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MANGSFERA INDICA LINN (AMACARDIACEAE)

Mangifera indica (Anacardiaceae), commonly known as Mango, is one of the most popular and delicious fruits bearing tree and probably most widely cultivated crop in almost every tropical country and sometimes rather beyond, e.g. Florida, USA, Egypt, etc.

VERNACULAR NAMESHindi: Am, AmbSanskrit: Amra, ChutaMarathi: AmbaGujarati. AmriTelgu: MarindiKannad: MavuMalyalam: Araram, Cutam, Mavu

HABITATThe genus Mangifera consists of evergreen trees distributed throughout

tropical and subtropical parts of south-east Asia, from India and Sri Lanka inthe west to the Phillipines and New Guinea in the east, from Yunnan (China) and Indo- China in the north, the Sunda and Sulu Archipelago in the south.

In India, it occurs wild or cultivated in tropical and subtropical hilly forests. It is common in the subtropical Himalayas, hills of western and eastern Ghats, and the forests of centra! India, Bihar, Orissa, Assam and Andaman Islands (Mukharjee, 1949).

Mangoes are grouped in two broad categories:(a) Seeding type (wild and cultivated)(b) Horticulture clones propagated by budding or

grafting.

A large number of Mango varieties estimated over 1000 grow in various parts of India, each having its own peculiar taste, flavour and consistency of

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pulp, stem bark is obtained from a large evergreen tree, ' 10-45 meters high with a heavy dome-shaped crown and straight stout bottom. Bark is thick, rough, dark brown to gray, flaking when old. The leaves are linear oblong or elliptic lanceolate, 10-30 cm long and 2-9 cm wide. When crushed they emit an aromatic rasinous odour. Inflorescence occurs large penicle containing some times more than 3000 flowers. Flowers are tiny, reddish white or yellowish and hermaphrodite born in the same panicle. The fruit is a large drupe exceedingly variable in form and size. Fruit skin is thick or thin, leathery, green, yellowish or red in colour. The flesh (rnesocarp) is whitish yellow or orange, soft or juicy, sub-acid or sweet, richly aromatic, fibres throughout the flesh, in some varieties either absent or very little in others. The seed is solitary, ovoid, oblong encased in a hard compressed fibrous endocarp (stone). Mango has been cultivated in India for at least 4000 years and recent studies on the genus indicate that it probably originated in Assam, Burma (Mynamar) and Thailand region vvhere truly wild mango trees, belong to both Mangifera indica and M. sylvatica, have been recorded (Burns et al., 1920).

CHEMICAL CONSTITUENTS 'Mango plant has been investigated extensively for the presence of volatile

oil constituents,triterpenes, sterois, phenolic compounds, carotenoids, lipid contents and nutritional components. Some important chemical constituents of Mango are described hereunder;

VOLATILE COMPONENTSNumerous studies on the volatile constituents of Mango cultivars have

appeared because each cultivar differs greatly in their attractive flavour characteristics to the same stage of ripeness. The flavour components of cultivar African (Sakho et al., 1985), Alphanso (Bandopadhyay et al., 1971; Gholap et al., 1986). Baladi (Engel et al., 1983 and Abd-EI Baki et al., 1981), Bowen (Bartley et al., 1987), Carabao (Bautista et a l, 1982), Carazon (Gonzaler et al., 1984), Dusehri (Kapur, 1983), Edward (Diaz, 1976 and 1980), Hindi (El-Nemr et al, 1986), Jaffna (MacLeod et al, 1984), Keitt (Diaz 1976 and MacLeod et al., 1985), Langra (Gholap et al., 1975), Pairy (Abd-EI Baki et al., 1981), Polmer (Diaz 1976 and 1980), Parrot (MacLeod

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et al., 1984), Tommy-Atkins (El-Nemr et a i. 1986), Totapari (Gholap et al., 1975), Venezulelan (MacLeod et al.. 1982), Kensington (MacLeod et al.,1988), Willard (MacLeod at ai, 1984), Zebda (Abd-EI Baki et al.. 1981), Zill (Diaz 1976 and 1980) and some others (Pickenhagen, 1981 and Pino et al.,1989) have been characterized.

Volatile components of Y -irradiated fruits (Eboul-Enein et al., 1983), head space (Bartley et al., 1987; Bartley, 1988), heated fruits (Sakho et al.,1985) at 90“C, leaves (Osman et al., 1972; Caraveire et al., 1980), epicarp (Osman et al., 1972), seed kernel (Osman et al., 1972) and latex (Gholap et al., 1977) have also been studied. Palmer and Zill varieties vêre richer in flavouring components than Edward and Kiett varieties and the green fruits had a higher number of volatile components than the ripe fruits (Diaz, 1976). Zebda mango was high in oxygenated terpenes and carbon compounds while Baladi mango was rich in esters, limonene and acids (Abd-EI Baki et al., 1981).

Monoterpenic hydrocarbons, acetophenone, benzaldehyde and dimethylstyrene were the important components in the essence of Venezuelan mangoes (MacLeod et ai, 1982). The major components of raw Alphonso and Totapari varieties odour were cis-ocimene and fl-myrcene which contributed to the green fruit flavour (Bandhopadhyay, 1983). Ocimene, linalool, furfural, alloocimene, fi-myrcene and lactones existed in the ripe Dusehri mango flavour (Kapur, 1983). The predominant aroma components of Sri Lankan Jaffna mango was cis-a-ocimene, whereas L-terpinolene was the major flavouring compound of Willard and Parrot cultivars (MacLeod et al., 1984). Mango varieties from Florida (Tommy Atkins and Kiett) possessed monoterpene hydrocarbons as the principal group of volatiles (MacLeod, 1985). Among 152 aroma substances characterized in Indian Alphonso mangoes, ocimene and 2,5-dimethyl-4-hydroxy- 3(2H)furananone were the prominent constituents (Engel et a!., 1983). The mainvolatile components of Australian Bowen mango were ethyl butanoate and terpinolene (Bartley et al., 1987). Cis-ocimene and fi-myrcene were responsible for characteristic aromas of latext of Alphonso and Batali cultivars (Gholap et al.,1977).

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Most of the volatiles are lost during storage and y-irradiation at higher doses of the fruits (Eboul-Enein et a i, 1983). The correlation of the ratio of palmitic:palmitoleic acids with aroma and flavour characteristics of mango fruits has been made in some varieties (Gholap et a i, 1975 and 1976). The distribution of volatile aroma compounds between pulp and serum of mango juice was investigated by means of analytical techniques (El-Nemr et at., 1986). Volatile components of mangoes preserved by deep freezing have been studied recently. Importance of some lactones and 2,5-dimethyl-4-hydroxy-3(2H) furanone to mango aroma was found out (Wilson et a/., 1990), The aroma compositionof canned mango juice has been investigated by means of chromatographic and spectroscopic methods (Proctor et ai, 1969). Limonene, B-myrcene, cis- and trans-ocimene were the major terpenic hydrocarbons of canned juice and methyl butanoate, ethyl-2-methyl butanoate, furfural, L-terpineol and y-octalactone were the most abundant oxygenated compounds (Proctor et ai, 1969). Aroma change in mango juice during processing and storage has been investigated (Corsano et a i, 1967).

TRITERPENOIDS AND STEROIDSTriterpenoids (Anjaneyulu et a i, 1985, 1989) are usually present in

mango resin (Corsano et a i, 1967; Mincione et a!., 1968), root bark(Anjaneyulu et al., 1982), seed kernel (Bajpayee ef ai, 1967; Gaydon et a!., 1984), leaves (Anjaneyulu, 1982; Osman et ai, 1973; Younes et a i, 1973) and panicles (Maheshwari et a i, 1975) (Table 1).

A pentacyclic triterpenic alcohol of unknown structure, named indicol, has been isolated from mango leaves. Other triterpenoids characterized in leaves (Anjaneyulu, 1982; Osman et ai, 1973 and Younes et al., 1973) are taraxone, teraxerol, friedelin, lupeol and ^.-sitosterol (Anjaneyulu, 1982 ; Osman et a i, 1973). The later steroid has also been reported in mango panicles (Maheshwari et a i, 1975) and seed coat (Subramaniam et a i, 1977). The fruit’s skin contained indicenol, a tetracyclic triterpene of unknown structure (Younes et a i, 1973). fJ-Sitosterol, stigmasterol and campesterol were the main sterols of mango kernel fat (Gaydon et a i, 1984). The stigmasterol/campesterol ratio was lower in the mango seed fat than in cocoa butter (Gaydon et ai, 1984). Lupenone,

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Resin „,em and root bark Seed kernel

Ambollc acid

Ambonic acid

Amyriri acetate

Cycloartanol acetate

(20S)-Dammar-24-en-3 (i-2 0 -diol

Erythrodiol

3-Hydroxycycloart-24-ene-27-al

3-Hydroxy-3’-keto- lanastan type dimer

Hydroxymangiferolicacid

3-Hydroxy'9-methyl-24-ene-lanostanol

3-Hydroxy-9-methyl-24-methylene-lanostanol

Isomangiferin

Lupeol acetate

Mangiferolic acid

Mangiferonic acid

a-Amyrin

a-Amyhn

Cydoartane-3B-24,25-triol

C yd oart-24-e ne-3G-26-dio I

Cycloart-25-ene-3f3,-24-diol,24-epimer

Cycloart-25-ene-3B-24,27-triol-C-24-epimer

Cycloartenol

Cycloart-23-ene-3S,25-diolCycioartane~3f2.,25-diol

Dammarenedioi II

3a,22-Dihydroxycyclo-art- 24E-en-26-oic acid

3fi-22-Dihydroxycycloart- 24E-26-oic acid

3B-23-Dihydroxycydoart- 24E-en~26-0(c acid

3a.27-Dihydroxycycloart- 24E-en-26“Oic acid

Friedelin-3fi.-ol

a-Amyrin

a-Amyrin

A'^-Avenesterol

A'-'-Avenesterol

CamesterolCholesterol

Citrostadenol

Cycloartenol

CyclobranolCyclosadel

Dammaradieno!

hriedelinolGermanicol

GramisterolLupeol

Luphenol

24-Methylene-cycloartenoi

Obtusifoliol

ft-Sitosterol

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Resin Stem and root bark Seed kernel

24-Methylenecy-cloartanol24-Methy!enecy-artan-3,27-diol

24-Methylenecyclo-3G-26-diolOleanolic aldehyde

3-Hydroxy-9-methyl-24-methylene-lanostanol24-Methylenecyclo-313-26-diol

Friedelin

3(l>-Hydroxycycloart-25-en-26-al

3B-Hydroxycycloart--24-en-26-al

Hopen-1,3,22~triol

Hydroxymangiferolic acidIsomangiferolic acid3-Ketodammar-24E-ene-20S-26-diol

3-Ketodammar-24E-ene-20,26-diol-26-monoacetateMangiferolic acidMangiferonic acid

24-Methylenecycloartane-3B-26-diol24-Methyienecycloartane- 3/S,26, diol24-epimer3(3)-22-Ditiydroxycloart' 24E-26-oic acid

3H-Hydroxycycloart-24-en-26-al

Mangdesistero!ManglupenoneMangsterol

Mangiferolic acidMethyl esterManglupenoneManghopanolTaraxerol

Mangoleanone

Stigmasterol

GramisterolLupeol

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o f sorne-Tri 3fMJ:SteroicIS"of

C O O HCOOH

Mangiferolic acidCOOH

M angiferonic acidGAc

21 , 20 24 26

19

10“' 8. 4 ^ 6 J 28

AcO

Isomangiferolic acid

1819

14

AcO 30 -29

CH.OAc

C-24 epim er of Cycloart - 25-ene- 3 p , 2 4 - d i o ! diacetate

OHCOOH

24 - M ethyelenecycloartane - 3 p, 26 - diol diacetate

3p , 22 (R or S) - dihydroxycycloart - 24 E - 26 - ioc acid

COOHOAc

3( , 23. (R or S) - Dihydroxy - cycloart - 24 E 26 - ioc - acid

A cO

Cycloartane - 3(i , 24, 25 - triol d iacetate

(C o n td ............ )

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H O

a - Amyrin p - Arnyrin

Taraxerane v|/ - Taraxastane - 3 f i . 20- diol

•OH

Hop - 1(3, 3p, -2 1 - triol Friedelan - 3p - ol

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lupeol, ursolic acid and glochidenol have been identified in different parts of mangoes. The fungal infection did not alter the production of these triterpenes (Ghosal et a!., 1965).

Six new tetracyclic triterpenoids have been isolated from the stern bark of Mangifera indica variety “Banganpalli" together with several known compounds (Anjaneyulu et aL, 1985). Phytochemical investigations on the stem bark of Neelum cultivar led to the isolation of four new triterpenoids and various earlierreported compounds (Anjaneyulu, et a!., 1989).

The stem bark of Chausa variety possessed the triterpenes manghopanal, taraxero!, friedelin, mangoleanone and C-24 epimer of cycloart-25-en-3B, 24,27-trioltriacetate (Sharma and Ali, 1994). A iupene-type triterpene, lup-20(29)- ene»3-one-30-ol, has been isolated from the Dusehri cultivar (Sharma and Ali,1993). The structures of a new sterol, isolated from the Desi cultivar along with taraxerol, 3R, 22-dihydroxycycloart-24E-en-26-oic acid and 3a, 27-dihydroxylcloart- 24E-ene-26-oic acid, have been established(Sharma and Ali ,1995).

PHENOLIC COMPOUNDSVarious phenolic components such as simple phenols, phenolic acids,

flavonold pigments, xanthones, and related compounds have been isolated from leaves (El. Sissi et a i, 1970; Tanaka et a/., 1984), bark (El-Sissi et a/.,1970; Saleh et al., 1966, Aritomi et at., 1970; Silla et et a/., 1980), seeds(Saleh et al., 1966), pulp (Saleh et ai, 1966; Sadana et al., 1949; Saeedet al., 1964), peel (Saleh et al., 1966, Yang et al., 1982; Cojcaru et al.,1986), panicles and some other parts of the mango plant (Ghosal et al., 1965; Saleh et al., 1966; Silla et al., 1983 (Table 2).

Mango is a rich source of mono- and diphenolics. The presence of mangiferin, tetra- and pentaoxygenated xanthones, despones, flavonoids and polyphenols has been reported in sound and infected parts of Mangifera indica. During the fungal infection of mangoes catechin, gallocatechin and cynidin are replaced by some polyphenols (Ghosal et al., 1965).

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'ftUbfe 3: Pltwi)s>llc v«)Lfipi>imii3 ftio.sd'iftt liif partî rtf Ms5Pf)jo

Parts of Mango

Plienois, Phenolic acids and related compounds

FlavO"rioids

Xanthones

All parts Ellagic acid Gallic acid Eythyl gallate

Catechin Mangiferin

Leaf ChinominGallocatechinIsochinominMethyl chinomin Protocatechuic acid

AstragallinCyanidinDelphinidinEpicatechin-3-O-gallate

IsomangiferinfVlangiferin-6’“0-gallateIVlangiferin-O”glucoside

Fruit m-Dlgallic acid Gallotannin Phloroglucinol Protoratechuic acid 1,2,3,4,-Tetra hydroxy benzene

1,2.3,5-Tetra- hydroxy benzene

Kaempfero!Myrlcelin

Bark Gallocatechin Protocatechuic acid

- Homomangifehn

Twig/Flower

GallocatechinC-GlucodepsideMethyl-6-(0"tri-methylgalloyl)-2,4,-dimethoxybenzoate

CyanidinFisetin

1.3.5.6.7-Pentaoxy- genated-xanthone1.3.6.7-Tetraoxy- genated-xanthone

Otherparts

m-Digallic acidGallotanninfi-Glucogallin5-[2(Z)-Heptadenyl]-resorcinol

Methyl gallate

5-Pentadecyl-resorcinol

ButinEpicatechinIsoauercitrinLeucocyni-din

Quercitin

85

tg 2: Strticfw''' ' c> riirnal*' o /inufiil.. of fyi Jioo,

,O H

HO.\ ,o.

OH

O H O

K aem pfero l

OH

HO

M yricetin

HO.

OH

'■OH

OH

C atechin

OH

Delphinidin chloride

OH

cr

O H

Leucocyanidin

Peonidin chloride

O H

Cyanidin chloride

O H

,O H

H O .

OH

OFisetin

OH

cr

cr

or

Petunidin chloride

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Among 20 Egyptian mango varieties studied, rnangiferin was the predominant compound of the leaves and twigs, while the gallotannin was the main constituent of the unripe fruits and seeds. Gallic acid, ellagic acid, methyl gal late and certain catechin are present in all parts, wfiite m-digallic acid has been detected only in unripe fruits. Fisotin is confined in the twigs while quercetin-3-g lucoside is found only in the leaves. Isom ang ife rin and homomangiferin were reported in the leaves and twigs only; the former was only detectable in the fruits of some varieties. fi-GIucogallin was only reported in the seeds, while gallotannin was found in all parts. The gallotannin obtained from Egyptian mangoes was different from that of the Indian varieties (Saleh e l a!., 1966). In young leaves, flavonoids were usually found in the epidermis while anthocyanins were always present in the parenchymal lacunal (Paris at a/., 1970). The dry seed coal contained catechins, epicatechins,ethyl gallate, leucocyanidin and gallotannins. The peel of Pairi mango fruit possessed more phenolic compounds than the pulp and these contents decreased on storage (Abou Aziz et aL, 1976),

Mamdapur (1985) characterized 5-2(Z)-heptadenyl--resorcinol in mango latex and considered it as mango dermatitis allegen. A mixture of 5'-12-cis-heptadienyl and 5-pentadecylresorcinol, isolated from the fruit peel, acts as a performed agent against a fungus Alternaria alternata responsible for the black spot disease of mango fruit (El Sissi et a!., 1971). The browning of mango peel was dueto oxidisable phenols present in it (Paris et a i, 1970). Mangiferin, dulcitol and a flavonol glycoside have been isolated from fresh Cuscuta reflexa, a parasite on mango tree (Subramaniam et a i, 1977).

Mangniferin has been reported to possess anti-inflammatory, diuretic, choleretic and cardiotonic activities (Shankarnarayan et a!., 1979) and displaysa high antibacterial activity against gram positive bacteria. It has been recommended as a drug in preventing dental plagues (Srinivasan et a i, 1982). Differences in the low and medium molecular weight phenolics and steroidal compounds in healthy and malformed florets of M.indica infected with Fusanum moniliforum are reported (Ghosal et a!., 1908), y-lrradiation on mangoesenhanced the synthesis of phenolic compounds (Lacroix et ai., 1990).

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MISCELLANEOUS COMPOUNDSA homogenous series of hydrocarbons like pentacosane, hexacosane,

and n-octacosanol, esters of C,g, alcohol and hexacosanic and tetracosanic acids have been identified as component of Mangifera indica (Khanna et a!., 1970). The presence of n-tetracosane, n-hexacosane and triacontane in Dusehri cultivar has been reported (Sharma and Aii, 1993).

Two saturated hydrocarbons, and from the leaves ofEgyptian mango as well as octadecane and n-octacosanol from mango panicles have been isolated (Maheshwari et al., 1975). Many gibberellin like substances have been characterized from the mango fruits (Chocke et al., 1970), seeds {Chocke et al., 1970; Ram of a!., 1979) and shoot lips (Pal et al., 1977).The shoot apex of Dusehri mango also contained auxins like indole-3-acetic acid and 3-indole acetonitrile (Lai et al., 1977). The presence of cytokinin like substances, some of them identified as zeatin riboside, zeatin and N®(A ~ isopentyl) adenine riboside, have also been reported in mango shoot tips (Agarwal et al, 1980), Fusarium monilrformae infected twigs and shoots (Ghosal et al., 1965), fruit pericarp and seeds (Ram et al, 1983 and Chen, 1983).

Reports are available concerning the presence of kinic and shikmic acids in leaves and barks (El Sissi et al, 1970), phthalic acid and bis-2-ethyl hexanyl phthalate in pannicles (Maheshwari et a l, 1975), lipoprotein in peel and pulp (Prabha et al, 1982), yellow pigments like 5-naphtho-y-pyrones, flavasperone, ribrofusarin, aurasperene-A, aurasperone-A monohydrate and aurasperene-D in the Aspergillus n iger infected mango fruits, mycoloxin, viz. zearalenone, diacetoxyresorcinol and T-2 toxin in malformed flowers and shoots of Mangifera indica infected with Fusarium moniliformae (Ghosal et a l, 1965).

An antifungal agent, consisting of mixture of 5-( 12-cis-heptadecenyl) and 5-pentadecylresorcin, was isolated from the peel of unripe mango fruits (Droby et a l, 1986). Pest harvest treatment of mangoes with abscisic acid hastened the process of ripening (Parikh et al., 1986). Mango fruit pulp was easily fermented to ethyl alcohol by Zymornonas molbilis (Di Biaggie et al., 1986). 1, 2-Dibromomethane was determined in mangoes by steam distillation with

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hexane (Suzuki et al., 1989). Malformed seedlings of mango showed a lower mean content of indole-3-acetic acid and gibberellic acid and a higher mean content of zeatin, abscisic acid and othylone than healthy seedlings (Singh et al., 1989; Stein et al., 1989). Methyl bromide residue is determined in fumigated mangoes. Abscisic acid levels in shoot apicis of mango have been determined (Kurian et al., 1991). The Chausa variety stem bark contained acyclic sesquiterpenes, viz. farnas-5, 15-olide and farnas-7-(14)-ene-9, 12-diol (Sharma and Ali, 1993). Mangcournarin was present in the stem bark of Dusehri cu ltivar (Sharma and Ali, 1993). The sesquiterpenic acid and mangeudesmenone were obtained from the stem bark of Desi variety (Sharma and Ali, 1995).

MEDICINAL USESThe ripe fruit is laxative, diuretic, diaphoretic, astringent, nutrient-,

invigorating, fattening and refrigerant, ft is useful in haemorrhage from uterus, lungs or intestine and in heat apoplexy. The juice along with aromatlcs is recommended as a restorative tonic (The Wealth of India, 1985).

The dried flowers are astringent and given in diarrhoea, chronic dysentery and catarrh of the bladder and gleet (Singh, 1960). The seed kernels have an astringent taste and are free from any toxic principles. These are anthelmintic, stimulant, tonic and given in diarrhoea, asthma and nasal bleeding (Hooker, 1972). The kernel powder is used as astringent in bleeding piles (The Wealth of India, 1985).

The leaves are given in the treatment of burns, colds and diabetes. Tender mango leaves are consumed as vegetables in Java and Philiipines. They are good source of ascorbic acid. The ash of burnt leaves is a household remedy for burns and scolds. The leaves are masticated to give tone to the gums. Fumes from the burning leaves are inhaled for relief from hiccups and infections of throat (The Wealth of India, 1985; Hookar, 1972). The stem exudes a gum resin which is sold in Indian markets and is considered as antisyphlitic. It is used in cracked feet dressing, in scabies and as a substitute of gum Arabic (MacLeod et al., 1988). The bark is astringent and anthelmintic.

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useful in haemoptysis, haemorrhage, nasal catarrh, malaena, diarrhoea, diphtheria, rheumatism and for lumbrici. It has also tonic action on the mucous membrane (The Wealth of India, 1985). The extracts of leaves, stem bark and unripe fruits exhibit moderate anti-bacteria! activity against Micrococcus pyogenes. The occurrence of antifungal miaoorganisms in ripe mango has been reported (Chopra et a!., 1956). The fragrant flovêrs are used in preparing anotto, Am attar. On steam distillation the flowers yield 0.04% of a pale brownish essential oil. The dried mango flowers are astringent. They are given in diarrhoea, dironic dysentery, catarrh of the bladdor and gleet (The Wealth of India, 1985; Ghalop et a!., 1977).

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EXPERIMENTALEXTRACTION AND ISOLATION OF CHEMICAL CONSTITUENTS OF STEM BARK OF MANGIFERA SNDICA C.UtTiVAR T A Z A L f

Plant material: Stem bark of M. indica variety "Fazali” was collectedfrom Laharpur, Sitapur, U.P.

Ejdractiori: The dried and powdered stem bark (4.0 kg) was extractedwith EtOH (95%) in a Soxhlet apparatus. The extracts were combined and the solvent evaporated under reduce pressure to obtain a dark brown viscous mass(260 g).

Isolation o f cliem icai constituents: The dried alcoholic extract wasdissolved in minimum amount of MeOH and adsorbed on silica gel to form a slurry. The slurry was air-dried and chromatographed over silica gel column prepared in petroleum ether. The column was eluted with petroleum ether, chloroform and methanol in order of increment of polarity to isolate the following compounds:

MANGLANOSTENOIC ACID AElution of the column with petroleum ether furnished white amorphous

powder of MF-1, purified by repeatedly coloum chromatography and preparative TLC, 82.0 g (2.05% yield), m.p. 140-141°, = 0.56 (petro leumether:benzene, 9:1), = 0 °(C 0.756, MeOH ),

UV 212, 236 nm (log e 5.2, 6.7).IR (KBr): 3380, 2955, 2865, 1700, 1690, 1615, 1455, 1380, 1355.

1260, 1195, 1155, 1125, 1085, 1015, 950 cm ’ .NMR (60 MHz, CCiJ: & 6.06 (1H, d, J = 5.5 Hz, H-1), 5.90

(1H, d, J = 5.5 Hz, H-2), 5.20 (2H, m, H-7, H-24),1.B7 (3H, brs, Me-26), 1.70 (3H, brs, Me-27), 1.20 (3H, brs, Me-29), 1.00 (3H, brs, Me-19), 0.83 (3H, brs, Me-28), 0.76 (3H, brs, Me-30), 0.70 (3H, brs, Me-18).

EIMS m/z (rel. in t): 452[M]^ (C3,H ,,0 3 ) (7.6), 437 (100),419(49.9), 393(6.9),378(1.5),311 (3.8), 302 (1.1), 296 (4,1), 287 (4.9), 256 (22.9), 243 (8 .6 ),

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229 (6,3), 216 (3.3), 215 (3,6), 202 (4.9), 188 (20.1), 166(20.3), 150 (3.2), 141(7.3), 136 (3,6), 121(44.0), 113 (22.9), 136 (23.0), 107 (47.4), 97 (19.8), 96(95.7), 83 (27.3), 70 (18.3), 69 {52.7). 55 (84.1), 54 (18,2).

-3,: 147.0 (C-1), 125.8 (C-2). 212.8 (C-3), 47.8 (C-4),46.7 (C-5), 24.3 (C-6 ), 118.1 (C-7), 147.0 (C-8 ), 53.2 (C-9), 36.2 (C-10), 21.1(C-11), 34.7 (C-12), 43,8 (C-13). 50.8 (C-14), 38.3 (C-15). 28,2 (C-16), 53.1(C-17), 15.8 (C-18), 19.2 (C-19). 35.1 (C-20), 173,6 (C-21), 34.6 (C-22),21.1 (C-23), 126.3 (C-24), 146,9 (C-25), 18.1 (C-26), 18.3 (C-27), 21.3 (C-28),22.2 (C-29), 21.5 (C-30).

off MF-1: Compound MF-1 (15 mg) was dissolved insolvent ether ( 1 0 ml), ethereal diazomethane added and left overnight.Evaporation of the solvent yielded monomethyl ester (MF-1 a), m.p. 76-77“ ,UV (MeOH) 212, 238 nm (log s 4.3, 5.7), IR 1725, 1710,1465, 1265, 1120, 980, 765, 750 \

reduction of MF-1: Compound MF-1 (15 mg) was dissolved inMeOH (5 ml), NaBH^ (250 mg) added and left overnight. Water (10 ml) wasmixed and the reaction mixture extracted with CHCI^ (3x15 ml). The organicphase was washed with HÔ (2 x1 0 ml), dried over Na^SO^ and evaporated tosecure 30>-hydroxy derivative (MF-lb), m.p. 158-160“.

UV (MeOH): 212, 237 nm (log s 4.5, 5.6),IR (KBr): 3350, 2890, 1675, 1620, 1545, 1450, 1370,

1200, 1175, 1005 cm-\o f TFA): 67.06 (2H,brs,H-1 ,H-2), 6,63IE

3

(2H,brs,H-7,H-24), 353 (1H,d,J = 9.0 Hz, H-3a), 2.03 (3H,brs,Me-26), 1,67(3H,brs,Me-27), 1.16 (3H,brs,Me-29), 1.13 (3H,brs,Me-19), 1.00 (3H,brs,Me-28), 0.93 (3H,brs.Me-30). 0.87 (3H,brs,Me-18).

Acetyiation of MF-1b: Compound M F-lb (5 mg) was treated with amixture of AcÔ (2 ml) and pyridine (1 ml). Usual work-up gave monoacetyl product (MF-lc), m.p. 114-115°, UV (MeOH) 212, 237 nm (log e 4.3, 5.2), IR y (KBr): 2940, 2890, 1720, 1695, 1645, 1370, 1254, 1025, 985 cm V

92

LiAlH Reduction of MF-1 : Compound MF-1. ' (20 mg) was dissolvedin dried solvent ether (10 ml). LiAIH^ (250 mg) was added in portions with shaking and left overnight. The reaction mixture was filtered and the filtrate evaporated to dryness to obtain 3a, 21-did derivative (MF~1d), m.p. 158-160°, UV (MeOH): 212, 237 nm (log s 4.5, 5,7).

!R (KBr): 3300, 2850, 1420, 1265, 1235, 975 cm-'.EIMS m/z (ret int): 468 [M]" (C2gH^Ô,,)(5.6), 453 (26.3), 437 (9.8),

422 (3.6), 306 (1.1), 285 (1.0), 247 (1.9), 219 (3.0), 204 (1.5), 203 (1.5),190 (2.1), 183 (1,0), 171 (16.4), 162 (2.8), 138 (1.0), 124 (1.1), 123 (1.1),110 (1.0), 95 (8,9), 83 (38.2), 81 (5.3), 111 (5,0), 70 (11.2), 69 (7.4), 55(20.5), 43 (38.2), 41 (12,2).

Acetylation o f Compound MF-1d (10 mg) was heated with amixture of acetic anhydride (3 ml) and pyridine (1 ml) for 1 hour and left overnight. The reaction was quenched by addition of water (10 ml) and the mixture extracted with CHCij ( 3 x 5 ml). The CHCI3 layer was worked up as usual to get monoacetyl product (MF-le), m.p, 78-80°, different spots on TLC plate from that of the original compound

NMR (100 MHz, CDCy: 6 5,35 (1H, brs, H-24), 5.27 (2H, d,J = 7.91 Hz, H-1), 5.13 (1Hz, J = 3.81 Hz, H-2), 4.05 (1H. d, J = 4.68Hz. H-3a), 3.31 (1H, d, J = 12.31 Hz, CH2 0 H-2 1 a), 3,22 (IN, d, J = 11,25Hz, CH2 0 H-2 1 b), 1,79 (3H, brs, Me-26), 1,71 (3H, brs, Me-27), 1.00 (3H,brs. Me-29), 0,96 (3H. brs, Me-19), 0.86 (3H, brs, Me-30), 0.80 (3H, brs,Me-29), 0.75 (3H, brs, Me-18).

MANGSTEROUDE (MF-2):Further elution of the column with petroleum ether furnished colourless

amorphous powder of MF-2, 1.20 g (0.03% yield), R, 0.71 in petroleumetherCghg (9:1), m.p. 85-86°. [a]^^°= - 8.0° C 0,9730, MeOH.

UV 212, 238 nm (log e 5.3, 6 ,8 ).IR (KBr): 3348, 2844, 2825, 1720, 1700, 1422, 1150,

1080, 1024 cm '.

9 3

3): 8 4.20 (1H, d, J - 6.5 Hz. CH,^-29a),4,05 (1H, d, J = 6.5 Hz, CH^-29b), 3.70 (1H, d, J = 6.5 Hz, CH2-2 1a), 3.60(1H, d, J = 6.5 Hz, CH2-21b), 2.40 (2H, brs, CH^-12), 2.25 (2 H. brs, CH^-2), 2.20 (2H, brs. CH^-4), 1,74 (3H, brs, Me-26), 0,92 (3H, brs, Me-19), 0.75(3H, brs, Me-18).

int); 468 [M]" (5.6), 453(26.3), 437 (9.8),422 (3.6), 306 (1,1), 285 (1.0), 270 (1.1), 255 (1.3), 247 (1.9), 219 (3.0), 204(1.5). 203 (1,5), 190 (2.1), 183 (1.0), 162 (2,8), 138 (1,0), 124 (1,1), 123 (1,4),111 (3.1), 110 (1.0), 95 (4.5), 83 (38.2), 81 (5.3), 70 (11,2), 69 (7.4), 55 (20,5), 41 (12.2).

Acetylafion off MF-2; Compound MF-2 (15 ml) was acetylated with AcÔ (3 ml) and pyridine (1 ml) at room temperature for 24 hours. After usual workup, a monoacetyl product (MF-2a) was obtained, m.p. 77-78°, IR 1725, 1715 cm^

Elution of the column with benzene furnished a white amorphous mass of MF-3, recrystallized from petroleum ether-CgH^ (1:1), 28.2 g (0.70% yield), R, = 0.33 (CgH^:CHCl3,9:1 ), m.p. 140-141°, = + 3.62° (C 0.306, MeOH).

(MeOH): 235 nm (log e' 6.3).IR (KBr): 3350, 2930, 1710, 1690, 1615, 1465, 1395, 1385,

1210, 1195, 1165, 1130, 1095, 1010, 955 cm''.m NMR (100 MHz, CDCg: 5 6.15 (1H, d, J 6.14 Hz, H-1), 6.08

(1H, d, J = 6.0 Hz, H-2), 5.30 (1H, d, J = 4.0 Hz, H-22), 1.91 (3H, brs.Me-21), 1.09 (3H, brs, Me-19), 1.05 (6 H, brs, Me-28, Me-29), 1.00 (3H, brs,Me-30), 0.95 (3H, d, J = 6.0 Hz, Me-27), 0.75 (3H, brs, Me-18).

ElMS m/z (rel. int.): 454 [M]^ (C3^H^g0 3 ) (21.6), 439 (100), 424(6.2), 421 (45.0), 410 (14.6), 393 (12.7), 297 (6.5), 256 (15.5), 190 (9.8), 187(14.3), 164 (6.9), 161 (16.2), 159 (19.4), 141 (5,6), 136 (18.4), 120 (28.1), 105(24.2), 96 (44.3), 70 (32,5), 54 (24.2),

9 4

-3):S 147.2 (C-1), 126,3 (C-2). 217.3 (C-3), 46.4 (C-4), 45.1 (C-5), 23.0 (C-6 ), 26.0 (C-7), 46.4 (C-8 ), 47.2 (C-9), 37.8 (C- 10), 22.1 (C-11), 32.0 (C-12), 45.2 (C-13), 47.0 (C-14), 32,0 (C-15), 29,1(C-16), 48.1 (C-17), 19.9 (C-18), 21.3 (C-19), 135.0 (C-20), 16.3 (C-21), 132,9 (C-2 2 ), 22.1 (C-23), 23.0 (C-24), 29.8 (C-25), 173.8 (C-26), 15.1 (C-27), 72.2 (C- 28), 23.0 (C-29), 21.2 (C-30).

-3: Compound MF-3 (15 rng) was treated withethereal CH^N^ overnight. Next morning the solvent was evaporated to get monomethyl ester MF-3a, m.p. 131-132°, different spots on TLC plate on comparison with the original compound (MF-3).

Further benzene eluants of the column furnished a white amorphous mass of MF-4, recrystallized from CHCIg-MeOH (1:1), 4.5 g (0.11% yield),R, = 0.18 in CgHg-CHCl3 (9:1), m.p. 150-151°. + 20.18° (C 0.9750,MeOH).

{MeOH); 230 nm (log s 6.7). m (KBr); 3100, 2955, 2880, 1705, 1680, 1635, 1457, 1361,

1266, 1197, 1156, 1077, 1000, 940 cm-\NMR f10O MHz, CDCg: 5 6.20 (1H, d, J = 6.0 Hz, H-1), 6.06

(1H, d, J = 6.0 Hz, H-2), 5.36 (1H, m, H-24), 1.96 (3H, brs, Me-27), 1.20(3H, brs, Me-29), 1.16 (6 H, brs, Me-19, Me-28), 1.10 (3H, d, J = 6.0 Hz, Me-19), 0.96 (3H, brs, Me-30), 0.86 (3H, brs, Me-18).

m/z (re t m l): 454 [M]" (3.4), 439 (7.3), 421(28.0), 410 (3.1), 257 (10.5), 252 (16.9), 203 (10.3), 189 (11.4), 185(22.5), 175 (13.3), 168 (15.7), 161 (18.2), 147 (18.5), 143 (20.1), 135(20.8), 129 (40.9), 105 (47,3), 95 (54.6), 91 (61.3), 73 (64,5), 57(100), 44 (68.1).

Methylation o f MF-4 Compound MF-4 (10 mg) was methylated with ethereal solution of CH^N^ at room temperature overnight for 1 2 hours.

9 5

Evaporation of the solvent yielded a monomethyl product (MF-4a), rn.p, 139-141°, different spots on TLC on comparison with the original compound.

MANGLANOSTENOIC ACID D PF-S};Further elution of the column with C^Hg furnished compound MF-5,

crystallized from CHClg-MeOH (1:1) 6.12 g (0.15% yield), R, = 0,250 (C^Hg-CHCI3, (9:1), m.p. 164-165°. [a^Q = + 1 3 .1 2 °, (C 0.6510, MeOH)

UV fMeOH): 257 nm (log s 5.6).(KBr); 3350, 1710, 1680, 1635, 1455, 1385, 1260, 1210,

1185, 1065, 955 c m \NMR |90 MHz, CDCI3); 5 6.13 (1H. d, J 5.0 Hz. H-2), 6.03

(IH. d, J = 5.0 Hz, H-1), 5.37 (2H, brs, H-6 , H-22), 1.87 (3H, s. Me-21),1.09 (3H. s, Me-28), 1.03 (3H, s. Me-29), 1.00 (6H, s, Me-19, Me-30), 0.93(3H. d, J ^ 6.0 Hz. Me-26), 0.80 (3H, s, Me-18),

ElMS OT/z (re t int.): 450 [M]^ (8.6), 435 (26.8), 418(15.0), 406 (1.1), 391 (6.0), 309 (4.1), 293 (3.4), 268 (52.6), 267 (7.6),255 (11.8), 252 (10.37), 241 (7.0), 227 (3,7), 214 (3.4), 213 (4.5), 200(8.1), 186 (8.2), 162 (3.8), 148 (12.7), 147 (26.3), 141 (6.3), 132 (22.1), 123(14.0), 109 (13.3), 108 (13.3), 96 (10.8), 95 (11.6), 94 (22.5), 82 (13.4), 81(10.1), 80 (20.9), 73 (19.0), 68 (31,9), 54 (47.7), 43 (100).

13C NMRf22.S MHz, CDCI3): 5 148.0 (C-1), 131.6 (C-2), 206.7 (C-3),44.9 (C-4), 144.5 (C-5), 115.0 (C-6), 56.8 (C-7), 132.0 (C-8), 134.6 (C-9),37.2 (C-10), 24.8 (C-11), 34.6 (C-12), 44.9 (C-13), 51.0 (C-14), 33.9 (C-15), 29.3 (C-16). 52.0 (C-17), 19.6 (C-18), 18,9 (C-19), 125.1 (C-20),18.9 (C-21), 145.2 (C-22), 26.7 (C-23), 34.1 (C-24), 35.9 (C-25), 19,8(C-26), 172.0 (C-27), 25.5 (C-28), 26,8 (C-29), 21.7 (C-30).

Methyiation of MF-5: Compound MF-5 (10 mg) was dissolved insolvent ether (10 ml), ethereal CH^N^ added and left overnight. The solvent was evaporated to get monomethyl ester (MF-5a) m.p, 148-149'’ , IR (KBr): 1735, 1710 cm-'.

9 6

P/4NGLANOSTENOIC ACID E fWfF»6):Further elution of column with C^Hg gave compound MF-6, recrystallized

from CHCI^-MeOH 35.5 g (0.88% yield), R, 0.435 (C^.Hg-CHCl3, 9:1),m.p. 139-141°, - + 2 . r (C 0.7520, iVleOH).

(MeOH): 241 nm (log e 8.1).IR (KBr): 3150, 2900, 2860, 1700, 1670, 1630, 1445, 1405,

1365, 1355, 1250, 1185, 1140, 1060. 975, 910 c m \'H NMR (90 MHz, CDClJi 5 6.05 (1H. d, J = 6.5 Hz, H-1), 6.00

(1H, d, J = 6 . 6 Hz, H-2), 5.30 (2H, brs, H-7, H-22), 2 46 (2H, m, CH,), 2.30(1H, m, CH), 2.10 (4H^, m, 2xCH^), 1.90 (3H, brs, CH3-2 I). 1.63 (6 H, m,2xCH^, 2CH), 1.43 (6 H, m. CH^, 4xCH). 1.07 (3H, s, Me-19), 1.03 (6 H. d.J = 6.0 Hz. Me-26. Me-30), 0.97 (3H. s, Me-2), 0.93 (3H, s. Me-29), 0.88(3H. d, J = 6.0 Hz. Me-18).

EJllS m/z (re!, int.): 456 [M]" (C3 H^g03) (5.0), 439 (11.3), 421(5.2). 410 (2.5). 392 (2.8), 313 (1.1), 298 (1.3), 286 (1.5), 272 (2.1),244 (2.8), 188 (7.5). 162 (7.8), 136 (9.8). 121 (5.3), 109 (5.5), 101(6.3), 96 (9.3), 83 (29.0), 81 (6 .6 ), 78 (13.2), 73 (7.7), 69 (10.8), 55(12.2), 54 (19.4), 44 (30.2).

13C NMR (CDCI3): 5 149.1 (C-1). 135.0 (C-2), 207.3 (C-3), 44.9 (C-4), 50.2 (C-5), 24.3 (C-6 ), 116.7 (C-7), 125.8 (C-8 ), 49.1 (C-9), 35.6(C-10), 22.8 (C-11), 35.0 (C-12), 37.1 (C-13), 34.8 (C-14), 33.8 (C-15),27,6 (C-16), 50.2 (C-17), 12.2 (C-18), 19.8 (C-19). 136.5 (C-20), 18.4 (C-21), 118.6 (C-22), 27.0 (C-23), 23.7 (C-24), 26.6 (C-25), 20.1 (C-26),173.1 (C-27), 12.8 (C-28), 23.2 (C-29), 21.8 (C-30).

M ethy la tion o f MF-6 : To compound MF - 6 (15 mg) etherealdiazomethane was added, shaken well and left overnight. Evaporation of thesolvent yielded a monomethyl ester (MF-6 a) which showed different spot on TLCplate on comparison with the original compound.

9 7

Sodium borohydride reduction of MF-6: Compound MF-6 (10 mg)was dissolved in MeOH (5 ml), NaBH^ (250 mg) was added in portions and left at room temperature for 24 hours. Water (10 m() was added and the reaction mixture extracted with CHCl^ (3x10 ml). The organic phase was washed with water, dried over anhydrous Na^SO and evaporated to secure 3(l.-hydroxy derivative (MF-6 b). m.p. 159-16r, IR (KBr): 3500, 2910, 2855, 1695,1625, 1465, 1370, 1230, 1145, 1040, 1025, 980, 735 cm'V

NMR (60 MHz, CDCI3): 5 6.10 (1H, m, H-2), 5.90 (1H, d, J = 6.00Hz, H-1), 5.53 (2H, rn, H-6 , H-7), 5,26 (1H, m, H-22), 3.26 (1 H, m, H-3a),1.93 (3H. brs, CH3-2 I). 1.00 (6 H, brs, CH3-29, CH3- I 9 ), 0.96 (6 H, brs, CH3-26, CH3-2 8 ), 0.86 (3H, brs, CH3-3O). 0.76 (3H, brs, CH3-I8).

ElMS m/z fre t M }: 456 [M]" (C3,H^g0 3 ) (3.0), 441 (12,5), 423(15.2), 320 (3.9), 300 (3,2), 278 (6,4), 255 (2.5), 246 (2.8), 241 (8.2),237 (5.8), 210 (11.2), 119 (5.2), 187 (11.3), 167 (1^.2), 164 (5.0),133 (26.1), 119 (12.2), 105 (67.9), 91 (27.6), 85 (30.2), 83 (100), 77(55.3), 55 (26.1).

MANGLANOSTENOIC ACID F |MF-7):Mother liquor of MF- 6 on recrystailization from CHCl3-MeOH (1:1) gave

MF-7, 2.25 g (0.05% yield), m.p. 172-174°, R, = 0.38 (C^H^:CHCl3, 9:1), [ct]p3o 0“ (C 1.035, MeOH).

IR (KBr): 3405, 2950, 2845, 1710, 1690, 1625, 1410, 1325,1225, 1000, 910, 890, 820 cm ’ .

NMR 1100 MHz, CDCg: 5 7,06 (IN, d, J = 9.52 Hz, H-1), 6,90(1H, d, J = 9.52 Hz, H-2), 5.32 (2H, brs, H-5, H-24), 2,79 (1H, d, J =5.13 Hz, H-7a), 2.59 (1H, brs, H-20), 2.30 (1H, brs, H-9), 2.27 (1H, brs, H-17), 2,13 (1H, d, J = 14.65 Hz, H^7b), 2.59 (1H, brs, H-8 ), 2.30 (2H, m,H^-ll), 2.13 (1H, d, J = 7,08 Hz, H-23a), 2.05 (2H, brs, H^-12), 1,84 (3H,brs, Me-26), 1,65 (1H, d, J = 10,26 Hz, H-23b), 1.54 (2H, brs, H-15), 1,43(2H, brs, H -16), 1,24 (2H, brs, H -22), 1,11 (3H, brs, Me-28), 1.05 (3H, brs.

9 8

Me-19), 1.00 (3H, brs, Me~30}, 0,92 (3H. cl, J = 8.3 Hz, Me-21), 0.89 (3H,brs, Me-29), 0,81 (3H, brs, iVle-18).

EliyiS m/x ( re l in t | ; 452 [M]^ (40.5), 437 (41.6), 420(29.4), 393 (29.0), 378 (2,1), 311 (6.1), 270 (4,8), 243 (5.6), 237 (4,5),229 (3.8). 223 (4.6), 216 (4.1). 215 (4.3), 202 (7.1), 188 (12.8), 162(8.8), 148 (8.3), 141 (3,1), 109 (13.5), 96 (9.7), 95 (29.1), 85 (7.4),81 (17.9), 67 (13.0), 54 (29.9), 44 (30.7).

o f MF-7: Compound MF-7 (tO mg) was treated withethereal solution of CH^N^ (10 ml) at room temperature for 24 hours. The solvent was evaporated on water bath to get monomethyl ester (MF-7a), m.p. 215-216°. IR 1735. 1710 cm".

Compound MF-7 (5 mg) was dissolved in MeOH (5 ml). NaBH^ (100 mg) was added in portions and left overnight. Water (10 ml) was mixed, shaken well and extracted with CHCI3

( 3 x 5 ml). After usual work-up fJ-hydroxy derivative (MF-7b) was obtained, different spots on TLC on comparison with the original compound MF-7,

GElution of column with CgHg-CHCIg (3:1) furnished colourless compound

MF-8, purified with preparative TLC (CgHg~CHCl3, 3:2), 11.3 g (0.28% yield), m.p. 170-171°, R, 0.65 (CHCl3-MeOH, 9:1), [a]^^° =+ 18.22“(C 0.92,MeOH),

UV (leO H ): 214, 245 nm (log b 6,7, 7,3).IR 2925, 2850, 1710, 1690, 1625, 1440, 1365, 1245, 1170,

1135, 1050, 910 cm-\NMR (60 MHz, CDCIJ: 5 6.23 (1H, d, J = 6.0 Hz, CH-2),. 6.00

(1H, d, J = 6.0 Hz, CH-1), 5.36 (2H, m, CH-6 , CH-24), 2.73 (1H, brs, CH-12), 2.50 (2H, brs, CH^-7), 2.37 (2H, m, CH-23), 2.06 (2H, m, CH2 -2 I),1.96 (3H, brs, COCH3), 1 . 8 6 (2H, m, H,-16), 1,73 (3H, brs, CH3-2 6 ), 1.63(1H, m, H-17), 1.53 (3H, brs, CH3, 27), 1.38 (3H, brs, CH3-2 I), 1.26 (2H, brs,CH^-IS), 1.16 (3H, brs, CH3-2 9 ), 1.03 (3H, brs, CH3-I), 0.93 (3H, brs.

99

CH3-28). 0.83 (3H, brs, CH -SO), 0.30 (1H, brs, CH.,-18a), 0.23 (1 H), brs,CH^-18b).

m i): 490 [M]" (4.9), 475 (10.4), 436(14.6), 420 (11.6), 321 (1.1), 308 (2.0), 29 1 (4.6), 280 (1,2), 255 (4.1),225 (7.9), 214 (1.3), 210 (2.9), 196 (3.1), 186 (3.4), 182 (4.3), 173(9.1), 169 (40.1), 163 (17.0), 159 (5.3), 155 (21.2), 148 (11,6), 143(3,7), 135 (25.0), 109 (16.9), 107 (19.0). 96 (15.4), 95 (35,6), 81(29,4), 67 (24.6), 55 (64.4), 54 (50.8), 43 (100).

I: Compound MF- 8 (15 mg) was heated with 2Nalcoholic KOH (5 ml) for 1 hour. Water (5 ml) was added and the reaction mixture extracted with CHCI3 ( 3 x 5 ml). The organic phase was washed with water, dried over Na^SO^ and evaporated to get deacetyl product (MF-8 a), m.p. 183-184°, IR Y 3410, 1720, 1630 cm '.' « max ’ '

reduction of MF-8: Compound MF-8 (10 mg) was dissolvedin MeOH (5 ml). NaBH^ (25 mg) was added in portions with shaking and left overnight. After usual work-up 3l3,,110.-dihydroxy derivative (MF-8 b) was obtained, m.p. 158-159°. IR (KBr): 3430, 2950, 1725, 1625, 1465, 1370, 1250,1235, 1155, 1025, 855 cm'.

(60 MHz, CDCI3): 5 6.26 ( -H, m, H-2), 5.96 (1H, d,J = 6.0 Hz, H-1), 5.26 (2H, m, H~6 , H-24), 3.30 (1H, d. J-4.5 Hz, H-3B),3.20 (1H, d, J = 8.5 Hz. H-12E), 1.93 (3H, brs, COCH3), 1.68 (3H, brs, CH3-26), 1.50 (3H, brs, CH3-2 7 ). 1.26 (3H, brs, CH3-2 I), 1.00 (3H, brs, CH3-2 9 ),0.90 (3H, brs, CH3- I 9 ), 0.80 (3H, brs, CH3-2 8 ), 0.76 (3H, brs, CH3-3 O), 0.20(2H, brs, C H -18).

Elution of the column with CHCI3 gave colourless amorphous powder of MF-9, recrystallized from CHCl3-MeOH (1;1), 18 g (0.45% yield), R, = 0.66 (CHCI3), m.p. 91-92°, [a]^3o = _ 20°(C 0.5635,MeOH).

100

flieOH): 228, 250 nm (log e 5.6. 6,2 ).IR (KBr): 3350, 1720, 1705, 1595, 1465, 1445, 1395, 1225,

1205, 1140, 990, 935, 720 cm',NMR (100 MHz, CDGI3)". 5 4.20 (1H, d, J = 6.84 Hz,

4.00 (1 H, d, J = 6.10 Hz, CH^-21b), 2.44 (2H, brs, -CH2-1 2 ), 2.38 (2 H, brs,CH^-2), 2.03 (2H, brs, CH^-23), 1.64 (3H, brs, Me-26), 1.55 (3H, brs. Me-27), 1.00 (3H. brs, Me-19), 0.87 (3H, d, J = 5.13 Hz, Me™29). 0.77 (3H, brs,Me~18).

454 [M]*- (12.1), 439 (18.8), 422 (10.3), 407(3.5), 392 (3.8). 354 (2.8), 285 (3.8), 270 (3.6), 255 (2 .0), 245 (3.8),231 (3,2). 227 (3.5), 217 (3.5). 215 (3.4), 212 (3.3), 204 (6.7), 203(6.9), 190 (5.3). 189 (5.8), 175 (9.3), 169 (7.3), 162 (7.3), 149 (7.4),147 (6 .8 ), 138 (2.7), 135 (16.7), 125 (7.7), 124 (7,8), 123 (7,9), 121(12.5), 110 (8 .8 ), 97 (5.1), 95 (17.1), 83 (24.8), 82 (7,3), 81 (10.2),70 (6 .6 ), 69 (12.9), 55 (17.8), 41 (12.1).

Acetylation o f MF-9: Compound MF-9 (10 mg) was heated with amixture of AcÔ (3 ml) and pyridine (1 ml) for 1 hour. Water (10 ml) was added and worked up as usual to secure monoacetyl product (MF-9a), different spots on TLC from that of original compound, IR 1735, 1720, 1705 cm-'.

101

EXTRACTION AND ISOLATION OF CHEMICAL CONSTITUENTS OF STEM BARK OF MANGSFERA INDICA CULT!VAR ’LANGRA'

Piaot material; Stem bark of M. indica variety “Langra” was collected from Laharpur, Sitapur (U.P.)-

Extraciion: The dried and powdered stem baric (4.00 kg) was extracted with EtOH (95%) in a Soxhlet apparatus. The extracts were combined and the solvent evaporated under reduced pressure to obtain a dark brown viscous mass (480 g). . ■

Isolation of chemical constituents: The dried alcoholic extract wasdissolved in minimum amount of MeOH and adsorbed on silica gel to form a slurry. The slurry was air-dried and chromatographed over silica gel column prepared in petroleum ether. The column was eluted with petroleu ether, chloroform and methanol to isolate the following compounds;

MANGTERPEWONEElution of the column with petroleum ether furnished colourless crystals

of ML-1, 3.2 g (0.08% yield). R, = 0,960 (petroleum ether), rn.p. 40-41°,+2.2 (C 1.3250, MeOH).

UV (CHCI3): 236 nm (log e 3.5).IR fKBr): 3445, 2905, 2855, 1720, 1595,. 1465, 1385, 1170,

1110, 725 cm-1.NMR (90 MHz, CDCy: 5 5.30 (1H, d, J = 5.0 Hz, H-10), 4.25

(1H, d, J = 5.0 Hz, H,-15a), 4.18 (1H, d, J = 7.3 Hz, H^-15b). 2.35 (2H. m,H^-3), 2.00 (1H, brs, H-5), 1.90 (1H, brs, H-11). 1.85 (3H, brs, H3- I 4 ), 1.60(2H, d. J = 5.5 Hz, H,-7). 1.25 (7H. brs, 2xCH^, Me-13), 0,90 (3H, t, J =6.0 Hz, Me-1), 0,77 (3H, d, J = 6.0 Hz, Me-12),

EliVlS m/z (ref. int); 254 [My (6.1), 225 (1.0), 211 (1,0),183 (2,2), 155 (1.0), 141 (1.0), 127 (5.7), 113 (1.5), 99 (1.2), 71(22.3), 59 (21.3), 43 (37.2).

102

Acetylatlon of ML-1: Compound ML-1 (10 mg) was acetylated withAc,0 (2 ml) and pyridine (1 ml) for 15 hours. Water (10 ml) was added and the reaction mixture extracted with CHCI3 (3x10 ml). The organic phase waswashed vi/ith water, dried over Ma^SO and evaporated to get rnonoacetylated product (ML-la). IR 1725, ,1715 cm-'.

MANGDITERPENIC ESTER (ML-2)Further elution of the column with petroleum ether furnished colourless

crystalline product ML-2, recrystallized from CHCl3-IVieOH (1 :1 ), 12.0 g (0.30%yield), R, =0.28, (petroleum ether)), m.p. 59-60°, = + 13.22 (C , MeOH).

UV (MeOH); 227 nm (log 's 6.1).IR 2920, 2850, 1735, 1710, 1640, 1465, 1415, 1375,

1260, 1205. 1185, 1115, 725 cm \m NMR {90 MHz, CDCy: 6 7.20 (1H, brs, H-3'), 6.42 (1H, brs,

H-2'), 4.10 (1H, brs. H-1a), 4.03 (1H, brs, H-1b), 2.30 (1H, brs, m, H-^').2.24 (1H, m, H-11), 2.00 (2H, brs, H^-IS), 1.90 (2H, m. H-15, H-3), 1.60 (2H.m, H-7, H-8 '), 1.22 (22 H, brs, llxH^), 1.16 (6 H, brs, 2xMe), 1,15 (3H,d, J = 6.0 Hz, Me). 1.03 (6 H, brs, 2xMe), 0.96 (3H, d, J = 6.0 Hz. Me),0.90 (3H, d, J = 6.0 Hz, Me), 0.83 (3H. brs. Me).

EIMS m/z (rel. int.): 478 [M]" (C^^H^gO ) (2.3), 421 (4.9), 407 (2.1),393 (2.9), 365 (3.1), 339 (2.8). 311 (3.2), 309 (3.1), 295 (3.1). 281(2,3), 253 (2.5), 239 (3.3), 225 (41), 211 (4.7), 197 (4.4), 183 (5.1),169 (11.3), 167 (56.0), 155 (5.6). 153 (20.4), 143 (27.1), 141 (11.2),139 (5.7). 133 (21.5), 127 (12.3), 117 (28,1), 113 (8,3), 99 (44,3). 91(100), 85 (19.5), 81 (28.3); 71 (21.3). 69 (24.3), 57 (32.6).

Hydrolysis of ML-2; Compound ML-2 (25 mg) was heated with 2N ethanolic KOH solution ( 6 ml) for 2 hours. Water (10 ml) was added and the reaction mixture extracted with CHCI3 (3x15 ml). The organic phase was washed with water (2 x 1 0 ml), dried over Na^SO^ and evaporated to get the alcohol (ML-2a), m.p. 71-72“, IR y 3450, 1710 c m \

103

The reaction mixture after extraction with CHCI^ was neutralized with dilute HCI and re-extracted with CHCI3. After usual work-up the acid (ML-2b) was obtained, m.p. 77-79°, IR y 3350, 1690 cm'.rtiiix

EICOSENYL E:STER (ML-3)Compound ML-3 was obtained as colourless amorphous powder from

petroleum ether eluants,recrystallized from CHCIj-MeOH (1:1), 4.1 g (0.1025%yield, R, = 0.028, (petroleum ether-benzene, 1:1), m.p. 95-96°,[ays + 9 8. (C 0.6120, MeOH).

IJV 222 nm (logs 6,1).IR 2920. 2850, 1730, 1705. 1600, 1465, 1420, 1380, 1315,

1260. 1205, 1185, 1115, 715 cm V’H NMR (90 MHz. CDCIJ: 5 7.20 (1H, brs, H-19), 6.43 (1H, brs,

H-18), 2.00 (4H, m, H^-e, H^-4), 1.23 (42H, brs, 2 IXCH2 ), 0.90 (3H, brs,CH3-1 1 '), 0.86 (3H. brs, CH3-IZ), 0.83 (3H, brs, CH^-I), 0.80 (3H. brs, CH3-10').

BMS m/z freL int): 492 [M]- (C3,Hg^03) (3.5), 477 (5,9), 463 (2.8),450 (2.7), 449 (2.7), 423 (12.6), 421 (1.8), 379 (2.3), 323 (4.3), 309(4.4), 307 (4.6), 295 (4.9), 281 (3.6). 279 (3.4), 267 (4.1), 253 (4.7),239 (5.3), 225 (6.3), 213 (3.4), 211 (3.4), 197 (6.4), 185 (7.9), 183(6 .8 ), 169 (11.2), 155 (6.2), 154 (13.1), 141 (11.1), 139 (9.6), 127(24.1), 113 (18.6). 111 (36.1). 99 (37.2). 85 (6 6 .8 ), 71 (52.6), 57 (100),42 (48.6).

Hydrolysis o f ML-3: Compound ML-3 (25 mg) was heated with 1Methanolic KOH solution (20 ml) for 6 hours. Water (10 ml) was added and the reaction mixture extracted with CHCI3 (3x10 ml). The organic phase was washed with water (2 x1 0 ml), dried over Na^SO^ and evaporated to get an alcohol ML-3a, m.p. 80-81°.

The reaction was neutralized with dilute HCI and re-extracted with CHCI3

(3x10 ml). After usual work-up the acid (ML-3b) was obtained, TLC comparable.

104

HEXACOSANYL ACETATE (fWL-4) '

Elution of the column with petroleum ether-CHCly (9:1) afforded colourless compound ML-4, recrystallized from CHCl^-MeOH (1 :1 ), 17.0 g (0.42% yield),

= 0.85 (petroleum ether-MeOH, 3:1), m.p. 102-104°, = -7.28. (C 1.2360,MeOH).

UV p e O H ): 210 nm (log e 6.5).IR (KBr): 2900, 2810, 1730, 1700, 1450, 1405, 1365, 1300,

1245, 1210, 1185, 1185, 1165, 1095, 700 cm \m m (60 MHz, CDCi^): 5 4.00 (1H, brs, H-26a), 3.85 (1H, brs,

H-26b), 2.40 (2H, m, H^-B), 2.20 (2H, m, H^-11), 2.06 (3H, brs, COCH^),1.70 (14H, brs, 7 x CH^), 1.26 (28H. brs, 14 x CFg, 0.55 (3H, t, J == 6.5 Hz,Me-1).

E!MS m/£ (re l in t): 438 [MY (2.0),. 395 (4.3), 381 (1.6),367 (1.0), 353 (1.0), 351 (1.0), 339 (1.0), 337 (1.1), 325 (1.3), 323(1.3), 311 (1.1), 309 (1.8). 297 (1.7), 295 (1.6), 283 (1.1), 281 (1.5),269 (1.6), 267 ( 1 .6 ), 255 (3.1), 253 (2.1), 241 (2.2), 239 (2.3), 227(2.8), 225 (2.9), 211 (5.6), 199 (3.0), 185 (4.6), 171 (4.8), 169 (7.5),157 (4.2), 155 (28.6), 143 (7.8).

A lkaline Hydrolysis of ML-4: Compound ML-4 was heated with 2Nalcoholic KOH (5 ml) for 2 hours. Water (15 ml) was added and the reaction mixture extracted with CHCI3 ( 3 x 1 0 ml). The CHCIj-layer was washed with HÔ (2 X 1 0 ml), dried over Na^SO^ and evaporated to obtain alcoholic product (ML-4a), IR 3450, 1700 cm ’ .

MANGOLACTONE (ML-5)Elution of the column with petroleum ether-CHCIg (9; 1) furnished white

amorphous mass of ML-5, recrystallized from CHCI^-MeOH (3:1), 6.5 g (0.16%yield), Rf = 0.55 (petroleum ether-CHCl3, 3:1), m.p. 77-78°, . 6.25. (C0.8650, MeOH).

105

(MeOH|: 248 nm (log e 5.6).3R (KBr): 3410, 2925, 2855, 1735, 1465, 1370, 1245, 1175,

1010, 725 cm-'.

m NMR (90 MHz, CDCI3): 6 5.26 (1H, brs, m, H-17a), 4.00 (1H, m,H-5). 2.73 (1H. m, H-19), 2.23 (2H, t, J = 7.2 Hz, H^-20), 1.97 (2H, m, H^-15),1.80 (4H, brs, H -A, H,-6), 1.26 (22H, brs, 11 x CH^), 0.97 (9H, brs, Me-1, Me-22. Me-27).

IreL irst): 424 [M]" (5.7), 409 (2.8), 395(14.6), 381 (3.8), 337 (2.8), 257 (17.9), 255 (6.5), 253 (2,1), 239 (3.3),229 (13.9), 227 (4.7), 213 (5.1), 211 (4.7), 209 (2.4). 199 (3.6), 197(2,3), 195 (2,8), 185 (6.3), 181 (3.5), 174 (13.1), 171 (6.2), 169 (2.6),167 (2.3), 157 (6.9), 153 (5.8), 145 (11,9), 143 (10.5), 139 (5,2),135 (13.9), 129 (2.6), 126 (3.5), 125 (16,5), 115 (7.0), 111 (29.3),101 (4,5), 97 (48.2), 87 (7.3), 83 (52.2), 71 (68.3), 69 (62,3), 57 (100).

of ML-5: A mixture of ML-5 (10 ml) was acetylated with AcÔ (3 ml) and pyridine (1 ml) for 24 hours at room temperature. The reaction mixture was poured into crushed ice and' extracted with CHCI^ (3x10 ml). The organic phase was washed with water (2x10 ml), dried over Na^SO^ and evaporated to get rnonoacetylated product (ML-5a), m.p. 80-81“ . IR 1735, 1725 cm-’ .

Compound ML- 6 was obtained from petroleum ether-CHCI^ eluants (3;1), crystallized from CHCi^-MeOH (1:1),8.0 g (0.2% yield), R, - 0.25 ( petroleum ether-CHCl3, 1;3), m.p. 80-81°, = + 1.25. (C 1,653, MeOH).

UV (MeOH); 238 nm (log s 4,5),IR (KBr): 3400, 2920, 1735, 1710, 1465, 1375, 1260, 1180,

1030, 725 cm-’ .NMR (90 MHz, CDCg: 5 4.10 (1H, d, J = 7.0 Hz, H^-17a), 4,00

(1H, d, J = 7.0 Hz, H^-17b), 3.60 (1H, d, J = 5.5 Hz, H^-1a), 3.53 (1H, d.

J = 6 . 0 Hz, H^-1b), 2.20 (2H, m, H,-2)', 1.90 (2H, brs, H,-3), 1,46 (4H, m,2 X H ), 1.20 (42H, brs, 21xH^), 0.93 (3H, d, J = 6,0 Hz, H ^-ll').

106

454 [M] (2.4), 423 (2.9), 395 (1.6),383 (1.4), 369 (1,4), 327 (1.4), 313 (1,4), 285 (1.6), 269 (1.7), 255(1,5), 241 (2,2), 227 (2.8), 213 (3,5), 199 (3.2), 185 (5.5), 171 (6,0),169 (13.9), 157 (4.5), 155 (14,1), 143 (9.0), 141 (16,8), 129 (11,2),127 (24.1), 115 (7.3), 113 (44.5), 101 (12.5), 99 (24,3), 85 (31,6), 83(55.2), 73 (8.1), 71 (43.2), 69 (65.2), 57 (100), 55 (70.3).

Acetyiation of ML-6; Compound ML-6 (25 ml) was acetylated with acetic anhydride (2 ml) and pyridine ( 1 ml) at room temperature for 24 hours. Water (10 ml) was added and the reaction mixture extracted with CHCI^ (3x15), The organic phase was washed with water (2x10 ml), dried over Na^SO^ and evaporated to get a gummy monoacetyl product (ML~6a) which was purified by preparative TLC, IR y (KBr) 1730, 1725 cm

k Compound ML- 6 ( 2 0 mg) was heated with 2M alcoholic KOH solution for 4 hours. The reaction mixture was diluted with water (25 ml) and extracted with CHCI^. The organic phase was washed with HÔ (3x10 ml), dried over Na^SO^ and evaporated to secure the alcohol (ML“6 b), m,p. 71-72°, IR y : 3450, 1710 cm ’ .

’ ' m a x '

The reaction mixture after extraction was neutralized with dilute HCI and re-extracted with CHCI . After usual work-up the acid (ML-6 c)was obtained, m,p. 83-84°, IR y : 3240, 1690 cm \

MANGTIRUCALLOIC ACID (ML-7) Elution of the column withpetroleum ether-CHCIg (1:1) yielded a white amorphous mass of ML-7, recrystallized from CHCI3 (1:1), 2.20 g (0.055% yield), = 0.55 (benzene- CHCI3, 1:1), m.p. 170-172°, = 0“ (C 1.0260, MeOH),

X : 227 nm (log e 6.5).ma)c V O' /IR (KBr); 3100, 2970, 2900, 1750, 1680. 1630, 1440, 1420, 1365,

1260, 935 cm'.’H NMR (100 MHz, CDCI3); 6 6.00 (1H, d, J = 6.0 Hz, H-1), 5.95

(1H, d, J^.O Hz, H-2), 5,16 (1H, d, J = 3.5 Hz, H-24), 1.80 (3 H, brs.

107

Me-27), 1.00 (3H, brs, Me-19), 0.97 (3H, brs, Me-29), 0.95 (3H, d, J = 6.0Hz, Me-21), 0,90 (6 H, brs, Me-28, Me-30), 0.70 {3H, brs, Me-18).

[M]^ (C3,H,,0.,) (14.5), 439 (100), 421(48.2), 410 (21.5), 393 (17.3), 313 (11.7), 287 .(17,2), 257 (16.7), 231(13.2), 189 (11.2), 187 (13.5), 185 (18,9), 175 (23,5), 161 (15,5), 159(27.8), 147 (27.1), 133 (42.8), 121 (24,3), 113 (12,3), 107 (40,3), 95(96.1), 81 (28.2), 69 (40,3), 55 (40,6), 44 (90.1),

o f ML-7; Compound ML-7 (10 mg) was methylated with ethereal solution of CH^N^ at room temperature for 12 hours. The solution was evaporated to get monomethyl ester (ML-7a),m.p, 164-165°, different spots on TLG on comparison with the authentic sample.

Elution of the column with CHCI3 furnished colourless crystalline compound iVIL-B, m.p. 139-140°, lit. m.p, 140°, 4.0 g (yield 0.1%).

fMeOH): 229 nm (log s 5.6), , ,m/z (rei. in i) : 414 [M]'" (CjgHgoO) (16,1), acetate m.p,

127-128°.

Compound ML-9 was obtained as colourless amorphous powder from CHCI3 eluants, crystallized from CHCI^-MeOH (1:1), 3,3 g (0.08% yield), R, = 0.33 (CHCI3), m,p. 100-102°, 18.2. (C 1.86, MeOH).

(MeOH): 221 nm (log e 5.6).IR (KBr): 3410, 2900, 2825, 1710, 1695, 1445, 1400, 1345,

1155, 700 cm '.NMR (90 MHz, CDCI3): 5 4.20 (1H, dd, J = 6.5, 5.5 Hz, H-7),

2.60 (2H. brs. H,-12), 2.47 (4H, brs, H^^), 1.93 (2H, brs, H^-2), 1.30(46H. brs, 23xH^), 0.95 (3H, brs. Me-32), 0.73 (3H, brs, Me-1).

108

in t); 494 [M]^ (3.9), 479 (12.1), 476(21.5), 465 (4.1), 437 (6.2), 423 (8.0), 419 (1,4), 409 (4.6), 395 (1.6),381 (2.7), 367 (2.0), 365 (1.4), 353 (1.4), 351 (1.4), 339 (1.6), 337(1.9), 323 (3.2), 311 (3.4), 309 (2.3), 297 (1.51), 295 (1.9), 283(1.6), 269 (2.5), 267 (2.5), 253 (1.3), 239 (4.9), 227 (3.6), 225 (8.3),211 (3.0). 199 (10.6), 197 (2.8), 183 (7.5), 169 (2.5), 155 (5.8), 141(6 .6 ), 129 (7.9), 127 (4.6), 113 (14.4), 111 (19.3), 99 (9.2), 97 (78.8),85 (45.3), 83 (81.3), 71 (79.2), 69 (76.1), 57 (100), 43 (61.3).

Acetyl alio n of ML-9: Compound ML-9 (25 mg) was acetylated with amixture of acetic anhydride (3 ml) and pyridine (1 .ml) at room temperature overnight. After usual work-up a gummy product was obtained. This was purified by preparative TLC to secure rnonoacetyi product (ML-9a), m.p. 88-89°, IR y ^ 1725, 1710 cm \

109

E3CTRACTI0N AND ISOLATION OF CHEMICAL CONSTITUENTS OF STEM BAF«1 OF MANGIFERA IMDSCA CULTIVAR "SAFEDA’

Piant materia!; Stem bark of M. indica variely'Safeda’ was collected from Bagpat, UP.

Extraction: The dried and powdered stem bark (3.00 kg) was extracted with EtOH (95%) in a Soxhlet apparatus. The extracts were combined and the solvent evaporated under reduced pressure to obtain a dark brown viscous mass (195 g).

isolation o f chemical constituents; The dried aicoholic extract was dissolved in minimum amount of MeOH and adsorbed on silica gel to form a slurry. The slurry was air dried and chromatographed over silica gel column prepared in petroleum ether. The column was eluted with petroleum ether, chloroform and methanol in order of increasing polarity to isolate the foil-owing compounds;

DIMETHYL TEREPHTHALATE {MSA)Elution of the column with petroleum ether furnished colourless amorphous

powder of MS-1, needles from ethyl acetate, m.p. 139-141° (lit m.p. 141-142°), 1.23 g (0.04% yield), R, = 0.21 (petroleum ether), = + ^75 (q o,998,MeOH).

UV X MeOH: 235 nm (log e 6.5).iR (KBr): 2965, 1720, 1440, 1410, 1385, 1285, 1195, 1110,

1015, 955. 875, 815, 715 crr)-\m NMR (90 MHz, CDCS3I: 5 7.23 (2H, s. H-2, H-6), 6.60 (2H, s.

H-3, H-5), 3.50 (6 H, brs, 2 x OCH3).

ElMS m/z (rei. int.): 194 [M]^ (C,^H,ÔJ(64.2), 179 (6.4), 164(25,0), 163 (100), 135 (44.1), 120 (14.5), 119 (9.3). 104 (17.7), 103(26.6), 92 (6.1), 77 (12.4), 76 (22.3), 75 (16.6),’52 (7.1).

110

Hydrolysis of MS-1: Compound MS-1 (20 mg) was refluxed with 10ml of 2N alcoholic NaOH. Usual work-up gave terephthalic acid, soluble inNa^COy solution.

NONANYL PROPANOATE |MS-2|:Further elution of the column with petroleum ether furnished colourless

amorphous powder of MS-2, recrystallized from CHCl,^--f\/leOH (1:1), 2.50 g(0.08% yield), R, = 0.42 (petroleum ether-benzene, 1:1), m.p. 72-73“ [ap^ ~ + 12,02. (C 1.62, MeOH).

UV (MeOH); 230 ■ nm (log e 6.5).IR fKBr): 2921, 2852, 1738, 1712, 1467, 1230, 1170, 723

cm'.’H NMR (90 MHz, CDCy^ 5 4.10 (2H, m, 2.33 (4H. brs,

2 X COCHj), 2.26 (2H, brs, COCH^), 1.96 (1H, m, H-15), 1.60 (6H, brs, 3 X CHj), 1.23 (22H, brs, 11 x CH^), 0.90 (3H, brs, Me-3'), 0.86 (3H. brs,Me-20), 0.82 (3H, brs, Me-1).

BMS m /i (re!, in i) : 368 [M]" (23.8), 353 (7.3), 339 (36.9),325 (6.3), 311 (6 .8 ), 297 (10.2), 283 (8.2), 255 (48.6), 241 (11.8), 239 (3.0), 227(13.1), 213 (26.5), 199 (12.1), 185 (26.0), 183 (4.5), 171 (21.2), 157 (18.1), 141(6 .8 ), 129 (54.4), 127 (8.9), 115 (23.8), 113 (11.5), 101 (17.5), 99 (25,8), 87(29.3), 85 (40.9), 73 (95.2), 71 (60), 57 (100).

Alkaline hydrolysis o f MS-2: Compound MS-2 (10 mg) was heatedwith IN alcoholic KOH solution (5 ml) for 2 hours. Excess of HÔ was added to the reaction, mixture, acidified with HC! to Congo red and extracted withCHCI3 (3 X 10 ml), dried over Na^SO^ and evaporated to secure the alcoholicderivative (MS-1 a), m.p. 77-78°, IR (KBr): 3450, 1710 cm''.

SAFEDASTEROL (MS-3)Elution of the column with petroleum ether-CHCIg (9:1) yielded a

colourless product MS-3 recrystallized from EtOAc to secure colourless crystals, 3.3 g (0.11% yield), R, = 0.18 (petroleum ether-CHCl3 ,4 : 1 ), m.p. 121-122°,[a] 25 = +22.3°, (C 0.754, MeOH).

111

|MeOH|: 240 nm (log e 6.2).IR fKBr): 3450, 2925, 2850, 1595, 1560, 1440, 1410, 1395.

1200, 1150, 1095, 795, 720 cm ’ .m NMR fiOO MHz CDCI3): 6 3.20 (1H, br, m, w 1/2 == 12.0 Hz,

H-3B),2.50 (1H, m, CH), 2.21 (2H, m, CH ), 2,00 (2H. m, Cl-g, 1,93 (4H,rn, CHj, 2 x CH), 1.70 (6H. m. 2 x CH , 2 x CH), 1,30 (3H, m, CH^CH),1.25 (2H, m, CH^). 1,16 (10H, m, 5 x CH^), 1.06 (3H, brs, Me-19), 0.96(3H, d, J = 6.0 Hz, Me-21), 0.90 (3H, brs, Me-29), 0.86 (3H, d, J = 6.5Hz, Me-27), 0.80 (3H, d, J = 6.5 Hz, Me-26), 0.76 (3H, brs, Me-18).

(C,,H ,„0) (7.8), 399 (4.2), 396(6.6), 381 (3.9), 329 (5.1), 302 (3.8), 273 (4.1), 271 (1.9), 257 (2.2),255 (4.1). 239 (2.3), 216 (2.3), 212 (6.9), 205 (2,1), 203 (10,1), 202(8.6), 201 (20.2), 192 (2.1), 191 (3,2), 188 (3.2), 187 (4.1), 178 (1.9),177 (2.6), 174 (3.6), 173 (4.1), 164 (3.1), 161 (5.0), 160 (3.0), 159(6.1). 147 (8.3), 146 (5.7), 133 (7,6), 126 (2.0), 122 (6,5), 112 (2,1),109 (6.5), 108 (10,8), 96 (3.6), 95 (12.1), 94 (9.7), 91 (12,7), 85 (4,1),82 (2.8), 81 (10.1), 72 (3.8), 71 (7.0), 69 (13.7), 68 (2.1), 67 (10.6), 61(100), 57 (14.7), 55 (20.5), 54 (2.1).

s~3; A mixture of AcÔ (2 ml) and dry pyridine (1 ml) was reacted with compound MS-3 (10 mg) for 24 hours at room temperature. The reaction was quinched by addition of cold water (10 ml) and the mixture extracted with C H C I 3 (3 x 5 ml). The organic phase was washed with HÔ ( 2 x 5 ml), dried over Na^SO^ and evaporated to get monoacety! product (MS-3a). IR (KBr): 1725, 1605 cm ’ .

O xidation o f MS-3: A mixture of Jones reagent was treated withcompound MS-3 in acetone for 2 hours at 4” C . Water (10 ml) was added. The reaction mixture extracted with C H C I 3 (3 x5 ml). The organic phase was washed with water, dried over Na^SO^ and evaporated to get 3-oxo. derivative (MS-3b). it gave positive Zimmermann test for 3-oxo steroids.

112

IViAWGTERPENOiC ACID |MS^):Elution of the column with petroleum ether-CHCI^ (4;1) afforded

colourless crystals of MS-4, recrystallized from MeOH, 42 g ( 1 ,4% yield), R, = 0.33 (CgHg:CHCl3, 4:1), m.p. 140-141°, =0“. (C 1.031, MeOH).

UV fMeOH): 222, 277 nm (log e 6.8, 6,1 ).IR (KBr|; 3457, 3307, 2977, 2835, 1705, 1690, 1619, 1534,

1469, 1410, 1384, 1315, 1255, 1197, 1039, 966, 866, 816, 762 cmNMR (60 MHz, DWlSO-cJ I: 5 7.13 (1H, s, H-20), 4.33 (1H, d,

J = 6.5 Hz, H-lOa), 4.20 (1H. d, J = 6.5 Hz, H-iOb), 3.70 (1H, brs,exchangeable. OH), 2.85 (1H, d. J = 6,5 Hz, H-8), 2.63 (1H, m, w 1/2 - 6.5Hz, H-6a), 1.50 (2H, brs. H2-4), 1.40 (3H. brs, fVle-7), 1.23 (2H, brs,H ~5).

ElMS OT/z (rei. in l | : 198 [Ml" (49,5), 183 (9.8), 170(22.8), 163 (5.4), 154 (18,1). 152 (100), 136 (3,5), 126 (12.1), 124(21.8), 107 (5,3), 93 (2.3), 79 (9,1), 69 (4,8), 58 (12,4), 45 (6.2), 44 (3,2),

Acetyiatiori o f MS-4: Compound MS-4 (10 mg) was acetylated withACjO (3 ml) and pyridine (1 ml) at room temperature. The excess of AcÔwas decomposed by addition of HÔ (10 ml) and the reaction mixture extractedwith C H C I 3 ( 3 x 5 ml). The organic phase was washed with HÔ (2 x 10 ml),dried over Na^SO^ and evaporated to secure monoacetyl product (MS-4a).IR fKBr): 3300, 1725, 1705 cm'’ ,

M etliylation o f MS-4: Compound MS-4 (10 mg) was dissolved insolvent ether (5 ml), ethereal solution of diazomethane added with stirring andleft for 24 hours. Evaporation of the reaction mixture yielded monomethyl ester(MS-4b), IR (KBr); 3455, 1735, 1705 cm \

iSOfVlANGTERPENOlC ACID (MS-5)Elution of the column with petroleum ether-CHCl3 (4:1) furnished

colourless crystalline product MS-5, purified by preparative TLC (CHCI^-MeOH,4:1), 12.2 g (0.40% yield, R, = 0.58 (CgHg-CHCl3, 3:1), m.p, 134-135°,[a]25 = +1 2 .6 , (C 1,003, MeOH).

113

fMeOH): 223, 279 nm (log e 6.7, 6.0),IR (KBr): 3460, 3290, 2980, 1710, 1690, 1617, 1535. 1469,

1384, 1315, 1251, 1196, 1036, 965, 867, 814, 762 cm'.|: 6 7,0 (1H, brs, H-2), 4.36 (1H, d.1S

6

J - 9.0 Hz, H-lOa), 4.20 (1H, d, J = 9.0 Hz, H-lOb), 3.33 (1H, brs, DÔexchangeable, OH), 2.86 (1H, d, J =: 6.0 Hz, H-8), 2,67 (IN, brs, m, w 1/2 = 13.0 Hz, H-6fi), 1.53 (2H, br, HÔ), 1.46 (3H, brs, Me-7), 1.23 (2H.brs, H^-5).

m t}: 198 [M]^' (46.1), 183 (9.6), 170(20.9). 163 (14.2), 154 (14.4), 152 (90.3), 147 (7.1), 138 (6.4), 136(5.2), 126 (9.9), 124 (20.3), 111 (13.3), 107 (6.3), 93 (2.1), 89 (5.7), 79(8.4), 69 (10.3), 65 (3.1), 58 (100).

of MS"5" Compound MS-5 (10 mg) was acetylated with AcÔ (3 ml) and pyridine (1 ml) for 24 hours at room temperature. After usual work-up monoacetyl product (MS-5a), was obtained, IR 3310, 1725, 1715 cm-^

i-5: Compound MS-5 (15 mg) was treated withethereal solution of CH^N^ (10 ml) overnight at room temperature. The solvent was evaporated to get monomethyl ester MS-5b, different spots on TLC on comparison with the original compound (MS-5), IR y ; 3455, 1725, 1715 cm ’ .

Elution of the column with CHCl^ gave colourless crystals of MS- 6, recrystallized from MeOH, 3.8 g (0.42% yield), R, = 0.11 (CHCy, m.p. 60-61°,

= - 18.5°. (C 0.972, MeOH).

(MeOH): 214, 237 nm (log Z 6,5, 6.9).IR (KBr): 3440, 2920, 2850, 1735, 1715, 1625, 1465, 1260,

1170, 1025, 820, 800, 720 cm'\NMR (90 MHz, CDCI3): 5 6.40 (1H, m, H-18), 5.30 (1H, d,

J = 5.0 Hz, H-19), 4.10 (2H, brs, H^-21), 3.90 (1H, brs, H-10), 3.60 (1H,

114

m, H-8), 2,30 (3H, m, H 9, H^-17), 2.00 (2H, d, J = 6.00 Hz, H,-12), 1.60(2H, brs, CH^), 1.20 (18H. brs, 9 x CHJ, 0.86 (3H, brs, Me-I).

int.); 368 [M]* (C^,H3^,0,; (11,1), 339 (9.7), 214 (4.5).195 (5,6), 177 (17.4), 173 (4.2), 167 (6.3), 165 (5.1), 143 (4.3), 142(4.2), 140 (6.3), 129 (11.8), 128 (4.4), 125 (8.7), 124 (4.3), 111(18.7), 110 (5.9), 99 (9.7), 98 (8.0), 97 (37.2), 85 (31,9), 83 (45.5), 71(50,3), 70 (21.9), 69 (54,5), 57 (100), 55 (84,7),

To a solution of MS-6 (10 mg) in dry pyridine(1 ml), AcÔ (2 rnl)was added and worked up as usual to get diacetyl product(MS-6a), IR Y„ : 1735, 1725. 1720, 1710 cm V

Elution of the column with MeOH gave colourless amorphous powder of MS-7, recrystallized from MeOH. 4.05 g (0.13% yield), R, = 0.61 (MeOH- Me^CO, 1:1), m.p, 280-282° (lit. m,p. 283-286°), [a]/® = +40.3 (C 0.45,CgH^N).

UV (MeOH): 221 nm (log £ 7.1). ■ •IR (KBr): 3450, 3400, 3300, 2920, 2850, 1600, 1430, 1355,

1240, 1110 cm-\): 5 5.50 (1H, d, J = 6.0 Hz, H-6), 4.731 !

(1H, brs, H-1'), 4.50 (1H, d, J = 6.5 Hz, H-5'), 4.20 (1H, brs, H-6'a), 4.17(1H, brs, H-6'b), 4.00 (1H, brs, H-2'), 3.86 (2H, brs, H-3' , H-4'), 3.63 (1H,brs, H-3), 1.10 (3H, brs, Me-19), 1,00 (3H, d, J = 6.0 Hz, Me-21). 0.97(6H, brs, Me-26, Me-27). 0.83 (3H, brs. Me-29), 0.76 (3H, brs, Me-18).

EIMS m /i (rel. int.): 576 [M]" (C3gHgÔg)(N.O.), 413 (38.3), 398 (6.1),396 (22.7), 381 (11.3), 329.(11.3), 315 (3.0), 272 (10.5), 255 (12.1). 341(3,4), 255 (23.5), 213 (15.5), 198 (7,8), 183 (11.5), 180 (4.5), 178(10.6), 163 (40.0), 159 (20.9), 153 (75.2), 145 (21.2), 141 (3.2), 105(29.3), 69 (53.4), 57 (100).

115

Acid hydrolysis of lViS-7: Compound MS-7 (50 mg) was refluxed with2M HCl-EtOH ( 1 : 1 ) ( 1 0 ml) on a steam bath for 4 hours. The reactionmixture was poured into crushed ice and the colourless crystals were filtered off, rn.p. and m.m.p. 139-140'’, TLC was compared with an authentic sample of fi- sitosterol. The filtrate was neutralized with Ag^CO, and filtered. The solution was evaporated to dryness under reduced pressure. It showed the presence of D- glucose (R 0.42) on comparison with an authentic sample on silica gel TLC developing in EtOAc-HÔ-MeOH-AcOH (6:1; 1:2).

Permethylalion and methanoiysis o f MS-7: To a solution of MS-7(10 mg) in DMSO (1 ml), powdered and dried NaOH- (25 mg) and Mel (0.5 ml) were added. The reaction mixture was shaken at room temperature for 2 hours and CHCl^ (1 ml) and water (1 ml) were added to the reaction mixture. The organic layer was washed thrice with water (5 ml), dried over Na^SO^, and evaporated to dryness. The residue was dissolved in 2N HCI in MeOH and refluxed for 2 hours. Excess of cold water (50 ml) was added and reaction mixture extracted with CHCL (3 x 10 ml). The CHCI-layer was washed with3 'jwater, dried over Na^SO^ and evaporated to get permethylated glucose, m.p., m.m.p. and TLC were compared with the authentic sample.

11 6

HYDRODISTILLATIOM OF FRUITS OF M. INDICA

Plant material: All the fruits were purchased from the local market ofDelhi.

ISOLATION OF VOLATILE OIL OF MAMGO CULTiVAR ‘BANARSI GOLA’The rind and pulp of M. indica fruit cultivar 'Banarsi Gola’ (855 g) was

added in a 2-litre flask and covered with water. An all glass Clevenger apparatus was attached and the contents were heated slowly. The collected oil was dried over anhydrous sodium sulphate and stored at 4°C in the dark. The yield was 0.25 ml (0.029% w/v) based on the fresh plant material.

ISOLATION OF VOl-ATiLE OIL OF MANGO CULTIVAR ^BOMBArThe fresh fruit pulp and rind (1.350 kg) were added to a 3-litre round

bottomed flask and water (1 litre) was added. A Clevenger apparatus was fixed and the contents were heated slowly. The volatile oil was collected time to time till it ceased to distil. The colourless distilate was dried over anhydrous sodium sulphate and stored at 4°C in dark. The yield was 0.44 ml (0.032% w/v).

ISOLATION OF VOLATILE OIL OF MANGO CULTIVAR *DESi’The fruit pulp and rind (780 g) were separated, water (500 ml) added

and the essential oils were obtained by steam distillation in a modified Clevenger apparatus in 6 hours. The oil was dried over anhydrous sodium sulphate and stored at 4°C. The yield was 0,23 ml (0.029% w/v),

ISOLATION OF VOLATILE OIL OF MANGO CULTIVAR ‘QALMFThe rind and pulp of M. indica fruits cultivar 'Qalmi’ (1,35 kg) was added

in a 2-litre flask and covered with water. An all glass Clevenger apparatus was attached and the contents were heated slowly. The collected oil was dried over anhydrous sodium sulphate and stored at 4 “C in the dark. The yield was 0,15 ml (0 .0 1 1 % w/v) based on the fresh plant material.

1 1 7

ISOLATION OF VOLATILE OIL OF MANGO CULTIVAR ‘LANGRA’The fruit pulp and rind (2.225 kg) were inserted in a 5-litre round bottom

flask, dipped with water and Clevenger apparatus was fixed. The contents were heated slowly and the volatile oil was collected from time to time when the oil content ceased to distil. The colourlessessential oil was dried over anhydrous sodium sulphate and stored at 4°C in the dark. The yield was 0.55 ml (0.023% w/v) based on the weight of fresh fruits.

ISOLATION OF VOLATILE OIL OF MANGO CULTIVAR ‘SAFEDA*

The fruit pulp and rind (860 g) were separated, water (500 ml) addedand hydrodistilled in a Clevenger apparatus for 6 hours. The oil was removed, dried over anhydrous sodium sulphate and stored at 4°C. The yield was 0,25 ml (0.029% w/v).

ISOLATION OF VOLATILE OIL OF MANGO CULTIVAR ‘TOTAPARrThe fresh fruit pulp and rind (1.150 kg) were separated, homogenized

with a blender and diluted with water. The mixture was hydrodistilled in a modified Clevenger apparatus. The volatile oil was collected every four hours over pentane sulphate. The yield was 0.35 ml (0.030% w/v).

118

DISCUSSION

STRUCTURAL ASSLJsUMEMT OF THE C H E M IC A L CONSTITUEISITS ISOLATED FROM THE STEWi BARK OF M. INDICA CULTiVAF^ TAZALl*

COMPOUND MF»1

Compound MF-1, designated as manglanostenoic acid A, was obtained from petroleum ether eluants of the column. It responded positive ly to Liebermann-Burchard, tetranitrortiethane, and Zimmermann tests and showed effervescences in sodium bicarbonate solution. It had a molecular ion peak at m /i 452 in its El mass spectrum relating to the molecular formula whichshowed nine degrees of unsaturation. The ion fragments in the mass spectrum at rn/z 437 [M-Me]\ 393 [M-!Vle-COJ\ 378 [393-!VIe]\ 141 sidechain, SC]^, 311 [M-SC]+ and 296 [311-Me]^ suggested the tanostenyi-typetriterpene with a Cg unsaturated side chain containing a carboxylic group. The ions at nVz 83 [ion f]+, 69 [ion f-CHJ"' and 55 [69-CHJ" reflected the existence of ‘2®’ unsaturation and C-21 carboxyl ic group. The peaks at m/z 70 [ion a] \ 54 [ion b]", 96 [ion c]', 136 [ion d]+, 302 [ion g]", 287 [ion g-Mej", 150 [ionh]^, and 97 [ion k]" supported the 3-oxo group and A’ olefinic linkage in ring A and another unsaturated bond at A^-position. The saturated nature of rings C and D was inferred from the ion peaks appearing at m/z 216 [ion i] " , 202 [ion i-CHJ\ 188 [202-CHJ", 243 [ion j]", 229 [ion j-CHJ" and 215 [229-CHJ"(Scheme MF-1).

The ■'H NMR spectrum of MF-1 displayed two one-proton each doublets at 5 6.06 and 5.90 with coupling interactions of 5.5 Hz assignable to H-1 and H-2, respectively. A two-proton multiplet at 5 5.20 was accounted to H-7 and H-24. Two downfield three-proton each singlets at 6 1.87 and 1.70 were associated with the C-26 and C-27 methyls located at C-25 olefinic carbon. Five tertiary methyl groups resonated as broad singlets at 5 1.20, 1.00, 0.83, 0.76and 0.70 ascribed correspondingly to C-29, C-19, C-28, C-30 and C-18 methyls, its NMR spectrum was compared with that of other Ianostone-type triterpenes. It showed the presence of one carbonyl group ( 6 212.8) and three-olefinic linkages at A -(5 147.0, 125.8), A’ (5 118.1, 147.0), and A^ - (5 126.3,

119

146,9) positions and a carboxylic group {6173,6),Conclusive evidence for the structure MF-1 was derived from the results

of chemical reactions. Treatment of MF-1 with diazomethane yielded a monomethyl ester (IVIF-la), IR (KBr) 1725 cm '(ester group). Sodiumborohydride reduction of MF-1 formed a 3R-hydroxy derivative (MF-1 b), 3350cm’’(OH. COOH). The 3-carbinol proton appeared as a doublet at 5 3.53 and its coupling interaction of 9.0 Hz indicated a orientation .of the proton. Acetylation of the reduced product (MF-lb) with acetic anhydride and pyridine mixture produced an acetylated product (MF-lc), IR 1720 cm '(COMe). Reduction of lViF-1 with LiAIH^ in dry ether formed 3a, 11-dihydroxy derivative (MF-ld), It showed 3-a carbinol proton at 6 4,05 and two one proton each doublets at 5 3.31 (J-12.31 Hz) and 3.22 {J=11.25 Hz) for C-21 hydroxymethylene group.

On the basis of these informations the structure of rnanglanostenoic acid A (MF-1) has been established as 20(R), 24(R)-3-oxo-9R-lanost-1,7.24-triene-21 - oic acid. This is a new lanostene-type triterpenic acid reported from any

natural or synthetic source for the first time.

COfMPOUND MF-2Compound MF-2, named mangsterolide, was obtained from petroleum

ether eluants. It responded positively to Liebermann-Burchard and Zimmermann tests for 3-0X0 steroids. The IR spectrum of MF-2 exhibited bands for hydroxyl group (3348 cm’'), a-lactone (1720 cm'') and carbonyl group (1700 cm’’ ). Its EIMS showed a molecular ion peak at 468 relating to a steroidal molecular formula, C gH gOg. The fragments appearing in the mass spectrum at m/z 453 [M-Me]\ 437 [M-CHÔH], 422 [437-Me]^ 183 [C,^H,g0 3 , Side Chain. SC] ^111 [CgHÔJ" and 285 p-SC ]^ 270 [285-Me]^ and 255 [270-Me]" indicated the steroidal-type carbon framework containing C^ side chain with one hydroxyl group and one S -lactone ring involving C-27 and C-29 carbons. The ion peaks generating at m/k 83 [ion a]+, 55 [ion a-CO]"-. 41 [55-CHJ\ 69 [ion a-CHJ*, 70[ion b], 138 [ion c]- , 124 [ion c-CH2] \ 62 [ion e] " and 110 [124-CHJ ^supported the saturated nature of rings A and B and the presence of keto groups at C-3. The location of keto group at C-11 was inferred from the fragments formed at 247 [ion d-SC]'^. 219 [ion d-2xCH^-SC] + , 306 [ion f]^,

1 2 0

C ,5 H,oO (ion i) m /z 2 16 (3 .3 )

Scheme MF-I: Mass Fragmentation Pattern of Manglanostanoic Acid A (MF-1

121

123 [ion f~SC]\ 95 [ion f-CO-SC] ' , 81 [ion f-CO-CH^-SC]', 203 [ion g]^ 204 [ionh] ' and 190 [ion h-CHJ' (Scheme MF-II).

The 'H MMR spectrum of MF-2 displayed one-proton each doublets with coupling interaction of 6.5 Hz at 5 4.20 and 4.05 assigned to C-29 oxygen-substituted methylene group of the lactone ring, and at 5 3.70 and 3.60associated with C-21 hydroxymethylene group. Three broad signals at 5 2.40,2.25 and 2.20, integrated for two protons each, were ascribed to C-12, C - 2 and C"4 methylene groups adjacent to the carbonyl groups. A three-proton broad singlet at 5 1.74 was accounted to C-26 methyl group attached to C-25 olefinic carbon. The C-19 and C-18 tertiary methyl groups appeared as three-proton each broad singlets at 5 0.92 and 0.75. Treatment of MF-2 with acetic anhydride and pyridine yielded a moncacetyl product (MF-2a). These evidences led to establish the structure of mangsterolide (MF-2) as stigmast-8(9), 24 (25)-diene-3, 11 -dione- 21-01-27, 29-olide, This constitutes the first Isolalion o f a steroidallactone from the Mangifera species.

COMPOUND MF-3Compound MF-3, named manglanstenoic acid B, was secured as a

white amorphous mass from petroleum ether;benzene (1:1) eluants. It gave positive Liebermann-Burchard and Zimmermann tests and effervescences in sodium bicarbonate solution. Its mass spectrum showed a molecular ion peak at m/z 454 corresponding to a triterpenic molecular formula, C3pH g0 3 . It indicated eight degrees of double bond equivalents. The ion peaks at m/z 439 [M-Me] \ 424 [M-2Mer, 147 [CHgH,3 0 3 ]^ 297 [439-SC]^ 256 [297-ring D fission]^ and 410 [M-COJ+ showed that the compound MF-3 was lanostene-type triterpene possessing a C- 8 unsaturated side chain with a cartXDxylic group. The ion peaks at m/z 70 [ion a]% 54 [ion b]^ ^ [ion c]"-, 136 [ion d]^ 164 [ion e]+ and 190 [ion f]+ suggested the presence of oxo-group in ring A which was placed at C-3 on the basis of biogenetic grounds and saturated nature of ring A (Scheme MF-II!).

The ’H NMR spectrum of MF-3 exhibited three, one-proton each doublets at 6 6.15 (J = 6.14 Hz), 6.08 (J = 6.00 Hz) and 5,30 (J ::: 4.0 Hz) assigned

1 2 2

, CH„

CH„0 ■CH.,

0C, HÔ (ion a) m /z83(38 .2 )

C „H „0 (io n b ) m /z 70(11.2)

O

C g H „ 0 (ion c) m/z 138 (11.0)

C,gH,s 0 ,

O C!-!.,

(ion d) [M]* m/z468 (5.6)

\ | /

C22H.0 O4 cl) m /z358(N .O )

C ,3 H „ 0 , (ion h) m/z 204 (1.5)

O '

C „H ,o O ( io n g) m /z203(1.5)

C „H ,^ 0 (ion e) m /2 162 (2 .8 )

0

0 ,3

0 , (ion f) m/z 306(1.1)

Scheme: MF-II: Mass Fragmentation Pattern of Mangsterolide (MF- I123

0

C,, Hg O (ion a) rn/2r70(32.5)

CH

CH

0C ^HÔ (ion b) m/Z54 (24.2)

Cg HgO (ion c)

C 3, H , , 0 3 ; [M r m/z 454 (21.6)

\

- Me

m/z439(100)

-Me-Sc m /z 424 (6 .2 )

-C O .

-C C l\K

m/z297(6.5)

m /z410(14.6) C 3„ H ,, 0 ,;(SC)

m/z 141 (5.6)

m/z-r in g

(ion d) m /z 136 (18.4)

C

H,g O (ion e)

m /z-164 (6.9)

0

C ,3 H ,, 0 (ion f) m/z 190 (9,8)

Scheme: MF-III: Mass Fragmentation Pattern of Manglanostcnoic Acid B (IVIF

124

correspondingly to H-1, H-2 and H-22. A Ihree-proton singlet at 6 1.91 wasattributed to C-21 methyl group attached to olefinic C--20 carbon. A doublet at 5 0,95, integrated for three protons, was associated to C-27 secondary methyl group. The remaining tertiary methyls appeared at 5 1.05 (Me--28, Me-29), 1.00(Me-30) and 0.75 (Me-18). The presence of methyl groups, attached tosaturated carbons, in the range 5 1.09-0.75 supported the lanostene-type skeleton. Comparison of NMR spectra! data with other lanostene-type triterpenes supported the location of one of the olefinic linkage at {5 135.0, C-20;132.9, C-22) in the side chain. Treatment of MF-3 with diazomethane yielded a rnonomethy! ester MF-3a, thus confirming the presence of the carboxylic acid in the molecule. On the basis of these spectral data analyses and chemical reactions the structure of manglanostenoic acid B (MF-3) has been established as 3~oxo-9fi-lanost-1, 20 (22)-diene-26oic acid. This lanostenolc acid is a new triterpenic acid and is being reported fo r the farst time from a natural or synthetic source.

COMPOUND MF~4Compound MF-4, namely manglanostenoic acid C, was obtained as

colourless amorphous mass from benzene eluants. It gave positive Liebermann- Burchard and Zirnmermann tests and effervescences with sodium bicarbonate solution. Its mass spectrum showed a molecular ion peak at m/z 454 relating to a triterpenic formula, C ,„K „0 ,. The mass fragmentation pattern was identicaloO v) iC

to that of MF-3 (Scheme MF-Ill) suggesting similar carbon skeleton of the compound. The NMR spectrum of MF-4 displayed one-proton each three vinylic signals at 6 6.20 (d, J = 6.0Hz), 6.06 (d, J = 6.0 Hz), and 5,36 (m,H“24) assigned to H-1, H-2 and H-24, respectively. A three-proton broad singlet at 5 1.96 was attributed to C-27 methyl group attached to C-25 vinylic carbon. The C-21 secondary methyl group appeared as a three-proton doublet at 5 1.10 (J = 6.0 Hz). The remaining tertiary methyl protons resonated at 5 1.20 (Me- 29), 1.16 (Me-19, Me-28), 0.96 (Me-30) and 0.86 (Me-18). The existence ofall methyl groups In the range of 5 1.20-0.86, except the vinylic C-27 methyl group, supported lanostane-type carbon skeleton of the molecule. Methylation of MF-4 with diazomethane yielded a monomethyl ester (MF-4a), thus confirming

125

the presence of carboxylic group in the molecule. On the basis of these informations the structure of manglanostenoic acid C (MF-3) has been elucidated as 20 (R), 24 (R)-3"OxO"lanost-1, 24-diene-26-oic acid. This is also a new laoostenoic acid isolated for the firs t time from the Mangsfera indica.

C0fflP0UNI3 MF=5Compound fVIF-5, named manglanostenoic acid D, was obtained as

colourless crystals from benzene fractions, it responded positively to Liebermann- Burchard test for triterpenes, and Zimmermann test for 3-oxo compounds and produced effervescences with sodium bicarbonate solution, It showed a molecular ion peak at m/z 450 corresponding to the triterpenic formula, Itindicated ten degrees of unsaturation. Its IR spectrum showed the characteristic absorption bands for carboxylic group (3350. 1680 cm ’), carbonyl group (1710 cm''') and unsaturation (1635 cm ’ ). Its mass spectrum displayed ion fragments of diagnostic importance at m/z 73 [ion a]"-, 141 [side chain, ion b]^ 435 [M-Me]+, 406 [M~COJ\ 391 [435-COJ", 418 [!Vl-2xCHJ", 309 [M-SC, ion c]',293 [309-Me]+, 267 [309-ring D fission]-" and 252 [267-Me]'". suggesting thepresence of an unsaturated side chain with carboxylic acid and the saturated nature of ring D. The ion fragments at m/z 214 [ion e]", 200 [ion186 [200-CHJ^ 95 [ion f]^ 82 [ion g]", 6 8 [ion g-CHJ^ 241 [ion h]*-, 227[ion h-CHJ", 123 [ion i]^ 108 [ion i-Me]^ 94 [108-CHJ" and 80 [94-CHJ" were indicative of the saturated nature of ring C. The location of a tetrasubstituted olefinic linkage at C- 8 (9) and a trisubstituted unsaturated bond at C-5 was deduced from the ion peaks appearing at m/k 162 [ion j]+, 148 [ion 147[ion kj]^ and 132 [ion k-Me]". The ion peaks at m/z 96 [ion 1] , 213 [ion m]",54 [ion n ]^ 255 [ion o ]', 109 [ion p]", 81 [ion p-CO]^ and 200 [ion q]" supported the existence of a di-substituted olefinic linkage at C-1 and 3-oxo group (Scheme MF-IV).

The ’H NMR spectrum of MF-5 displayed the presence of four vinylic proton signals as one-proton each doublet at 5 6.13 and 6.03, vîth coupling interaction of 5.0 Hz each, assignable to H-2 and H-1, respectively,and one two- proton broad singlet at 5 5.37 associated with H- 6 and H-22. A three- proton singlet at 81.87 was attributed to C-21 methyl group attached to C-20

1 26

m/z 435 (26 ,8) — ^

COOH

391 (6.0)

-> CHjCHCHOOH C ,,H ,0 , (ion a) m/z 73 (15.0)

H3C COOH

c H .g O j (ionb) m /zU ^ (4.3)

(M)+ m/z 450 (8 .6 )

- CH, m/z + CH,^--------- no C~

m/z94 (22,5)

-CH.

109 V (13.3)

-C H

C ,H,^0(ionc) rn/zZm (4.1)

m/z 80 (20.9) C3 H ,^ 0 (ion i) m/^ 123 (14.Q)

\k

C ,,,H , ,0 ( io n h ) mA 241 (7.0)

-O H ,

m/z 227 (3 ,7 )

C,3H2,0(iond) m/^ 268 (52.6)

C,aH„0(ione) m ^ 2 1 4 (3.4)

C , H „ 0 (ion f) m/i: 95 (11.6)

Contd.

127

CH,O

C ,,H ,,0 ( io n i) m/7. 162 (3,8)

* CH..

m/z 148 (12.7)

C ,,H , ,0 ( io n k ) m/z 147 (26,3)

-C H „

m/z 132 (2 2 .1 )

O \

C,AO(ionl) m/z 96 (10.8) C ,3 H^, (ionrn)

m/z 213 (4.5)

OC3H2 0 (ion n) m/z 54 (47.7) C,gH,„(iono)

m /i 255.(11.8)

0C ^H gO (ion p) m/z 109 (13.3)

CO -^m /z 81 C isH jo (ionq) m/ 2 2 0 0 (8 .1 )

Schemc MS-V: Mass Fragmentation Pattern of Manglanostenoic Acid D (iVIF-

128

unsatLirated carbon. A three-proton'double!: al 5 0,93 (J = 6.0 Hz) was asaibed to C-26 secondary methyl group. The five tertiary methyl groups appeared as broad signals at 5 1.09 (Me-28), 1.03 (Me-29), 1.00 (Me-19, Me~30), and 0.80 (Me-18). An euphane or tirucallane carbon framework for MF-5 was ruled out on the basis of the 'H NMR, where except for a vinylic methyl group at C-25, no methyls appeared more downfield than 5 0.97 nor upfield of 0,70 (Itoh et al., 1976; Chiang and Chang, 1973). Its NMR spectrum demonstrated the presence of thirty carbon atoms along with one carbonyl signal at 5 206.7 (C-3) and one carboxylic group at 5 172.0 (C-27). The assignment of carbonchemical shifts were made by comparing their values with similar compounds (Monaco et al, 1973). The compound MF-5 y ie ld e d a monomethyl ester (MF-5a) on treatment with diazomethane. On the basis of these spectral data and chemical evidences the structure of manglanostenoic acid D (MF-5) has been established as 3-oxo-lanosta-1, 5, 8 (9), 20 (22)-tetra-ene-27-oic acid. This is also a new laoostenoic acid.

COMPOUND MF-6Compound MF-6, designated as manglanostenoic acid E, was obtained

as colourless crystalline mass from benzene fractions. The compound responded positively to Liebermann-Burchard and Zimmermann tests and showed effervescences with NaHCOg solution, it had a molecular ion peak at m/z 456 in its mass spectrum corresponding to Cg^H^gO . It showed eight degrees of undsaturation. Its IR spectrum demonstrated the presence of carboxylic acid (3150, 1670 cm-^), a,fi-unsaturated ketone (1700 cm’ ) and olefinic bond (1630 cm-''). The mass spectrum showed important ion fragments at m/z 439 [M-Me]+, 410 [IVI-CO l , 313 [M-side chain]^ and 298 [313-Me]"^ supporting the presence of carboxylic functional group and a C-9 side chain. The ions at m/z 73 [ion i]^ and 101 [ion h]^ indicated the presence of the carboxylic group at C-26/C-27 and one of the olefinic linkage in the side chain at C-20 (22). The ions at m/z 54 [ion a]^ 96 [ion b ]^ 136 [ion c]", 188 [ion d]" and 81 [ionj]" further indicated the existence of carbonyl group at C-3 and olefinic linkages at 0-1 (2) and C- 6 (7). The saturated nature of rings C/D and 13, 14-secowere deduced from the ion peaks appearing at m/z 244 [ion e]', 286 [ion f]",272 [ion f-CFIJ* and 313 [ion g]" (Scheme MF-V).

129

0

CH

C3 H j 0 (ion a) m.'i 54 (19.4)

OCj. O (ion b) m/z 96 (9,3)

OC ,,H ,jO ( io n c ) m/z 136 (9.8)

/ N 8

21 9 I j COOH

-V

/>

C,3H,gO(iond) m/z 188 (7.5)

m/z 272 (2.1)

0c , H ^ O (io n j) m/z 81 (6 .6 )

C ,H ,0 ^ ( io n h ) m/z 101 (6.3)

Scheme MF-VI: Mass Fragmentation Pattern of Manglanasteroic Acid E (MF-

130

The 'HNMR spectrum of MF- 6 displayed two one-proton each downfield doublets at 5 6,5 (J == 6.5), 6.00 (J - 6.5 Hz), assigned to H-1 arid H-2 anda two-proton broad signal at 5 5.30 (brs) attributed to H-7 and H-22, A three- proton broad singlet at 5 1.90 was ascribed to C-21 methy! group attached to the C-20 unsaturated carbon. Another four sharp singlets at 6 1.07, 0.97 and 0.93 integrated for three protons each, were accounted to C-19, C-28 and C- 29 tertiary methyls, respectively. A six-proton doublet at 5 1.03 with coupling interaction of 6.0 Hz was due to C--26 and C-30 secondary methyl groups. The C-18 secondary methyl group resonated as a doublet at 6 0.88 (J = 6.0 Hz). The remaining protons appeared in between 6 2.46-1.43. The NMR spectrum of MF- 6 exhibited 30 carbon signals. The signals a t '5 149.1, 135.0, 125.8,116.7, 136.5 and 118.6 were due to olefinic carbons. Treatment of MF - 6 with diazomethane yielded a monomethyl ester (MF-6 a). Sodium borohydride reduction of MF-6 gave a 3fi-hydroxy derivative MF~6 b. It showed typical IR absorption for hydroxyl group at 3500 cm ' and the 3a-methine proton at 5 3.26 (m). Its EIMS displayed a molecular ion peak at /v/z 456 relating to Fromthese data the structure of manglanostenoic acid E (MF-6 ) has been elucidated as 3-oxo-lanosta-1, 6, 20 (22)-triene-13, 14-seco-27-oic acid. This is also anew Irlterpenic acid o f ianostene-type.

COMPOUND MF-7Compound MF-7, designated as manglanostenoic acid F, was obtained

from mother liquor of MF-6 . It showed Liebermann-Burchard test for triterpenes, Zimmermann test for 3-oxo compounds and sodium bicarbonate test for carboxylic acid. Its IR spectrum exhibited absorption bands for carboxyl ic group (3405, 1690 cm ’ ), carbonyl group (1710 cm'^) and unsaturation (1625 crrr^). The El mass spectrum of MF-7 displayed a molecular ion peak at m/z 452 corresponding to the triterpenic formula, C3qH^ ^ 0 3 along with the important ion peaks at m/z 437 [M-Me]", 393 [437-COJ", 378 [393-Me]^ and 311 [M-C^H.Ô^ side chain] \ The ion fragments at m/z 54 [ion a]^ 96 [ion b ] \ 162 [Ion c ]^148 [162-CHJ\ 109 [ion g l^ 81 [10&-C0]^ 67 [81-CHJ^ and 95 [109-CHJ'suggested the location of olefin ic txDnds at C-1 and C-5 and the carbonyl group in ring A which was placed at C-3 on the basis of biogenetic considerations. An ion at m/z 85 [ion f]+, generated due to cleavage of linkage, supported

’ 131

Schemc : Mass Fragmentation Pattern of Manglanostenoic Acid F (MF-

132

the presence of the carboxylic group at C-27. The saturated nature of rings Cand D was deduced from the ion peaks appearing at m/z 216 [ion d] ' , 202 [iond-CHJ", 188 [202-CHJ^ 270 [Ion e ]\ 243 [ion h]', 229 [ion h^CHJ\ 215[229-CHJ-", 237 [ion i]^ and 223 [Ion i-CHJ" (Sciieme MF^VI).

The ’H NMR spectrum of MF-7 showed two one-proton each downfield doublets at 5 7.06 (J = 9.52 Hz) and 6.90 (J = 9,52 Hz) assigned to H-1 and H-2, respectively. A two-protons broad singlet at 5 5.32 was ascribed to the oleflnic H-5 and H-24. A three-proton singlet at 5 1.84 was accounted to C-26 methyl group attached to the vinylic carbon. The C-21 methyl group appeared as a doublet at 5 0.92 (J = 8.3 Hz). The four tertiary methyl groups resonated as broad singlets at 5 1.11 (Me-28), 1.05 (Me-19), 0.89 (Me-29) and0.81 (Me-18). The signals in between 5 2.79-1.24 were associated to theremaining methylene and methine groups. An euphane or tirucallane carbon framework for MF-7 was ruled out on the basis of *H HMR spectrum, where except for a vinylic methyl group at C-25, no methyls appeared more downfield than 5 0.97 nor upfield of 6 0.70 (Itoh et. a/., 1976; Chiang and Chang,1973). The geometrical relationship between the C-24/C-25 double bond and the carboxyl ic acid was established from the chemical shift of H-24 (55.32) and itscomparison with that of methyl angelate (55.97, COOH and H-24 trans) and methyl triglate (5 6.72, COOH and H-24 cis) (Jackman et a i, 1960). Conclusive evidence for the structure MF-7 was derived from results of chemical reactions. Treatment of MF-7 with diazomethane yielded a monomethyl ester (MF-7a). Sodium borohydride reduction of MF-7 produced a 3R»-hydroxyl derivative (MF-7a). On the basis of these observations the structure of manglanostenoic acid F (MF-7) has been formulated as 20 (R), 24 (R)-3-oxo-9ft-lanosta-1, 5, 24-triene-27-oic acid. MF-7 is a new member o f lanostane- type triterpene and firs t such member from M angifera indica.

COMPOUND MF-8Compound MF-8 , named manglanostenoic acid G, was was obtained as

colourless amorphous powder from benzene-chloroform (3:1) eluants. It had the composition of C3 2H^ 2 0 4 (M* 490) established on the basis of electron impact mass spectrum and proton count of the ^HNMR spectrum. It responded positively

133

to the Liebermann-Burchard and Zimrnermann tests ind icative of a 3"Oxo4riterpenoid skeleton. Its IR spectrum exhibited carbonyl (1710, 1690 cm-1) and unsaturation (1625 cm“ ) absorption bands. Its ’H NMR spectrum isconsistent with the proposed structure and clearly showed olefinic protons as one- proton each doublets at 5 6.23 (J = 6.0 Hz, H-2), 6.00 (J = 6.0 Hz, H-1)and as a two-proton multiplet at 5 5.36 (H-6 , H-24). Three broad singlets at 5 1.96, 1.73 and 1.53, integrating three protons each, were associatedcorrespondingly with the acetyl group and C~26, C-27 methyl groups attached the C-25 olefinic carbon. The four tertiary methyl groups appeared as three-proton each singlets at 5 1.16 (CH3-29), 1.03 (CH3-I), 0.93 (CH3-2 8 ) and 0,83(CH3-3 O). Two one-proton each upfield broad singlets at 5 0.30 and 0.23 wereascribed to C-18 methylene group.

In the mass spectrum of MF- 8 (Scheme VII) the significant fragment ions at m/z 475 420 [475-C^H,, fission]" and 321 [M-C10H17O2, side chain]-' reflected the presence of C,Q-side chain with the acetylgroup and one of the olefinic linkage at position. The presence of 3-0x0 group at C-3 and unsaturation in rings A and B at C-1, 5 and 8 (9) wereinferred from the ion fragments appearing at m/z 436 [ion a]", 54 [ion b]^. 109[ion c]+, 81 [ion c-CHJ, 96 [ion d]+, 225 [ion e ] \ 173 [ion g ] \ 159 [ion g-CHJ", 163 [ion h]-" and 148 [ion h-CHJ".

The location of another carbonyl group at C-11, saturated nature of ring D and the presence of C-12 (18)-tricyclic ring were deduced from ion peaksgenerating at m/z 214 [ion i ] \ 186 [ion i-CO]^ 135 [ion j]^ 107 [ion j-CO]^,308 [ion k ]\ 294 [ion 280 [294-CHJ^ 210 [ion \ ] \ 155 [ion196 [ion 1-CHJ" and 182 [196-CHJ. Hydrolysis of compound MF- 8 produced a 20fS-hydroxy derivative (MF-8 a), IR 3410, 1720 cm'. Sodium borohydridereduction of MF- 8 yielded 3ft, IIR-dihydroxy derivative (MF-8 b). The 'H NMR spectrum of MF-Bb exhibited two signals for hydroxymethine protons at 5 3.30 (d, J = 4.5 Hz, H-3R) and 3.20 (d, J = 8.5 Hz, H-12ft). These data indicated that the structure of manglanostenoic acid G (MF-8 ) must be lanost-1,5,8 (9), 24-tetraene-20ft-acetoxy-12ft,18-tricyclo-3,11-dione. This is a new Ianostene-type triterpene reported for the firs t time from any natural or synthetic source.

134

C , , H „ 0 , (ionk) m/z 308 (2.0) (iong)

m/z m (9.1)

OAc

\

C „ H , , 5 0 , (ion i) m/z 2 U (1 3 )

oc

C ,3 H „ 0 , (ionI) m/z 210 (2.9) C ,H ,,5 0 ( io n j)

m/z 135 (25.0)

Scheme MF-VIII: Mass Fragmentation Pattern of Manglanostenoic Acid G (MF‘8)

135

COMPOUND MF-9

Compound MF-9, named mangstigmasteryl ester, was obtained from chloroform eluants. It responded positively to the Liebermann-Burchard and Zimmermann tests, indicative of a 3-oxo steroidal/triterpenoid skeleton. It had the composition of established on the basis of the EIMS and proton-countin the 'H NMR spectrum. Its IR spectrum exhibited characteristic bands for hydroxyl (3350 cnr’ ) and carbonyl groups (1720, 1705 cm ') and for unsaturation (1v595 crrr’ ). Its 'HNMF? spectrum is constistent with the proposed structure and clearly shov/ed C-21 hydroxy methylene protons as one-proton each doublets at 5 4.20 (J - 6.84 Hz) and 4.00 (J = 6.10 Hz). Three broad singletsat 5 2.44, 2.38 and 2.03, integrated for two-protons each, were associated withthe C-12, C-2 and C-23 methylene groups adjacent to the carbonyl groups. Two three-proton each broad singlets at 5 1.64 and 1.55 were accounted correspondingly to C-26 and C-27 methyls attached to vinylic carbon, The C-29 secondary methyl group appeared as a three-proton doublet at 5 0.87 (J “ 5.13 Hz). Two upfield broad signals at 5 1.00 and 0.77, integrated for three-protons each, were ascribed to C-19 and C-18 tertiary methyl functionalities, respectively. Absence of any signal beyond 5 4.20 suggested the tetra-substituted nature of the olefinic bonds.

The most significant evidence for the structure elucidation of MF-9 was its mass spectrum (Scheme MF-VIII), apart from the molecular ion, thesignificant ion fragments appeared at m/z 439 [M-Me]', 422 [M-CH3OH]' , 407[422-Me]^ 392 [407-Me]\ 169 SC]^ 285 [M-SC]", 227 [270-ringD]*- and 212 [227-Me]^ suggesting the presence of a unsaturated side chain with one hydroxyl and one carbonyl groups and two carbonyl groups in the carbocyclic framework. The ion peaks appearing at m/z 70 [ion a]" , 83 [ion b ]\55 [ion b-CO]", 69 [ion b-CHJ-, 138 [ion c] ' , 124 [ion c-CHJ", 110[124-CHJ^ 215 [M-70-SC]\ 175 [M-110-SC]^ and 147 [M-138-SC]^ supportedthe existence of one of the carbonyl group at C-3 and saturated nature of rings A and B. The location of C-11 carbonyl group and A***®’ olefinic linkage was inferred from the ion peaks generating at m/z 203 [ion d ] \ 189 [ion d-CHJ",175 [189-CHJ^ 204 [ion b]^ 190 [ion e-CHJ, 162 [190~CHJ\ 95 [M-190-SC]\ 123 [M-162-SC]\ 245 [C, ,^-0,^ fission]' , 231 [245-CHJ* and 217[231-CH ]■". The ion peaks at m/z 125 [ion f] * and 83 [ion g ]' were indicative

136

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OC ,,H ,„0 (ionc) m/z 138 (2.7)

C „ H „ 0 (iond) m/z 203 (6.9)

C,3H„0,(ione) m/z 204 (6.7)

Scheme: WIF-IX : Mass Fragmentation Pattern of Mangstigmasteryl Ester (MR 9)

137

of one of the carbonyl groups at C-22 and olefinic linkage at Thecompound fV1F-9 yielded a monoacetyl product (MF-9a) on treatment with acetic anhydride and pyridine. These data indicated that the structure of rnangstigmasleryl ester (MF-9) must be stigmast-8(9), 24(25)-diene-3,11,22-tnone- 2 I -0I. T liis is a new stigmasterol derivative isoiated from a natural source.

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STFÛCTURAL ASSESSMENT OF THE CHEfVIICAL CONSTITUENTS ISOLATED FROM THE STEM BARK

OF M. INDICA CULTIVAR LANGRA

COMPOUND ML-1Compound ML-1, named mangterpenone, was obtained as colourless

crystals from petroleum ether eluants. It had a rnolecuiar ion peak at m/z 254 corresponding to an unsaturated cyclic sesquiterpenic fomiula, C,5H2q03. Its IR spectrum displayed typical absorption bands for hydroxyl group (3445 cm ''), carbonyl group (1720 a ir'’) and unsaturation (1595 cm’’ ). Attachment of various groups in the acyclic chain was concluded by interpreting the EIM spectrum. The ion fragments at m/z 59 [ion a] '- and 99 [ion b] ' suggested the location of hydroxyl group at C-15 and olefinic linkage at C-9. The presence of carbonyl groups at C-8 and C-4 was inferred from the ion peaks appearing at m/z 155 [ion c] + , 71 [ion g]"-, 211 [ion h]* and 43 [C3H.J" (Scheme ML-I).

The 'H NMR spectrum of ML-1 exhibited a one-proton doublet at 5 5.30(J = 5.0 Hz) assigned to H-10 vinylic proton. Two one-proton each doubletsat 5 4.25 (J = 5.0 Hz) and 4.18 (J-7.3 Hz) were ascribed to C-15hydroxymethylene group. A three-proton broad singlet at 6 1.85 was associated with C-14 methyl group attached to olefinic C-9, The methylene groups adjacent to 0-4 and C-8 carbonyl groups appeared at 5 2,35 (m, CH^-S) and 1.60(d, J = 5,5 Hz, CHj-7), Two three-protons signals at 5 0.90 (t, J - 6.0 Hz)and 0.77 (d, J = 6.00 Hz) were accounted correspondingly to C-1 and C-12methyls. The remaining protons resonated at 6 2.00 (1H), 1.90 (1H) and 1.25(7H). Acetylation of ML-1 with acetic anhydride-pyridine yielded a rnonoacetyl derivative (ML-Ia), (IR 1725 cm'') supporting the presence of one acetylable hydroxyl group in the molecule.

On the basis of these results, the structure of mangterpenone (ML-1) has been elucidated as 5, 9-dimethyl dodec-9-ene~15-ol-4, 8-dione. This is a new sesquiterpene and isolated fo r the first time from a natural source.

159

0 0

H O 'C ^ H „ (ionb) m/z 99 (1,2)

C JH , (0 )

O , (ion c) rn/z 155 (1,0)

/H O ,

C .,H ,0 (ion a) m/z 59 (21.3)

m/i 211 (1.0)

C3H,m/z 43 (37.2)

HOC ,H ,,O j( io n d ) m/z 127 (5,7)

0

o

H., 0 (ion g) m/z 71 (22.3)

Scheme; ML-I: Mass Fragmentation Pattern of Mangterpenone (M L-I

160

COMPOUND ML-»2

Compound ML-2, named mangditerpenic ester, was obtained as colourless crystalline product from petroleum ether eluents. Its IR spectrum demonstrated the presence of characteristic carbonyl, group (1710 cnr') and unsaturation (1640 cm’' ), It had a molecular ion peak at m/z 478 corresponding for a molecular formula C3,Hj.gO , The spectrum showed a prominent ion peak at m/z 167 [ion e]+ relating to -acetyl group and an ion fragment at rn/z 311 [ion f]" due to an acyclic diterpenic diain containing a carbonyl group. The ion peaks at m/z 113 [ion a ] \ 365 [ion b]-, 139 [ion c ]" and 339 [ion d]"indicated the presence of the olefinic linkage at C-2'{3'). The ion fragments at m/z 183 [ion g ] \ 295 [ion h]*, 99 [ion \ ] \ 71 [ion j]" and 407 [ion kj*supported the presence of carbonyl group at C-12 (Scheme ML-II).

The M-l NMR spectrum of ML-2 displayed two one-proton each broadsinglets at 5 7.20 and 6.42 assigned to C-2' and C~3' vinylic protons. The oxygen substituted methylene protons appeared as broad singlets at 6 4.10 and 4,03. The secondary methyl protons appeared at 6 1,16 (2Me), 1.15 (Me),1,16 (Me), 1.03(2lVle), 0,96 (Me), 0.90 (Me) and 0.83 (Me), The remainingmethine and methylene protons appeared in between 5 2.30-1,22. Alkaline

hydrolysis of ML-2 yielded a C-11 carboxylic acid and a diterpenic alcohol ML-2a, Based on these spectral data analyses and chemical reactions the structure of mangditerpenic ester has been characterized as 3,7,11,15-tetramethyl hexadecan- 12-one-l-yl 4',8'-dimethyl non-2~ ene-1'-oate. This contributes the firs t report o f the isolation of a diterpenic ester from Mangifera indica.

COMPOUND ML»3Compound ML-3, designated as eicosenyl ester, was obtained as

colourless amorphous mass from petroleum ether eluants. Its IR spectrum exhibited charateristic absorption bands for ester (1730 cm ') and carbonyl groups (1705 cm-'). It had a molecular ion peak at m/z 492 in its mass spectrum corresponding to The presence of a long aliphatic chain wasconfirmed from the mass spectrum which had a uniform difference of 14 mass units with regard to a larger number of fragments. More intense clusters of peaks corresponding to (e.g, 71, 85, 99, 113, 127, etc) in comparison to that

161

o

C gH ,, (ion a) ffl/z 113

C „ ,H „ ( io n c) m/2 139 (5.7)

C , ,H , , ,0 ( io n e ) m/z 167 (56.0)

CHO

0CjoH33(0) Cj Cô^«0;(ionO

m/>. 311 (3,2)

m/z 183 (5.1)H-

C „ J H „ 0 ^ io n h ) m/z 295 (3.1)

C ,H „ O (ion i) m/z 99 (44.3)

m/z 71 (21,3)

O

C,,H47 0 3 (ionk) m/^ 407 (2.1)

Scheme; ML-II: Mass Fragmentation Pattern of Mangditerpenic Ester (ML-2 §162

corresponding to (e.g. 69, 83, 97, 111, 125, etc.) supported the acyclicnature of the compound (iViishra et ai, 1989). The; presence of [M-Me] ’ ionat tn/z 477 suggested branched chain nature of the compound (Stoianova-Ivanova et al., 1969). The branching at C-T was deduced from the intensified ions appearing at m/z 127 [ion a]-, 169 [ion b]', 154 [ion b-Me]’ , and 139[154-Me] '. The ion peaks at m/z 307 [ion c]* , 185 [ion d ]‘ . 213 [ion e]* and 279 [ion fj' indicated the ester linkage at C-20. The olefinic linkage at C-18(19) and carboxyl group at C-5 was inferred from the ion fragments appearingat m/z 239 {ion g] ^ 253 [ion h]*, 85 [ion i] and 57-(ion jj" (Scheme MUII).

iVlcLafferty rearrangement of M 1*3 formed ions at m/z 450 and 42 (Scheme ML-IV) supporting the presence of the carbonyl group at C-5.

The ■'H NMR spectrum of ML-3 showed two one-proton each broadsinglets 5 7.2 and 6.43 assigned to C-19 and C-18 vinylic protons, respectively.The methylene protons appeared at 6 2,00 (4H) and 1.23 (42H). Four breadsinglets at 5 0.90, 0.86, 0.83 and 0.80, integrated three protons each, were assigned correspondingly to C-11', C-12’, C-1 and C-10' methyl functionalities.Alkaline hydrolysis of ML-3 yielded a tertiary alcohol ML-3a and an unsaturated keto acid (ML-3b). On the basis of these spectral data analyses and chemical reactions the structure of eicosenyl ester {ML-3) has been elucidatd as T^T-dimethyl decanyl“eicos-18-€ne"5-one-20-oate, This natural product has been isolated for the first time.

COMPOUND ML-4Compound ML-4, named hexacosanyl acetate, was obtained as

colourless product from petroleum ether-chloroform (9:1) eluants, It did not give colour with tetranitromethane suggesting its saturated nature. The IR spectrum of ML-4 showed typical absorption bands at 1730 cm ’ (ester linkage) and 1700 cm ’ (CO). Its mass spectrum exhibited a molecular ion peak at m/z 438 consistent with the molecular formula, C^gH^Ô.,. The spectrum showed the presence of C^H^ and ions reflecting aliphatic nature of themolecule. Most of the ion peaks were separated by 14 mass units and decreased in intensities and abundance with increasing the molecular weight of

163

o

CH3~(CH,);CgH,g(iona)m/z 127 (24.1)

m/z 139 (96)

- Me

m/z 154 (13.1)

" Me

CH,,-(CH ) -C(CH.,)’ ,C,^Hj^(ionb)m/z 169 (11.2)

C - CH = CH ■■ C.,,. 0 „ (ion c)m/z 307 (4.6)

CH., - (CH„)

Chi.I

C - 0 ’I

CH.,

C^.,H„.,0(iond) m/z 185 (7.9)

CH. 3 0 4 6 0 7

CH., -- (CH,)J - C J O -! C .ICH = CH -- |CH,) U C - 1(CH ),, CH

■ ' i n ' I ' I I

C 3, O 3 ; [M] m/ 2 492 (3.5)

O

C,^H,,OCO' C r jH js O ^ (io n e ) m /z 213 (3.4)

O

C , , H 3 ,, 0 (io nf)

m/z 2 7 9 (3.4)

C,^HjgOC-CH = CH 0 ,5 O ^ (io n g )

m /z 2 3 9 (5.3)

+o

C , , H ^ ,3 O (io nh )

m/z 2 5 3 (4 .7)

O

C ,H ,0 ( io n i)

m /z 85 (6 6 .8 )

C^Hg(ioni) m/z 5 7 (10 0 )

Scheme ML - III: Mass.Fragmentatlon Pattern of Eicosenyl Ester (ML -

o\

CH., - (CH^)„ - C - O C .. CH - CH - (CHy,,, - C

Cl-i

H

CH - CH„

CH.,CH..

CH., O

CH.

OH

CH„

+ CH = CH - CH3 m/z 42

C ,, H ,, O 3

m /z 450 (2.7)

Scheme ML - IV: McLafferty Rearragment of Eicosenyl Ester (ML-3

165

the long-chain hydrocarbon (Budzikiewicz ef a/., 1965; Pankh, 1974; F^hyag and Stegagen, 1963). The absence of [M-Me]" ion disclosed the unbranched nature of the compound (Stoianova-Ivanova et a!., 1968), Elimination of acetyl groupfrom the molecular ion peak generated the prominent ion peak at m/z 395 [ion a]' . The presence of the carbonyl group at C-10 was inferred from the prominent ion peaks appearing at m/z 155 fion c], 283 [Ion b ]’ , formed dueto B-fission, and at m/z 127 [ion e] + , 311 [ion d ] ‘ arose due to a-fission(Scheme ML-V).

The NMR spectrum of ML-3 showed two one-proton each broadsinglets at 5 4.00 and 3.85 assigned to oxygen-substituted C-26 methylene group. Two three-proton each signals at 5 2.06 (brs) and 0.55 (t, J = 6.5 Hz) were attributed correspondingly to acetyl and C-1 methyl groups. The remaining resonances at 5 2.40 (2H), 2.20 (2H, 1.70 (14H) and 1,26 (28H) wereassociated with the methylene functionalities. Alkaline hydrolysis of IViL-4 yielded a keto alcohol (ML-4a). Based on these observations the structure of the new aliphatic ester, hexacosanyl ester (ML-4), has been established as hexacos-10- one-26-yl acetate. This is a new aliphatic ester isolated from a natural source.

COMPOUND ML»SCompound ML-5, named mangolactone, was obtained as colourless

amorphous mass from petroleum ether eluants. Its IR spectrum showed distinctive absorption bands for hydroxyl (3410 cm-') and ester groups (1735 cm '). It had a molecular ion peak at m/z 424 in its mass spectrum corresponding to a molecular formula The spectrum exhibited the presence of

and ions reflecting aliphatic nature of the molecule. The intensitiesand abundance decreased with increasing the molecular weight of the long-chain hydrocarbon (Budzikiewicz et al., 1965; Parikh, 1974; Rhyag and Stegagen, 1963). The absence cf [M-Me]^ ion disclosed the unbranched nature of the compound (Stoianova-lvanova et al., 1969). The existence of functional groups was determined due to appearance of intensified fragment Ions in the spectrum. The ion peaks at m/z 126 [ion a]^ 169 [ion b]* and 255 [ion c]* suggested the presence of 6-lactone possessing a pentyl group at one of the terminal end. Elimination of m.u. 197 [ion d]* from [M]" and formation of [ion e]" at m/z 227

166

o

CH3 CO -

2 3II O1 I

O - H C - (CH,),.CH3

[M] - m /i 438 (2.0)

■CM, CO0 -(C K ,)„.-C -(C H ,,

m/7 395 (4.3)

CH., COO (CH,,)

C„H3„0,(ionb) nVz2&3 (1.1)O

C-(CH.,),,-CH3

C „H .„0 ( io nc ) rn/z 155 (28.6)

CH_, COO (CHJ ,,, - C

C,.,H ,,0 3 (iond) rn/z 311 (1.1)

(CH,)e-CH3

CgH„,(ione) nVz 127 (10,9)

Schemc ML - V : Mass Fragmentation Pattern of Hcxacosanyl Acctate (ML-4)

167

suggested the carbonyl group in the molecule at C-16. Subsequent removal of methylene groups from [ion e]' formed ion peaks at rn/z 199, 185, 171, 157, 143, 129, 115 and 101. The presence of hydroxyl group at C-5 was deducedfrom the ion fragrrients appearing at rn/z 337 [Ion f] ' , 87 [ion g ]‘ . 57 [ion h]' and 69 [ion g-HÔ]', Formation of ions at rn/z 209 [ion e -H ,0 ]’ and then gradual elimination of methylene groups from m.u. 209 generating ion peaks at m/z 195, 181, 167, 153, 139, 125, 111 and 97 supported the existence ofhydroxyl group in the molecule (Scheme ML-Vl).

The 'HNMR spectrum of ML-5 showed three one-proton each multiplet at 5 5.26, 4.00 and 2.73 assignable to H-17a, H-5 and H-19, respectively. A two- proton triplet at 5 2.23 (J = 7.2 Hz) was accounted to C-20 methylene group adjacent to carbonyl function. The remaining methylene groups appeared as broadsinglets at 5 1.80 (4H) and 1.26 (22H). A nine-proton singlet at 5 0.97 wasaccounted to C~1. C-22 and C-27 methyl functionalities. Acetylation of ML-5 formed a monoacetyl product (ML-5a) indicating the presence of one acetylable hydroxyl group in the molecule. On the basis of these findings the structure of mangolactone (ML-5) has been established as 16-methyl-l 9-pentyl heneicos-5- ol-17, 21-olide. This is a new 5-lactone Isolated from a natural source for the first time.

COMPOUND ML-6Compound ML-6, named heptadecanyl ester, was obtained as colourless

amorphous mass from petroleum ether-CHCIj (3:1) eluants. It did not respond to tetra-nitromethane indicating saturated nature of the molecule. Its IR spectrum showed the presence of hydroxyl (3400 cm ’ ) group. It had a molecular ion peak at m/z 454 in its mass spectrum corresponding to the molecular formula

A large number of fragment ions recorded in the mass spectrumexhibited a uniform difference of 14 mass units, thus confirming the presenceof a long-chain aliphatic compound. Furthermore, the presence of a peak at m/z 423, formed due to expulsion of a 31 mass units, from M^ suggested the presence of hydroxymethylene group in the molecule. More intense clusters of peaks corresponding to (®-9- 57, 71, 85, 99, 113, 127, 141, etc.)in comparison to that corresponding to , (e.g. 55, 69, 83, 111, 125,

168

139, etc.) supported acyclic saturated nature of the compound (Silverstein and Bassler, The absence of [M-15]' ion confirmed the straicjht chain carbonframework of the molecxile (Stoianova-lvanova and Hadljieva, 1969). The location of functional groups was deduced from the suddenly intensified ion peaks in the spectrum. The ton fragments at m/z 141 [ion a ]’ , 313 [ion b ]\ 169 [ion c]*, 285 [ion d ]‘ , 185 [ion e]* and 269 [ion f ] ’ indicated the pre^sence of a C-10 ester group at C-17 of the alcohol. The presence of the carbonyl group at C"2 was deduced from the ion peaks appearing at rri/z 395 [ion g] " and 423 [ion h]^ generated due to a- and fi~fission of the carbonyl group, respectively (Scheme VII). From these data it was evident that the compound ML-6 was the ester of decanoic acid with 2-keto-aIcohol. Firm evidence in support of the proposed structure of this new ester was obtained from its ’H NMR spectrum which displayed four one-proton each doublets at 5 4.10 (J = 7.0 Hz) and 4.00 (J = 7.0 Hz) assignable to C-17 ester substituted methylene group and at 5 3.60 (J = 5.5 Hz) and 3.53 (J = 6.0 Hz) associated with C-1 hydroxyi-methylene group. The methylene protons appeared at 5 2.20 (2H), 1.90 (2H), 1.46 (4H)and 1.20 (42H). A three-proton doublet at 5 0.93 (J = 6.0 Hz) was accountedto C-11 terminal methyl group. The compound ML-6 formed a monoacetyl derivative (ML-6a) with acetic anhydride and pyridine. Alkaline hydrolysis of ML-6 yielded an alcohol (ML-6b) and an acid f\/IL-6c. On the basis of foregoing account compound ML-6 was characterized as heptadec-1-ol-2-one-17~yl undecanoate. This is a new aliphatic ester isolated from a plant source.

COMPOUND ML^7Compound ML-7, named mangtirucalloic add, was obtained as a white

amorphous mass from benzene-chloroform (1:1) eluants. It responded positively for unsaturated 3-oxo-triterpenes with the typical reagents. Its mass spectrum had a molecular ion peak at m/z 454 consistent with the molecular formula, C3pH^p.j. The mass fragmentation pattern was identical to that of MF-3 suggesting lanostene /terucallane-type carbon framework with C^-unsaturated side chain containing a carboxylic group. Its 'H NMR spectrum displayed three vinylic doublets at 5 6,00 (J = 6.0 Hz), 5.95 (J = 6.0 Hz) and 5,16 (J = 3.5 Hz) accountable to H-1, H-2 and H-24, respectively A 3 -proton broad signal at 51.80 was associated with 0-27 methyl functionality attached to C-25

170

CHg -(CK,) (CH.,)'

C,„H2, (iona) m/z 141 (16.8)

O

0C-O-(Chg,g-C-CHj0H

C , „ H „ 0 , ( io n b ) 01/Z313 (1,4)

(CH,J,,CO'*"

O (io n c ) m/z 169 (13,9)

'• 0(CH.,),,, COChipH

0 ,,( iond ) m/z 285 (1,6)

4 5I 0

OH

m/z 313 (1.4)

O

CH3 (CH,), -C-0(CH,),,C0

C „H „ O3 (ion h) n\/z A22 (2.9)

N/ O

C H 3-(C H ,),-C -0 (C H ,);

Oa (iong) m/z ZQ5 (1.6)

CH3-(CH )^C00 *

Oj(ione) m/z 185 (5.5)

•' (CH3),, -C0CH,0H

C,,,H„ O, (ionO ni/z269 (1.7)

Scheme ML ■ V II: Mass Fragmentation Pattern of Heptadecanyl Ester (ML-6)

171

unsaturated carbon. The remaining five tertiary methyls rc-)sonated at a 1,00 (Me-

19), 0,97 (Me~29), 0.90 (Me-28, Me-30) and 0,70 (Me~18). A doublet at 60.95 (J = 6.0 Hz) was ascribed to C~21 secondary methyl group. The appearance of all the methyl signais in the range ft 1.00-0.70, except the vinylic methyl at C-25, supported the tirucallane-type skeleton of the molecule. Methylation of fV!L-7 yielded a rnonomethyl raster (ML-7a), thus confirming the presence of carboxylic group in the molecule. On the basis of these observations the structure of mangtiruralloic acid (ML-7) has been established as 20 (R), 24 (R)-3-oxo-tirucalla-1 (2),24-diene-26-oic acid.This constitutes the firs t report of the presence of tiaicallane-type acids In Mangifera species.

COMPOUND ML-3 ■ ■Compound ML-8 was identified as ^.-sitosterol on the basis of m.p.,

m.m.p,, co-TLC, mass fragmentation pattern and acetate formation.

COMPOUND ML-9Compound ML-9, designated as rnangoketone, was obtained as

colourless amorphous powder from chloroform eluants. Its IR spectrum showed characteristic absorption bands for hydroxyl (3410 cm ') and carbonyl (1710, 1695 cm-’ ) groups. It had a molecular ion peak at m/z 494 in its mass spectrum corresponding to the molecular formula The mass spectrumexhibited and , ion peaks typical of aliphatic compounds.The spectrum also showed separation of most of the fragments by 14 mass units and decreased in abundance with increasing molecular weight of long straight chain hydrocarbon. The presence of an intensified ion fragment at m/z 476, generated due to removal of water molecule from M \supported the existence of hydroxyl group in the molecule. More intense clusters of peaks corresponding to C^ H^ ^ ( e . g . 71, 85, 99. 113, etc.) in comparison to that corresponding to C^H^^, (e.g. 69, 83, 97, 111, 125,etc.) suggested the acyclic and saturatednature of the molecule (Misra et al., 1989). The ion fragments at rn/z 227 [ion a]", 267 [ion b]", 295 [ion c] ' and 199 [ion a-CO]' indicated the presence of one of the carbonyl group at C-13. The location of the hydroxyl group at C-7 was deduced from the ion fragments appearing at m/z 365 [ion d ]', 129[ion e ] \ 111 [ion e-HÔ]'. 395 [ion h] and 99 [ion i ] \ Two ion peaks at

172

m/z 437 [ion f ] \ 57 [ion g ]' and 419 [ion f-H^,0] ‘ attested the presence ofanother carbonyl group at C-3 (Scheme ML-VIH). The 'H NMR spectrum of ML-9 showed a one-proton double doublet at 5 4.20 (J = 6.5, 5.5 Hz) assignable to C-7 carbinol proton. The methylene protons adjacent to carbonyl groups appeared as broad singlets at 5 2.60 (H2-12), 2.47 H,-4) and 1.93(H2-2). A broad singlet at 6 1,30. integrated to 46 protons, vyas associated with the remaining methylene groups of the rnolectile. The terminal methyls resonated asi three-protons each broad singlets a t5 0.95 (Me-32) and 0.73 (Me-1).Acetylation of ML-9 yielded a monoacetyl product ML-9a.

These data led to formulate the structure of this natural productrnangoketone (ML-9) as dotriacont-7-ol-3, 13-dione. It is being reported for the fis-st time from a natural or synthetic source.

173

o

C,,,H j,0,{iona) m/z 227 (3,6)

-f-

0-, (io'ib) m/z 267 (2.5)

- CO-> m/2 199 (10.6)

O

CH3(CH,),^C^

C,„H.,,0(ionc) nVi 295 (11.9)

O

C.„_H,,^0(icuKJ) m/z 365 (1.4)

C H , ( C H J , „ - C ^ - ( C H j ,

OH O

/ \ O , (ione ) rn /i 129 (7.9)

I o OH O

CH3-(CH,,),g! - cJ (CH ) , CH -l(CH^), C-CH^-CH3

C ^ , H „ 0 , [MJ * m / /494 (3,9)

N/

c,, H,3 (O)CHOH

C2eH5,0,(ionh) /rvfe 395 (1,6)

OH-f

C,, H,, (O) CH (CH,)3- ^ m/^4W (1.4)-HÔ

C „H „O j (ionf) m/^ 437 (6.2)

(CH,), 0(0) H,

CgH,,0(ioni) m/z 99 (9.2)

C-C,H,C3 H,_ O (ion g) m/i 57 (100)

Scheme ML - V III: Mass Fragmentation Pattern of Mangoketone <ML - 9)

174

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M L - 6 c

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M L - 7 a R = CH„

O 032 II 1 3 II 2 1CH., - (CH ),„ - C - (CH,), - CH - (CH,)3- C •• CH, .-CH,,

M L - 9 : R = H

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Mass Spectrum of Mangterpenone (ML-1)

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Pass Spectrum of Mangditerpenic ester (ML-2)

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Wiass Spectrum of Mango!actone (ML-5|

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Mass Spectrum o f Heptadecanyl e s te r (ML-6)

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?50 4(10100 150 200 250 3*0

I Mass Spectrum of Mangtirucalloic acid (ML-7)

45(1 500

Mass Spectrum of B-Sitosterol (ML-8)

Mass Spectrum of Mangoketone (ML-9)188

S lk l jC T iJ l lA L ASSEf>SMEi^JT OF THE C H EM IC AL C O M frriTU E N TS ISOLATED FROM THE STEM BARK OF M. iNDICA CULTIVAR 'SAFED.A'

COIVlPOUND IViS~1:

Compound MS-I was obtained from petroleum ethejr eluants. It had a molecular ion peak at tn/z 194 cxsrresponding to the molecular formula Its IR spectrum disclosed the presence of ester group (1720 cm ') and aromatic ring (955, 875, 815 cm ’ ). The ’H NMR spectrum of MvS-1 displayed the presence of a downfield signal at 6 7.23, integrating for two protons, assigried to H-2 and H-6 of the aromatic ring. Another two-proton signal at 6 6.00 was attributed to H-3 and H-5 protons. Two methoxy functions resonated at 5 3.50 as a broad signal. These data indicated that the im pound was a dimethyl ester of terepfithalic ester.

Further information about the structure of the compound was obtained from the ion fragmentation patterns in its mass spectrum, it exnibited the presence of ions of diagnostic importance at rn/z 179 [M -M e]", 163[M-2 X M e ]\ 135 [M-COOMe]% 119 [164-COJ* and 76 [ilS -CO ^]*'.Hydrolysis of MS-1 with alcoholic sodium hydroxide solution at refluxed temperature yielded terephthalic acid. These data led to characterize the compound MS-1 as dimethyl terephthalate.

COMPOUND MS-2:Compound MS-2, named nonanyl propanoate, was obtained as

colourless amorphous powder from petroleum ether eluants. It did not respond to tetranitromethane indicating saturated nature of the molecule, fts (R spectrum showed characteristic absorption bands for ester (1738 cm ') and carbonyl groups (1712 cm ’ ). Its mass spectrum exhibited a molecular ion peak at m/z 368 consistent with the molecular formula, A large number offragment ions recorded in the spectrum showed a uniform difference of 14 mass units, thus confirming the presence of a long aliphatic compound. More intense clusters of peaks corresponding to , , supported the acyclic ester chain of the compound. The generation of a peak at m/z 353, formed due to expulsion of a methyl group from M*, indicated branched nature of the molecule

189

o ooII

»C-0-C^„H„(0)

f/i/z 339 (36,9)

C j H, C + (ion b)

m/z 57 (100)

+ CH„

0 - ( C H , ) , - C H - C , , h j , , ( 0 )

C .o H 3.,0 ,( io n c )

m/z 311 (6.8)/K

2

1 2 3I

O CH,

C H , • C H , ■■ C ■■ O

C,,H, 0_, (loncJ)

m/z 73 (95,2)

O

C H ,-^ C H , L C L 0 -1- (C H J , 1 CH -I (C H ,), i C - ( C H , ) , ............. CH,,■ I 1 I ~ I i I

C„H^,03;(M1+^^!/^ 368 (23,8)

O CH.

C,H,~C-0-(CH,),-CH + CgH,,0, (iong) m/z 157 (18.1)

C,H,-C-0»C^3K, C,,H3,0,(ionf) n /z 255 (48.6)

OII ^

(ione) m/z 129 (54.4)

Scheme MS-1: Mass Fragmentation Pattern of Nonanyl Propanoate (MS-2)

190

(Stoianova-lvanova and Hadjieva, 1969). The mcjss speJcU'um of MS■-2 also showed intense ion peaks at rn/z 339 ion a]*, 57 [ion b] ■, 311[ion c]' and 73 [ion d]" suggesting the estenfication of propyl group at C-19 of the aliphatic chain. Another prominent ion peaks at m/z 12.9 [ion e]' and 157 (ion g]*' indicated the presence of methyl branching at C--15. The location of the) carbonyl groups at C-7 was deduced from the intensifi€5d ion peaks appearing at m/z 255 [ion f]", 113 [M-ion fj', 183 and 185 [M-183]" (ScfiemeMS-1). From these data it was evident that the compound v- as a propyl ester of methyl nonadecane.

Further evidence in support of the proposed structure of this new ester was obtained from its 'H NMR spectrum which displayed a two-proton broad multiplet at 5 4,10 assignable to C-19 oxygen substituted methylene group and three broad singlets for methylene groups attached nearby to the carbonyl groups at 6 2,33 (4H) and 2.26 (2H). The methyl groups appeared as three-protonseach broad singlets at 5 0,90 (Me-3'), 0.86 (Me-20), and 0.82 (Me-1). Theremaining methine and methylane functionalities appeared at 5 1.96 (1H, rn,H-15), 1.60 (6H, brs, 3 x CH.J and 1.23 (22H, brs, 11 x CHJ. Alkalinehydrolysis of MS-2 yielded an alcohol; 3450, 1710 crrr'. These accumulative evidences led to establish the structure of MS-2 as 15-methyl nonadec-7-one- 19-propanoate. This is a new aliphatic ester and is -being reported from M. indica fo r the first time.

COMPOUND MS-3Compound MS-3, named safedasterol, was obtained from petroleum

ether-chloroform (9:1) eluants. It responded positively to steroidal tests. Its IR spectrum showed characteristic absorption bands for hydroxyl group (3450 cm ’ ) and unsaturation (1595 cm ’).

The compound had a molecular ion peak at m/z 414 in its mass spectrum consistent to a steroidal formula, C gHgÔ. It indicated five degrees of unsaturation; four of them were adjusted in four rings of the steroidal framework and the remaining one in olefinic linkage.

191

CH„

CH.,HO

HO

'CH.,

C,H, H,, O (ion b) m/z 72 (3 ,8)

Cj HgO (ion a) m/z 85 (4.1)

C. H,, 0 (ion c) m/r. 112 (2.1)

H.. O___m/z 94 (9.7)

I iT' n L ' \ , \C.oH,,

C. 2H3,-,0(iond) m/z 302 (3.8)

C,3H,^0(ionh) m/z 192 (2.1)

CH,C,oH„

CH-HO

C.eHj, (ion 0 m/i: 250 (N.O)

C„H ,^0(ione) m/z 164 (3.1)

C„Hj,0(iong) m/z 205 (2.1)

Schcme MS-II: Mass Fragmentation Pattern of Safadasterol (MS-

192

The ’H NMFi spectrum of MS-3 displayed one proton broad multiplet at 5 3.20 assigned to C-3 carbinol proton and its half-widtt-) of 12,0 Hz indicate^d its B"Oriantation. Two broad singlets at 6 1,06 and 0,76, integrated for three protons each, were attributed correspondingly to ttie C-19 and C-18 tertiary methyls. The secondary C~21, C-26 and C-27 methyl functionalities appearedas three- proton each doublets, respectively, at 5 0.96 {J = 6.0 Hz), 0.86 (J = 6.5 Hz), and 0.80 ( j = 6.5 Hz), A three-proton t,)road singlet at 6 0.90 was ascribed to C-29 methyl which suggested an ethyl group in the side chain located at C-24 on biogenetic ground. The remaining methine and methylene groups resonated from 5 2.50 to 6 1.16. The appearance of all the methyls in the region 6 1.06-0.76 suggested that these groups were attached on saturated carbons. The 24S methyl resonance (0.90) was more deshielded as compared to 24R configuration (0.82). The absence of any signal beyond 6 3,20 supported tetrasubstituted nature of the olefinic linkage.

The electron impact mass spectrum of MS-3 (Scheme MS-il) exhibited diagnostically important ion peai<s at m/z 399 [M-Me] ", 396 [IV1-'H,,0} * , 381 [396-Me]^ 273 [M^,oH^,, side chain, SC]^ 271 [273-2H] ' , 255 [273-H,0]' and212 [255-ring C f is s io n ]T h e ion fragments at nVz 85 [ion a]", 67 [ion a-HÔ]', 71 [ion a-CHJ", 72 [ion b]*, 201 [M~SC~ion b ] \ 112 [ion c | \ 94 [ion 302 [ion d]^ 161 [ion d-SC]^ 147 [161^CHJ-, 133 [147-CHJ\ 126 [273-147]^ 108 [126-H20]^ and 122 [273^133-H.p]" indicated the presence of saturated nature of rings A and B and the existence of the hydroxyl group in ring A which was placed at C-3 on biogenetic analogy. The presence of A®*'” tetrasubstituted linkage was deduced from the ion peaks appearing at m/z 164 [ion e]", 146 [ion e-H20]\ 91 [ion f-CH^-SC]-, 205 [ion g] * , 192 [ion h]"and other subsequent ion peaks generated due to removal of methylene and water groups. Acetylation of MS-3 with acetic anhydride and pyridine yielded a monoacetyl derivative (MS-3a). Treatment of MS-3 with Jones reagent produced 3 -0X 0 derivative (MS-3b) which responded positively to Zimmermann test confirming the presence of 3-hydroxyl derivative.

On the basis of these findings, the structure of safedasterol (MS-3) has been elucidated as 24a-ethyl cholest-8(9)~ene-3a-ol. This is a new steroidal co n s titu e n ts and con tribu tes the firs t re po rt o f occurrence of the 3a-hydroxysterol in Mangifera species.

193

COMPOUND lViS-4

Compound MS-4, named rnangterpenoic acid, was obtained from petroleum ether-chloroforrn (4:1) eluants, !t produced effervesasnces with sodium bicarbonate solution. An IR spectnjm examination of MS-4 showed the presence of hydroxyl (3457 crrr'), carboxyl (3370, 1690 cm ') and carbonyl groups(1705 cnr') and unsaturation (1619 crn-’ ). Its mass spectrum exhibited a molecular ion peak at m/z 198 relating to a formula of a monoterpenic acid, C,oH.,40 . The Ion fragments at m/z 183 [M-Me]*, 170 [M-CO]', 152 [183-C H p H ]\ 124 [152-CO]-, 154 [iVl^COJ^ 126 [M^C0^4-i,0]' and 136H jO ]”* supported the existence of methyl, carbonyl, carboxylic and hydroxymethylene groups in the molecule. The ion peaks at in/z 107 [ion a ] \ 93 [iona~Mep , 65 [93-CO]^ and 79 [107-CD]"' indicated the presence of methyl andcarbonyl groups in the ring. The ion fragments at m/z 45 [ion b-COJ" and 58 [ion attested the presence of the C3-unit with hydroxyl and carboxylicgroups attached to the cyclohexane hng (Scheme MS-Ill)-

The 'H NMR spectrum of MS-4 showed a one-proton downfield singlet at 5 7.13 assigned to H-2. The C~10 hydroxy-methylene function appeared as one-proton each doublets at 6 4.33 and 4.20 with coupling interaction of 6.5 Hz, A DÔ exchangeable hydroxyl proton resonated as a one-proton broad singlet at 5 3.70. A broad multiple! at 5 2.63 with w 1/2 6.5 Hz was associated with H-6 a. The remaining protons appeared as a doublet at 6 2.85 (J = 6,5 Hz, H~8 ), and as broad signals at 6 1.50 (CH2-4 ), 1,40 (Me-7) and 1,23 (CM,-5). Acetylation of MS-4 with acetic anhydride and pyridine yielded a monoacetyl derivative (MS-4a), thus confirming the presence of one acetyiable hydroxyl in the molecule. A monomethyl ester derivative (MS-4b) was formed on treatment of MS-4 with diazomethane. From these informations the structure of mangterpenoic acid (MS-4) has been determined as 3-m ethyl-6fi- (10 -hydroxymethylene ethanoic acid)-cyclohex-2-ene-1 -one. This constitutes the first report o f the presence of the monoterpenic constituent from the stem bark of M. sndica.

194

0'

COOH

OH

[M] m/z 193 (49.5)

-Me C H . OH

A f ? i / z 1 8 3 ( 9 i

- rn/7. 170 (22,8)

m/z 152 (100)

-- CO

m/z 124 (21.8)

-CO,.~>m//154

(18.1)>m/z 126 (12,1)

"H„0

m/i 136 (3.5)

C, - C, fission

0

Hg 0 (ion a) m/i136 (3.5)

-Me

m/z 93 (2.3)

-CO

m/z 65 (4.8)

,-COm/z 79 (9,1)

CH- COOH

CHjOH

C3Hj,0 3 (]onb) m/z 89 (N.O.)

./CO,

m/z 45(6 .2 )

,-GHÔH

m/z 58(12.4)

Scliemc MS-Ill: Mass Fragmentation Pattern of Mangterpenoic Acid (MS

195

COMPOUND iVIST)Compound MS-S, designated as isornangterpenoic acid, was secured as

colourless crystalline product from petroleum ether-chioroforrn (4:1) eluants. It gave effervescences with sodium bicarlDonate solution. Its If spectrum displayed charaderistic absorption bands for hydroxy {MGO crn '), carboxyl (3290, 1690crn-'), carbonyl (1710 cm-') groups and unsaluration (1617 c m ’ ). Its mass spectrum exhibited a molecular ion peak at rr?/z 198 consistent w ith the molecular formula, The mass fragmentation pattern of MS-5 coincided with thatof MS-4 (Scheme MSW) suggesting identical carbon framework. The 'H NMR signals of MS-4 also coincided with those of MS-5. Thus, the structures of MS-4 and MS~5 differ only in the identity of H-6 signals, in the NMR spectrum of MS-5 the H-6 appeared as abroad multiple! at 6 2,67 with half width of 13.0 Hz which was assigned to B-configuration. Therefore, the C^-unit is a-orientated, The compound MS-5 formed a rnonoacetylated product with acetic anhydride and pyridine and a rnonomethyl ester with diazomethane. From the spectral data analyses and chemical reactions the structure of isornangterpenoic acid (MS-5) was proposed to be 3-rnethyl-6cx (10-hydroxymethylene ethanoic acid)-cyclohex-2-ene-1 -one. This is also a new natural product.

COMPOUND MS-6Compound MS-6, named eicosanyl lactone, was obtained from

chloroform eluants. Its IR spectrum exhibited characteristic absorption bands for hydroxyl group (3440 crrr^), ester/lactone (1735 cm ’ ), carbonyl group (1715 cm'), unsaturation (1625 cm’ ) and long aliphatic chain (820, 800 and710 cm-'). Its mass spectrum exhibited a molecular ion peak at rn/z 368 corresponding to a molecular formula, C2 ,H3gOg. A large number of fragment ions recorded in the mass spectrum displayed a uniform difference of 14 mass units and decreased in abundance with increasing the molecular weight supporting the aliphatic long chain nature of the compound. Furthermore, the absence of [M-Me] * peak suggested its straight chain nature. More intense clusters of peaks corresponding to C^H^^, (e.g. rn/z 69, 83, 97, 111, 125, etc.) in comparisonto C^Hj^^, peaks (e.g. nVz 85, 99, 113, 127, 141, etc.) indicated the acyclicand unsaturated nature of the compound. The ion peak at m/z 97 [ion a]* was formed due to generation of an unsaturated 8-lactone moiety. The ion peaks at

196

0

COOH

HO

m/z 198 (46,1)

- Me CH, OH

.A m/i 183(9.6)-

--- CO 4 m/z 170 (20,9)

m/z 152 (90.3)

- C O

m/z 124 (20.3)

CO.,,*n/z154 (14,4)- ->ni/z 126 (9.9)

Me

m/z 138 (6,4)

H.,0

- Cj., fission

C. Hj, O (ion a) m/z 107 (6,3)

-M e

m/z 93 (2.1)

-C O

m/z 65 (3.1)

-C O

ni/z 79 (8.4)

rn/z 136 (5.2)

CH COOHICHjOH

C,,Hg0 3 (ionb) m/z 89 (5.7)

-C H ,O H

m/z 58 (100)

Scheme MS-IV: Mass Fragmentation Pattern of Isomangterpenoic Acid (MS- i197

m/z 167 [ion b]'. 195 [ion c]', 70 [ion t>97]’ and 98 [ion o97]* suggessted theexistence of carbonyl groups at C-11. The presence of the hydroxyl groups at C-10 and C-8 was deduced from the ion peaks appearing at ni/z 173 [ion d ] ', 140 [ion d-2H20]', 143 [ion e]*, 125 [ion e4J/3 j‘ , 129 [ion f | \ 111 [ion f-

99 [ion g]', 98 [ion c-97]', 128 [Mnon e]*, 110 [128+1,0]-. 142 [Mnonf]'- and 124 [142-Hp]" (Scheme MS-V),

Firm evidence in support of the structure of this new substituted o'-lactone was obtained from its ’H NiViF spectrum of MS-6 wfiich displayed two one-proton each downfield signals at 5 6.40 (m) and 5,30 (d, J = 5,0 Hz) assigned toH-18 and H-19, respectively. A ^vo-pôton broad singlet at 5 4.10 was ascribed to oxygen-substituted methylene group of 5-lactone. Two one-proton ead'i signals at 6 3.90 (brs) and 3.60 (m) were associated correspondingly wifh H-10 andH-8 carbinol protons. A three-proton broad singlet at 5 0.86 was accounted to C-1 methyl group. The remaining methine and methylene functionalities appeared at 6 2.30 (3H, m, H-9, H^), 2.00 (2H, d, J '= 6,0 Hz H,-12), 1.60(2H, brs, hy and 1.20 (18H, brs, 9 x CH.J. Acetylation of MS~6 yielded adiacetyl product (MS-6a).

Therefore, the structure of eicosanyl lactone (MS-65) is formulated as eicos-18-ene~8,10-diol-11--one-20,21-olide. This is a new 6-iactorie product and contributes the first report o f isolatlori of a lacto iiic compound from the Mangifera species.

COMPOUND fVIS"7Compound MS-7, namely li-sitosterol glucoside, was obtained as

colourless amorphous powder from methanol eluants. It responded positively to steroidal glycoside tests. Its IR spectrum showed characteristic absorptions for hydroxyl groups (3450, 3400, 3300 cm ’)- Its mass spectrum displayed ion fragments of diagnostical importance at nv^ 413 [iVI-C ,H;,,OJ *, 396 ,255 [396-C,^H^3. SC]^ 213 [255-ring D fission]\ 198 [213 (213-^VIe]^ 183[198-Me]\ 163 [C^H^,OJ*, 180 and 141 side chain, SC]',suggesting glucosidic nature of the steroidal molecule which possessed C,, , saturated side chain.

198

o.0

C,, H, (ion a) m/z 97 (37.2)

O

rn/r. 167 (6.3)

0o

o

C,,, H,,03(ionb) m/z 167 (6.3)

/ \ /]OH OH

C H - CH., ■■■ CfH - ( C H ; ) ,_ -C H 3

C ,^ ,H , ,0 , ( io n d )/Ji'i? 173 (4.2)

- 2 H, O

m/z 140 (6.3)

0.^ .02 3 4I O 1 OH I OH

I (CH,),| - C I CM I CH, I- CH -|(C>i,),, CH3

C , ,H 3 ,0 . , ; [M ]n n ^ 368 (11.1)

6 \ /

(CK)^-ch,3 '

C ^H ,^ (ion g) n v i 99 (9.7)

OH

4 CH,-CH-(CH,)g-C> C , H „ 0 ( f o n e ) ' ’ '

m/z 143 (4,3)

OH

CH^(CH,),-CH3

C ^ H „0 ( io n O ni/z 129 (11.8 )

■'H,0

/7vk 111 (18,7)

m/z 125 (8.7)

Scheme MS • V ; Mass Fragmentation Pattern of Eicosanyl Lactone (MS-

199

The 'H NMF? sp6}ctrum of MS-7 showed a one-proton doublet at o 5 50 (J = 6.0 Hz) assigned to C-6 olefinic proton. A one-proton broad singlet at ft3.63 was ascribed to C-3 c:iarbino! proton. Tiie sugar protons appeared as broad singlets at 6 4.73 (H-T), 4.20 (H-6'a), 4,17 (H-6’b), 4,00 (H-2‘), 3,86(H-3' and H-4') and as a doublet at 6 4,50 (1H, J 6,5 Hz, H*5'), Themethyl signals resonated at 5 1.10 (Me-19), 1.00 (d, J = 6.0 Hz, Me-21),0.97 (Me-26, Me^27), 0,83 (Me-29) and 0.76 (Me-18). The appearance ofall these signals in the region 5 1.10 and 0.76 reflected that these functionalities were attached to the saturated caiiDons. Appearance of C-'29 re sonance? at 6 0.83 suggested 24F configuration of the ethyl group since 24S-ethyl group appeared slightly in downfield region at 5 0.88 (Fûbinstein et a!., 1976). Acid hydrolysisof MS~7 yielded a sugar moiety and fi-sitostero!. Thei sugar moiety was identified as D-giucose by direct comparison with the authentic sample. F'êrmethylation of lViS-7 with methyl iodide and then acid hydrolysis of the methylated produd gave permethylated glucose and (3-sitosterol.

On the basis of these findings the compound MS-7 was identified as fi- sitosterol--D-glucoside.

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ASSESSMENT OF VOLATILE COMPOMENTS OF M. fNDiCA FRUITS CULTIVAR ‘BANARSI GOLA’

the volatile components of fruits of 'E3anarsi (3ola’ <3re listed in Table 7 The method of identification and the percentage are given and the? components arranged in order of GC elution on DE3-1 column. Overall. 41 components were eluted, Quantitatively, as indicated from total peak arcea, the oil waej characterized by a high amount of rnonoterpenic constituents. In aii, 21 cxinstituents, comprising ca 96.22% of the isolate were positively identified. Among 15 rnonoterpenes, ca 82.33%, there were 11 hydrocarbons (ca 35,14%), 3 alcohols (46,02%), 1 ester (ca 1.17%) and 1 aromatic hydrocarbon (ca 2.04%), Terpinen-4-ol was the predominent characterized constituent (ca 30.32%), followed by 1-«,-terpineol (ca 13.65%), a-pinene (ca 13,98%), G-pinene (C3 5,44%} and l-lirnonene (ca 5.02%). Only two sesquiterpenes (ca 3,28%), tvuo aliphatic carboxylic acids (ca 8.33%), one aliphatic amine (ca 2.06%) and benzophenone (ca 0.22%) were present in the oil. There were 18 partially characterized components. The isolc-jted constituents were identified on the basis of their mass spectra in combination with Kovats in accordance with the literature (Jennings and Shibemioto, 1980; Swigar and Silverstein, 1981; Adams, 1995; Libery, 1991; Vernin and Petitjean, 1982; Andersson and Falcone, 1969). Two components remained unidentified.

ASSESSMENT OF VOLATILE COMPONEfsJTS OF M. INDICA FRUITS CULTIVAR ‘BOMBAY’

The identified components, percentage and method of identification of the volatile components of the fruits are listed in Table 8 and arranged in order of GC elution on DB-1 and SE-30 chromatographic columns. Analysis of isolates resulted in the complete identification of 44 compounds, comprising ca 93,28% of the total volatiies. In the table, the general elution sequence is confirmed by Kovats retention indices reported in the literature and to that of authentic samples (Jennings and Shibamoto, 1980; Swigar and Silverstein, 1981).

By far the most abundant class of compounds was the 16 monoterpenes

and 23 sesquiterpenes identified, comprising 39.65% and 38,27%, respectively.

216

... Essential oii "composition of fruits of■Mangifem mdica ciiftivar ' Banarss Goia'

Component RMD8-1

Rt-2 IK A re a

a-Pinene 4.72 3.54 928

Camphene - 3,73 93S

Dehydrosabinene - 3,96 948

Sabinene 5.65 4.01 959

E-Pinene 5.77 4.10 9 36

Myrcene - 4.21 976

A'’-Carene 6.69 4.82 1001

cx-Terpinene - 4.68 1006

p-Cyrnene 7.11 4.73 1008

l-Limonene 7,26 4.90 1015

V'T erpenene 8.24 5.42 1040

9.31

10.31 -

10.66 -

1130 ~

Borneol 12.43 7.71 1146

Terpinene-4-ol 12.92 8.04 1151

L-a-Terpineoi 13.48 8.32 1162

Octanoic acid 13.66 8,50

Bornyl acetate 17.50 11.00 1259

C H 20.31 13.3915 24trans-Caryophyllene 23.28 15.37

29.38 -

13.98

0,61

0,12

0.92

5,44

0.54

4.82

0.37

2.04

5,02

1,28

0,75

0,77

1,06

7,8

2.05

30.32

13,65

0.38

1.17

0.55

3.34

0.97

IT

GC4K, MS

GC-IK

GC4K

GC-IK MS

GC-IK, MS

GC-IK

GC4K, MS

GC-IK, MS

GC4K, MS

GC-IK, MS

GC-IK, MS

MS

MS

MS

MS

GC-IK, MS

GC-IK, MS

GC-IK, MS

GC4K, MS

GC-IK, MS

GC-IK, MS

GC-IK, MS

MS

217

Component RMDB-1

F*?t»2 IK Area !T

C,sH,.0 29.43 1,22 iViS

Spathulenol 29.87 19,77 0,94 GC4K, MS

ESenzopienone 31.34 19,91 0,22 GC4K, MS

32,11 1,96 MS

C.J'i,.,1 / 32.26 0,76 M53

33,27 0.76 MS

34.02 2.26 MS

37.16 1,98 MS

38,03 1,20 MS

40,79 1.63 MS

41.11 0,64 MS

1-Octadecanarnine 44.06 35,12 2.06 GC, MS

C„H3.0, 44.15 0,13 MS

44.22 - 1.23 MS

Hexadecanoic acid 44.95 - 7.95 MS

46,54 - 1.83 MS

Unknown 48.31 0,18 -

Unknown 48,37 0.25

Rt = Retention time on DB-1.

Rt-2 ” Retention time on DB-2.

IK = Fte4ention index according to authentic standard.

IT ■-= Identification tecl'inique.

GC“tK = Retention index acrarding to reported authentic compounds,

MS = Mass spectrum fragmentation pattern.

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GC-MS Analysis of Volatile Oil of Fruits o f M. indica Cultlvar‘BanarsI Gola’ (Retention time from 6.00 to 12.00)

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GC-MS Analysis o f Volatile Oil of Fruits of M. indica Cuitivar ‘Banarsi Goia’(Retention time from 12.00 to 19.00)

223

GC-MS Analysis of Volatile Oil of Fruits of M. indica Cultlvar ‘Banarsi Gota’(Retention time from 20.00 to 30,00)

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GC-MS Analysis of Volatile Oil of Fruits of M. Indies Cuitivar ‘Banarsi Gofa’(Retention time from 30.00 to 46.00}

225

of the total volatiles. Among the monoterpenes, six vêre the hydrocarbons (ca 32%) and six possessed bydroxy or carbonyl groups. Of particular interest arethe (/"pinerK'} (26.57%), B-pinene 2.1%), l~lirnonene (1,44%) and trans-vefbenol (1.95%). It is noleworthy that although five monoterpene alcohols were preserit, none of Iheir ester derivatives was identified.

The .single most abundant compound idc^ntified in ttiis .study was caryophyllen(-3 epoxide (ca 14,54%) arnong the 9 sesquiterpene I'lydrocarbons (ca 8,39%), 3 epoxides (ca 22,26)%} and 3 alcohols (ca 2.62%). The other prominent components identifie^d are humulene epoxide (7,33%), ft-selinejne (2.02%), a-copaene (1.38%) and caryophyl(enoi-2 (1,29%),

ASSESS.MENT OF VOLATILE COMPONEIMTS OF IW. MD/CA FRUITS CULTIVAR *DESr

The volatile oil corr^ponents (Table 9) were characterized by conparing Kovats indices and mass spectra. About 43 individual constituents, comprising 90,95% of the total isolates, were completely characterized.

Quantitatively, as assessed from total peak areas, the isolates possessed a high amount of monoterpenic hydrocarbons (ca 51,38%). p-Cyrnen-8-ol was the predominent identified component (ca 28.56%) of the volatile oil. The other important monoterpenes were A-’-carene (5.73%), p-cymene (2.88%) and 1- limonene (2.57%). The remaining 21 monoterpenes occur in trace? amounts.

Among 18 aliphatic constituents, compnsing 10.93% of the essential oii, the important compounds were (Z,Z)-3,4“dimethyl”2,4~hexadtene (5..17%o), (E:)-3- isopropyl-6-oxo-heptanol (2.59%) and (Z)-3,4,4,-trimethyl-5-oxo-2-hexenoic acid (2.95%).

The 10 benzene derivatives, identified were accounted for 41.3% of the total isolate. Although this figure was largely due to p-cymen-8-ol at 28.56%, the single most abundant compound identified among the volatile components of Dcssi variety in this study. The other main aromatic compounds were 1 "methyl-2

226

Tabie 8: Volatile oil coiTiponents of Miingif&'a incSica fruits cultivar liom bay ’

Compound m-GL Rt-MS IK YiekI%

Method

3-Hydroxy'2-biitanane 2.142,?r'Dihydro4-niettiyl furan 2.272-Butenol 3,35it-Pinene 3,55

ji-Octen-3-ône 3.732-lVlethy!-3-buten-2-o! 4.11

!',-Pinene 4,11I’l'Myrcerie 4.20

1.5-Heptadien-3.4-diol 4.28

p-Cyrnene 4.74l-L.irnonen€5 4.91E-fe-Ocimene 5.113.3.5-Trimethyl-1,5-lieptadiene 5.18

3-Methyl butyl propanoic acid 5.18

f>- Cy m e n €i- 8 - ol 6.063-(4-Methyt-3-pentenyl)fijrun 6.214-Methyl-1-(1-methylethyl)-bicycio

1M2,40

3.194.76

5.055,475.80 6.10 6.85 7,14 7.29 7,53 8,04

8,12 9.63

9.80

911925

937964964970976100510141 (324

102710271070

1070

0 41 0 04

0,10 26,57

0.65 1.05 2.10 0,80 0,1 0,84 1.44 0,25 0,14

0.13 0.83 1.34

GC-RT, MS G C ^rr, MS GC-RT, MS GC-fRT. MS

g c -r t , ms

GC4"?T, MS GC,:-RT, MS GC-RT, MS

GC-RT, MS GC-R'T, MS GC-F?T, MS GC-RT. MS

GC-F-rr, MS MSGC-RT, MS GC-RT, MS

[3.1.0] tiexan-S-ol 6.34 10,03 1084 0.57 GC-RT, MS

3-Methyl furan 6,49 10.15 1091 0,38 GC-RT, MS

((-Campholene aldo^hyde 6.63 10.75 1098 0,83 GC-F^T, MS

trans-Pinocarveol 7.09 11.30 1116 0.77 GC-Trr. MS

cis-Verbenol 7.09 11.30 TI16 0.77 GC-RT. MS

irans-Verbenol 7,20 11.55 1120 1.95 GC-RT, MS

l-ii-Terpineol 8.32 13.43 1162 0.96 GC-RT, MS

Verbenone 8,57 14.17 1171 Q.88 GC-RT. MS

Butanoic adcl,3-oxo~ 1,1-

dimelhylethyl ester 9,02 14.55 1188 0,37 GC-F^T, MS

3-{Acetyloxy)~2-propenenitrile 10.96 18,19 1258 2.40 GC-RT, MS

4-Methyl pentanarnide 11.27 19.11 1269 2,02 GC-RT. MS

13.41 20.68 1341 0.28 GC-Frr, MS

(i,-Copaene 14.12 2 1 .47 1364 1,38 GC-RT. MS

traris-Caryophyllene 15.36 23,32 1403 0,81 GC-RT, MS

227

Comf)otnuj

c ,„n ,„o

u-Guaicno

(.'■I'ltirnulene GJermacrene 0

S-Se!inene a-Selinene

(■>-(?iuaiene

1 -S-C is-C a I a me ne neCan/ophyllene epoxide isomer

LedoiCaryophylk?ne epoxide

u-Sanfalol Hurnulene epoxide

CEiryophyllenol-2

Tetradecanoic acid

Hexadecanoic acid

Rt-GL F«-IV!S IK Yield%

Method

- 23.B7 1 15 MS24,14 1419 0,5 fvIG

15,94 24.44 1421 0.4 1 GC-RT, MS16,34 24,75 1434 0 89 GC-RT, MS16.99 25,74 1454 0,67 GC-RT, MS17,29 26.14 1464 2,02 GC-F?T\ MS

17.59 26.50 1473 0,40 GC-RT, MS17.70 26,69 1476 0,53 GC-RT, MS

17,88 26.96 1482 0,63 GC-RT, MS

18.17 27,64 1491 0,56 GC-RT, MS19,05 28.83 1521 0,39 GC'RT, MS19.71 29.78 1543 0,91 GC-RT, MS19.91 30.14 1550 14,54 GC-Rl", MS

20.19 30.71 1560 0.42 GORT. MS20.64 31.15 1575 7.31 GC-FRT. MS

20.64 31.27 1575 0.82 GC-F-?T, MS21,27 31.88 1597 0,81 GC-RT, MS

21.92 32,88 1619 0.74 GC-RT, MS22.39 33,56 1635 1.29 GC-RT, MS

23.59 35.53 1677 10.39 g c -f?;t , MS

23,62 35.67 1679 0.32 GC-f^T, MS25.39 37.14 1741 0.29 GC-RT, MS25.87 38.60 1759 1.12 GC-FIT, MS

28.15 43.60 1842 0.80 GC-RT, MS

30.21 44.08 1923 0.40 GC--RT, MS

Rt-GL = Retention time on SEI-30.

Rt-MS -■= Retention time on D B-1.

IK = F?etention index according to autl-icsntic standards on DB-1and SE-30 chromatographic columns.

GC-F^T = Identical retention time and Kovat's index to authentic sample.

MS = Mass spectrum fragmentation pattern.

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232

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GC-MS Analysis of Volatile Oil of Fruits of M. indica Cultivar 'Bombay'(Retention time from 30,00 to 44.00)

233

(2-propenyl)-benzene (3,42%), p-OBSol (2.57'%) and 44.rirnethy! b(?nzene methanol (2,52%), The furan derivative occurred oniy in trace amount. Among 12 sesquiterpenes, comprising 11,23% of the volatile oil, the important ones wore caryophyllene; (3,29%),fl-serinene (148%) and I'lurnulene (1,40%)

A'jM JnliW ENT OF VOLATILE COMPOMEzNTS OF M. ilMDiCA FRLJirS CIJLTIVAF? ‘QALM!’

The volatile components of fruits of M. indica cultivar Qalnn are listed ■ Table 10. The identifie^d components, percentage and method of ;denltificatiO:-i are given and arranged in order of GC elution on DB-1 column. Analysis o: the isolate resulted in the identification of 56 components. Quantiiaiiveiy, as assessed from total peak aretes, the oil was characterized by a high :irnount of monoterpenic constituents. In ail, from 33 rnonolerp£?ne components, comprising ca 91.45%) of the isolate, there were 13 hydrocarbons (ca 76.4%), 10 alcohols (ca 7.08%), 4 ketones (ca 2,21%), 2 alde^hydes (ca 1,4.5%), 4 phenols (ca 1,95%) and 1 ester endoboryl acetate (0,36%). a~Prne?ne v ass the predOiTiinent characterized constituent (ca 34,54%), followed by sabineine (ca 13.94%), fj-pinene (ca 12.55%) and l~lirnonene {ca 9.06%o). With ih€?ir terpenthinate flavour^ the rnonounsafurated bicyclic hydrocarbons a- and (l-pjnenes contributed to the arorna of a variety of oils. Only three sesquiterpenes, viz.longipinene (ca 0.70%), spathulenol (0.28%) and caryophyffene oxide (ca 0.44%)) were present in the oil. Of 56 components in the oii, 41 of them, comprising 93.58%> of the sample, were identified on the basis of their mass spectra in combination v /ith Kovats indices in accordance with the literature (Jennings and Shibarnoto, 1980; Swigar and Silverstein, 1981; Adams, 1995; Libery, 1991; Vernin and Petitjean, 1982; Andersen and Falcone, 1969), Fifteen components remained unidentified. A majority of the components, i.e. 29 constituents, were found in concentralions below 1,0%. Among the compounds with miscellaneous structures, methyl cyclopentane and n-decane have been de tec ted fo r the f irs t time in Magnifera species.

It is remarkable to note tfiat the separation of monoterpene hydrocarbons IS generally most efficiently performed on non-polar columns in which these compounds have a greater liquid phase solubility. Lirnonene co - eluates with 1,8"phellandrene on the carbowax 20M column. Since the fruit volatiles does neither contain 1,8-cineoie, nor (i-phellandrene, the quantitation of lirnonene was not a problem,

234

Tabie 9: Volatile oil cofiiporierits of Manglfwa iridiQs fruits C4,ilt!¥ar 13asi’

Component

2,5'Hexadiene

(x-Pinene

Dehyclrosabinene

3-Propy! cycSopentene

B-Pinene

fl-Myrcene

,A‘‘-Carene

/\'^-Careoe

p-Cymene

l-Limonene

p~Cresol

1 ~Methyl--2-{2-pfopenyl) benzene

3-Methvl phenol

4-Ethenyl-1,2<jirnethyl bejwene

1-Ethenyf-3,5-d)metfiy( benzene

Cyclooctanone

C„H„0C„H„0^>Cymen-8-ol

4-Trimethyl benzene methanol

Octanoic acid ethyl ester

C,oH„p,3-(2-Furanyl)~v'i-penten-2-one

(E)-3,7-Octadien-2-one

{2,Z)-3,4-Dimethyh2,4.-

hexadiene

Rt^GL Rt-MS IK Yield%

Method

2.11 4,40 0,10 GC..f^rr, MS

3-55 4,74 9,25 1,26 GC-RT, MS

3,72 5,62 937 0,07 ’■'C-RT. MS

3,95 5,95 953 0.79 (7. :-Frr, MS

4.11 f»64 0,19 G ': RT, MS

4,21 6.10 971 1.53 Gc >rr, MS

4,46 6.43 988 0,92 GC.f<T, MS

4,S3 6.73 1000 5,73 GC-R i', MS

4,74 7.14 1005 2,88 GC-.R'T, MS

4.91 7,29 1014 2,57 GC-RT

5,96 8.88 1065 0.61 GC4^T, MS

6.06 9,35 1070 3.42 GC-RT, fulS

- 9.72 , 0.22 MS

„ 9,91 0.44 MS

10,20 - 0.45 MS

6„96 10,85 1111 0.26 GC4-?T. MvS

- 11,12 - 0.17 MS

- 11.54 1119 0.92 MS

- 12.83 0.32 MS

8,01 13.40 1150 28,56 GC-RT, MS

8.41 13.54 1185 2,52 (3C4^T. MS

9.02 13.72 1188 0,60 GC..P.T, MS

14.00 „ 1,90 GC-RT, MS

9.53 15.96 1207 0,13 GC-RT, MS

10.30 16,90 1213 0,23 GC-RT, MS

10-71 17.45 1249 5,17 GC-f^T, MS

235

Corriponetif

3,4- D i rn e t h y 1 - 2,4 - h o x a die .n e

trans-2-E;thyl-1 ,1 '-bicyclohexy!

6-Mefhyiene spiro [4 .5] decane

3-(1,1-Dimethylethyl)phenol

{1 ,1-Dime!hyiethyl) methyl

benzene

{E)-3-iso propy 1-6-oxo-■2-h e pte n a I1-fv1ethyl“4~(2-rTietfiyioxy)-'

7-oxabicyclo [4.1,0] heptane

(E,Z)-3,4-Dimethyl-2,4-hexadiene

(Z)-3.4,4-Trimet:hyt--S--oxo-2--

hexenoic acid

2-Methyl-3-(1-rnethylelheriyl)

cyclohexariol acetate

tranS'ft-Caryophyllefie

C ,oH ,,0 ,

(E)-3-lsopropyl~6-oxo~lieptenai

fi-Selinene

C„>i„

f«-£3L Rt-MS IK%

Method

11.16 17,66 1265 0 53 GC-R17 MS

11.80 18,00 1282 0,21 GC-RT, MS

12 02 18,11 1296 0,32 GC-F^T, MS

12.30 18 51 1305 i,,:vi GC RT, MS

18,64 1,73 V71S

18.97 0 24 MS

-19,6719.79

1,760,89 K' •

12,50 20.21 1312 8.39 GC - n \ MS

13.95 20,74 1352 0,52 MS

14,14 21,16 1,364 0.39 GC-R i . MS

- 21,44 0.34 MS

14.41 21.82 1373 1.46 Gc-Frr, r,'is

14.57 22.52 1378 1,18 GC4^T, MS

22,63 - 1.26 MS

- 23,31 1393 1.87 MS

- 23,67 - 2.95 MS

.. 24.32 . 0,63 MS

24,56 - 0,55 MS

15.35 24,75 1403 0.29 GC-RT, MS

16,05 25.38 1425 1.14 GC-RT, MS

■■ 25,S7 2,59 MS

16.52 26,14 1439 1.48 GC-RT, MS

17,30 26,27 1464 1.37 GC T' T, MS

-- 26.42 1.16 MS

18 00 26,93 14,8f3 0.17 GC-R1", MS

236

Componens

C,,H„

C a ry 0 phyll e n e e po xi (I a

HurriuJene cjpoxide

TetradectMioic acid

C„H,,0c.,K„o1 "Hexadeone

Hexadecanoic acid

P e n t a methyl disilo xa n e

b(s(2-Ett'iylhexyl)^1,2-

bfjnzenedicarboxylic acid

Rt-GL Rf-MS IK Vsoid%

Method

18,57 28 33 1 504 0.18 GC.R'T.

18 78 29,26 1511 0 1 4 GCRT. MS

19,92 30,12 1,550 3 29 GC7RT, MS

20,35 30.70 1565 0.10 3C-RT, f,4S

20.65 31.14 1575 1.40 ."1C-RT, MS

21,27 35,50 1597 0,19 - : : -RT. MS

21,50 37.09 1604 0.36 '-RT, MS

22.40 38.56 1636 0.60 G r RT , MS

38.85 0.39 G C 'T, MS

23.6 41,13 1677 0.84 G C - - ' MS

25.90 4185 1760 0,48 GC-^R : MS

26.53 44.04 1783 0.13 G C - R T fwlS

27.29 44.40 1810 0..')6 G C - R T , >,1S

28.13 45.80 1827 0.68 G C - R T , r-’S

28.30 46.39 1847 1.52 G C - R T , MS

bi s(2 , fc th yl h exyI) b e nzen e-

dicarboxylic acid isomer

Rt-GI„ = Fêtention time on SE--30.

F t-MS = Ftetention time on DB-1,

IK = Ftetention index according to auihentic standard's

on DS-1 and SE~30 cFtrorriatogr'aphic columns

G C -R T ~ Identical retention time and Kovat’s index to authentic compound.

MS = Mass spectrum analysis.

237

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GC-MS Analysis of Volatile Oi! of Fruits of M, md/ca Culfivar ‘Desi’(Retention time from 0.00 to 45.00)

239

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GC'MS Analysis of Volatile Oil of Fruits of M, ind ica Cultivar ‘Desi’(Retention time from 2.00 to 6.00)

240

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GC-MS Analysis of Volatiie Oil of Fruits of M. ind ica Cultivar 'DesF(Retention time from 2.00 to 6,50)

241

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GC-MS Analysis of Volatile Oil of Fruits of M. indica Cultivar ‘Desi'(Retention time from 2,00 to 12.00)

242

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GC-MS Analysis of Volatile Oil of Fruits of M. indica Cultivar ‘Desi’(Retention time from 2.00 to 14,00)

243

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GC-MS Analysis of Volatile Oil of Fruits of M. ind ica Cultivar ‘Desi’(Retention time from 13.00 to 20.00)

244

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GC-MS Analysis of Volatile Oii of Fruits of M. mc/Zca CuUivar ‘Desi’(Retention time from 13.00 to 28.00)

245

Ta?-)fc' ■fD.' ",Cli8micat eonstltiients of ti© volatile oil of fruits of MatigifBm imiics Ciiltivar T almF '

Compound Rt-1 Rh-2 K

Meihyicyclopentane

Benzene

(i-Tbujerje

(i-Pinene

Carnphene

Dehydrosabinene

Sabinerie

(J-Pinene

B-Myrcene

G „H„

n-Decane

A-'’-Carene

(/-Terpinene

p-Cymene

3,46

3,56

3.75

3,89

4.04

4.14

4,22

4,29

4,48

4.64

4.75

1,59

1,71

4,58

4,85

5.10

5,29

5,78

5.91

6.12 6,21 6,33

6.74

6.91

7.18

918

928

939

948

959

966

976

976

990

10011006

1008

Area

0,44

0,20,89

34-54

1.020.0213,94

12,55

0.84

0.020.212,09

0,103,06

Mefhod

iVtSMSGC-IK. MS

GO IK, MS

Gc -S'C, y s

GO--;':, (vis

GC-IK, MS

GC-1K, MS

GC-!K, f,lS

GC-1K, r.^3

GC-1K, fv S

GC-1K. IVIS

GC-1K, MS

!-Lirnonene 4.93 7,39 1015 9.06 GC-IK, MS

7-Terpinene 5,44 8.25 1040 0.11 GC !K. MS

ds-Sabinene hydrate 5.55 8.62 1045 0,57 GC-iK, iMS

trans-Sabinene hydrate 6.17 ,9.77 1075 0,67 GC4K. y s

C,oH,3 6,35 10.06 1084 0.30 C5C-1K. MS

<i-Campholene aldehyde 6.64 10.77 1088 0.57 GC-iK, MS

cis-p-Mentha-(x-en-1 -o( 6.74 11.21 1110 0.22 GC-IK, MS

cis-Verberiol 7.09 11,37 1116 0.93 GC-IK, MS

1-Methyl-2-1-methylethyl) benzene 7.18 - 3.06 M S

trans-Verbenol 7.21 11,67 1120 1.44 GC-!K, MS

Sabinacetone 7.46 12.07 1130 0.42 GC-IK, MS

Pinocarvone 7.71 12.25 1139 0.71 GC-IK, MS

246

..........—----- ---- - ... .........—.. ...- ........ -..... .......Compound Rr-2 IK Araa !VlGtS'!CK!

Borneo! 7,91 114(3 0,58 75 C - .'

Terpinen-4-ol a,04 12.96 1 151 1,56 3C IK. M::

rn-Cymen-8-ol 8 15 13 16 1155 0,08 ■,1C If- , Ml

13 26 0,52 f 1' S

S>Cymetv8~ol 8.26 13.31 1159 0,67 r.'M K

1 -/-Terpinenol 8,32 13,53 1162 0,18 G', IK

Myrtenal 8.42 13,68 1106 0,83 Gc: 4<

Myrtenol 8.49 13,32 1168 0,88 GC '■

Myrtenotie 8.57 14,31 1171 0,79 GC-i-'

trans-Car'êol 9.01 14,75 1183 0,58 GC4r-

Carvone 9,50 15,69 1206 0.29 GC4K

C,oH,.0 9.69 16.04 1213 0 14 GC4K

(E)-2-Decenal 9.99 16.99 1223 0 21 GC4K

5ndo'borny! acetate 11.00 17.53 1259 0.36 GC4K

Thymol 11.11 17,90 1263 0,14 GC-1K

Ck,h ..o - 18.26 1,33 MS

Crol 11,59 13-80 1270 0,71 GC4K

C,oH,A - 19.07 1270 2,62 MS

C,oH,,.P 11.30 19.33 12,81 0,24 GC4K

11,88 19.,5 7 1288 0,52 GC4K

C,oH,.0 ■■ 20.22 1,16 MS

Longipinene 13,96 20.34 1258 0,70 GC4K

21.73 0,45 MS

C.oH„0 22.01 1,56 MS

Cu,H,A 22,41 0,49 MS

26,44 1,62 MS

Spathulenol 19.91 29,98 1544 0,2,8 (3CîK

Caryophyllene oxide 20,63 30,40 1550 0,44 GCMK

247

Compound FU-1 Hi~2 JK Ars?a IVlG'Uiod

2-Nilrophenol 46.30 • 1 06

■ 4 6 , 3 8 1 01 ' /IS

R{-1 -- F?x'tention time on SE-SO,

Rt-2 Retention lim e on DB-1,

IK = F?elentioii index accordmy to aulluintic stamJatds

GC-IK Identical retention time and Koval’s uidox to auttnMitir; compound.

M S = Mass spectrum fragmentation pattern.

248

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GC-MS Analysis o f Volatile Oil of Fruits of M. indica Culfivar ‘Qaimi’(Retention time from 5,00 to 45.00)

250

ASSESSMENT OF VOLATil„E COMFÔMEMTS OF M. INDiCA r-TllJlTS CULTIVAIl ‘LAMGRA’

The resultant isolate of the fruits of M. indica cultsvar Langra on appropriate dilution possessed strong distinct !tirpen!inc3'-like odour.

The components identified?lhe percentage of eacti aarnponent and method of identification are listed in Tab!e 11, The components are arranged in order to GC elution on silicone columns SE-30 and DB-1, Analysis of the volatile by GC and GC-MS resulted in the complete identification of 34 com ponents comprising ca 95.75% and six tentatively characterized compounds (ca 4.25%).

Fused silica SE-30 capillary column was used, containing either bonded phase carbowax 20IV1 or .silicone OV-1. Kovats retention irsdict^s o f the components are also listed in the Table 11 and confirrnejd the general esiution sequence. Quantitatively, as assessed from total peak areas thej voiatile or! vva,s characterized by a high amount of monoterpenic hydrocarbons (ca 76.66%), car- 3-ene was the predorninent characterized constituent (ca 61.71%) of the isolate. The notable presence of car-3-ene is a distinguishing facet yvtndi is a weil-known aroma component of P/nus species.

a-Terpinolene (ca 4,32%) and l-limonene (ca 2,43%) were the two other important monoterpenes. Among 10 sejsquiterpenes comprising ca 8.21%, there were eight hydrocarbons (ca 7.46%) and two alcohois (ca 0.75%). The major sesquiterpene was B-selinene foHowed by ti~ans-caryophyHene and cx-humulene^. Among 21 aliphatic compounds, amounting about 25.38%, there were 13 esters (ca 15.59%), 5 acids (ca 8.09%), 2 ketones (ca 0.76%) and 1 aldehyde (ca 0.70%).

The prominent aliphatic component was 9--hexadecenoic acid followed by octanoic acid ethyl ester, hexanoic acid ethyl ester, hej,xadecenoic acid ethyl ester £)nd tetradecanoic acid ethyl ester,. The levels of B--myrcene. a-terpinolene and 9-hexadecenoic acid were almost identical.

2S1

Table'll: Perceritag© coffiposiliori of tlie volatile oil of Mang/fera imiica fruits ciillivar *l.afigra*

Compound Rt~GL Rt-MS IK

E5u!anoic acid ethyl ester

2-'Melhvl'propeny! 1 -methylethvi

2.24 2,54

Yield %

900 012

2,2’-Melhylene bis [5-rnethyl]

Method

GC'RT IV1S

acetate 3.05 0.16 MSri-Pinene 3„56 4.76 925 0 40 GC-F?T, MSB-Myrcene 4,20 6.13 959 4 15 GC4?r. MS

Hexanoic acid ethy! ester 6,33 969 3,11 MSl-F^hellandrent? 4.50 6.57 991 0,54 GC-RT,, MS

\'‘-Carene - 6.84 10(31 61,71 MS1-Lirrionene 4-92 7,32 1014 2.43 GC-RT, MS

2-Methyl pfopionic acid 5.20 e.14 10.23 4,32 GC-RT, MS

ct-Terpinolene 6.06 928 107(3 0.47 GC -RT, MS

EW'iyi c;is4"(X';tenoate a.2 7 13.38 1160 0 23 GC-RT, iViS

Octanoic acid, ethyl ester 8,49 13.79 1168 3,69 GC-RT, MS

furan 13.10 17,13 1331 0,08 Gc-F-rr, MS

Ethy! cis-4-clecenoate 13.85 21,67 1355 0 14 GC-RT. MS

E-3~(3-Methyl'1-butenyl)-

cyclohexene 14.13 21,76 1364 0.16 GC-RT, MS

'Decanoic acid, etiiyj ester 14,38 22,42 1372 0.99 GC -RT, MS

trans-Caryophyllene 15.36 23,39 1403 2,10 GC-RT, MS

ii-Hurnulene 16.35 24,81 1434 1,05 GC-RT, MS

y-Gurjiinene 16.84 25.46 1450 0,3 GC-F?T, MS

Gerrnacrene 17,13 25,96 1459 0,13 GC-RT. MS

(.ii-Selinene 17.30 26.23 14.64 3.15 GC-RT, MS

Valencene 17,52 26.46 1471 0.16 GC-RT, MS

1/,-Salirjene 17.60 26.55 1473 0.37 GC-RT, MS

F’entadecane 18,03 26.73 1487 0.15 GC-F^T, MS

trans-Octahydro-1 H-inden-1 -one 29.56 0,30 MS

Dodecanoic acid 29,80 0,66 MS

Dodecanoic acid, ethyl ester 20.39 30.68 1566 1.21 GG~RT, MS

Ledol - 32,77 . 0,27 MS

252

Cotnpou nd R.t-GSL iK Yield Metlitxi

Veridiflorol - 32,93 0 4H

2-Tndecanorie 23,70 34,60 1B30 0 24

Tetradecanoic acid „ 37 49 - 1 84

Tetradecanoic acid, ethyl ester 26.03 38,29 1764 2 32

Octadecanal 38,91 ■ 0,70

7-Hexadecenoic acid ethyl ester 28.16 41,24 1842 0 51

2-Heptadecanone 28,35 41,93 1849 0 46

14-Methyl pentadecanoic ,acid,

mefhyl fjster 28,84 42,74 1866 0,20

9-Hexaclecenoic acid 43.78 4.03

Ethyl 9-hexad€3cenoate 30,38 4.4,32 1928 0.28

hlexadecanoic acid 30,38 44.78 1928 1.09

Hexo-decanoic acid, ethy! ester 31.22 45,23 1967 2.47

FJt-GL = fêtenlion time csn SE-30,

Rt-MS = Fêtention time on DE^d

IK = Retention index according to autherilic .standards on

MSGC'.RT, MG

M S

GG-Frr. MS

MSiSC-RT, MS

GC.RT, MS

GC-RT. MS

MSGC-RT,, MS

GC-FH', MS

GC-F^T. MS

DB-1 and SE-30 chrorTiafographic columns.

GC-F?T = Identical retention time arsd ,Kovat';,', if'sde,x to authentic cornpound.

MS = Mass spectrum analysis.

253

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GC-MS Analysis of Volatile Oil of Fruits of M. //jc/ZcaCultivar‘Langra'(Retention lime from 0,00 to 45.00)

255

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GC-MS Analysis of Volatile Oil of Fruits of M. /wi/ca Cultivar 'Langra'(Retention time from 2.00 to 10,00)

256

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GC'MS Analysis of Volatile Oil of Fruits of M. mof/ca Cultivar ‘Langra’(Retention time from 10.00 to 26.00)

257

. \ b u n d a n c e

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GC-MS Analysis of Volatile Oil of Fruits of M. mcfica Cultivar ‘Lancjra’(Retention time from 28.00 to 38.00)

258

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GC-IVIS Analysis of Volatile Oil of Fruits of M. ind lca Cuitivar *Langra’(Retention time from 39.00 to 46.00)

255

ASSESS WENT OF VOLATILE! CO MP U i'U Û'V, f f\ M. INDiCA FRUITS CULTIVAR ‘SAFEDA’

'ThG com ponents ider'itified, the p-ercenlage composition o f eacti cxjnstituent,

and method of identification are stirnmarized in 'Tabie 12, Anaiysis of lh€5 isolate by Kovais retention indices; and GC-fvIS analysis ressult-ed in the idenlification of50 components comprising 89,73% of the total volatilcis. Quantitatively, as assessed from total peak areas, the isolates v/ere characterized by a high amourii of sesquiterpenes (ca 51.08%). AiTiong 22 sesquiterpenes 11 cornporienls (46.23) were completely idesntified and the predorninent crjmponent was caryophyllene oxide (15,0%), It was followead by fi-seiinene {13.16%), hum ulene

epoxide (8.0%), transoryophyllene (2.69%), a-hum ulene (1.91%) and y-gurjunene (1.44%). Only four rnonoterpenes, comprising 3.68% of the volatile oil, we-re identified as a-pinene (3.88%), trans-ocimene (1.75%), B-myrcene and cis-ocirnene.

Of greater relevance was the positive identification in thi.s work of .28 aliphatic constituents representing as a proportion as 22.25% of the? Safeda sample. Among these constituents there were 5 aliphatic ketones (0.71%), 4 aldehydes (11.41%), 3 alcohols (1.09%), 2 amines (1.04%), 2 acids (1,04%) and10 esters (8.4%). The important feature of these constituents was the presence of sulphonyl bis methane, 2,2'”(methylimino)”bis ethanol and pentarDide.

Bisaboiene occurred in trace amount. This compound was originally isolated from the essential oil of cotton buds. It exhibited an apple blossom- like odour. Aromatic compounds identified were N-ethyha-methyl benzene ethanarnine; 2,3-dihydro-4~methy! furan was the only furan derivative.

ASSESSMENT OF VOLATILE COMPONENTS OF M, iNDlCA FRUITS CULTIVAR TOTAPARf

By means of Kovats retention indices and GC-MS fragmentc^tion pattern,51 components comprising 75.98%, were identified in the fruit pulp and rind, The011 contained 23 aliphatic constituents (15.63%), 23 rnonoterpenes (41.4%), 25 sesquiterpenes (29.87%) and 14 compounds with miscellaneous structures were

260

found for the first time as constituenets from mango fruit ctiltivar T'otapari. The chromatographic and spectr0scx)pfc data of these compounds are represenfed in Table 13. The constituent..s are arranged in order to GC elution on silicone colurrins SE-30 and DB~1„ Fused silica SE-30 capillary column was used, containing either bonded phase rarbowax 20iVI or silicone OV-1.

Quantitatively, rnonoterpenes played a decisive role among the Tbtapari fruit volatiles. In ail there were six rnonolerpenc} hydrocarbons (7,55%);,three aldf^hycies (3,37%) and one ketone (103%). The predorninent rnonoterpene was a-pinene (19.74%) followed by a-terpineo! (3.26%), ft-pinene (2,46%), rx-copaeno(2.36%), limonene (%) and verbenone (1.03%). The oil contained about 15 sesquiterpene hydrocarbons (12.04%) and 3 epoxides (12,67%). The major sesquiterpene was aaryophyllene epoxide (7.81%) fdiowed by hurnulefie epoxide (4.60%) and fi-selinene (3.0%), Among aliphatic constituents there were 3

alcohols (3.36%) and 5 carbony! compounds (2.82%),

261

Tabfe.i2: Volatile oil compositiofi of Mariglfera Imfica ffiills ' CLiltiVar ,‘Safecla*

Compound Rt-GL RI'-MS IK

3--Hydroxy-2-bulanone2-Me!hyf-2-buter('t-ol2.3-Difiyci rO'4 -rTie!, hyl fu ra nButonoic ack! elhyl ester2-PentanoneHexane2-PentanoriePenlanal2,2-Dimelhyl'3~bulenoic acid2-8utenaiSulphonyl bis methane a-F-’mene3-Ethoxy-3,4-dimethyl-1-hexyrt€) Methyl butenol1 -Methylene-4“(1 -rneihylethenyi)cyclohexaneR-PineneS-Myrcene2,2'-(Methylirriino) bis-eihano!Butanoic acid 2-hexenyl estercis-Ociinenetrans-Ocimene(E)-2,6-Dimethyl-octatriene-3-one(E)-6-Methyl-3,5-heptadien-2-oneOcianoic acid ethyl ester C,oH„,02-ethy( butanal N-EthylHx-methyl benzene ethanamine (Z)2'Butenediamide 1-Octadecanamine

C,aH„07--Hydroxy-3,7-dimethyl-octanalPentanamide

Decanoic acid ethyl ester tra{is-(?r-CaryophyllGiie

Ck,HmO

2.14

2,212.472.702.93

3,32,3,54

4,114.20 4.74

4,915,16

7.21

8.32 8.48 8.57 9.64

9.7910.1210.4210.9411,1512.32 13.4014.33 15.35

1,902.32 2,41 2,54 2,61 2,70 2„81 2,38 3.04 3.20 4,23 4,75 .5,05 5.43

5.80

6.116,296.857,537.89

9,62

9.8913.6914.17 15.64

15,9216.07r/,3518.1919.1819.3220.6922.33 23.32 24,44

8008038348S4

909924

S64,9701005

10141026

1162 t168 1171 1211

121612281243125712651306134013701403

Yield

% ....0,300,170,521,690,250,100,160,420,770,750,423,880,520,82

0,220,510,700,100,670,721,75

1120 0,45

0.390.570,322,12

2,370.510,293,363.211,390,690,4,92.692,26

y e t hod

MSGC'-RT, MS GC-RT. MS GORT. MS GC -RT, MS GC'-RT. MS GC-RT, US GC-RT., MS MSGC-RT, MS GORT, MS GCO-?T, MS 1V1S

MSGC-R'T. MS GC-RT. MS GC-RT. MS MS(5C-F?:r, MS GC-RT. MS

GC-RT, MS

GOFTf, MS GC-RT. MS GC-RT. MS GC-RT. MS

GC-RT, MS GC-RT, MS GC-RT, MS GC-RT. MS GC-RT, MS GC-RT, MS GC-RT, MS GC^RT, MS GC-F?T, MS MS

262

Compound Rt-GL Rt~IV!S

(i-Humuleney-GurjuneneS-Se!ineneValencf3nei/,-S€;!inene

CaryophylleDe epoxide isomer

CuryophyllenQ oxide Dodecanoic acid etfiy! ester

i;t"Bisabolol Humulene epoxide

C„H„P

Caryophyllenol-2

1-Dodecanamine 2,3-Dirnethoxy pcapionic acki methyl eater

Tefradecanoic acid elhyl ester 7-Hexadeceno(c ethyl ester Etliyl-9'-hexadecenoate Hexadecenoic acid Hexadec€!noic acid ethyl ester

16.3416.83 17,29 17,52 17,5517.83 18,09 19,04 19,23 19.9220.35

20.64 20,80 21,29 21.42 .21,82 22..3 9

23.5823.96

24.7624.97 26 .,02 28,17 30.4030.58 31.21

24.7624.4325.1526.43 26,51 27,42 27.75 28.83 29,59 30,09 30,58 30,7031.0131.1231.89 32.05 32,19 33.04 33,5733.89 35,5436.01

,37,0137.1337.1341.1644.13 44,2745.01

IK

14.34 1449 1464 1471 1471 1481 1489 1520 1527 1550 1565

157515801597160216161635

16761689

1718172617641842192919371966

YiGfd7„

1 91 1 44 13,16 1 11 M l 0.42 1.00 0 30 0 2615.00 1.24 0,38 0.443.00 0.31 0.71 0,34 0-461.07 0 671.07 0.37

0210..331,660,670.190.271.D1

ftflethod

GC-.R1', MS GC-RT. MS GC-flT, MS GC-RT. m GC-RT, MS GC-RT, MS GC-RT, MS GC-FTT’., MS GC-RT, MS GC-RT, MS GC-r?T, MS MS MSC5C-T?T, MS (SC-RT, MS GC-RT, MS GC-RT, fvIS C5C-RT, MS GC-RT., MS GC-RT, MS GC-RT. MS MS

IV1SfvISC3C-RT, MS GCT-RT, MS GC-f-rr, MS GC-RT, MS GC-F^T, MS

R t-G LRt-MSIK

GC-RT

Retention time on SE-30.Retention time on DB-1.Retention index according lo authentic statidards on DB-1 anci SE-30 chromatographic columns.Identical retention time and Kovat’s index to authentic compound. Mass spectrum analy;.ys.

263

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264 Q Z ' S l J ' i ' T S 6 / 6 B / 3 2 i D 3 f H I y

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GC-MS A n a lys is of V o la tile Oil of Fruits of M. ind ica Cuitivar ‘S a feda ’(Retention time from 0.00 to 45,00)

265

2 .J5 4 3 l l ' C 0 I

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GC-MS A n a lys is of Volatile Oil of F ru its o f M. /ric/fca Caltivar ‘Safeda'(Retention time from 2.00 to 8.00)

266

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23 0000

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GC-MS Analys is of Volat i le Oi l o f Fru i ts o f M.. /V)d/caCui l iva r 'Saf©da ’ (Retent ion time from 8.00 to 20.00)

267

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GC-MS Analysis of Volatile Oil of F-'ruits of M, i/ id ica Cultivar ‘Safeda’(R e ten tion tim e from 22.00 to 30.00)

268

SEundarice^

27 0000

260000

250000

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, 2 3Q000

; 2 2 0 0 0 0

: 210000ij 2 0 0 0 0 0

I 190000

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: 160000

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GC-MS Analysis of Volatile O il o f Fruits of M. indica Cuitivar ‘Safecia’(Retention time from 31.00 to 37.00)

269

34 0 0 0(3

i 3 3000 0

3 200 0 0

Abundance

3 5 0 0 0 0 -TIC;" ' INI3IA- C. D

41 16 44:. i.:i 4 5 i 0 1

,3100 0 0 38 .1

300000

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; 2 80000 ;1

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200000

190000

180000

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160000

150000

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: GC-MS Analysis of Volatile Oil o f Fruits o f M. incffca Cultivar 'Safeda' (Retention time from 37,00 to 45.00)

270

Talit#:'f3:: Voiatite -oil components o f MamjifBra indies ' fryfis c tiliiva r Totapari*

CornpoLind

2,3-Dihyd rtJ-4-methyl furan

N-2-Dirnethyl 1 -propanarnine

Butanoic acid ethyl ester

Fêntana!

N-(2-'Methylpropyl)- fon'namide

2-Octanamine

M-fvlethyi ethanarr«ne

3 -■ M 6t hy I -2 (5 H) -f u ra n o e

a-Pinene

2-Propenoic acid octy! ester

1,1-DirnetSioxy heptane

G.-Pinene

B-Myrcene

2-Methylamino ethanol

Butanai

3-Penten-2-ol

p-Cyrnene

Liinonene

trans-O cim ene

3,3,5-T fimeJhyl-1.5-hepladiene

Hornarine

4-Cyclohexyl 2-butanone

2,3,5,6-Tetramethyl phenol

3-(4-M ethyl~3-pentenyl)-fura/i

R t -G L Rt -MS

2,15

2,22

3,55

3.73

4.1

4.20

4.62

4.73

4.90

5,12

5.62

5.85

6.20

2.39

2,53

2.58

2.71

2,86

2.92

3.01

3.18

4.72

5.05

5.50

5.77

6.08

6 .1?

6.50

6.89

7,11

7,25

7.50

8.02

8.75

9.34

9.59

9.77

IK

%

800

803

872

925

937

963

970

976

999

1005

1013

1024

1049

1060

1070

1077

Yieid

0 25

0.39

0.51

1.09

1.31

0.52

1.10

2,81

19.74

0.56

1.24

,2,47

0,45

1.27

0.16

1.2.1 0.98

1,03

0.17

0,20

0.10

0,70

0,15

1.95

Mettiod

MS

GC47T. MS

MS

f'4S

MS

MS

MS

GC-RT. MS

GC-R'r, MS

C:3C-RT, MS

GC-RT, MS

GC-flT. MS

MS

MS

MS

GC-R17 MS

GC-RT. MS

QC-RT, MS

GC-RT, MS

GC-RT., MS

GC-RT, MS

GC-RT, MS

271

Cornpou ncl C3LC

RM3L Rt4/lS G C M S

iK%

Yh?! M e th o d

O clan at

c is -3 -Cyclopentene-1,2-cliol

C „H O’ 0 !Carnptiolenal

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6,34 10.03 1084 (,} 3 1 GC-Rl'3 MS

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10.31 ■■ 0 44 MS

6,63 10,72 1098 0 83 GC-RT. MS

7,08 11.29 1115 0,68 GC-RT. MS

7,20 11,54 1120 1.86 MS

3.04 13.19 1151 1 OR GC-RT, MS

8,32 13.43 1162 3.2fi GC-RT. MG

8,48 13.58 1168 0.25 GC4R'r, MS

13.65 0.36 MS

8,56 14.18 1171 1.03 MS

9,02 14.70 1188 2 48 GCM^T, MS

9.16 14.97 1193 0,82 C3C-R"T. MS

9,66 16.32 1211 0.30 GC- RT, MS

9.98 17.08 1223 0,44 GC4?T, MS

10,78 17.23 12S2 1 07 GC-RT. MS

11,02 17,48 1260 0,88 GC-m\ MS

11.32 18.22 1,271 0.56 GC--RT, MS

12.45 18.37 1310 0,62 GC-Frr. MS12.80 1926 1321 0,30 GC-RT. MS

13,21 19.76 1334 0,15 MS

13,44 20.79 1342 0 62 MS

14,11 21.41 1363 2,35 GC-RT, MS

23.57 .. 0 56 MS

1535 - 1403 0.17 GC-RT, MS

15.67 23.93 1413 0.65 GC-RT. MS

272

Com poundGLC

Rt*GLGCMS

Rf-MS !K%

Ymki Mfsfhod

15,02 24,06 1421 0 44 GC RT, MS

4,5-Dimethyl-2-u nd ecene 16.32 24.50 1433 0-18 C5C..-R17 MS

16.83 24.70 1449 0,27 GORT. MS

16.99 24.96 1454 0,70 GC-F^T, MS

25.63 0,47 MS

- 25.81 0,68 MS

(J-Selinene 17.2S 26.Q8 1463 3.00 MS

a-Selinerie 17.59 1473 0.4-2 GC-RT, MS

24 17,69 26 .,64 1476 0,58 MS

&-Guraiene 17,87 26.91 1432 0.50 GC4^T., MS

C,sH„ ~ 27.17 „ 2.20 y s

1~S-cis-CaiaiTienene 18.17 27.60 1491 0,56 GC-Ri;, MS

Caryophylierje epoxide isomer 19.04 1520 0.26 GC-RT, MS

- 28.32 .. 1,14 MS

Ledol 19,69 29.72 1542 1.16 GC-RT, yS

C aryophyllene epoxide 19.90 .30,38 1550 7.81 GC'-RT, MS

20.62 30.67 1559 0.61 MS

Hurnulene epoxide 20.62 31.09 1574 4.60 GC-RT, MS

21.27 31.24 1597 0,30 GC-RT, MS

21.93 33.04 1619 0.45 MS

Caryophyllenoi-2 22.40 33.57 1636 0.52 GC-F?T. MS

„ 34.30 0 49 MS

23.59 35.52 1677 0.34 GC-RT. MS

, 35.68 0.74 iViS

- 36,01 0.44 MS

- 36.35 - 0.32 MS

Tetradecanoic acid . 37,19 , 1,40 MS

273

Compound R W L Rt-P lS iK Yield M ettiodGLC GCMS %

- 37.35 0,10 ÂS

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„ 38.30 - 0,5-1 MS

- 38,61 1759 1,19 MS

28.15 41.32 1842 0,37 GC-RT, MS

31,05 42.42 1959 0.28 GCMTf. MS

32,15 43.39 2009 1,86 MS

35.84 43.55 2163 1,74 MG

Rt-GL = F?etention time on SE-30.

Rt-M S = Retention lime on DB-1,

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Retention in d e x according to ERjIherjijc starsdards

on D8-1 and SE-30 efirotriatographic cojunins.

Identical retention time and Kovat’s index to auiherdic

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Mass spectrum analysis.

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