Alkaloid With Some Isolation

INTRODUCTION Two main source of medicine are, one is synthetic and another is naturally occurring. Synthetic drugs show rapid onset of action but having more side effects in comparison to naturally occurring drugs. The modern trend re back to choose natural medicine against synthetic medicine. Natural source of drugs are plants, animals, or minerals. About 8000 plants are listed in medicinal uses. In this 1800 in Ayurveda, 1100 in Shiddha, 750 in Unani, 300 in Tibetan and 4700 plants are used as traditionally ethno medicinally. The world over the total trade of medicinal plants about 8800-lakh dollar, of which contribution of India in world trade less then 1%. Out of this majority of plants are yet to be study photochemical, estimated of such pants to the extent of 5000 to 6000. Phytochemistry is branch of Chemistry, which deals with the study of chemistry of plants. Where the term phytochemistry is comes out from phyta + chemistry (phyta⇒ plant). Pharmaphytochemistry The word pharmaphytochemistry is derived from pharmakon⇒drugs, phyta⇒ plants i.e. chemistry of medicinal substance inside the plants. Alkaloids Chemistry: Sertuerner in 1806 laid the foundation of Alkaloids Chemistry. It is the branch of Pharma Phyto Chemistry, which deals with the study of Alkaloids. He reported isolation of Morphine from opium. What is Alkaloids: Alkaloids means Alkali likes. The Pharmacist W.Meissner proposed the term Alkaloids in 1819. Acc. to him "Alkaloids (alkali = base, oid=like sub) are basic nitrogenous compd. of plant origin which have complex molecular structure & many pharmacological activity." Acc to Landenberg "Alkaloids are defined as natural plant compounds that have a basic character and contain at least one nitrogen atom in a heterocyclic ring and having biological activities." Acc to characteristic features Alkaloids are basic nitrogenous plant origin, mostly optically active & possessing nitrogen hetero cycles as there structural units with physiological action.

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This definition not fully correct because not follow on all alkaloids for e.g. Colchicine: Colchicine is regarded as an alkaloid although it is not Heterocyclic and is scarcely basic. Thiamine: It is heterocyclic nitrogenous base but not as a alkaloid because it is universally distributed in living matter. Nitrogen as side chain: Some compound is classed as in alkaloids but they do not contain nitrogen in heterocyclic ring, but contain nitrogen inside the chain e.g. ephedrine, hordenine, betanine, choline, muscarine, strychnine & tryptamine etc. Naturally occurring open chain basic compound: These compounds have physiological activity but do not class in alkaloids e.g. Cholines, amino acid, phenylethylamines etc. Piperine: It is neither basic character nor possessing any physiological activity but include in alkaloids. Those compound, which fully satisfy the definitions, like physiological active, heterrocyclic basic nitrogenous ring but they do not classed in alkaloids e.g.- Thiamine, caffeine, purine, theobromine, and xanthenes. Acc. To Pelletier 1983 “an alkaloids is cyclic compounds containing nitrogen in negative of oxidation state. Which is of limited distribution in Living organisms”. "From above discussion, a conclusion can be drown quit safely that it is still difficult to define an Alkaloid"

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OCCURRENCE OF ALKALOIDS Alkaloids are chemically nitrogenous heterocyclic basic compound occur in nature, about15% of vascular plant & widely distributed in higher plant e.g.. -Apocynace, papaveraceae, papilanaceae, rananeulaceae, solenaceae. They are present in the form of salts of organic acid, like acetic acid, oxalic acid, malic, lactic, tartaric, tannic, aconitic acid, few are with sugar e.g. Solanum, veratrum groups. Acc. to parts of plants: Leaves: Nicotine Bark: Cinchonine, Quinine. Seeds: Strychnine, Nibidine. Roots: Rawelfinine, Glycyrrhizin, Punarnavine I & II NOMENCLATURE There was no systematic nomenclature. But there are some methods for nomenclature are mention below. 1. According to their source: There are named according to the family in which they are found. e.g. papavarine, punarnavin, ephedrin. 2. According to their Physiological response: There are named according to

their physiological response. e.g.. Morphine means God of dreams, Emetine means to vomit. 3. According to there discover: There are named according to there discover. e.g.. pelletierine group has been named its discoverer, P.J. Pelletier. 4. Prefixes: There are named by some prefixes are fix in nomenclature of alkaloids. e.g. epi, iso, neo, pseudo, nor- CH3 group not attach to Nitrogen.

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CLASSIFICATION Alkaloids are classified as: A) Taxonomic based: According to their family e.g. solanaceous, papilionaceous without reference their chemical type of alkaloids present & another according to genus. e.g.. ephedra, cinchona etc. B) Pharmacological based: Their pharmacological activity or response. For example: 1. Analgesic alkaloids 2. Cardio active alkaloids etc. Do not have chemical similarity in their group. C) Bio Synthetic based: According to this alkaloids are classified on the basis of the type precursors or building block compounds used by plants to synthesise the complex structure. e.g.. Morphine, papaverine, narcotine, tubocurarine & calchicine in phenylalanine tyrosin derived base. D) Chemical classification: This classification is universally adopted & depends on the fundamental ring structure. According to these two main groups. 1. Non-heterocyclic Alkaloids: In this group of alkaloid not has any one

Heterocyclic ring in their structure. e.g.- Hordinine (Hordeum vulgare), Ephedrine(Ephedra gerardiana) Genateceae. 2. Heterocyclic Alkaloids: According to heterocyclic ring the alkaloids are sub divide in following: 1) Pyrrole-Pyrrolidine: This type of alkaloids contains pyroll or

pyrrolidine ring in their structure e.g.. Hygrines Coca sp. 2) Pyrrolizidine: Alkaloids containing Pyrrolizidine Heterocyclic ring in their structure e.g.. - seneciphylline Senecio sp.

Pyrrole

Pyrrolizidine

Pyridine

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3) Pyridine & Piperidine: Alkaloids containing Pyridine Heterocyclic ring in their structure e.g. Nicotine, Lobaline, Piperidine, Ricinine. 4) Piperidine (Tropane): Alkaloid containing tropone ring. Hyoscyomine, Atropine Hyoscine- Solanceae Cocain- Coca sp.

Tropane

Quinoline

Iso Quinoline

Nor Lupinane

e.g..-

Indole

5) Quinoline : Those Alkaloids containing quinolin ring in their structure e.g..- Quinine, Quinidine. (Cinchona bark) Cinchonine, Cinchonidine & cusparin -(cusparia bark 6) Iso Quinoline: Alkaloids containing iso quinoline ring in thier chemical structure e.g. Papavarine, Narceine Emetine & cephaline. (Cephalis sp Rubiaceae). 7) Reduced isoquinoline (Aporphine): The alkaloid contain reduced isoquinoline ring in their structure e.g. Baldine, (Peumus Baldus) (Manioniaceae) 8) Nor Lupinane: Alkaloids present in leguminoceae plants e.g. spartine, lupanine. 9) Indole Alkaloids: Alkaloids containing indole ring. e.g. Yohimbine, Aspidospermine (Apocynaceae) Vinblasine, vincristine (catheranthus roseus). EXTRACTION AND ISOLATION Purification or isolation of alkaloids from a plant is always difficult process because an alkaloids bearing plant generally contains a complex mixture of several alkaloids with glycoside organic acid also complicate the process. Following steps are involved in isolation process. 1. Detection of presence of Alkaloids: First of all confirm the presence of alkaloids in raw material or crud drugs by various reagents called Alkaloids reagents e.g.

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I. Mayer (Cream Co lour) Test II. Marquis (Conc. HCHO) Test. III. Erdmann (Conc. HNO3) Test IV. Hager's (Yellow Colour) Test V. Frohdes (Molybdic acid) Test 2. Extraction: - The plants is dried, then finally powdered and extracted with boiling methanol. The solvent is distilled off and the residue treated with inorganic acids, when the bases (alkaloids) are extracted as their soluble salts. The aqueous layer containing the salt of alkaloids and soluble plant impurities is made basic with NaOH. The insoluble alkaloids are set free precipitate out. The solid man (ppt.) so obtained is then extracted with ether when alkaloid pass into solution and impurity left behind. Flow Chart of extraction Powdered Drug/ Macerated Plant Light Petroleum ether. Filtration. Filtrate Plant residue Evaporate. 1. CH3OH (72 hrs. extraction) 2. Filtration. Fat 3. Evaporate. Crude Plant extract 1. Dissolve Water. 2. Acidify to pH-2. 3. Steam distillation. 4. Filter. Filtrate Ether. Ether soluble Evaporate.

Acid Solution Ether + NaOH

Basic Material Aqueous residue

Ether solution Evaporate. (Crude Alkaloids) 6

3. Separation of Alkaloids: After detection of next step is separation of a

relatively small percentage of alkaloids from large amount of crude drugs. E.g.- Opium contains 10% Morphine, Chincona contains 5-8 % Quinine, Belladona- 0.2% of Hyoscyamine. The required alkaloid is separated from the mixture from fractional, crystallization, chromatography and ion exchange method. PHYSICAL-PROPERTY I)

They are colorless, crystalline solid. Exception - Berberin (Yellow), Nicotine Coniine (liquid).

II)

They are insoluble in water (exception liquid alkaloids soluble in water), soluble in organic solvent ( CHCl3, Ethyl alcohol ether)

III) Taste: They are bitter in taste. IV) Optically active, Most of levo ratatory but few are -Dextro rotatory e.g. Coniine, some inactive- e.g.- papaverine. CHEMICAL TEST OF ALKALOIDS 1. Mayer's Test: Specimen with Mayer's reagent give Cream or pale yellow ppt. 2. Dragendroff Reagent Test: Specimen with Dragendroff Reagent give orange ppt. 3. Wagners Test: Specimen with Wagner's Reagent give brown or reddish brown ppt. 4. Hager's Test: Specimen with Hager's reagent give yellow ppt. (Special Type) 5. Amonium Rinker Test: Specimen with Ammonium Rinket solutions with

HCL give flocculent pink ppt.

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GENERAL METHODS FOR STRUCTURE DETERMINATION OF ALKALOIDS Molecular formula of majority of Alkaloids is complex so very little achievement in their elucidation of structure. During 19th Century. Now general procedures for elucidation of structure of alkaloids are adopted. 1. Molecular formula molecular weight: A pure specimen of alkaloids its empirical formula and molecular weight by elemental or combustion analysis. No. Of double bond is determined by double bond equivalent method. 2) Number of Double bond: - Number of Rings present in an alkaloids can be determine by following formula- Ca Hb Nc Od Then number of double bond present in Ring= a-b/2 + C/2 + 1 3) Functional group Analysis: a) Functional Nature of Oxygen: - Oxygen presents in alkaloids as: - OH (Phenolic/ Alcoholic), - OCH3 Methoxy, - OCOCH3 (Acetoxy), - OCOC6H5 ( Benzoxyl), -COOH (Carboxylic),- COOK (carboxylate),>C=O (Carbonyl),= C-O-O (Lactones Ring).It can be determined by infra red or organic analysis method. (1) Hydroxyl group: - Formation of Acetate on treatment with Acetic anhydride /Acetyl chloride or benzoate on treatment with Benzyl chloride. R- OH + (CH3CO)2 O R- OH + CH3COCl R- OH + C6H5COCl

ROOCCH3 + CH3COOH ROOCCH3 + HCl ROOCC6H5 + HCl

If Primary amines are present in an alkaloids also give this test. Then Hydroxyl group is can be determined by zerewitinoff method. NaOH R-OH +CH3COCl

R-OCOCH3-

R- OH +CH3COONa

Excess of Alkali is estimated by titration with standard HCl. Number of -OH group can be calculated from the volume of Alkali used for Hydrolysis. Grignard-method.

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- OH + MeMgI - NH + MeMgI

- OMgI + CH4 - NMgI + CH4

1- OH= 1>NH group= 22.4 liter of N2 STP

10, 20-OH, 30 -OH group. By oxidation or dehydration to unsaturated compound. If OH group is phenolic, and then Alkaloid is - Soluble in NaOH - Re precipitated by CO2 - Giving coloration with FeCl3

(2) Carboxylic group: - soluble in aqueous solution sodium carbonate or ammonia on treat with alcohol form ester. Number of -COOH group can be determined by volumetrically by titration against a standard. Ba(OH)2 solution by using phenolphthalein as an indicator.

(3) Oxo-group: - On treatment with Hydroxylamine. Semicanbezide, phenylhydrazide ,oxime ,semicarbazone phenyl Hydrazine >C=O + H2NOH

>C=N-OH

>C=O +H2NNHCONH2

>C=NNHCONH2

The distinction between aldehyde and ketone is done by oxidation or reduction, also by NMR, IR, and UV techniques.

(4) Methoxyle group: - BY Zeisel determination method. When methoxy group present in a alkaloids treated with HI at 1260C perform methyl iodide which can treated further with silver nitrites to perform silver iodide precipitate. Which estimated gravimetrically e.g.. Papavarine. HI C2OH4OO4N

AgNO3 4MeI

4AgI

(estimated gravimetrically)

(5) Methylenedioxy group: - On heated with concentrated with HCL or H2SO4 to form formaldehyde and further formation of dime done derivative, which estimated gravimetrically. -OCH2 O-

heat/HCl/H2SO4

HCHO

dime done derivative (Estimated gravimetrically)

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b) Ester Amide Lacton & Lactum group: These groups are identified by the estimation of product. >CONH2 + NaOH

Heat

-COONa + NH3

>COOR

Heat

-COONa + ROH

+ NaOH

c) Nature of Nitrogen Majority of nitrogen presence in alkaloids are secondary and tertiary: If tertiary when treated with H2 O2 (50%) form. =N +H2O2

=N

O +H2O

If alkaloids react with one molecule of methyl-iodide to form N-methyl derivative, it means secondary e.g. (C8H16O4) NH + CH3I

(C8H16O4) NCH3 + HI

General reaction of alkaloids with acetic-anhydride, methyl-iodide, and nitrous acid are often showing the nature of nitrogen If react with one molecule of methyl-iodide to form crystalline quaternary salt this indicates that nitrogen is tertiary e.g. N= (C10H24)= N + 2CH3I

IH3C+N= (C10H24)= N +CH3I-

N-methyl group: On distillation with soda lime if methylamine is produce show the presence of N-methyl group for e.g. Soda-lime (C10H14N)=N-CH3 CH3NH2 Nicotine CaO Determination of Number of CH3-groups Attached to N-atom: By HerzigMayer’s method when a sample is treated with hydrogen iodide at 1500-3000C and than treated silver nitrate, which form silver iodide, which estimated gravimetrically.

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N- methylamine + HI

1500-3000

>NH + MeI

AgNO3

AgI

Estimated gravimetrically

d) Estimation of C-Me Group by Kuhn –Roth Oxidation: When treated with K2Cr2O7 or H2SO4 an acid is produce, which estimated gravimetrically. K2Cr2O7 or H2SO4 -C-Me

MeCOOH Estimated gravimetrically

e) Degradation of Alkaloids: For discovering the structural system which incorporate these substituents groups & is tackled by degradation of the molecules by following methods:

1) Hoffmann's exhaustive methylation: - This is a composite reaction of alkaloid (Heterocyclic amines). This involves following steps: a) The alkaloid is treated with excess of CH3I to form quartertionarey -ammoniumiodide.

b) 40-ammonium iodide is converted to the hydroxide and heated. The -OH of

hydroxide extracts hydrogen atom from beta position and eliminate a water molecule and also the ring is cleaved at the N-atom to give an open chain 30amine.

c) The step Ist and IInd are repeated when a second cleavage at the N-atom

given an unsaturated hydrocarbon which isomerase’s to conjugated derivative.

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The exhaustive methylation of an alkaloid is an important method for the investigation of the nature of the C-skeleton in the heterocyclic system.

2) Emde Degradation Method: If alkaloids do not contain beta Hydrogen atom then Hoffmann exhaustive degradation method is failed. In such cases Emde degradation are applied, in this final step involve the reductive cleavage of quartertionarey ammonium salt either by Na-Hg or NaNH3 or by catalytic hydrogenation e.g. Na-Hg or NaNH3

RCH2NR3X

RCH3 + NR3+ HX

2) Von Braun's Method: - This method is of two types: A. Tertiary amine, which contains at least one-alkyl substituents, is treated with cyanogen bromide. The results in cleavage of an alkyls nitrogen bond to give an alkyl halide and a substituted Cyanamid. R3N + CN - Br --

R - Br + R2N - CN

This method is often applicable to such compounds that do not respond to Hofmann's method e.g. – Cocaine B. Secondary cyclic Amine is treated with Benzoyle chloride in presence of NaOH to yield the Benzoyle derivative which on treatment with phosphorus followed by distillation under reduced pressure yield di Halo compund.

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Br2

NaOH

R2NH+ C6H5COCl

R2N-COC6H5

Distillation

R2NCBr2C6H5

Br-RRBr + C6H5CN

3) Reductive degradation & Zinc dust distillation: In some case ring may open by Heating up to 3000C with Hydroiogic acid e.g. Coniine C8H17N

Zn dust /Heat

+ 6H C3H7

4) Alkali fusion: This is very drastic method, which is often employed to break down the complex of complex alkaloids molecule to simpler fragment. The nature of fragment, which will give information type of nucleus present in alkaloid molecule e.g. Adrenaline, Papaverine, Cinchonine Adrenaline

KOH Fusion

5) Oxidation: - This method gives quite information about the structure of alkaloids by varying the strength of the oxidizing agents it is possible to obtain a variety of product e.g. A. Mild oxidation- H2O2, HI in ethanolic solution & alkaline potassium ferriccyanide. B. Moderate oxidation: KMnO4 (Alkali) Cr2O3 (Acetic Acid) C. Vigorous oxidation: K2Cr2O7, (H2SO4), Cr2O3 (H2SO4), conc. HNO3, MnO2 (H2SO4) C10 H14N2

K2Cr2O7 (H2SO4) C6H5COOH

6) Dehydrogenation: - When Alkaloid is distilled with catalyst such as sulphur, selenium or palladium dehydrogenation takes place to form relatively simple & easily recognizable product which provide the clue to gross skeleton.

7) Synthesis: - The structure of the alkaloids arrived at by the exclusive analytical evidence based on going method is only tentative. The final conformation of the structure must be done by the unambiguous synthesis.

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BIOSYNTHESIS OF ALKALOIDS As more and more structure of alkaloids were elucidated, it become increasingly probable that the precursor in the biosynthesis of many alkaloids were amino acids and amino-aldehyde or amines derived from them, Woodward 1948 proposed a biosynthesis of strychnine. Because the great diversity of structure of alkaloids, it not possible to develop only one hypothesis of biosynthesis of alkaloids. Thus many pathways have been proposed, each one accounting for the biosynthesis of a number of alkaloids of related structure. The most common aminoacids that act as precursor in biosynthesis of alkaloids are: i. Ornithine H2N (CH2) CHNH2COOH ii Lysine H2N (CH2)3 CHNH2COOH iii. Phenylalanine (R=H) RC6H5CH2CH(NH2)COOH iv. Tyrosine (R=OH) vi. Methionine MeSCH2CH2CH(NH2)COOH vii Trytophen Some common reactions are: Decarboxylation : formation of amine RCH (NH2) COOH

RCH2NH2 +CO2

Oxidation: formation of aldehyde RCH (NH2) COOH

RCOCOOH

Shiff base formation: R1CHO +R2NH2

R1CH=NR2

PHYSICAL METHOD

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RCHO

These following physical methods are applied to elucidate the structure of alkaloids: • • • • • • •

U.V. Spectroscopy IR Spectroscopy Nuclear Magnetic resonance spectroscopy Mass spectroscopy Optical rotatory dispersion & circular dichroism. Conformational Analysis X-Ray diffraction

1. ATROPINE This is the most important alkaloid of tropane group and occurs in the roots of deadly nightshade (Atropa belladonna), thron apple (Datura stamonium)n and many other member of solanaceous family together with lhyoscyamine which is optically active ( [<]D = -220 ). Atropine is the racemic form of l-hyoscyamine which readily racemises to atropine when warmed in an ethanolic alkaline solution, thus atropine is ( ± )-hyoscyamine ISOLATION Atropine is extracted either by from belladonna root or from juice of datura plant. In practice, the juice which contains hyoscyamine is heated with potassium carbonate solution when hyoscyamine is racemised to atropine. Then, this atropine is extracted with chloroform. The chloroform is recovered by evaporation and the residue is then extracted from the residue with dilute sulphuric acid. The solution is made alkaline with potassium carbonate when atropine is precipitated out. The precipitated atropine is extracted with ether and purified by converting it in to an oxalate or a sulphate. STRUCTURAL ELUCIDATION Molecular formula- C17H23NO3. Melting point- 118oC. Structure15

UV ( Ethanol ): λmax 246, 251.6, 257, 263.5, 271 nm ( c 147.6, 175.1, 209.8, 143.3 and 24.6 respectively ) IR ( KBr ): Vmax 3070 (OH- Hydrogen bonded), 2930 ( CH stretch ), 2810 ( N- CH3 stretch ), 1725 ( O-C=O ), 1595, 1580 ( C=C, Ar ), 1155, 1030 ( CC-O ), 770,725 and 690 ( monosubs Ar ) cm-1. 1 H NMR ( CDCl3 ): δ 7.23 ( 5H, s, Ar-H ), 4.96 ( 1H, t, H-3 ), 3.9 ( 1H, m, H10 ), 2.93 ( 2H, bs, H-5 ), 1.66 ( 8H, m, H-2,4,6,7 ). 2. MORPHINE Morphine was the first alkaloid to be isolated from serturner plant ( 1806 ). In opium, it is present in a quantity of 10-23 percent along with other substances like fats, resins, proteins, carbohydrates, mineral salts, meconic acid about 20 or more alkaloids. Codeine and thebaine are the other closely related alkaloids to morphine. Theses three are commonly known as morphine alkaloids and from a sub-group of the opium alkaloids. In all morphine alkaloids, phenanthrene nucleus is present. Due to this, these are also known as phenanthrene alkaloids. The morphine alkaloids have been studied comparatively more due to the following reasons: (i) These are widely used as analgesic agents, and (ii) These undergo a wide variety of molecular rearrangements. ISOLATION Morphine is extracted from opium by involving the following steps : 16

(i) First of all, the raw opium is extracted with cold dichloromethane repeatedly. Papaverine, narcotine and gum go into the dichloromethane layer whereas morphine and other substances remain in the insoluble residue. Dichloromethane layer is separated. (ii) The dichloromethane layer obtained from step (i) is evaporated to dryness to yield a residue which is extracted with hot dilute HCl, then treated with the charcoal and finally filtered. The filtrate is neutralized with ammonia when papaverine and narcotine are precipitated out. When precipitate is shaken with hot alcohol, the papaverine goes into alcohol. From this, papaverine is precipitated out as oxalate and then purified by recrystallisation. The crude narcotine present in the residue from the alcohol extraction is similarly purified. (iii) The residue obtained from the dichloromethane extraction is agitated with lime water at temperature below 20oC. morphine, codeine and thebaine are present in lime water which when extracted several times with benzene remove codeine and thebaine in a benzene layer. Then, the pH is raised to 8 when the crude morphine is precipitated out. The filtrate still contains morphine. This filtrate, when evaporated in vacuum and then extracted with amyl alcohol, yields further amount of crude morphine. the two sample of crude morphine are mixed. (iv) The crude morphine obtained from step (iii) is dissolved in dilute HCl and filtered through alcohol. Then the filtrate is neutralized with ammonia followed by the addition of alcohol. This results in precipitation of morphine. The precipitated morphine is dissolved in minimum quantity of dilute HCl and the resulting solution so obtained on concentration and cooling yields crystals of morphine hydrochloride. (v) The benzene extract obtained from step (iii) is evaporated to yield a residue. This is then treated with hot alcohol, followed by cooling and filtration. The filtrate when treated with sulphuric acid yields a precipitate of codeine sulphate which is filtered out. To the resultant filtrate, tartaric acid is added to precipitate out thebaine as thebaine acid tartrate. STRUCTURAL ELUCIDATION Molecular formula- C17H19NO3. Melting point- 254-256oC. Structure-

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UV ( Ethanol ): λmax 286, 250 and 298 nm ( log c 3.256, 3.275, 3.360 respectively ) IR ( KBr ): Vmax 3480, 3350 ( OH ), 3210 (OH bonded), 2940, 2920 ( CH stretch ), 2840( N- CH3 stretch ), 1640 (C=C alkene ), 1605 ( C=C, Ar ), 1250, 1090( X-O stretch), 760 ( monosubs. Ar ) cm-1. 1 H NMR ( HCl ): δ 6.76 (d, H-1 ), 6.68 ( d, H-2 ), 5.75 ( d, H-7 ), 5.40 ( d, H8 ), 5.06 ( d,H-5, J5,6= 2Hz ), 4.37 ( m, H-9 ),4.20 (d, H-14 ), 3.0 (s, N-Me ), 2.16 (d, H-10 )

3. EPHEDRINE It is an important drug which occurs along with five other alkaloids, namely pseudoephedrine, methylephedrine, methylpseudoephedrine, norephedrine and norpseudoephedrine in the genus Ephedra; a species which occurs in temperate and subtropical regions of Asia, America and Mediterranean. The base ephedrine was first isolated from the Ephedra in 1887 by Nagai and much later Gulland and Virden obtained a very small amount from the Yew ( Taxus baccata L. ). The Chinese have used the herb Ma Huang. ( a Chinese drug ) which contains ephedrine and is said to have been used medicinally in china for 5000 years. Ephedrine has been used successfully in the treatment of bronchial asthma, hey fever and other allergic conditions. It also increases blood pressure. It is a much stronger stimulant than caffeine. ISOLATION

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1 kilo of powdered Ma Huang was extracted with cold benzene in the presence of dilute Na2CO3 solution, and the benzene extract was shaken up with a sufficient quantity of dilute HCl to remove the basic substances. The acid solution was made alkaline with solid K2CO3 and the liberated base was then extracted with chloroform. The chloroform solution, when dried over anhydrous Na2SO4 and distilled, gave 2.6 g of crude base. Preparation of Ephedrine HCl by Fractional Crystallization The crude base obtained as above was taken up with about twice its weight of alcohol and neutralized with concentrated HCl diluted with twice its volume of alcohol. Nearly pure ephedrine hydrochloride crystallized out on standing. After filtering it was washed with a mixture of alcohol and ether, and then with pure ether, and dried. A further quantity of ephedrine hydrochloride may be got by concentrating the mother liquors and washings. The final mother liquor was kept for the isolation of pseudoephedrine (see below). Ephedrine hydrochloride crystallized out from alcohol in prismatic needles, mp 216°C, [α]D22 -32.5° . The salts prepared by fractional crystallization show no change in the melting point when recrystallized seven times. In many of our experiments the salts were recrystallized twelve times. STRUCTURAL ELUCIDATION Molecular formula- C10H15NO. Melting point- 38.1oC. Structure-

UV (Methanol): λmax 250, 256 and 262 nm (log c 170, 20, 165 respectively) IR ( KBr ): Vmax 3330 ( -OH stretching ) 2480 ( NH + 2 stretching ), 1490, 1450 (ArH ) 750 and 699 ( C-H out of plane deformation ) cm-1. 1 H NMR ( D2O ): δ 7.53 (5H, s, Ar-H ), 5.28 ( d, OH ), 3.60 (m, CHNH2 ) 2.9 ( s, NH-CH3 ), and 1.20 ( d, CH- CH3 ).

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4. RESERPINE Reserpine is the main constituent of Rauwolfia species, particularly R. serpentina and R. vomitoria. Other important alkaloids presents in these species are yohimbine, ajmalicine and ajmaline. All these alkaloids are hypertensive and sedative reagents. Reserpine is mainly used for the treatment of hypertension, headache, tension states, asthma and dermatological disorders. ISOLATION Mature root bark (264 g) was extracted with methanol in a Soxhlet for 30 hr, the extract concentrated to remove the methanol, and the residue treated with NaHCO, solution and ether. Any resinous material separating at this stage was dissolved in a little methanol and retreated in the same way, this process being repeated thrice or until no more resin formation occurred. All the aqueous solutions were extracted four times with ether and the combined ether extracts washed with 2% acetic acid (3~100c.c.) then 2% HC1 (2 x 100 c.0.). The combined acid extracts were then Basification with NH, gave a resin which would not dissolve in ether. A solution of this material in a little methanol gradually deposited a crystalline precipitate which after several recrystallizations from methanol (Yield 0.07 g). A further quantity was obtained on longer standing. STRUCTURAL ELUCIDATION Molecular formula- C33H40N2O9. Melting point- 264-265oC.

Structure-

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UV: λmax 296, 267, 216 nm (c 9660, 15700 and 55700 respectively ) IR: Vmax 3480 ( N-H stretching ), 2840-3030 ( C-H stretching ), 1732 and 1713 ( C=O stretching ) cm-1. 1 H-NMR (CDCl3 ): δ 7.85 ( s, NH ), 6.7-7.3 ( indole Ar-H ), 7.34 ( 2H, s, trimethoxybenzene Ar-H ), 5.05 ( H-18, m ), 4.43 ( H-3, t ), 3.92 ( 6H, s,OMe X 2 ), 3.29 ( 3H, s, CH-OMe ), 3.79 ( 3H, s, C16- OMe ), 3.46 ( s, C17 OMe ) 5. VINCRISTINE Vincristine is an indole alkaloid obtained along with the Vinblastine from the catharanthus roseus family. Apocynaceae. This plant was previously known as Vinca roseae L. Vincristine is one of the cytotoxic drugs and used in treatment of acute leukemias particularly in children and in other cases such as lymphosarcoma, reticulum sarcoma, neuroblastoma wilms tumor and tumors of the brain, breast and lung. ISOLATION The mixture of dried ground plant of Vinca rosea and dilute tartaric acid is extracted with benzene. Collect benzene extract and evaporate till concentrate. The concentrated extract is then steam distillated to obtain insoluble residue. Dissolve the residue by mixing it with dilute tartaric acid and hot methanol, and then distill out the methanol from it .Carryout the extraction of the bottom product using dilute solution of ammonia. Collect the extract, evaporate and dry. The dried powder of Vincristine is dissolved in benzene and chromatographic separation is carried out on alumina column using eluting solvents as benzene, benzene + Chloroform, Chloroform and Chloroform + Methanol mixture. STRUCTURAL ELUCIDATION Molecular formula- C46H56N4O10. Melting point- 273-281oC. Structure21

UV: λmax 296, 290, 275, 255, 221 nm (c 15600, 14000, 11400, 15400 and 47100 respectively ). MS : m/z 824 ( M +, negligible ), 806 ( M+ - 18 ).

Functions Alkaloids functions • As reservoir of nitrogen • As reservoir for protein synthesis • As detoxicating agents • As toxicating agents • As harm one for many activity of plant. 22

Alkaloids have many physiologically biological and pharmacological properties

Conclusion It can be concluded that: 1. Alkaloids are naturally occurring heterocyclic complex compounds. 2. Alkaloids have indefinite definition. 3. Alkaloids have mainly nitrogen in heteroatom. 4. Alkaloids have complex molecular structure. 5. Alkaloids are bio & physiologically and pharmacologically active.

Reference: 1) Qadry J.S (Twelth Edition), Shah and Query’s Pharmacognosy, B. S. Shah Parkashan. 2) Shah C.S. &. J.S Qadry, 11th edition, A Text Book of Pharmacognosy, B. S. Shah Parkashan. 3) Handa S. S., A Text Book of Pharmacognosy, Vallabh Parkashan, New Delhi.

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4) Gupta A.K. Vol-1, Quality Standards of Indian Medicinal Plants, ICMR, New Delhi. 5) Evans W.C., (Fifteenth edition) Trees & Evans Pharmacognosy, W.B. Saunders. 6) Agarwal. O. P., Vol-2, Chemistry of Natural Product, Goel Publication Meerut. Page 193-311. 7) Finar I. L., Vol-II Vth edition, Organic Chemistry (Stereochemistry & Chemistry of Natural Products) ELBS. , Page 696702 8) Robert MF, M Wink, Alkaloids (Biochemistry, Ecology, Medicine application) Plenum Press, N. York. 9) Brunton Jean, 2nd edition, Pharmacognosy Phytochemistry of Medicinal Plants, Levoisier Paris. Page 783-799.

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