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
1
Two of these alkaloids namely didecarbomethoxytetrahydrosecamine ,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.
Introduction
The 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 ArRazi , 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, elliptic–lanceolate, 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-urahman 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
2
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 it҆ 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()
16–Decarbomethoxytetrahydrosecamine 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.
O-
N
+
N
1,2-dehydroaspidospermidine –N oxide
3
N
N
N
H
didemethoxycarbonyltetrahydrosecamine
N
R1
N
N
R2
R3
R1COOCH3
N
H
R2H
R3H
16–Decarbomethoxytetrahydrosecamine
Experimental work
GENERAL EXPERIMENTAL CONDITIONS
Physical 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.
4
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 condition
System GA ( used for analysis of yohambine type indole alkaloid) of GC
MASS
Characteristic of GA system of GC MASS
GA packed column :3%SE 30 or OV-1 on 80-100 mesh chromosorb GHP
(acid washed and dimethyldichloro silane treated),2m×2mm. glass column it
is essential that the support be fully deactivated.
Column temperature normally between 100and 300 (in our experiments we
don҆t exceed 200οC )for isothermal condition on approximate guide to
temperature is to use the RI
.
Carrier gas nitrogen at 45 ml/min
Capillary column 10 to 15m ×0.32 or 0.53mm id 100% dimethyl- psx(x-1)
with a 1.5 to3 ʍm film thickness.
Carrying gas helium.Temperature programming 4 min 135 3/min to200 .
5
EXTRACTION
1.25 kg powdered root of R. stricta was deffated by maceration with 4.00
litres of petroleum ether (60-80 ο 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 .
6
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 (6×300
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, 60230 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%meth
anol: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.
7
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(60-80ο 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 (60-80ο 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
8
Unknown RA1 :(yield 8mg)
SPECTRAL DATA
UV ‫גּ‬max MeOH nm (log€ ): 224(2.16),284 (1.45) and 290 sh(1.38); ‫גּ‬min:
255(1.31) and 325(1.10).
IR ʋmax 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 ‫גּ‬max (MeOH) 224 nm (log £ 2.16 ), 284 nm (log£ 1.45),
290 nm (sh, log£ 1.38) and ‫ גּ‬min 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
9
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 C 9H18N units are
attached. The molecular ion peak at m/z 564 readily lost C 7H5N 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
10
Mass spectara of didecarbomethoxy Tetrahydrosecamine
11
Unknown RA2
( yield 8mg)
SPECTRAL DATA
UV ‫גּ‬max MeOH nm (log€ ): 220 (5.90),283 (5.40)and 291 (5.22) ;
‫ גּ‬min:
250(5.72) and 289(5.35).
IR ʋmax 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 ‫גּ‬max at
220 nm , 283 nm and 291 nm and ‫גּ‬
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
16R,16 decarbomethoxytetrahydro-
The mass spectrum of
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
12
m/z 126 which is characteristic of the tetrahydrosecamine system containing
a saturated 3-ethyl piperidine ring . The molecular ion also lost C 7H14N 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
N
R1
N
N
R2
R3
R1COOCH3
N
H
R2H
R3H
16R,16-decarbomethoxytetrahydrosecamine
13
16R,16-Decarbomethoxytetrahydrosecamine mass spectra
14
Mass Fragmentation pattern of 16 Decarbomethoxy
Tetrahydrosecamine from622 m/e
15
Unknown RA3
(1.3 mg)
SPECTRAL DATA
UV ‫ גּ‬maxMeOH nm (log € ): 222 (4.50) and260 (3.80) ) : ‫גּ‬min: 245 (3.60)
IR ʋmax 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
characteristic of indolenine chromophore with ‫גּ‬
max
N-oxide
was
222 nm, 260nm and ‫גּ‬min
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
16
1,2-dehydroaspidospermadenine Mass spectra Figure
17
Discussion of the experiments
The 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.5
Most alkaloids are not very soluble in petroleum ether. But this extract should
always be checked for the presence of alkaloids using one of the alkaloidprecipitating 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.
18
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
19
20
21
22
23
24
25
26
27
28
29
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
30
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.
31
32
he main skeletal types of indole alkaloids can be drived
33
Strychnos type
Corynanthe type
dehydrosecodine
eburnamine
connect Strych/ Corynanth
tetrahydrosecamine
secodine intermediate
Aspidosperma
Scheme 2
Vindoline
acrylic ester
formyl ester
enamine
9 4
secologanine
Wieland Gumlum
epiajmaline
Stemadenine
Iboga
aldehyde
Cathranthene
8
3
5
7
secamine
Strychnim
6
Formyl strictamine
Aspidosperma
spiroindole
akuammicine
vindoline
ajmalicine
vincoside
secologanin
Catharanthine
Tabersonine
peraksine
perakine
Tubotaiwine
Stemadenine
secodine
Antirhine
Catharanthine
Iboga
preakuammicine
seco intermediate
didecarbomethoxytetrahydroseca
mine
epiajmalicine
hydroxynerol hydroxygeraniol
Loganin
Nerol
geraniol
isovincoside
strictosidine
Cathenamine
geissochizine
Tetrahydroalstonine
34
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