal for R. stricta in Arabic countries ( as reported in flora of Iraq ).There is a plethora of studies on the phytochemical constituents of the leaves, fruits, legumes and roots of R.stricta (for an extensive review see Atta-ur-ahman et al., 1989). Table 1 summarizes the data obtained from some selected studies. Unfortunately, only few studies have investigated the biological activities of the isolated constituents. Several types of alkaloids and a few flavonoids have been isolated and their structure elucidated, mainly from the leaves but also from other parts of R. stricta found in India, Pakistan, Saudi Arabia and the United Arab Emirates.The anticancer activity of some of it s alkaloids is also reported() We have previously reported a number of new alkaloids from the plant () including didemethoxycarbonyltetrahydrosecamine from the roots() which was found to be cytotoxic against Eagles KB carcinoma of the nasopharynx in cell culture() 16Decarbomethoxytetrahydrosecamine was previously reported from the roots of Amsonia Tabernaemontana and from the rootbark of Aspidosperma excelsum but it was not determined whether the isolated compound possessed 16 R or 16 S-streoisomer.1,2-Dehydroaspidospermidine N-oxide() has previously been reported and its characterization by spectroscopic methods described() .Attemped deoxygenaton by reduction with phosphorus trichloride and by treatment with sodium borohydride led to formation of 1,2-dehydroaspidospermidine along with an indolic base, the structure of which is under investigation.() Alkaloid N-oxide are abundant in indole and indoline alkaloids, but their occurrence in the indolinine seriese is rare() . Thus 1,2-dehydroaspidospermidine N oxide is a new addition to the naturally occurring N- oxide in the indolenine series.

1,2-dehydroaspidospermidine N oxide

didemethoxycarbonyltetrahydrosecamine

16DecarbomethoxytetrahydrosecamineExperimental work GENERAL EXPERIMENTAL CONDITIONSPhysical constants: Melting points were determined in glass capillary tubes using Gallenkamp melting point apparatus and are uncorrected . Optical rotations were recorded on JASCO DIP-360 digital polariment in chloroform . The pH value were measured on model -25 pH meter (Shanghai Kanchuan Metal factory ). People Republic of China. Ultra-violet (UV) spectra : were determined in methanol on a Pye Unicam SP 800G or Shimadzu UV-240 (Shimadzu Corporation Kyoto, Japan) Instruments .Infrared (IR) spectra: were recorded in chloroform on a JASCO IRA-1 (JASCO International Co. Ltd ., Japan ) or JASCO A-302 (Japan Spectroscopic Co. Ltd .) spectrophotometers.Mass Spectra (GC MASS)GC conditionSystem GA ( used for analysis of yohambine type indole alkaloid) of GC MASSCharacteristic of GA system of GC MASSGA packed column :3%SE 30 or OV-1 on 80-100 mesh chromosorb GHP (acid washed and dimethyldichloro silane treated),2m2mm. glass column it is essential that the support be fully deactivated.Column temperature normally between 100and 300 (in our experiments we dont exceed 200C )for isothermal condition on approximate guide to temperature is to use the RI.Carrier gas nitrogen at 45 ml/minCapillary column 10 to 15m 0.32 or 0.53mm id 100% dimethyl- psx(x-1) with a 1.5 to3 m film thickness.Carrying gas helium.Temperature programming 4 min 135 3/min to200 .EXTRACTION 1.25 kg powdered root of R. stricta was deffated by maceration with 4.00 litres of petroleum ether (60-80 C) overnight with occasional shaking and the process was repeated . After removal of the solvent by filteration the marc was air dried at room temperature .The bulked filterate was concentrated under reduced pressure and the concentrated extract monitored by TLC on silica gel layers using different solvent systems and spray reagents but no alkaloids were detected .The concentrated extract was evaporated to dryness under reduced pressure.The dried powder was extracted with 2.0 litres of methanol employing overnight maceration with occasional mechanical stirring . The solvent was removed by filteration and the filterate evaporated to dryness under reduced pressure .The marc was again extracted using 2.0 litres methanol-ammonia (2% concentrated ammonia in methanol) and two further extractions were carried out using 2 litres volumes of solvent . The extracts were recovered by filteration and the filterates dried under reduced pressure each time and bulked . Maceration of the marc was under taken using 2.5 litres of 1% solution of sodium hydroxide in methanol overnight and the resultant liquid filterated .A final extraction was carried out using another 2.5 litres of the same solvent . The extract was recovered by filteration and the two filterates were combined and dried under reduced pressure .The combined residues from the whole extraction weighed 187 gm .The resultant dry extract was shaken with 500 ml N/1 hydrochloric acid solution and the mixture filtered . The residual insoluble material was further extracted with successive 250 ml quantities of N/1 hydrochloric acid .The acidic layers were bulked and shaken with six successive portions of chloroform (300 ml); the combined chloroform layers were collected together and evaporated to dryness to yield weakly basic fraction (5.2 gm). The aqueous phase was rendered alkaline with dilute ammonia solution ( PH 4.00 ) and extracted with chloroform (6 300 ml portions). The resultant chloroform extracts were bulked and evaporated to dryness under reduced pressure to produce the intermediately basic fraction (8.6 gm). The residual aqueous phase was rendered more alkaline with 5% sodium hydroxide solution (PH6.0 ) and similarity extracted with chloroform (6300 ml) to give the strongly basic fraction (1.8 gm). The alkaloidal extracts containing strong and intermediate basic fractions were bulked together (10.4 gm PH6, PH4 ) because they were found to be chromatographically similar with different solvent systems .The bulked extract was further fractionated by column chromatography using a column (5.0 cm in diameter ,80 cm in length) packed with the silica gel(210 gm, 60-230 mesh size) with increasing polarity of methanol in ethylacetate (500ml of each mixture) as an eluent . the fractions obtained on elution with (20%methanol:80%,ethylacetate),(30%methanol:70%ethylacetate),(40%methanol:60%ethyl acetate),(50% methanol: 50% ethylacetate), all these fractions was subjected to preparative thin layer chromatography using precoated silica gelGF 254 plates in the following solvent systems.1.Petroleum ether :acetone(7:3) 2.Ethyl acetate :isopropanol:ammonia (16:3:1)3. chloroform :methanol (19:1) The similar fractions were grouped together to give three fractions (total weight 10.4 gm) designated as fractions A-C. Each fraction was subjected to preparative TLC .Fraction A , was fractionated using the solvent system chloroform :petroleum ether(60-80 C):methanol (7:1:2) four band were isolated .Each band showed on checked TLC , the presence of many spots. The low yield of isolated bands beside the prescence of crowded spots in each, hundered further purification .Therefore no pure compound was obtained .Fraction B , was fractionated using the solvent system ethyl acetate :isopropanol: methanol(7:2:1).Four bands were isolated.which was subjected to preparative TLC using the solvent system chloroform :methanol : ammonia (8:2:0.5) The low yield of isolated bands beside the prescence of crowded spots in each, hundered further purification two compound were obtained RA1 2.2mg, RA2 1.9mg.Fraction C,was separated using solvent system ethyl acetate :isopropanol: methanol:petroleum ether (60-80 C)( 6:2:1:1)three isolated band were obtained and subjected to three steps of preparative TLC using chloroform : methanol (9:1). . compound RA3 2mg were isolated

Unknown RA1 :- (yield 8mg)SPECTRAL DATAUV max MeOH nm (log ): 224(2.16),284 (1.45) and 290 sh(1.38); min: 255(1.31) and 325(1.10).IR max CHCl3 cm-1 : 3500 (N-H), 2880(C-H stretching), 1360 and 1140 and 1015.MS m/z (relative intensity % ): 564 (0.43), 451 (0.54) ,438 (0.33),293 (3.65), 283(1.12),126(100)and112(0.87).The UV spectrum was characteristic of the indole chromophore showing absorption maxima at max (MeOH) 224 nm (log 2.16 ), 284 nm (log 1.45), 290 nm (sh, log 1.38) and min at 255 nm (log 1.31) and 325 nm (log 1.10 ). The IR spectrum afforded peaks at 3500 (N-H), 2880 (C-H stretching), 1360 ,1140 and 1015 cm -1 but did not reveal any absorption peaks in the carbonyl region .The mass spectrum showed a weak molecular ion peak at m/e 564. The most prominent feature of the mass spectrum was a very strong base peak at m/e 126 which was about 20 times stronger than any other peak in the spectrum .This is characteristic of the tetrahydrosecamine system containing a saturated 3-ethyl piperidine ring which also display an unusually strong signal at m/z 126(45). Accurate mass measurement of the M+ signal, the exact mass 564 (C36 H52N4. Calc. 564.4191). This showed the presence of 15 double bond equivalents in the molecule .Other fragments in the mass spectrum appeared at m/z 451.3016 (C31H37 N3), 438.2908(C30H36N3), 295.2182 (C20H22N2),283.2138 (C19H27N2), 126.1289 (C8H16N), 112.1180 (C7H14N). The mass spectrum indicated that didecarbomethoxytetra - hydrosecamine contained two indole units to which two C9H18N units are attached. The molecular ion peak at m/z 564 readily lost C7H5N to afford the ion at m/z 451 (C31H37N3, ion I) . Another loss of C8H16N from the molecular ion afforded the ion at m/z 438 (C30H36N ,ion II. The fragment at m/z 295 (C20H27N2, ion III) was shown to arise from the molecular ion by the loss of C18H25N2 . The fragment at m/z 283 (C19H27N ,ion IV) arose by the loss of C19H25N2 from the molecular ion .The intense base peak in the mass spectrum at m/z 126 (C8 H16N, ion V) arose by the loss of C30H36N3 from the molecular ionic peak as well as from the fragment at m/z 283. The molecular ion lost C31H38N3 to afford the ion at m/z 112 (C7 H14N, ion VI). The fragmentation pattern of didecarbomethoxytetrahydrosecamine (89) is shown in scheme . The intensities and structures of the proposed ion are present in the table The unknown RA1 was suggested to be didecarbomethoxy - tetrahydrosecamine

Mass spectara of didecarbomethoxy Tetrahydrosecamine

Unknown RA2 ( yield 8mg) SPECTRAL DATAUV max MeOH nm (log ): 220 (5.90),283 (5.40)and 291 (5.22) ; min: 250(5.72) and 289(5.35). IR max CHCl3 cm-1 : 3650 (N-H), 2900 (C-H stretching), 1720( saturated di esterC=O),1600(C=C),1430 and 1010.MS m/z (relative intensity % ): 622 (1.85),578(0.31),509 (0.22),369(0.26),313(0.44),239(0.54), 126(100) and 69 (4.54).The UV spectrum of 16R,16-decarbomethoxytetrahydrosecamine was identical to those reported for secamine (164,187,333,386) revealing max at 220 nm , 283 nm and 291 nm and min at 250 , 289 nm . The IR spectrum showed absorbtions at 3650(N-H), 2900 (C-H stretching), 1720 (ester C=O), 1600 (C=C), 1340 and 1010 cm1 The mass spectrum of 16R,16 decarbomethoxytetrahydro- secamine afforded the molecular ion peak at m/z 622.4576 corresponding to the formula C40H54N4O2, indicating the presence of 16 double bond equivalents .Other fragments in the mass spectrum appeared at m/z 564 (C38H52N4), indicating the loss of ester group from the molecular ion . Another fragment at m/z 496 arose by the loss of piperidine moiety from the molecular ion . Other significant fragments appeared at m/z 451 (C31H37N3), 293 and 126 (C8H16N).A prominent feature of the mass spectrum was astrong base peak at m/z 126 which is characteristic of the tetrahydrosecamine system containing a saturated 3-ethyl piperidine ring . The molecular ion also lost C7H14N unit to afford the ion at m/z 510 (C33H40N3O2 ). The formula of the ion were established by computer monitored high resolution mass measurement.(89) The unknown RA2 was suggested to be 16R,16decarbomethoxy- tetrahydrosecamine

16R,16-decarbomethoxytetrahydrosecamine

16R,16-Decarbomethoxytetrahydrosecamine mass spectra

Mass Fragmentation pattern of 16 DecarbomethoxyTetrahydrosecamine from622 m/eUnknown RA3(1.3 mg)SPECTRAL DATA UV maxMeOH nm (log ): 222 (4.50) and260 (3.80) ) : min: 245 (3.60)IR max CHCl3 cm-1 :2830 (C-H stretching) and 1710 (C=N)MS m/z (relative intensity % : 296(1), 280(46),251(23),210(37),194(21),169(9),168(12),156(12),149(33),124(32),97(42)83(48) and 56(100)The UV spectrum of 1,2-dehydroaspidospermadenine N-oxide was characteristic of indolenine chromophore with max 222 nm, 260nm and min 245nm .The IR spectrum showed a peak at 1710 cm-1 indicate the presence of a C=N group at 2830 cm-1 (C-H) ,mass measurement gave the mass of molecular ion to be 296. in agreement with the formula C19H25N2O .The intense peak at m/z 280. (C19H24N Ion I) corresponded to loss of 16 mw (o) indicate the presence of N-oxide function in the molecule . Other peaks were at m/z 210.(C15H16N2 Ion II) ,251,(C14H12N) and 124.(C8H14N, ion III) ,97,83 and 69. The fragmentation pattern was remarkably similar to that reported for 1,2-dhydroaspidospermadenine and related alkaloids ( ) and suggested that the substance was the corresponding N-oxide. In contrast to the 2,3-double bond of vincadifformine which facilitate the cleavage of ring C which afford a major fragment m/z 210 (ion II)formed from ion at m/z 296. The fragmentation of same of the ions are present in scheme

1,2-dehydroaspidospermadenine Mass spectra Figure Discussion of the experimentsThe plant material, often contain quite substantial quantity of very nonpolar fat and waxes .Because these compounds frequently cause problems due to emulsions when they are subjected to partition, they are often removed from the plant material as an initial step by percolation of the plant material with petroleum ether.5Most alkaloids are not very soluble in petroleum ether. But this extract should always be checked for the presence of alkaloids using one of the alkaloid-precipitating reagents described previously . After defatting, several procedural choices are available . The plant material may be extracted with water ,with methanol (procedure 2,3) or ethanol (procedure1), with aqueous alcoholic mixture , or with acidified aqueous alcoholic solutions(procedure1). Most alkaloid occur in plants as organic salt, and these salt are normally soluble in 95% ethanol. Pigments ,sugars and other organic secondary constituents are almostly completely removed with alcohol. But many of the more complex organic and inorganic salts are only partially removed . This usually reduce the problems of precipitation and emulsification in the next step.The alcohol solution is evaporated to a thick syrup and the residue partitioned between an aqueous acid solution and an organic solvent.Emulsion or precipitate are frequently observed at this stage .After repeated extraction with organic solvent ,the aqueous phase is made basic with sodium carbonate or ammonia.The basic aqueous solution is then extracted with a suitable organic solvent , normally chloroform or ethyl acetate .The extraction method are done in some like acidic medium (pH4,pH6) than the ordinary basic medium which are done usually in alkaloid extraction

All t The crude alkaloids were extracted from the roots (1.25 kg) of Rhazya stricta (experimental, Scheme A and B ).Extraction into chloroform on the basis of differential basicity afforded a number of fractions. The fraction (()) obtained by extraction into chloroform at PH 8 was subjected to preperative thin layer chromatography using silica gel (GF245) plates in petroleum ether :acetone (7:3) as the solvent system to afford a number of alkaloids . The band at Rf=0.36 was scraped off and elution with petroleum ether : acetone (8:2) gave a pure alkaloid (mg) which gave a characteristic orange coloured reaction with Dragendarff s reagent.

The crude alkaloid from the roots of R. stricta were obtained by extraction with aqueous methanol. They were subjected into column chromatography on silica gel. Elution with increasing polarities of petroleum ether ,petroleum ether ethyl acetate, ethyl acetate , ethyl acetate methanol and methanol resulted in several fractions. Fraction ()(experimental) obtained on elution with ethyl acetate : methanol (85:15) was subjected to preparative TLC to afforwd a new alkaloid ((mg)). The alkaloid gave orange coloured reaction with Dragendorff s.

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