Coumarins

“Coumarou” is a Caribbean word Utilized as a vernacular name of tonka bean (Diteryxodorata, Willd, fabaceae) [J. Bruneton and etal 1999]. Similarly ”Coumarou” term was also subjected to trivial name of tonka tree (Coumarouna adorat Aubl)[Guillemette, 1835]. Coumarin is now well known as a collective trivial name for naturally occurring aromatic lactones which posses a 2H-1-benzopyran-2-one nucleusas a functional structural unit. Coumarin (1) as in fig. was the pioneer member of respective series, isolated from the natural source and got the structural characterization. Several researchers independently deduced the structure (1) for coumarins.

[Fittingand etal, 1868] [Strecker and etal, 1968] [Tiemann, F. and etal 1977]

Coumarins are well distributed in all parts of plant. Synthesis of coumarins generally occurs in leaves but predominantly fruits have higher coumarin content followed by root, stem and leaves. A total of 107 plant families are known to produce coumarins. Coumarins are found free or as heteroside among the dicotyledonous families like Apiacea, Fabaceae, Asterreacea, Morraceae, Roseaceae, Rubaceae and Solonaceae [Wienmann and etal 1997] [Matern, U. and etal 1999]. Rutaceae and umbelliferae are rich sources of coumarins. From Rutaceae above 800 naturally occurring coumarins have been recorded [40 no ref of nighat sultana thesis]. Among the monocotyledonous families Gramineae and orchids are representatives of coumarin content.

Classification of Coumarins

Coumarins are diverse and wide naturally occurring compounds belonging to benzopyrane group in which benzene ring joins a pyrone. In natural coumarins oxygen functionalities are present at one or more of the six available nuclear positions. But exception is present like a case of coumarin (1) and 4methyl thio-5-methylcoumarin (2). The oxygen atoms can be ethereal, glycosidic or phenolic groups. C-7 is the oxygenated position in most coumarins. 7-hydroxy coumarin is the most abundant member marked as Umbelliferone (3) is also regarded as a biogenic precursor of more complex coumarins. Classification phenomena is on the basis of fused ring system and linked functional groups.

1-Simple Coumarins

This class contains Alkoxylated like herniarin (4), alkylated like 4-methyl thio-5-methyl coumarin (2) and hydroxylated like umbelliferone (3) derivatives of coumarins and their respective glycoside.

2-Furano coumarins

This class contains a furan ring fused to an aromatic ring like a linear type like Psoralen (5) or an angular type like angelicin (6) and their respective derivatives.

3- Pyrano coumarins

As the name of class suggests a pyran ring fused to an aromatic ring in a linear type like xynthylitin (7) or as an angular type like seselin (8).

4- Substituted pyrone ring type coumarins

In this class a substituted pyrone ring like 4-hydroxy-coumarins (9) and 3-phenyl coumarins like Coumesterol (10) are found.

Biosynthesis of coumarins

Coumarins are typical metabolites of higher plants rarely occur in microorganisms. Biosyntesis of coumarins like 2-benzopyrones is derived via shikimic acid, chrosmic acid, phenylalnine and cinnamic acid pathway along with an enzyme system capable of o-hydroxylating cinnamate, responsible for synthesizing coumarins. During the process the ultimate carbon source is carbon dioxide. [90, Brown and Towers 1960]

Formation of shikimic acid starts as D-erythrose 4 phosphate and phosphoenol pyruvic acid (PEP) undergo aldol condensation phenomena. Reaction is catalyzed by an enzyme DAHP synthetase giving a 7 carbon sugar product, 3-Deoxy-D-arabionoheptulosonic acid 7-phosphate. Reaction is a type of nucleophilic addition to the tetrosephosphate carbonyl group, methylene group of PEP in a concerted mechanism act as a nucleophile with the hydrolytic cleavage of bond between the phosphoric acid and enolic oxygen. Intermediate formed having an absolute configuration with C-1 and C-5 assymetric centers, stereospecific enzyme controlled steps I and step2 give this result, reaction of sp2 prochiral carbon atoms are involved. C-4 configuration correlates to that of precursor D-erythrose at position 2.

In the next step intermediate undergoes dehydration than carbonyl functions subjected to reduction, obtained product is shikimic acid. In this product Shikimic acid there is a new chiral centre C-3 is generated by stereospecific reduction of corresponding carbonyl group while loss of C-1 assymetric centre of dehydroquinic acid takes place.

The shikimic acid undergoes phosphorylation at C-3 attachment at a 3 carbon side chain, here one molecule of phosphoenolpyruvate (PEP) is fascilitated than 1-4 elimination of phosphoric acid elements produce chorismic acid.

Step 2- Chrosmic acid converts to coumarin

Chrosmic acid is considered as a key compound because of its multifunctional structure capable of providing variable pathways to develop various aromatic compounds.

a- Formation of perphenic acid

Phenomena of vascular plants while higher plants have an important role in their metabolism

In L-phenylalanine deamination stereochemical mechanism is involved which can be explained by Hoffman elimination type, prefers involvement of one of the 2 diastereoscopic hydrogens C-3(Hs) methylene.

E2 type reactions, on the basis of stereoelectronic requirements discriminate 2 prochioral hydrogens. Among the E2 type reaction at the transition state the leaving groups are anti periplaner, together with(s) configuration of C-2 assymetric centre with stable configuration of the transition state. Newman formula describes transition state where NH2 groups and pro-S hydrogen are anti periplaner. Geometrical representation of transition state is an evidence of trans configuration of produced compound i.e. Cinnamic acid.

Step 3- Cinnamic acid leads to coumarin formation

This stage, Cinnamic acid formulation, a branch point in the total biosynthesis of Coumarins, Aflatoxins, Furanocoumarins, Phenylcoumarins, Pyranocoumarins, Miscillineous microbial coumarins and simplecoumarins, as shown by different classes of coumarins.

Simple coumarins have the entire carbon skeleton, without any additional ring, and a benzopyrone nucleus. Brown utilized tracer experiments and evaluated biosynthetic route of coumarin formation. [ ] Biosynthetic path for 7 oxygenated and nonoxygenated coumarins differ but an overall common pathway is there.

Scheme elaborates ( ) biosynthetic route of

Scheme elaborates ( ) biosynthetic route of

Scheme elaborates ( ) biosynthetic route of

The common phenomena in plant based coumarin biosynthesis is an isomerization of cis and trans glucoside forms.

Heierochloe adarata (Graminacea) and Milieatus alba (Lusiminaceae) were trailed to study coumarin biosynthesis. (aa)--- (dd) as sequence of scheme 3 was truly reported as described. C-7 is the hydroxylation position inn maximum coumarin cases.

Lavander (Lavandula officinalis) studied by Tracer distinctly revealed in experiments an efficient incorporation of ,”Cinnamic acid, Glucose, Phenylalanine in both coumarins ( ) and ( ).

o-coumaric acid --------------------o-comaryl glucoside----------------- coumarins

p-coumaric acid---------------------2-Glucosyl ox-4-methoxy Cinnamic acid

Umbelliferne Herniarin

In M. alba gene controlled ortho-hydroxylation is employed on trans Cinnamic acid . C14 labelling proved the formation of intermediates, ( ) o-glucoside as in the table and ( ) o-coumarinyl glucoside and an enzyme beta-glucosidase causing specific hydrolizatio of cis-glucoside in M. alba. In case of Herniarin p-hydroxylation of Cinnamic acid occurs. Isolation of B-glucoside enzyme is possible by crushing cells of M. alba, affording an aglycone, o-coumaric acid ( ), giving spontaneous cyclization to give coumarin ( ).

Flavonoids“Flavus” a Greek word means yellow drive’s the word Flavonoid a name representing a wide spread and most diverse natural products belonging to a large and important group of poly phenolic compounds. Flavonoids in general have a universal distribution among higher plant accompanied by their occurance lower plants notably in Liverworts and Mosses, rarely in Fungus but no reports till now from Bacteria [Harborane, 1999, Zeenester, 1957]. 2 percent of all photosynthesized carbon is converted in to these biologically active and important plant ingredients.

Autoxanthin term Kis used for flavonoids as they owe colouring copigments quality, giving a wide range as red to blue colour, in flowers, fruits and leaves. Others, like flavones, truly colourless providing”Whiteness” of white flowers otherwise it is translucent [Swain, 1976]. So brilliant natural colour in flowers and fruits indicates presence of one or more flavonoid pigments. Few black and brown natural pigments are outcomes of oxidation phenomena of flavonoids and relevant phenolic compounds [Swain, 1976]. On the other hand flavonoids play different roles in growth and development of plants. e. g. Leaf flavonoids deposited at epidermal cells or at upper waxy surface of leaves manage protection against Ub-B radiation potential damages.

Classification

Presentation of flavonoids is by means of a variety of structures is available. Truly all flavonoids have a parent nuclei of 15 carbon atoms and exhibit a common structural phenomenon of 2 phenyl rings linked by a 3 carbon chain (diphenyl propane derivatives). Skeleton of 1,3diphenyl propane is a presentation of chalconoids. A tri carbon chain linking two phenyl rings can form a third ring (may be five or six membered) by an oxygen on one of phenyl rings generate a tricyclic system. Auronoids are tricyclic compounds having a five member heterocyclic ring. Flavanoids and Flavonoids are tricyclic compounds having a six member heterocyclic ring as in fig…..( ). Tricyclic compounds of isoflavoniods, isoflavanoids and 3-phenylcoumarins are originated from 1,2diphenyl propane system and neoflavonoids are of 1, 1-diphenyl propane origin as in fig…… ( ).

Saturation of central heterocyclic ring raise 2 categories of flavonoids. i. e. presence of unsaturation like orthocyanin case while in flavones and flavanol molecules is of planar type.

Occasional distortion in planarity occurs. e. g. 2/hydroxyl substitution in a 3-o-methylflavanol provides bathochromic shift during spectral evaluations. In case of saturated flavanoids like flavans and flavanones involvement of one or more chiral centers make optically active forms one or more. Infact they generally take up a conformation with tw].o benzene rings in right angles. Glycosidic substitution in flavinoids can raise optical activity [Harborane, 1999].

Flavonoid tricyclic compounds like Auronoids and isoflavonoids have ring labelling like ABC while individual carbons (atom/atoms) denoted by numbering system in that, ring A and C marked by ordinary digits (1, 2, 3 etc) and by primed digits (1/, 2/, 3/ etc) for ring B. As given in fig. ( ). Although referring to C-9 and C-10 in flavonoids as C-8a and C-4a is present.

For chalcones A ring iswritten at left, denoted by prime numbers and B denoted by ordinary numbers. Carbons of exocyclic type are managed by alpha and beta signs in relation of their carbonylfunction.

Naturally occuring flavonoids and isoflavonoids are in oxygenated condition with hydroxyl and or methoxyl substituents. In scheme ( ). naturally occuring classes of monomeric flavonoids is described by nomencleature of represented structure.

An additional carbon in main skeleton is a tendency of hamoflavonoids belonging to tribe flavonoids. Occurance of flavonoids as 0-glycoside is reported where one or more hydroxyl groups belonging to flavonoid nuclei are bound to single or more sugar by an acid labile hemiacetal bond. In/, flavonoid C-glycoside the sugar is C-linked and linkage is acid resistant [ ]. Glycosylation as an effect render flvonoids to be more water soluble and less reactive.

Biosynthesis of Flavonoids

Generally phenyl propanoid pathway leads to synthesize Anthocyanins, Flavanols, Flavanones, Flavones, Isoflavones and Lignins, all of those are secondary metabolites. Flavonoid biosynthesis is supposed to occur in the cytoplasm (also regarded as a cytoplasmic product) in an enzyme catalyzed enviorment. Enzymes are associated with membrane of endoplasmic reticulum and here large macromolecular complexes are formed by them. Biosymthetic origin determines the classification of flavonoids. Certain flavonoid classes serve like intermediates as well as like products during the biosynthesis of flavonoids as they are accumulated in plant tissues. Chalcone, an example of that kind.. Biosynthesis of flavonoids involve a central C-15 intermediate Chalcone, both biosynthetic paths, either shikimic path or acetate path are leading to form that particular and basic precursor to produce flavonoids. Number of investigations has justified the central role of chalcone in flavonoid biosynthesis [Wang, 1965, Grisebach, 1965]. Chalcone synthase, an enzyme of key importance which conducts chalcone formation. Malonyl co enzyme A and 4-coumaryl coenzyme A also termed as hydroxycinnamic acid co A ester are derived from carbohydrates both are flavonoid precursors. Malonyl co A is a product of glycolysis intermediate acetyl co A carboxylase. 4-coumaryl co A synthesis is complex occurs via shikimic acid pathway dealing with major path way to form amino and aromatic acids, phenylaline and tyrosine in higher plants.

D erythrose-4-phosphate and phosphoenol pyruvic acid undergo condensation to start biosynthesis and give shikimic acid as in scheme…. ( ).

Some individual steps in Flavonoid classes

Flavanone formation

Phenomena of Isomerization; Intra molecular cyclization of chalcones leads to the formation of flavanones. Invivo and invitro evidences narrate the equilibrium between chalcones and flavones formed via this path [56]. Chalcone isomerase catalyze in vivo inter conversion of flavanones and chalcones. Stereo specificity as apparent in (s) chirality of C-2 in flavanone derivatives is an important feature of cyclization of chalcone isomerase. That is the evidence of naturally occurring flavones having (S) configurationat C-2 and levorotatory. The available hydroxyl group plays an important role in the stability of equilibrium as Chalcones with C-2 and C-6 free hydroxyl groups tend to shift the equlibrium rapidly towards flavanone. Similarly hydrogen bond between carbonyl group and orth-phenolic hydroxyl group reflects its role on stability, equlibrium state and rate of interconversion. In case of single available hydroxyl group either for cylisation or hydrogen bonding an open system is recorded like chalcone form [Ref, 56 super visor]..

Scheme 15 of supervisor.

Step 4- Formation of Flavone

Firstly in parsly plants invitro flavone to flavones formation was studied. [ ]. Later results were observed in depth via parsly cell suspension cultures [ ] and in Antirrhium flowers.

The reaction of naringenin (flavone) to apegenin (flavone) is an enzyme catalyzed process in both the above mentioned cases ( i. e. parsly plant case and Antirrhium flower case). Double bond’s mechanism of formation is yet to be clarified. While a purposal of 2-hydroxy flavanone formation at an initial step and elimination of water (H2O) by means of dehydrase exists.

[Britsch and etal 1981] [stoz, g. etal 1981]

Sceme 16 of super visor

Step 5-Isoflavone formation

Isoflavones are related with flavonoids biogenetically although they have distinct class because of rearranged C15 skeleton, derived from 1,2 diphenylpropane. Isoflavone constitute the largest group of natural products. Due to that natural flavanoid derivatives have thousands of aglycones. e. g. (a) Simple isoflavones

(b) Complex isoflavones

© Isoflavone glycosides

2,3 migrations of aryl side chain of a flavone chalcone intermediate is an important and key point in Isoflavone biosynthesis.

It is recorded recently that the transformation of naringenin (flavone) in to genistein by an enzyme activity catalyzing this phenomena, in microsomal preparations from elicitrol-challenged soya bean cell suspension cultures. 2 steps of an enzyme catalyzed process are reported. Firstly oxidation and rearrangement make 2-hydroxy2, 3 dihydrogenistein from

naringenin, provided with a must needed NADPH and molecular oxygen. While the next step involves water elimination from 2-hydroxyisoflavanone in soluble fraction. i. e. Soya bean cell suspension culture and this require a full characterization till now. [Dr. Itrat Anis].

Step 5- Flavanol formation

From parsley cell suspension cultures, an enzymatic conversion like dihydroflavanols to Flavanols are also discovered. Flavanol biosynthesis catalyzed by a soluble enzyme 2-oxoglutarate-dependen oxygenase.

Flavanol biosynthesis occurs by a 2-hydroxyintermediates like 2-hydroxydihydrokaempherol with subsequent dehydration produces a respective Flavanol as illustered in scheme ( ). Like parsly plant studies 2-oxoglutarate-dependent dioxygenase (a soluble enzyme) catalyze Flavanol formation in flowers, Flavanol synthase detection in floral extracts of Matthiola and petunia [63, 64].

Glycosylation

Flavonoid glycosides occur naturally in vast numbers showing a wide range of glycosyl transferases with different specifications of their substrates. Anethin cell cultures [67], chrysosplenium shoots [65, 66], pisum flowers [64] and tulip anthers [63] revealed an enzymatic preparation of novel Flavanol 0-glycosyl transferases. Among such enzymes pronounced specificity execution regarding substrate, the position and transfer of sugar. E. g. High specificity displayed by7-o-glycosyl transferase (an Isoflavone) fro cicer [68].

Chrysosplenium extracts gave 2 enzymes glycosylating B ring of methoxylated (highly) flavanols at positions 2/ and 5/ [69]. Silene pratensis and Silene dioica were studied for an extensive genetic and biochemical evaluation of Glycosylation of isovitexin(6-c-glycosyl apegenin)resulting an interesting situation. Functional 11 allaels spread over six loci, as identified now, coding different glycosyl transferases, catalyzing Glycosylation, galactosylation and xylosylation of 77-hydroxyl group or Glycosylation arabinosylation, rhamosylation and xylosylationof 2hydroxyl group of carbon bond glucose in isovitexin case.

Methylation

In case of flavanoid structures methyl transfer from S-adensylmethionine to its various hydroxyl groups, catalyzed by many enzymes. Requirement of an obligatory co-factor as Mg+2 type is recorded in these cases. An enzyme inhibitor like S-adenosylhomocystein is formed during the reaction. From flowers of Lotus corniculatus 8-hydroxy flavanol 8-o-methyl transferase is recorded. [jay1983, Jay1985]. 3 and3/-o-methyl transferase is also found in Lotus, can cause methylation of Flavone (Luteolin) and Flavanol like quercetin, znyricetin at relevant positions.

Terpenes

“Terpentine” is a term used for volatile oil coming form pine tree, with a basic alpha piene chemical used in oil paintings was later frequently subjected to water insoluble volatile compounds having resiny smell from plants, lead to the word Terpenes [clayden, J, etal, 2001]. Among natural products Terpenes are wide spread and most of the times with interesting chemistry with C-5 isoprene unit arrangements. Each isoprene unit is represented by C-5 (a monomeric unit) regarded as nature's favourate bulding block in terpene biosynthesis [1998].

In 1884 Tilden assigned a structural fragment of a common five carbon unit in monoterpenoids, on the basis of structure similarity in them and isoprene formation after their thermal decomposition. Similar structure unity was the base of isoprene rule, expressed by Ruzicka in 1921, I. e. Among terpenoids carbon skeleton is in the form of isoprene units having regular arrangements [Ruzicka, 1953, Eschemmoser, 1955]. “Biogenetic isoprene rule” supposed rearrangements in biosynthesis to overcome apparent exception [Barton and etal, 1979]. Irregular terpenoids biogenesis were later justified by Ruzicka in 1953. I. e. Terpenoid owe C-5 (isoprene units) but enzymatic modification of formed structures revealed different ways to produce the skeletal forms. Later on improved rule justified no of naturally isolated terpenoids [Ruzicka, 1953]. According to biogenetic isoprene rule terpenoids are combination of isoprene units and aliphatic substances. I. e. furnesol, geraniol, squelene etc.

So derivative of terpenoids occur by cyclization and cylised rearrangements of aliphatic precursors [Borton, 1979]. Classification is as

1- 1 C-5 unit (C5) Hemiterpene

2- 2 C-5 Unit (C10) Homoterpene

3- 3 C-5 units (c15) Triterpenes

“Head to tail” bonds,supplementry bonds, polymerization of isoprene units are the consequences of a common biosynthetic origin of terpenes [Pa Manito, 1981, Newman, 1981].

In variety of microorganisms and eukaryotes squalene and 2,3 oxidosqualene undergo stereo selective cyclization and rearrangement of skeleton by an enzyeme (one enzyme only) catalyzed reaction to give tetra and penta cyclic triterpenoids. Tritepenoids resulted by sqalene and 2,3epoxysqualene are categorised in sub-groups in accordance of the enzyme catalyzing them.

---- Oxidosqualene cyclases catalyze's OS oxy squalene formation.

---- Squalene cyclases catalyze's Squalene formation.

As literature narrates terpenoids resulted from dehydration, hydrolysis, isomerization, oxidation and reduction like phenomenas during biosynthesis so classified as under

1- Arborinol also known as ferneol or mortenol group.

2- Cycloratane group

3- Dammarane group.

4- Euphane group.

5- Hopane group.

6- Lantostane group.

7- Lupane group.

8- Sqalene group

9- oleane group.

10- Ursane group

11- Tetrahymanol group.

Biosynthesis of Triterpenes Polycyclic triterpenes have 3 following steps during the mentioned proces.

1- IPP and MAP Isoprene unit biosynthesis

2- Epoxy, prenylogues and squalene biosynthesis

3- Polycyclic triterpene biosynthesis via cyclization and rearrangement.

Acetyl coEnzyme A

6CO2, 6O2 undergo photosynthesis to form glucose molecule C6H12O6. C6H12O6 undergo glycolysis to give pyruvate which is transferred in to Acetyl coenzyme A as

IPP and DMAPP Isoprene units C-5

Natural products of 30-carbon skeleton are triterpenes, supposed to be derived from mevalonic acid (MVA) through 2,3 epoxy squalene mostly and through squalene as well. Squalenes double bonds multiple cyclizations,are resposible for various triterpene structures, mediated by cyclases (an Enzyme) capable of exrting rigrous stereochemical control. Cornforth studies 1959 reveals the 2 active isoprene forms IPP and DMAP obligatory for terpene synthesis among plants. Different enzymes catalyzed incorporation of IPP and DMAP and respective intermediates to terpenes [Cornforth, 1966, Cornforth, 1969].

Mevalonate Pathway

Biosynthesis of squalene starts with the dihydroxy mevalonic acid (MVA) so same respect is for triterpenoids. At first step clasien condensation provide acetyl coenzyme A which in turn reacts with another molecule of Acetyl coenzyme A.

Than aldol condensation phenomena gives 3-hydroxy3-methyl glutaryl coenzyme A (HMG_CoA). HMG_CoA reacts with NaDPH (reduced form of NADPH2, nicotinamide) under the catalyses of an enzyme hydroxymethyl glutaryl-CoA reductase (HMG_CoA) to give Mevalonic Acid(1).

Interconversion between mevalonic acid form and mevalonolatone form is ctalyzed by an enzyme sigma-lactonase having heat labile nature[Cornforth, 1969].

Mevalonate kinase (an enzyme) catalyse's 5-phspho mevalonic acid formation in an ATP required reaction from mevalonate. % phosphomevalonic acid undergo phosphorylation while an enzyme phosphomevalonate kinase act as a catalyst to form R-5-diphospho mevalonic acid(1b) in the presence of diphosphomevalonate decarboxylate give, IPP(2) when decarboxylative elimination occurs, isoprene unit like a biological form exists. In the next stepan isomeric form of IPP, DMAPP is formed under the influence of isopentenyldiphosphate triangle-isomerase [Dewick, 1999]. scheme 1.

Squalene Naturally occurin prenyl transferases have different types, each of that capable of catalyzing single or more reactions as in scheme ( ). Namely fornesyl pyrophosphate (FPP), Geranyl fornesylpyrophosphate (GFPP), Geranyl geranyl phosphate (GGP) and Geranyl pyrophosphate (GPP) are the obligatory intermediates to synthesize terpenes and curstenes, are free pyrophsphate esters formed from isoprene units. In open chain intermediates elaborations like cyclization gives structural varities of mono to sester terpenes. In case of higher terpenes like C-30 triterpenes and crotenes instead of derivation from 2 hydrocarbons , as squalene and phytoene produced by FPP and GFPP respetively., a complicated phenomena of tail to tail dimerization recorded [Manito, 1981].

Tail to tail dimerization via reductive form takes place and fornesyl pyrophosphate (FPP) forms squalene. In FPP molecule addition of 2 hydrogen atoms liberates 2 molecules of phosphoric acid to form one molecule of squalene. An intermediate (PSAPP) procceds the reaction as isolated from yeast microsomal preparation when added with FPP [Wrilling, 1969]. An assymetric conversion occurs here. Squalene formation in most organisms,but an immediate transformation in to 2,3 epoxide, takes place.

Cyclization and rearrangement giving polycyclic triterpenes

Fundamental triterpenes are derived via cyclization of squalene at 2,3 epoxide position. Approximately 20 known kinds of triterpenes depend upon the squalenes tendency with six double bonds, it posses, to procced or undergo no of cyclizations and division in to subgroups according to their origin, as under

a- Oxidative b- Non oxidative c- Oxidative-nonoxidative

In cyclization initialy an inicipient carbocation formation at the tertiary carbon of the end double bond in poly isoprene unit/chain. Cyclization can be promoted via an oxidation agent leading cyclic compounds with ring A having a hydroxyl group or ketonic group at 3rd carbon. In case of squalene conversions to 2,3 epoxysqualene. Followed by protonation and Markonikoff's opening at epoxy ring as in path A of scheme ( ) or in OH+ (protonated oxene). Case 2,3 double bonds are attached as in path B of scheme.

The nonoxidative cyclization is intilized via an attack of proton as illustered in scheme ( ). Path C oxidative cyclization of squalene and 2,3 epoxysqualene is a preffered path in case of fundamental triterpenes.

Squalene or 2,3 epoxy squalene usually undergo oxidative cyclization in case of fundamental triterpenes.

Oxidative cyclization

originates at a single terminal of squalene or derived from a molecule of 2,3 epoxy squalene, at an active enzyme site in chair-chair-chair-boat or chair-boat-chair-boat conformations. Cyclization, successive hybrid series and shifts of methyl groups is interruptionless depending upon the tendency of C-13, electrophilic center, to attack penultimate double bond of squalene in accordance with Markownikoff regiospecificity.

Non oxidative cyclization

Rarely occur as a result of protonic attack on 2-3 squalene double bond. Such cyclization involve formation of different skeletons derived via 'chair-chair-chair-chair-chair” squalene conformation. Cyclization of that kind extendable triterpenes nonhydroxylated at C-3 position.

Oxidative & non oxidative cyclizations

In such kind, squalenes involve their both sides in cyclization phenomena. An independent electrophilic attack on both sides of squalene ( ). In case of oxidative e. g. Onocerin and non-oxidative e. g. amberin scheme ( ) [Eschenmosser, 1990] are given to justify the biosynthesis of triterpenes.

Epoxy squalene, squalene, prenyiogues

In first step the IPP and DMAP couples and condense under an influence of pre-enyl transferase enzyme to give GPP and its isomer. GPP again condenses with IPP, catalyzed by same pre-enyl transferase enzyme to produce FPP. Condensations are conducted in such a manner that produce IPP's superficial alkylation-deprotonation takes place. Basically it is a concerted addition-elimination sequence or in other words trans addition than trans elimination phenomena is going on. DMAPP can only behave as foundation stone, loaded with building IPP bricks, obtained product prior to C-5 addition, posses some tail structure and reactivity equivalent to DMAPP.

Simple SN-2 mechanism type inversion of configurationat C-5 (carbon atom)of geranyl pyrophosphate, coressponding to allylic alcohlic group of DMAPP>

IN IPP, retention of Hc pro R-H from C-4 of mevalonic acid is accompanied by product configuration. I. e. Geranyl ester of C-4 and 2,3 E double bond. Scheme ( ).

In sceme ( ) Cornforth's 2 mechanisms give chemistry of geranyl pyrophosphate in coupling of isoprenyl units DMAPP and IPP.

Geranyl pyrophosphate chemistry is given by scheme ( ) as discussed earlier. For coupling of isoprenyl units via 2 mechanisms, as proposed by cornforth.

Non Mevalonate pathway

step 1- Dxp synthase catalyze's the condensation of pyruvate and D-glyceraldehyde 3-phosphat to give 1deoxy-D-xylose 5-phosphate as a product.

Step 2- Isomerase and reductase enzymes convert Dxp to MBP via an anonymous path.

Step 3- Mep undergoes citidilyl transferase catalyzed reaction in the presence of CTP to give CDPME

Step 4- CDP-ME kinase cnverts Cdp-ME to CDP-ME 2P where ATP converts to ADP as well.

Step 5-

Step 6-

Biosynthesis of Oleanes and Ursanes

Generation of oleanes and ursanes is due to action of an enzyme (oxide squalene-beta-amyrin cylase) on oxido squalene [Ref-2] and justified conformation prechair-prechair-prechair-preboat. Mechanism is as illustered in scheme [Seo, 1975, Curey, 1959, Attaullah, 1975]. Schematic presentation of mechanism starting from oxidosqualene involving formation of intermediates like (1) – (8). Routes proposed by Ruzicka(1921) are evidently proved by formation of rings D and E in oleananes scheme.

Consistant labelling patterenas loss of 12 alpha proton (7) and than cyclization-rearrangement mechanism via hydride shift scheme ( ).

Radi labelled mellolanic acid and proved Ruzicka purposal as ursolic acid and 2 alpha hydroxyursolic acid followed same biosynthetic route up to for mation of intermediate 7 [23 Ref]. Instead of 12 alpha proton, 12 beta proton removal is an only exception.

Biosynthesis of Lupeol

As described earlier in part ( ). Lupeol having the same route. Differece at final step is just that, the intermediate (6) eliminates the proton of terminal methyl of the isoprpyl residue located at C19.

Launaea nudicaulis Hook, f.

Scientific classification

Kingdom; Plantae

unranked;

unranked;

unranked;

unranked; Asterales

Family; Astereaceae or Compositaea

Genus; Launaea

Species; nudicaulis

Synonymous names

a- Launaea procumbens (Roxb.) [Ramayya and Rajgopal, 1969]

b- Launaea fallax (Jand S) [O. Ktze, 1891]

c- Microrhynchus fallax (J and S 1847-50)

d- Prenanthus procumbens (Roxb) [1832]

e- Paramicrorhynchus procumbens (roxb) [Krip, 1964]

f- Launaea procumbens (Roxb) [Amin, 1956]

g- Launaea nudicaulis Hook, f. [Hooker, 1881]

Local names

In punjab (Pakistan and India); Bathal, Dadglak, Dudhalk,

Spudukei, Tarizha.

Rajhastan (India); Aleria, Ban jobi, Jangli gobi

Kharan; Maharikka, Lasvela; Bhattri, Bombay; Pathari etc.

Occurance

Afghanistan, Indo-Pak region, Westward to atlantic. As a common weed throughout indian plains. In Pakistan Karachi, throuhout Sind, Baluchistan, Wazir and Kurram agency, NWFP (Currently named as Khyber Pakhtonkhaw), Lower Hazara, Punjab, Jammu, Lasvella.

Flowering season/Bloosming season---- October to December

Botanical description

Launaea is a glabrous perenial herb containing yellow juice, grows at moist places at sides of channals under shades as well as weed of cultivated crops and gardens. Launaea's stem is trufted, usually decumbent,have 6-24 in variable branches. Roots have yellow juice, naked stem, Flower clusters maybe with few leaves. Leaves 2-10 by 1-3in., usually sessile, sinuate lobed, pinnatifid or runcinate; lobes irregularly lobulate and sharply toothed, oftenly white cartilagenous teeth. Usually very numerous stems are flowering, 6-24 in. long , spread in all directions, slender are stout, simple or branched. ½,1/2 in long heads, clusters of 2-5 or about 10, rarely soltary, forming much interrupted racemes or crowned together at branched ends. Involucer-bracts overtopping the pappus (collot), ½ in, very pale,polymorphous, inner some times as if compressed of , 4 thick ribs, outer slightly curved and flattened, with a thick ventral and several thick dorsal ribs, all smooth and abscurely uneven. Pappus is ½,1/2 in, very dioiduous, hairs are of equal length straight and soft as well. [ Kirtikar, k. R., 1918, Ishaque , M. A. M., 1999].

Family Thymeleaceae Plant family Thymeleaceae contains perenials, shrub or low trees , a few herbs or shurblets, distributed in 500 species with 50 representative genera, loving temprate and tropical regions. They are notified because of their scent flowers and poisnous berries. Africa in general and South Africa in particular regions of that family, while it is less common in s. America and pacific

islands. Reports of its occurance in Afghanistan, Australia and Meditranian regions are there. In Pakistan 7 species of 5 different genera from this family are found. [1,2]

Daphne the Genus Daphne is a Greek word spoken as or vocally delivered as “Daefni” means laurel.

Habit- There are shrubs ranging small to medium sizewith flexible shoot.

Leaves- Branches have clusters at their ends.

Flowers- Vary in colour, either in clusters or axillary spikes or in terminal heads.

Fruit- Small berry, drupe or nut.

Daphnes are not usually attractive tolivestocks. Nearly all species are poisnous except few newzealand records.

Occurance- App. 70 species of genus Daphne are well distributed in Asia, IndoMalaya, Phillipines, Meditranian region, N. Africa, Australia and Europe. In our home land mentioned 3 species are recorded.

a- Daphne oloides

b- Daphne papyracea

c- Daphne retusa

Earlier Daphne oloides was studied with great interest and authority in our university.

Daphne retusa Hemsl. Scientific classification

Kingdom; Plantae

Sub kigdom; Angiosperms

Class; eudicots

Sub class; Rosids

Order; Malvales

Family; Thmeleaceae

Genus; Daphne

species; retusa

Occurance

Daphne retusa belongs to genus daphne of family Thymeleaceae. It is well ditributed in westren China and also found in eastren China province Zhejang. While in Pakistan this species was reported from Musa (Muzafar abad) growing at an altitude of 3-4000m .

Botanical Description

Daphne retusa is a very rare plant in Pakistan. Shrub with leaf size 3-5cm in length. Stem is glabrous, tips of shoot are pubscent. Oblong lanceolate, apex retuse, coriaceous, subsessile. Flowers 2cm long, in terminal heads, white, rosy red outside; tube, 1-2cm long, lobes slightly smaller, obtuse, Ovary glabrous. Fruit is of red colour and ovoid like with length of app. 8mm.