Pharmacognosy by alaa mohamad khalil b. Sc Pharmacy 2003 Supervised by dr. Abdul-Mutalib Abdul- ghany to my latent father and sister




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Phytochemical study of some alkaloids of Iraqi Rhazya stricta Decaisne

THESIS

SUBMITTED TO THE DEPARTMENT OF THE PHARMACOGNOSY AND THE COMITTE OF GRADDUATE STUDENTS IN THE COLLEGE OF PHARMACY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN PHARMACY

( PHARMACOGNOSY )

BY

ALAA MOHAMAD KHALIL

B.Sc Pharmacy 2003)

Supervised BY

Dr.Abdul-Mutalib Abdul- Ghany

TO MY LATENT FATHER AND SISTER

CONTENTS


ABSTRACT

PART ONE

INTRODUCTION PAGE


  1. . Historical introduction 10

  2. Systematic and chemotaxonomy of the genus Rhazya 14

  3. Distribution and botanical description of R.stricta 27

  4. Pharmacological activities of rhazya stricta 28

  5. .Chemical studies of the genus rhazya 29

5.1 A.Chatterjee and co worker 29

5.2 Sclinose and co worker (Massachusetts Group) 30

5.3C.F.Smith and co worker (Manchester Group) 30

5.4A.R.Battersby and co worker(Liverpool Group) 31

5.5 Siddiqui and co workers (Krachi Group) 32

5.6 H.Kaneco and co worker (Osaka Group) 32

5.7G.N.Smith and co workers (Manchestor Group) 32

5.8Cordell and co workers (Manchester group) 33

5.9Zsadon and co workers (Budapest Group) 33

5.10Atta-urRahman and co workers(Karachi Group) 33

5.11J. Stoeckigt and coworkers (Munich Group ) 35

5.12GA.Cordell\Mukhopadhyay and Coworkers (Chicago group) 35

6.SPECTROSCOPICAL DATA OF ALKALOIDS OF THE GENUS RHAZYA 36

A-Ultraviolet Spectroscopy 36

B-Infrared spectroscopy 38

C Mass spectroscopy (MS) 39



  1. Corynantheine and related alkaloids

  2. Sarpagine and related alkaloids .

  3. Aspidosperma alkaloids .

  4. Eburnamine and related alkaloids.

  5. Secodine type.

  6. Secamine and Presecamine type.

G- Miscellaneous

Objective of the present work 64

CHAPTER 2

PLANT MATERIAL 66

EXTRACTION METHOD 1 74

EXTRACTION METHOD 2 79

CHAPTER 3

IDENTIFICATION OF ISOLATED COMPOUNDS 85

DISCUSSION OF EXPERIMENT 105

INTERRELATIONSHIP OF ALKALOIDS OF RHAZYA STRICTA FROM ROOTS 108

SCOPE FOR THE FURTHER WORK 115

BIBLIOGRAPHY 116

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 pharma-

cological 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.Akuammidine

3.Didecarbomethoxytetrahydrosecamine

4. 16-RDecarbomethoxytetrahydrosecamine

5 .O-Methyl eburnamine

6. Dihydrocorynantheol

Four of these alkaloids namely didecarbomethoxy- tetrahydrosecamine ,16R-decarbomethoxytetrahydro

Secamine,omethyleburnamine ,dihydrocorynantheol is considered a new alkaloids isolated from Iraqi Rhazya,

While akuammidine and 1,2 dehydroaspidospermidine were isolater 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.

Chapter 1

1. Historical introduction

The plant kingdom had long served as prolific source of useful drug ,and particular plant gained reputation as specific curing agents .among such plants were those of the family apocynaceae. (1)

` The family Apocynaceae consists of approximately 250 genera and 2000 species of tropical trees, shrubs, woody climbers, and herbs. The cardinal botanical features are an exudation of an abundant milky latex; simple,, opposite or whorled leaves; and showy, often pure white, salver-shaped and slightly fragrant flowers with five contorted lobes, with fruits in pairs.(2) The most remarkable characteristic is the plant’s ability to elaborate a series of dimers of monoterpenoid indole alkaloids including, notably,vinblastine and vincristine, from Catharanthus roseus . Vinblastine is particularly useful in treating Hodgkin’s disease whereas vincristine sulphate is used to treat acute leukemia in children.(3) A classical example of apocynaceae is Rauwolfia serpentine . The root of this plant has been used in India for a very long time in the treatment of insomnia and certain forms of insanity. In Western medicine, Rauwolfia serpentine tablets and powdered Rauwolfia serpentina(U.S. National Formulary , 1965) consisting of the dried, finely powdered roots have been used to treat hypertension and migraine.(4) Rauwolfia serpentine contains reserpine, an indole alkaloid which blocks the adrenergic transmission by depleting norepinephrine from sympathetic neurons.(5) Ibogaine is a psychostimulating alkaloid from Tabernanthe iboga, which protects the N-methyl-aspartate neuron receptors against the excessive release of excitatory amino acids, and represents, therefore, a potential therapeutic agent for the treatment of Alzheimer’s disease, Huntington’s chorea, and other brain conditions.(6) An additional interesting feature of Apocynaceae is the production of cardiotonic glycosides, steroidal alkaloids, and iridoids. finally strophanthusource kombe is the source of the cardio active glycoside ouabine , which also available.

An example of these alkaloids are illustrated in figure (1)





Figure (1)

Example of bioactive natural products from the family Apocynaceae.

The genus Rhazya (family Apocynaceae) comprises two species (3,4), namely Rhazya stricta Decaisne and Rhazya orientalis (5) . Rhazya spp 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)



Rhazya 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 10cm × 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 Rhazya stricta in Arabic countries ( as reported in flora of Iraq ).

Rhazya stricta is an important medicinal species used in indigenous medicinal herbal drugs to cure various ailments in Pakistan, India and Afghanistan, Saudi Arabia, Qatar, United Arab Emirates (UAE), Iran and Iraq (7).Itis reported as , mostly in the form of decoctions, for a variety of unrelated illnesses that include diabetes mellitus, fever, sore throat, inflammatory conditions and helminthiasis (8). In the rural areas of Saudi Arabia, the leaves of Rhazya stricta are used in folk medicine as a reputed bitter tonic and a curative for syphilis (9), chronic rheumatism, and similar types of pains , powder of dried fresh leaves is taken with water for pimples and acne problem of face. Fresh leaves are kept in shoes and put under feet for foot burning. Branches are used as toothbrush for teeth ache . The paste of soaked seeds with butter used for achenes and the removal of the heat effects (10).

The juice of the leaves of Rhazya stricta is given in the indigenus system of medicine with milk to children(8) . The infusion of leaves is useful for the sore throat and is reputed as a bitter tonic for fever and is used in general debility(12).The leaves are also used in the preparation of cooking bitter infusion , it is used as curative for chronic rheumatism . The leaves have also been reported to be poisonous. It possesses anticancer properity (8)

A good amount of work regarding pharmacological, phytochemical, toxicological and to some extent biological activities of Rhazya stricta has been reported (Atta-ur-Rahman and Fatima, 1982; Atta-ur-Rahman et al., 1989; Chatterjee et al., 1974; Ali et al., 2000)(13). But more work needs to be done regarding its medicinal importance, taxonomic and ecological aspects. As the plant of Rhazya stricta has immense potential as an anti-microbial due to the rich source of phytochemicals it possesses, therefore, such studies on biological activities anti microbial are recommended in various parts of the countries of Arabian and the Indian (14).

2.Systematic and chemotaxonomy of the genus Rhazya

The genus Rhazya is a member of sub family Plumerioideae,(23) tribe:plumerieae,subtribe:Euplumerieae belonging to the botanical family Apocynaceae , this family belongs to the sympetalous order Gentainales(23). Which also include the families; Rubiaceae,Loganiaceae,Asclopidaceae and Gentianaceae. Most of the plants of these families with the exception of asclepiadaceae species ,had been found to produce precursore of indole alkaloids if not the alkaloids themselves (23).

The family Apocyanaceae according to Pichon , are divided into 3 subfamilies which are described as follow

Subfamily Cerberiodaea : It is represented by small group of plants , reported to contain monoterpene alkaloids ,beside to types of piperidine alkaloids . The cardioactive glycosides of Cerbera (including Tanghinia) and Thevetia are well known .

Subfamily Echitoideae : Steroidal cardioactive glycosides are found in many genera of this subfamily , for example , Apocynum , Adenium, Beaumotia,Nerium and Strophanthus. In addition to the cardiac glycoside , some genera namely Funtumia, Kibatalia , Nerium and Strophanthus contain steroidal alkaloids known to have similar structure to the glycosidic Strophanthidine .

Subfamily Plumerioideae : It is the largest subfamily , represented by over 80 genera which are divided into seven tribes ; Alstonieae ,Allamandeae ,Ambelanieae, Carisseae, Chilocarpeae ,Rauvolfieae and TabernaemontaneaeThe plumerioideae produced a vast range of indolic alkaloids . Among the genera associated with alkaloid production are Alstonia ,Aspidosperma , Catharanthus ,Hunteria eiocarpa,Tabernaemontana, Rauwolfia ,Rhazya ,Vinca and Voacanga (23)



2.1 Indole alkaloid

More than 3000 terpenoid indole alkaloids are recognized making this one of the major groups of alkaloids in plants. In terms of structural complexity, many of these alkaloids are quite outstanding, and it is a tribute to the painstaking experimental studies of various groups of workers that we are able to rationalize these structures in terms of their biochemical origins . Many of the steps have now been characterized at the enzymic level, and appropriate genes have been identified. In virtually all structures, a tryptamine portion can be recognized. The remaining fragment is usually a C9 or C10 residue which are monoterpine, and three main structural types are discernible according to the arrangement of atoms in this fragment. These are termed the Corynanthe type, as in ajmalicine and akuammicine, the Aspidosperma type, as in tabersonine, and the Iboga type, exemplified by catharanthine. The C9 or C10 fragment was shown to be of terpenoid origin, and the secoiridoid secologanin was identified as the terpenoid derivative which initially combined with the tryptamine portion of the molecule. Furthermore, the Corynanthe, Aspidosperma and Iboga groups of alkaloids could then be related and rationalized in terms of rearrangements occurring in the terpenoid part of the structures. Secologanin itself contains the 10-carbon framework that is typical of the Corynanthe group. The Aspidosperma and Iboga groups could then arise by rearrangement of the Corynanthe skeleton as shown. This is represented by detachment of a three-carbon unit which is then rejoined to the remaining C7 fragment in one of two different ways. Where is C9 terpenoid units are observed, the alkaloids normally appear to have lost the carbon atom (Scheme 1).(24)






Figure (2)

the Corynanthe type, as in ajmalicine and akuammicine, the Aspidosperma type, as in tabersonine, and the Iboga type, exemplified by catharanthine

Indole alkaloids with a C9 or C10 monoterpenes moiety can be classified into the following main groups:


  1. Corynanthean Type : e.g. ajmalicine .

  2. Aspidosperma or Plumeran Type : e.g tabersonine and vindoline

  3. Iboga Type : e.g catharanthine

  4. Vincosan Type : formed by condensation of tryptamine or tryptophan with secologanin followed by ring formation lead to vincoside or other e.g peraksine and perakine

  5. Vallesiachotamine Type : derived from the common precursor of the Corynanthean and vincosan groups e.g antirhine

  6. Strychnan Type : e.g akuammicine

  7. Aspidospermatan Type : derived from stemmadenine e.g tubotaiwine

  8. Eburnan Type : derived from vincadifformine like a precursor of the aspidosperma group and having a rearranged secologanine skeleton e.g eburnamine

  9. Seco Type :in this type and Iboga has become fragm entated into seven and three (or two) carbon unit e.g secodine

10-The binary indole alkaloid : two alkaloid each belonging to one of the above classes, are linked to one another e.g vinblastine and vincristine

All the main skeletal types of indole alkaloids can be derived from () and they can be divided biogenetically into two main groups .



  1. Non –rearranged secologanin moiety , represented by corynanthean , vincosan , vallesiachotamine , strychnan and aspidospermatan.

  2. Eburnan,plumeran ,iboga and seco type alkaloids with rearranged secologanin unit.

2.2 Biosynthesis of indole alkaloids

Experimental evidence for the biosynthetic pathway came in 1965, when several groups showed that mevalonate was incorporated into alkaloids belonging to the three groups postulated byThomas and Wenkert(25,26).

Adminstration of [3-C14], [4-C14] and [5-C14] mevalonate (29,30,31) gave the same radio active alkaloids and confirmed that mevalonic acid played a part in the biosynthesis of indole alkaloids. Radioactive ajmalicine , reserpine (Corynanthean type), vindoline(Aspidosperma type) and catharanthine (Iboga type) were isolated , when sodium(2-C14)- mevalonate were feed to Catharanthus roseus shoots . The fact that most of the radioactivity was located at the ester carbonyl function of ajmalicine vindoline and catharanthine isolated from Catharanthus roseus showed that mevalonate was the a precursor of the C 9-10 unit in the Corynanthe/Strychnos , Aspidosperma types of indole alkaloids. Degradation of labelled catharanthine and dehydroaspidospermidine gave results in agreement with the head- to- tail combination of the two C5 units (30,32).

The usual biosynthetic precursor of mevalonate is acetate which would be expected to be a precursor to indole alkaloiods . But this fact was not established by labeling experiments . The experiments resulted in randomization of acetate radioactivity in Catharanthus roseus (32). DL-[-2-14C]- alanine was however found to be incorporated into the isopentenyl pyrophosphate – derived moiety of geraniol in large amounts.

In the experiments described above the incorporation of mevalonic acid led to mono, di-, tri- and sesqui- terpenoidal systems and thus suggested the terpenoid origin of indole alkaloids . The product of condensation of isopentenyl diphosphate and 3,3-dimethylallyl diphosphate is gerainol which was shown to be involved in the biosynthesis of indole alkaloids (Scheme 1 ) .

Thus administration of [2-14C] geraniol to Catharanthus roseus resulted in the incorporation of radioactivity into ajmalicine ,catharanthine, vindoline and serpentine () . Similarity nerol,an isomer of geraniol may act as a precursor . In labelling experiments in Catharanthus roseus with [9-14C ]-10-hydroxnerol, [9-14C]-10-hydroxygeraniol were incorporated into loganin , ajmaline ,vindoline and catharanthine.Similar results were obtained when[1-3H]-10-hydroxygeraniol and [1-3H ]-10-hydroxynerol were asdministered (35).

Similar experiments by Argon(30) and Scott(33) confirmed that geraniol may act as intermediate in the biosynthetic pathway for the Corynanthe, Aspidosperma , and Iboga classes of bases.



(Scheme 1)

the incorporation of mevalonic acid led to mono, di-, tri- and sesqui- terpenoidal systems and thus suggested the terpenoid origin of indole alkaloids

Of the various labelled iridoids only loganin was found to be incorporated into all three types of indole alkaloids(34) .This strongly supported that loganin playes a key role as intermediate in the biosynthesis of the three major classes of indole alkaloids comprising Yohombine, Aspidospermine and Iboga bases.

The fission of the cyclopentane ring is required in generation the non-tryptamine unit indicating that a seco- loganin type compound could be involved before condensing with tryptophan (or tryptamine ) to afford indole alkaloids. In vitro condensation of secologanin with tryptamine , afforded vincoside and isovincoside (identical to strictosidine isolated from Rhazya stricta Decaisne(36) ).

The pathway of strictosidine which lead to ajmalicine and other Corynanthe alkaloids requires no skeletal rearrangement, but enzymatic cleavage of glucose by glycosidase enzymes. Strictosidine is converted via a series of reactive intermediate (scheme 2) to 4,21-dehydrocorynantheine aldehyde (scheme 3) .This aldehyde then isomerise to which by cyclization lead to ajmalicine ,19-epiajmalicine ,geissoschizine and tetrahydroalstonine Scheme . The intermediate could be converted in plant to Yohimbine (scheme 3)38,39,40.





Figure 2

Vincoside ,isovincoside (identical to strictosidine isolated from Rhazya stricta Decaisne) possible intermediats



( Scheme 2)

The pathway of strictosidine which lead to ajmalicine and other Corynanthe from series of reactive intermediate

(Scheme 3)



The intermediate converted in plant to yohimbine,ajmaline, Tetrahydroalstonine ,geissochizine

Seeding of Catharanthus roseus [Ar – 3H]geissoschizine(40) and [OMe-3H] geissoschizine (43) are incorporated into akuammicine (96) (Strychnos type),ajmalicine (Corynanthe type ),and catharanthine (Ibogi type) . This providing evidence geissoschizine being a precursor to alkaloids having rearranged skeleton of the non- tryptamine unit. The conversion of geissoschizine to akuammicine require a rearrangement of the carbon skeleton.(Scheme)

Further evidence for such a conversion was obtained in Strychnos nux vomica L.() by the incorporation of geissoschizine and Wieland -Gumlich aldehyde into strychnine(Scheme). This indicate that the biosynthesis of strychnine (Strychnos type ) follows the following route Geissoschizine –Wieland –Gumlich aldehyde-strychnine

It can be postulated that geissochizine could be converted by one electron oxidative coupling to formylstrictamine rearrangement of the compound would first afford preakuammicine which lead to formation of akuammicine (44)

Wenkert had proposed that Strychnos alkaloids could serve as the precursors for the Aspidosperma and Iboga bases().This proposal was supported by feeding experiments in which the Strychnos – type alkaloid stemmadenine (O-methyl-3H,11-14C) was observed to be incorporated into Aspidosperma bases tabersonine and vindoline as well as into the Iboga base, catharanthine in Catharanthus roseus(). This mechanism involves the initial isomerization of stemmadenine to the enamine to generate the secodine intermediate which by intermolecular cyclization can give rise to tabersonine and catharanthine as given in Scheme 13

Wenkert proposed the contribution of C3-C7 'seco' species linking the Strychnos alkaloids with the Aspidosperma-Hunteria and Iboga alkaloids . According to his proposal the stemmadenine type intermediate could be converted to the α-formyl ester which by reduction followed by dehydration can afford the acrylic esters and 133,134 . These could then react by Michael and Mannich type reactions to afford Aspidosperma and Iboga classes of alkaloids respectively Scheme 15

The Manchester group of worker have isolated the dimeric alkaloids , secamine from Rhazya orientalis (45,46) which gave evidence for the existence of "seco" intermediate such as 133 in Scheme 15 . Subsiquent studies led to the isolation of a secodine derivative from Rhazya stricta. Decaisne and Rhazya orientalis (41,42). This further led some worker to assume the role of these compounds as an intermediate between Strychnos . Aspidosperma and Iboga type alkaloids .Incorporation with[14CO2,CH3 ,19H] secodine in Catharanthus roseus helped to isolate labelled vincoside

Subsquently a number of other seco have been demonstrated to occur, isolation of tetrahydrosecamine from Rhazya stricta(47) and decarbomethoxytetrahydrosecamine from Tabernaemontana cumminsa (48) , vinca minor and Rhazya stricta further supported the proposal by Wenkert. Secodine alkaloids have also been demonstrated to be present in Rhazya orientalis (49). These studies clearly show that the Aspidosperma and Iboga alkaloids rise in nature by rearrangement of the C-10 unit after condensation of secologanin with tryptophan

Subsequently a number of other seco alkaloids have been demonstrated to occur, isolation of tetrahydrosecamine from R. stricta and A.elliptica (45) and decarbo-Methoxytetrahydrosecamine from Tabernaemontana cumminus (), vinca minor(45) and R. stricta (67)further supported the proposal by Wenkert. Secodine alkaloids have also been demonstrated to be present in R.orientalis by dilution analysis(24).

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