R/IA,JOR ESSENTIAL O I L CONSTITUENTS IN

Piper Linn.

INTRODUCTION

Phytochemistry has gained an important status as a contributoly discipline in taxo-

nomic and evolutionary studies (Naik, 1992). Chemical characteristics may have a particu-

larly high taxonomic value when they are stable and unambiguous. These evidences are

of great significance from the lower groups of plants to the highly specialised angiosperms.

The results of the investigations in the chemical constituents of plant groups are applied

mainly for two purposes. Firstly, to provide taxonomic characteristics which may improve

existing plant classifications, i.e., a strict taxonomic purpose and secondly, to add to the

existing knowledge of phylogeny or an evolutionary relationship.

Most of the plant products are metabolic by-products and they are important

taxonomically. Phenolics, betalains, oils, fats, waxes, proteins etc, are some of the best

known plant products the presence of which serve as reliable taxonomic features.

The term essential oil is open to wide interpretation and may include not only

products obtained by traditional methods of distillation but also those obtained by solvent

extraction or by mechanical extraction. These essential oils, the natural products of com-

merce (Hegnauer, 1982), are a heterogeneous group of complex mixtures of organic sub-

stances. There is barely any group of naturally occurring substance in which the number

of possible components are as great as the essential oil constituents.

Essential oils, the odor~ferous volatile material of plant origin (Ames and Mathews,

1968), found in intimate association with resins as well as gums are synthesised in special

secretary structures. These oils contain numerous chemicals, the relative proportions of

which are usually characteristic of a given genus. Sometimes they may vary significantly

depending on the plant species, its geographical source and the environmental conditions

associated with its growth, harvesting and predistillation handling (Gerhardt,l972 ; Green

et a1.,1980).

Essential oils are found in all the distinctly aromatic plants, particularly in families

like Asteraceae, Lauraceace, Myrtaceae, Rutaceae, Geramiaceae, Poaceae, Fabaceae,

Caesalpiniaceae etc. (Samba Murthy and Subrahmanyan, 1989). Considerable data are

available to show that chemical constituents in a number of aromatic plants like Mentha

and Ocirnurn are genetically controlled (Sobti,l973 ; Sobti and Pushpangadan,l976;

Pushpangadan et a1.,1975 ; Singh and Sharma,l98O).

The economic importance of the family Piperaceae is due to the genus Piper.

Different species belonging to this genus are rich in aromatic constituents (Guenther, 1952;

Nambudiri et a1.,1970 ; Stahi,1972 ; Watt,1892 ; Anonymous,l969). Various authors (Chopra

et a1.,1969 ; Ghoshal et al.1996 : Babu et a1.,1996) have enumerated their application in

pharmaceutical industry.

The genus Piper has been widely investigated chemically, from the point of view

of its organoleptic, medicinal and insecticidal properties. Dhar and Atal (1967) identified 5-

hydroxy-3' 4 '7' trimethoxyflavone as the major component of the fruits of P peepuloides,

Royle. Sito-sterol was isolated by Desai et al. (1975) and a cyclohexane di epoxide by

Singh and Atal (1969) from P hooker;. Gupta et al. (1972) isolated methyl piperate from

the fruits of P officinarurn.

The uses of long pepper (P longurn) in the Ayurvedic and the Unani systems of

medicine go far back to ancient times. Synge and Wood (1956); Manavalan and Singh

(1979): Atal and Ojah (1965) and Atal et al. (1966) have undertaken the phytochemical

aspects of this species. Shankaracharya et al. (1995) analyzed two samples of P cubeba

for their physico-chemical characteristics. Jose (1983) reported a high content of oleoresin

and essential oil in Pcubeba.

F! betle (Pan) or betel-vine is widely cultivated in India and South East Asian

countries, mostly for its leaves. Chewing pan is stimulating and exhilarant. It increases the

117

flow of saliva, alleviates thirst and even hunger. It gives a feeling of general well being. It

is aromatic, carminative, stomachic, astringent and stimulant. Tannins, sugars, starch, dia-

stase and vitamin A and C are present in the leaves (Anonymous, 1969) The amount of

essential oil varies from 0.5 to 4.2 percent depending upon the variety, time at which the

leaves are plucked, the part of the vine from which they are removed etc. (Ghani and Sial,

1952). The oil contains phenols and terpenoids like eugenol, cardine, eugenol methyl ether,

estragol, chavicol and caryophyllene (Ghani and Sial, 1952).

Black pepper, referred to as 'The King of Spices' and as 'Black gold', is the whole

dried fruit of P nigrum, while white pepper is its dried fruit from which mesocarp has been

removed. The most widespread use of black pepper is as a spice in cookery. The alkaloid

piperine is considered to be the major constituent responsible for the biting taste of black

pepper. Other pungent alkaloids occurring in pepper in smaller amounts are chavicine,

piperidine and piperettine.

Aroma and flavour characteristics are important quality attributes of spices, which

are based on the nature of essential oil in them. The characteristic aromatic odour of

pepper is due to the presence of a volatile oil in the cells of the pericarp. The aromatic

properties of black pepper are due to the monoterpene hydrocarbons in its essential oil.

On steam distillation, crushed black pepper yields up to 4.8 percent of the oil, the yield

depending greatly upon the age of the dried fruits subjected to distillation (Anonymous, 1969).

There are different varieties of P betle and P nigrum in India. They differ in the

quality and quantity of their chemical contents, which characterise their taste. Several workers

have undertaken investigations on the chemical contents of P betle and P nigrum earlier

(Guenther, 1952 ; Ghani and Sial, 1952 ; Anonymous,l969 ; Nambudiri et al., 1970 ; Stahi,

1972; Ganguly and Gupta, 1974a ; Jose. 1983; Zachariah and Gopalam 1987; Zachariah,

1995). However, informations on the major essential oil constituents in the species P betle

and P nigrum are meagre.

The present study is undertaken to find out the percentage of essential oil in four

varieties of i? betle and nine varieties of F! nigrum and also to identify the major compo-

nents in these oils. These data are used to draw conclusions regarding the inter specific

and intra specific relationships of this genus. Attempts have also been made to correlate

these findings with the taxonomic grouping and to study the variation in yield and quality

of the essential oil.

MATERIALS AND METHODS

Fresh mature leaves of four varieties of P betle were collected from the experi-

mental Botanical garden at Sacred Heart College. Thevara and mature berries of nine

varieties of P nigrum were procured from the germplasm collection of Indian Institute of

Spices Research, Calicut. These materials were cleaned and shade dried at room tempera-

ture Shade drying reduces one third of the fresh weight and maximizes the oil yieid without

affecting the quality of essential oil (Hazra et a1.,1990).

Materials investigated

P betle var. aluva

P betle var. nadankodi

P betle var. salem

P betle var. thulasikodi

P nigrum var. aimpiriyan

P nigrum var, chumala

P nigrum var. kalluvalli

P nigrum var. karimunda

P nigrum var. kottanadan

P nigrum var. neelamundi

P nigrum var. panniyurl

P nigrum var. valiakaniakadan

P nigrum var. vellanamban

Isolation of Essential oil

Separation of volatile oils from dried, powdered berries and leaves was conducted

by hydrodistillation in a Clevanger apparatus (Clevanger, 1928) for 4- 5 hours as prolonged

extraction normally increases the yield (Gildermeister and Hoffman, 1961). Extraction was

carried out at ambient temperature to necessitate economy (Guenther, 1949). The percent-

age of essential oil was calculated on a dry weight basis to avoid faulty estimations that

may arise due to the different water contents of the tissue analyzed each time (Von

Rudloff. 1972). The isolated oil was then dried over anhydrous sodium sulphate and stored

at 4-6 OC.

Qualitative analysis

Gas chromatographic evaluation of the oils were carried out in the Perkin Elmer Auto system Gas

Chromatograph equipped with a flame ionization detector (FID) connected to a PE.Nelson 1022

GC plus integrator. The GC was carried out on a OV -1 7 column. Nitrogen was used as carrier gas

at 10 psi (inlet pressure) with aflow rate of thirty ml per minute. Temperature programming in oven

was performed at 70 OC to 220 OC at the rate of 5 OC per minute. Major components were identi-

fied by retention time (RT) analysis (Finar, 1975) and peak enrichment by co-injection with authen-

tic standards (Jeffery et al., 1989) and by comparison with literature data.

Quantitative analysis

In GC, the quantification of the peak areas was done by thePE. Nelson 1022 GC plus

integrator having a built in computer. The quantitative data obtained thereby were based on com-

puter integrated peak area calculations

OBSERVATIONS

Essential oils extracted from thirteen cytotypes of Piperaceae exhibited wide varia-

tion in their yield. The percentage of essential oil ranged from 0.95 percent in F! betle

var. salem to 4.6 percent in F! nigrum var. kottanadan (vide Fig. 44). As regards the

chemical exploration, the principal components mainly fall under monoterpenoids,

sesquiterpenoids and phenols. The taxa with an yield of two percent and above were

considered as oil rich and the chemotype of the different plants were determined on the

basis of their major component. The name of the major component was given for the

respective chemical races when the component occupied a significant amount in the

total composition. Others were designated as mixed chemotypes. Those taxa which

showed co-dominance of two components were also considered as mixed chemotypes.

In the present study, all the varieties of P nigrum and two varieties of P betle (aluva and

nadankod~) were mixed chemotypes whereas, P betle varieties salem and thulasikodi were

eugenol chemotypes.

A clear delimitation between the two genera on account of the presence or

absence of the major essential oil component was noticed. A comparative study of the

chemical components are given in table-45. Compounds present in less than four percent-

age were considered to be present only in traces. The gas chromatograms (vide Fig.18-

23, Fig. 30-35, Fig. 42 ) represent qualitative analysis and pie charts (vide Fig. 24-29, Fig.

36-41, Fig. 43) represent quantitative estimation of essential oils.

The major chemical components identified in the present study along with their

respective chemicd classes are given below:-

SI. Name of the taxa Essential oil Class

No: components

1. P betle var. aluva

2. P betle var nadankodi

3. P betle var. salern

Pinene Monoterpene

Sabinene

Limonene

Cineole Oxide

Linalool Alcohol

Chavibetol Phenol

Eugenol

Sabinene Monoterpene

Lirnonene

Cineole Oxide

Linalool Alcohol

Chavibetol Phenol

Eugenol

Pinene Monoterpene

Sabinene

Limonene

Cineole Oxide

Linalool Alcohol

Chavibetol Phenol

Eugenol

SI. Name of the taxa Essential oil Class

No: components

4. P betle var. thulasikodi Pinene Monoterpene

Sabinene

Limonene

Linalool Alcohbl

Chavibetol Phenol

Eugenol

5. I? nigrum var. aimpirian

6. P nigrum var chumala

7. i? nigrum var karimunda

8. P nigrum var, kalluvalli

Pinene Monoterpene

Myrcene

Limonene

Caryophyllene Sesquiterpene

Pinene Monoterpene

Myrcene

Limonene

Caryophyllene Sesquiterpene

Pinene Monoterpene

Sabinene

Limonene

Caryophyllene Sesquiterpene

Pinene Monoterpene

Sabinene

Myrcene

Limonene

Linalool Alcohol

Caryophyllene Sesquiterpene

SI. Name of the taxa Essential oil Class

No: components

9. P nigrum var kottanadan

10. P nigrum var. neelamund~

11. P nigrum var. panniyurl

12. P nigrum var. valiakaniakadan

13. P nigrum var, vellanamban

Pinene

Sabinene

Limonene

Linalool

Terpineol

Caryophyllene

Pinene

Sabinene

Myrcene

Limonene

Linalool

Caryophyllene

Pinene

Sabinene

Myrcene

Limonene

Caryophyllene

Pinene

Sabinene

Myrcene

Limonene

Linalool

Caryophyllene

Pinene

Sabinene

Myrcene

Limonene

Caryophyllene

Monoterpene

Alcohol

Sesquiterpene

Monoterpene

Alcohol

Sesquiterpene

Monoterpene

Sesquiterpene

Monoterpene

Alcohol

Sesquiterpene

Monoterpene

Sesquiterpene

Distribution of essential oil components in the taxa investigated Components

Name of taxa Eu Ch Ca Li Sa LI Pi Ci My Tp

P betle var. aluva + ++ - + + tr tr + - -

P betle var. nadankodi + ++ - + + tr - tr - -

f! betle var. salem + + + tr tr tr tr* tr - -

P betle var. thulasikodi ++ + tr tr tr tr - - - -

P nigrum var. aimpirian . - + + - + - + + - I

P nigrum var. chumala - - ++ + . + - + -

P nigrum var karimunda - - + - + + + + - - -

- P nigrum var kalluvalli - - ++ + + tr + tr -

- P nigrum var. kottanadan - + + + + tr + - - tr

- i? nigrum var neelamundi . - + + + ++ tr tr tr -

P nigrum var panniyurl - - + + ++ - + - tr -

I? nigrum var. valiakaniakadan - - + ++ + + + - + -

1 P nigrurn var. vellanamban - + + + + - tr .. tr - 1 Eu - Eugenol Ch - Chavibetol Ca - Caryophyllene LI - Linalool

Sa - Sabinene Li - Limonene Pi - Pinene Ci - Cineole

My - Myrcene Tp - Trepineol ++ - Major component '+' - Present

tr - Traces '-' - Not detected

tr*- As the percentage is less than 1, not represented in the pie chart (Fig.26).

126

Table-46

Previous reports of major essential oil constituents in Piper nigrum

Constituents References

Monoterpene hydrocarbons

carnphene Ikeda et al. (1 962), Nigam and Handa (1 964), Lewis et al. (I 969b)

Richard et a1.(1971), Richard (1972), Russell and Else (1973)

Debrauwere and Verzele (1 976)

D-3-carene lkeda et al. (1 962), Wrolstad and Jennings (1 965)

Richard and Jennings (1971), Richard et al. (1971), Richard (1g72),

Russell and Else (1973), Debrauwere and Verzele (1976)

p-cymene lkeda et a1.(1962), Nigam and Handa(1964), Wrolstad and Jennings (1965

Lewis et a1.(1969b), Richard and Jennings (1971),

Russell and Else (1973), Debrauwere and Verzele (1976)

lirnonene lkeda et al. (1962), Nigam and Handa (1964), Jennings et al. (1968)

Richard et a1.(1971), Russell and Else(1973), Richard and Jennings (1971)

Salzer (1975 a), Debrauwere and Verzele (1976)

Zachariah and Gopalam (1987), Gopalakrishnan et al .(1993)

Zachariah (1 995)

myrcene lkeda et al. (1962), Nigam and Handa (1964), I 1 Wrolstad and Jennings (196% Jennings et al. (1968), Lewis et al. (1969b),

Richard and Jennings (1 971), Richard (1 9721, Russell and Else (1 973)

127 (Contd.. .)

Constituents References .

myrcene Debrauwere and Verzele (1 976), Zachariah and Gopalam (1 987),

Zachariah (1995)

cis-ocimene Russell and Else (1973), Pino (1990)

a-phellandrene Hasselstrom et al. (1957), Nigam and Handa (1964),

Wrolstad and Jennings (1965), Richard and Jennings (1971),

Russell and Else (1973), Debrauwere and Verzele (1976),

b -phellandrene Nigam and Handa (1964), Wrolstad and Jennings (1965),

Richard and Jennings (1971), Richard et al. (1971),

Russell and Else (1 973)

a-pinene Hasselstrom et al. (1 957),lkeda et al. (1 962),

Wrolstad and Jennings (1965), Richard and Jennings (1971),

Richard et al. (1971), Russell and Else (1973),

Debrauwere and Verzele (1 976)

I b-pinene lkeda et at. (1962), Nigam and Handa (1964),Jennings et al. (1968)

Lewis et al. (1969b), Richard and Jennings (1971), Russell and Else (197

Debrauwere and Verzele (1 976), Mc Carron et a1 (1 995)

sabinene lkeda et al. (1962), Nigam and Handa (1964),

Wrolstad and Jennings (1965), Jennings et al. (1968)

(Contd. 128

Constituents References

sabinene Lewis et al. (1969b), Richard and Jennings (1971), Richard (1972),

Salzer (1975 a), Zachariah and Gopalam (1987), Zachariah (1995),

Gopalakrishnan et al .(1993)

a -terpinene lkeda et al. (1 962), Wrolstad and Jennings (1 965),

Richard and Jennings (1971), Richard (1972), Russell and Else (1973)

g-terpinene lkeda et aI.(l962), Nigam and Handa(l964),Wrolstad and Jennings (1965),

Richard and Jennings (1971), Debrauwere and Verzele (1976)

terpinolene lkeda et al. (1962), Wrolstad and Jennings (1965) ,Richard et al. (1971)

Richard and Jennings (1971),Russell and Else (1973)

Debrauwere and Verzele (1976)

a -thujene lkeda et al. (1962), Wrolstad and Jennings (1965),

Richard and Jennings (1 971), Richard (1 972), Russell and Else (1 973)

Sesquiterpene hydrocarbons

a-cis-bergamotene Muller et al. (1968), Lewis et al. (1969b), Russell and Else (1973)

a-trans-bergamotene Muller et al. (1968), Lewis et al. (1969b), Russell and Else (1973)

b-bisabolene Muller et al. (1968), Richard et al. (1971), Richard (1972),

Russell and Else (1973), Debrauwere and Verzele (1976)

d-cadinene Muller et al. (1968), Debrauwere and Verzele (1976)

129 (Contd ...)

Constituents References

g-cadinene Debrauwere and Verzele (1 976)

calamenene Muller et al. (1 968), Debrauwere and Verzele (1 976)

b-caryophyllene Hasselstrom et al. (1957),Nigam and Handa (1964),

Muller et al. (1968), Lewis et al. (1969b), Richard et al. (1971),

Richard (1972), Russell and Else (1973), Debrauwere and Verzele (1976)

a-copaene lkeda et al. (1962), Richard et al. (1971), Richard (1972),

Russell and Else (1 973), Debrauwere and Verzele (1 976)

a-cubebene lkeda et al. (1962), Richard et al. (1971 ), Richard (1972),

Debrauwere and Verzele (1 976)

P-cubebene Debrauwere and Verzele (1 976)

ar-curcumene Russell and Jennings (1969)

d-elemene Muller et al. (1968), Debrauwere and Verzele (1976)

b-elemene Muller et al. (1968), Richard et al. (1971), Richard (1972),

Russell and Else (19731, Debrauwere and Verzele (1976)

b-elemene Muller et al. (1968). Richard et a1.(1971), Russell and Else (1973),

Debrauwere and Verzele (1 976)

(Contd ...)

130

Constituents References

b-farnesene Muller et al. (1968), Richard et al. (1971), Russell and Else (1973),

a-guaiene Debrauwere and Verzele (1976)

a-hurnulene Nigarn and Handa (1964),Muller et al. (1968)

Richard et al. (1971), Russell and Else (1973)

Debrauwere and Verzele (1 976)

isocaryophyllene Muller et al. (1968)

g-rnurolene Muller et al. (1968), Russell and Else (1973),

Debrauwere and Verzele (1976)

a-santalene Muller et al. (1968), Russell and Else (1973)

a-selinene Muller et al. (1968), Lewis et al. (1969b), Richard et al. (1971)

Richard (1972), Debrauwere and Verzele (1 976)

b-selinene Muller et al. (1968), Lewis et al. (1969b), Richard et al. (1971),

Richard (1972)

Oxygenated rnonoterpenes

borneol Debrauwere and Verzele (1 975)

camphor Debrauwere and Verzele (1 975)

cavacrol Debrauwere and Verzele (1975)

cis-carve01 Russell and Jennings (1969), Richard and Jennings (1971)

(Contd.

Constituents References

carvone Russell and Jennings (1969), Richard and Jennings (1971),

Richard et al. (1971), Richard (1972),

Debrauwere and Verzele (1 975)

carvetonacetone Debrauwere and Verzele (1975)

1.8-cineole Debrauwere and Verzele (1 975)

cryptone Hasselstrom et al. (1957), Russell and Jennings (1969)

p-cymene-8-01 Russell and Jennings (1969),Richard and Jennings (1971),

Debrauwere and Verzele (1 975)

p-cymene-&methylether Debrauwere and Verzele (1 975)

dihydroca~eol Hasselstrom et al. (1 957)

dihydrocarvone Debrauwere and Verzele (1 975)

linalool Russell and Jennings (1969), Richard and Jennings (1971),

Russell and Else (1973), Debrauwere and Verzele (1975)

cis-2,8-menthadien-2-01 Richard and Jennings (1971)

3,8(9)-p-menthadien-1-01 Debrauwere and Verzele (1 975)

1 1 (7),2-p-menthadien-6-01 Debrauwere and Verzele (1975)

1 1 (7),2-p-menthadien-4-01 Debrauwere and Verzele (1975)

1 1,8(9)-p-menthadien-5-01 Debrauwere and Verzele (1975)

(Contd..

132

Constituents References

1,8(9)-p-menthadien-4-01 Debrauwere and Verzele (1 975)

cis-p-2-menthen-1-01 Richard and Jennings (1971)

rnyrtenal Debrauwere and Verzele (1 975)

myrtenol Debrauwere and Verzele (1 975)

methyl cawacrol Debrauwere and Verzele (1975)

trans-pinocaweol Richard and Jennings (1971), Debrauwere and Verzele (1975)

b-pinone Debrauwere and Verzele (1 975)

cis-sabinene hydrate Russell and Jennings (1970), Richard and Jennings (1971)

I -1erpinen-4-01 Russell and Jennings (1969), Richard and Jennings (1971),

Russell and Else (1973), Debrauwere and Verzele (1975),

Pino (1 990)

I-terpinen-5-01 Debrauwere and Verzele (1 975)

a-terpineol Russell and Jennings (1969), Richard and Jennings (1971),

Richard (1 972), Debrauwere and Verzele (1 975)

I ,I ,4- Debrauwere and Verzele (1975) trimethylcyclohepta- 2,4-dien-6-one

Phenyl ethers

eugenol Richard and Jennings (1971)

methyl eugenol Russell and Jennings (1969), Richard and Jennings (1971)

(Contd,

133

Constituents References

rnyristicin Russell and Jennings (1969),Richard and Jennings (1971),

safrole Russell and Jennings (1969),Richard and Jennings (1971)

Oxygnated sesquiterpenes

5,l O(15)-cadinen-4-01 Debrauwere and Verzele (1 975)

ca~o~h~lla-3(12).7(15)-dien-4- Debrauwere and Verzele (1 975)

caryophylla-2.7(15)-dien-4-ol Debrauwere and Verzele (1975)

b-caryophyllene alcohol Debrauwere and Verzele (1 975)

caryophyllene ketone Richard et al. (1971)

caryophylllene ox~de Debrauwere and Verzele (1975)

epoxy-dihydrocaryophyllene Hasselstrorn et al. (1957)

nerolidol Russell and Jennings (1969), Richard and Jennings (1971)

4,10,1O-trimethyl-7-methylene- Debrauwere and Verzele (1 975) bicyclo-(6.2.0)decane-4- carboxaldehyde

Table-47

Previous reports of major essential oil constituents in Piper betle

Zonstituents

Wonoterpenes : a-thujene, p-ocimene, bornylene, p-pinene ,camphene, a-pinene,

trans p-ocimene , y-terpinene, terpinolene, Limonene, a110 ocimene, p-cymene, p-myrcene

a-Terpinene, p-phellandrene, 2,6,6 Trimethyl, I-methylene cyclo hex 2-ene, sabinene,

jesqui terpenes : y-cadinene, A-cadinene, u-cakinene , y-elemene, p-salinene, p-elenene,

cis-caryophyllene, trans-caryophyllene, aroma dendrene, a-cubebene, p-cubebene,

Alcohols : linalool, a-terpineol ,terpinen-1-01, a-costol, A-cadinol, geraniol,

2,7,11,15 TetraMethyl-2-hexadecan-1-01,

Aldehydes : decanal (Capric aldehyde), decanal (laural aldehyde), stearaldehyde

Acids : hexadecanoic acid

Oxides : 1,8 cineole, caryophylleneoxide.

Phenol : eugenol, isoeygenol, chavicol IChavibetol.

Phenolic ethers : methyl eugenol, methyl chavicol, anethole , I ,3 Benzodioxole (5) -2-Propenyl

Esters : eugenol acetate, methyl benzoate

References - Rawat et a1 . (1989); Balasubrahmanyan and Rawat (1990) ; Garg et al. (1996)

s. Solvent 7. Pinene 9. Sabinene 13. Limanene 15. Cinede 16. Linalool 28. Chavibetd 30. Eugend

Fig. 18. Gas chromatogram of R betk var. alum

S. Sobant 9. Sabinene 1 1. honene 14. Cineole 16. L i n M 27. Chavibetd 29. Eugend

Fig. 19. Gas chromatogram of P be& uar. nadankodi

S. Sdv8nt 9. Pinene 10. Sabmene 14 Limonew 16. C i m k 17. LinaEool 30. Chavibetol 31. Eugenol

Fig.20. Gas chromatogram of l? betle var. salem

L

Fi.21. Gas dlromatugram of I? be& var. thutasikodi

Fii.22. Gas chromatogram of F! nigmm var. ain7pirian

S. Solvent 8. Pinene 10. Myrcene 12. Limonene 31. Caryophyllene

S. !?abent 8. PFnene to. Myroene 12. tirnonene =.mm*

Fig23. Gas chrornatogram of F nigrum var. c h u m

6 Chavibetd R Eugend Linalod L i e n e Pinene R S a m e

Fi.24. Compositbn U F major essential oil components in /? 6ez.k var. slluva

Fig.25 Composition of major essential oil components in P betle var. nadankodi

Fg. 26. Canposition of major essential oil components in I? be& var, sakm

F~.27. Cornpositin of major essenW oB components in P bet% var. thuksikodi

Fg.28. Composition of major essential oil amponants in R n&wn var. aimpi&

Fg.29. Composition of major essent'il 01 components in R nignrm var. chu&

S. Soh/ent 7. Pinene 9. Sabinene 1 1. Umonene 28. Caryophyllene

Fig.30. Gas chromatogram of F! nigwm vac. kan'nmda

S. solvent f 1. Anene 13. Sabinene 14. Myrcene 15. Limonene 18. Linalod .,32. Catyophyllene

Fi.31. Gas chromatogram of I? nigmrn vat. kaluvani

S. S o h t 10. Knene 13. s a b i i 15. Urn- 19. tinalool 26,Tepineol 33. Caryophylbne

Fi.32. Gas chromatogram of I? nignrm var. koitamdan

Fig.33. Gas chromatogram of /? nigrum var. neekmmd

, -

Fi.34. Gas chromatogram of R nignrm var. pannwl ..

S. Solvent 4. Pinene 6. Sabinene 7. ~ c e m 8. honene 21. c w w m

S. solvent 2. Pinene 4. Satinme 5. Myrcene 6. Limonene 8. Linalool 15. CeryophylW

Fig.35 Gas chromatogram of F! n i g m var. vakkmMdan

Fi.36. Composition of mjor essential oil components in I? nigrum var.karimunda

Fi.37. Composition of major essential oil components in l? nigrum var.WWli

Fg.38. Composition of major essential oil components in F! nigrum var. kothnadan

Fig.39. Composition of m w r essential oil components in I? nigrum var. n e e m i

Limonene I Unidentified

Fig.40. Composition of major essential oil components in l? nigmm var. panniyuf?

Caryophyrlene W Sabnene Limonene Pinene a Linald Dblymene IUnidentified

Fig.41. Composition of major essential oil components in /? nigrum var. valk&aniakam

S. Solvent 3. Pinene 5. Sabinene 8. AAyrme 19. L i m m 25. Caryophykne

Fig.42. Gas chromatogram of I? nigrum var. veIknamban

Fig.43. Composition of major essential ail components in P njgrum var. v e i m b a n

Fig.&. Comparison of the percentage yield of essential oil in various taxa studied

-p. - 1.Pbetlevar.aluva g! . 2. P be& var. nadankodi 3. f? be& var. Salem w - 4, P be& var . thuksikodi 5. F? nigrum var. aimpirian 6. f? nignrm var. chumala

7- Mmm Vat. mu^ 8. P nigrum var. kalluvalli 9. P nlgrum var.ko&mdan

10. f? nigrum var. mlamundi 11. l? nignrm var. panniyurl 12. P nigrum var.valth.!m~alan

13. f? nigrum var. velku~mban

DISCUSSION

Four varieties of Piper betle and nine varieties of Piper nigrum were evaluated for

the quantity of essential oil and chemical constituents. The quantity of essential oil varied

from 0.95 percent to 2.2 percent in P betle and 2.5 percent to 4.6 percent in P nigrum.

(vide Fig. 44). Pinene, limonene, linalool, terpineol, sabinene, cineole, caryophyllene,

myrcene, chavibetol and eugenol were the constituents identified in them (vide ~ i b . 18-43).

The presence of characteristic secretary structures is an important feature of

Botanical families (Heath,l986). The specific aroma of black pepper (P nigrum) is due to

the presence of volatile oil in the cells of the pericarp. In the fruits of P nigrum, oil-bearing

cells are found mainly in the skin and towards the tip of the cortex. (Narayanan and

Mathew, 1985) The essential oil is stored in large thin-walled cells ranging in size from 50

pm to 55 pm. These cells are distributed mainly in the inner part of the skin. A small

percent of essential oil was also seen in the core towards the stigma end and around the

embryo. (Mangalakumari and Mathew, 1986)

It was Dumas (1835) who first examined the composition of black pepper

oil. Subarain and Capitaine (1840) recognised a high proportion of oxygenated compounds

in the oil. Later, Eberhardt (1887) ; the Schimmel Co. (1890) and Schreiner and Kremers

(1901) identified terpinhydrate, alpha-phellandrene, dipentine and beta caryophyllene, as

components of the oil. Further progress on the detailed composition of black pepper oil

was made only after the advent of gas chromatographic analysis techniques. Consequently

more than sixty major and minor components were identified in the genus Piper.

Previous reports on the essential oil constituents of Piper are given in tables 46

and 47. Hasselstrom et al. (1957): Lewis et al. (196913); and Pangborn et al. (1970) have

carried out investigations on the aroma and flavour characteristics of pepper. According to

Hasselstrom et al. (1957) the specific odour of the oil is due to the presence of small

amounts of the oxygenated compounds. Lewis et al. (196913) opined that large proportions

of monoterpene hydrocarbons are necessary for strong peppery topknots. Pangborn et al.

(1970) has suggested that alpha bergamotenes and alpha santalene are important com-

ponents, which contribute to the distinct odour of black pepper. The major constituents of

the oil are alpha and beta pinenes, sabinene, myrcene, limonene, caryophyllene and

humulene (Jennings et al., 1968). These constituents impart specific flavour to the oil

individually and collectively like turpentine, pleasant, lemony etc. (Zachariah and Gopalam,

1987).

The physico-chemical properties of individual oil samples can vary considerably

depending on the cultivar, geographical origin, age and quality of the material used and

also to some extent the distillation procedure employed (Guenther.1952). Lewis et al. (1969b)

and Richard et al. (1971) screened seventeen cultivars of pepper grown in Kerala. Accord-

ing to Lewis et al. (1969b) pepper oil is a complex mixture of hydrocarbons, monoterpenes

and sesquiterpenes. Small amounts of oxygenated compounds are also present. Oil of

pepper is an almost colourless to slightly greenish liquid (Hasselstrom et al., 1957) with a

characteristic odour of pepper and also of phellandrene. Although the characteristic odour

of pepper oil is due to these oxygenated compounds (Hasselstrom et al., 1957), to some

extent, the mono and sesquiterpenes are responsible for the flavour of pepper. (Lewis et

al., 196913). This is the main reason for the spicy flavour of freshly ground pepper.

Zachariah (1995) evaluated forty-two accessions of black pepper germplasm for

essential oil and chemical constituents. He identified constituents like pinene sabinene,

limonene, caryophyllene and myrcene. Good variability was observed among the acces-

sions for flavour and quality.

Gopalakrishnan et al. (1993) reported beta-caryophyllene, limonene, beta-pinene,

sabinene, and alpha-pinene from four new Indian genotypes of P nigrum namely panniyurl,

panniyur2, panniyur3 and Culture-239). A few new sesquiterpenes previously not

137

characterised in black pepper oil were also identified. The odours of the four oils were

ranked. The oil of panniyurl was found to be inferior to those of the other three. Panniyurl

had less peppery and spicy flavour than either panniyur 2 or panniyur 3 Culture-239 con-

tained lower levels of sabinene, limonene, and beta-pinene and higher levels of myrcene,

p-cymene, and alpha-phellandrene. Culture-239 was found to possess a more refreshing,

piney, green pepper-like odour .It also possessed a citrus or lemon-like aroma.

Pino et al. (1990) analysed the chemical composition of black pepper oil. A total

of 46 compounds were identified of which beta-ocimene, delta-guaiene, farnesol,

delta-cadinol and guaiol were reported for the first time. Sabinene and terpinen-4-01 ap-

peared to be the most important contributors to the characteristic odour of black pepper

oil. In the present study, the monoterpene hydrocarbon group exhibited the most striking

variation in the composition pattern. In P nigrum, the highest percent of monoterpenes was

in the variety valiakaniakadan (64.26%) and next to it there was the variety aimpirian (57.44%).

The variety vellanamban possessed the lowest percent of rnonoterpenes (20.64%). The

different components of monoterpenes varied greatly in different varieties. Out of the nine

varieties of P nigrum, the variety aimpirian (vide Fig. 28) contained the highest percent of

myrcene (25%). Sabinene was the maximum (21%) in the varieties karimunda (vide Fig. 36)

and panniyurl (vide Fig. 40) and the highest percent of limonene (21%) and pinene (17%)

were present in the varieties valiakaniakadan (vide Fig. 41) and chumala (vide Fig. 29)

respectively. Lirnonene was the major component in P nigrum var. valiakaniakadan while in

P nigrum var, aimpirian, it was rnyrcene. In P nigrum var. karimunda and panniyurl sabinene

occured as the principie chemical component.

McCarron et al. (1995) compared the essential oils of green and black berries of

P nigrum of Indian and Sri Lankan origin. The monoterpene hydrocarbons of Indian oils

were similar to those of corresponding Sri Lankan oils but the oils differed with regard to

their sesquiterpene and oxygenated components. beta-Pinene and calyophyllene occurred

in all the oils and sabinene in the Sri Lankan oils only. Sumathykutty et al. (1990) reported

that there was a difference in the percentage of oil content and the constituents among

the different grades of pepper of the same cultivar. Salzer (1975a) proposed distinction

between Sarawak, Malabar, Tellichery, Brazillian and Lampong black pepper oil, on the

basis of relative abundance of sabanine, beta-pinenes, delta-3-carene, limonene and

caryophyllene.

In the present study, caryophyllene was found to be one of the major components

in all the investigated varieties of P nigrum. Caryophyllene was the maximum in the variety

vellanamban (vide Fig.43). In P nigrum varieties chumala, kalluvalli and vellanamban this

was the major component (vide Fig.29, 37, 43). Richard et al. (1971) and Russell and Else

(1973) demonstrated the presence of beta-caryophyllene as a major constituent in many

samples of Kerala. Sumathykutty et al. (1990) also reported that caryophyllene is one of

the major components in different grades of P nigrum.

Sumathykutty (1984) studied the essential oil composition of the fruits of pepper

at six stages of development. The youngest fruits (pin head grade) yielded the least oil

with sesquiterpenes and higher polar compounds as the main components, while the later

developmental stages yielded 2.5-4.25 percent oil with 47-64 percent monoterpenes and

30-47 percent sesquiterpenes and higher polar compounds. The oxygen containing com-

pounds of black pepper oil was separated into free acids, esters, carbonyl compounds,

alcohols and oxides. Debrauwere and Verzele (1976) identified fifty-one substances, which

were recorded for the first time in black pepper oil. The alcoholic groups like linalool and

terpineol were present in traces in the materials investigated.

There occurs different varieties of P betle with different taste and have dif-

ferent qualitative and quantitative features. (Ganguly and Gupta, 1974a,b.) The most impor-

tant factor determining the aromatic value of the leaf is the amount and particularly, the

nature of the essential oil present. The oil of P betle is bright yellow to dark brown liquid

possessing an aromatic, pungent, sharp taste. The yield of oil depends upon the type of

leaves, the time of plucking and the nature of the material distilled.

In the present study, the phenolic compound eugenol or chavibetol was found to

be the major constituent in the four varieties studied (vide Fig. 24-27). The percent of

eugenol in the oil varied from 25 in P betle var. aluva to 52 in P betle var. salem. The

percent of chavibetol was the highest in P betle var aluva (41) and least in P betle var.

saiem (4). Other constituents of the oil are limonene, cineole, pinene, sabinene and linalool

in traces. Betle oil of Indian type contains eugenol as the predominant phenolic constitu-

ent. The oil consists of phenol and turpenes, their relative proportion varying with the

origin of the leaves. The higher the proportion of phenol in the oil, the better the quality

(Anonymous, 1969).

P betle varieties salem and thulasikodi are eugenol chemotypes (vide Fig.26 and

27) while P betle variety aluva and nadankodi are mixed chemotypes of eugenol and

chavibetol (vide Fig. 24 and 25). Studies by Jantan et al. (1994) and Garg et al. (1996)

revealed that in some cultivars eugenol was the major component while in some others it

was chavicol. Constituents of the essential oil of F! betle were used as determinants to

distinguish between cultivars. (Rawat et al., 1989). He made investigations on five different

cultivars of P betle and found that eugenol was one of the major components in all the

cultivars, In the cultivar bangla the dominant compound was eugenol (64%). The highest

content of 1,3-benzodioxole (5)-2-propenyl was in the cultivar desawari (45%). The cultivar

meetha could be distinguished from others by the presence of anethole (19.31%) and

cis-caryophyllene. Among the 5 cultivars investigated, kapoori appeared to be a distinct

genotype. This cultivar is marked by the large number of specific compounds such as

alpha-thujene, trans beta-ocimene, terpinolene, allo-ocimene, delta-cadinene, terpinen-1-01,

alpha-costol, delta-cadinol, methyl-2-hexadecan-1-01, geraniol, hexadeconic acid and methyl

benzoate. Sanchi was characterised by the presence of stearaldehyde which was absent

in other cultivars. The less pungent nature of desawari and kapoori leaves was due to

lower eugenol concentrations in them. The sweet taste and fennel-like odour of meetha is

due to the presence of anethole

Balasubrahmanyam and Rawat (1990, 1992) carried out biochemical investigations

on five betel cultivars (bangla, desawari, kapoori, meetha and sanchi). Eugenol Gas found

to be the major constituent in all the five cultivars. Sanchi oil was marked by the presence

of stearaldehyde, which was, absent in other cultivars. It was suggested that the charac-

teristic heavy clove-like aroma of bangla and sanchi leaves were due to the phenolic

compounds, including eugenol. The sweet fennel-like taste of meetha leaves was due to

anethole (19.31%).

Jose and Sharrna (1983) reported a remarkable correlation between chromosome

number and chemical constituents in different species of Piper. They have reported that the

contents of oleoresins and essential oils were the highest in the varieties of P betle with

the chromosome number 2n=78. Species with chromosome numbers higher or lower than

2n=78 had low quantity of oleoresin and essential oils. One remarkable feature reported

by them was the high content of oleoresin and essential oil in a low diploid species of

Piper cubeba (2n=24). This observation leads to the assumption that during evolution of

species, the increase in chromosome number might have led to the decrease of chemical

constituents.

In the varieties of P nigrum (2n=52), the differences in the oil content were attrib-

uted to the differences in the karyotypes (Jose and Sharrna. 1983). Undoubtedly, in the

present investigation also, the karyotypes differed even though the chromosome number

remained the same. Regarding P nigrum all the varieties studied showed the chromosome

number 2n=52 and the total percentage of oil differed from 2.5 to 4.6. But the extent to

which these karyotype differences could be responsible for the slight differences in their

chemical constituents, seems doubtful. The chromosome number of P betle also did not

show any selective value with regard to the chemical constituents. Regarding the total

chromosome length also there was no correlation with the percentage of oil content.

The present study on the chemical constituents of the essential oil of Piper is in

conformity with the earlier reports (vide Table-46, 47) and showed that pepper oil is a

complex mixture of hydrocarbons, monoterpenes and sesquiterpenes. Harborne (1991)

had studied the ecological role of plant terpenoids. According to him, their volatility and

intense pungent odour characterise terpenoids. Plant terpenoids had been a topic of im-

portance in chemical ecology. They were recorded as phytoalexins, insect antifeedants and

defense agents. Zerngue (1987) reported the monoterpene myrcene as signal molecules.

He has reported that myrcene given off by the leaves of one cotton plant is capable of

triggering of an induced synthesis of insecticidal C25 terpenoid aldehyde in another adja-

cent cotton plant. Pine oil, which is a mixture of monoterpenes such as alpha and beta-

pinene, limonene, myrcene and several monoterpene alcohols, is an effective feeding re-

pellent to voles and snowshoe hares. (Bell and Harestad, 1987). Monoterpenes are toxic

to microorganisms and have allelopathic effects on plant tissues (Fischer et al., 1988).

Monoterpenes are also toxic to most insects (Wada and Munakata,l97land Sinclair et

a1.,1988). Extracts of the seeds of i? nigrum and the ground seeds themselves were found

to be toxic to Aedes larvae (Novak, 1974). The therapeutic use of the essential oil of F!

nigrum was discussed by Recsan et al. (1997) and the antibacterial property by Mar-Mar-

Nyein et al. (1996) Studies on the tolerant factors of Piper betle L, cv. 'kapoori' to some

fungal pathogens (Tripathi et a1..1985) showed that the essential oil with terpinyl acetate,

eugenol and 1,8-cineole was the factor responsible for this tolerance. Dubey and Tripathi

(1987) conducted studies on antifungal, physico-chemical and phytotoxic properties of the

essential oil of i? betle. Eugenol was isolated and identified as the antifungal principal of

the oil.

Betel leaves are chewed alone or with other plant materials for their

mouth-freshening, digestive and aphrodisiac properties. The leaves are also reputed to

possess laxative and antihelmintic properties. A decoction of leaves is used for healing

wounds. The leaf oil has been found to possess antiseptic properties and as a result has

some use in the treatment of respiratory catarrhs. A gargle consisting of the juice or the

essential oil from the leaves mixed in warm water or the inhalation of leaf oil vapour has

been recommended in the treatment of diphtheria. The leaf essential oil of P betle was

highly active against different strains of bacteria and fungi. The essential oil was more

effective against tapeworms and hookworms than the synthetic antihelmintics piperazine

phosphate and hexylresorcinol. (Garg et a1.,1992). Studies on the biological activities as-

sociated with sesquiterpenes are many and varied (Tang et al.; 1988; Carter et al.; 1989;

Howard et al., 1989 and Richardson et al., 1989) Caryophyllene, one of the major com-

ponents, in the oil of P nigrum, is reported to protect the plants from insect attack because

of their anti microbial properties (Hubbell et al., 1983). Low herbivore damage on the

leaves was significantly correlated with higher concentrations of this particular constituent.

Flake and Turner (1973) evaluated the utility and potential value of various volatile

constituents as taxonomic characters. Chemotaxonomic evaluation based on volatile oils in

various plant taxa has been conducted by Hegnauer (1962,1978.1982 ) ; Pugialli (1996)

;Mandi and sharma (1994); Harborne and Turner (1984); Stace (1980). Terpenoids are of

great significance at the sub familial level and intra generic level. (Cole, 1992)

The main difference observed between P betle and P nigrum was that caryophyllene

was present only in the varieties of P nigrum, whereas eugenol and chavibetol were present

only in P betle. This supports the taxonomic grouping of these taxa under different spe-

cies. The rest of the components were common in both the species supporting their

intrageneric relationship. However, a detailed study incorporating all the compounds in the

essential oil will help in selecting the different species of Piper for further improvement of

this genus.

SUMMARY

The quantity of essential oil varied from 2.5 percent to 4.6 percent in P nigrum.

The variety kottanadan contained the highest percentage of oil and the variety vellanamban

contained the lowest percentage. The different components were caryophyllene, terpineol,

linalool, limomene, pinene, sabinene and myrcene. The components of monoterpenes varied

greatly in different varieties. Out of the nine varieties of P nigrum, the variety aimpirian

contained the highest percent of myrcene. Sabinene was the maximum in the varieties

karimunda and panniyurl and the highest percent of limonene and pinene were present in

the varieties valiakaniakadan and chumala respectively. In P nigrum var. aimpirian myrcene

was the major component. In P nigrum var. karimunda and panniyurl sabinene occured as

the principle chemical component.

P betle var aluva contained the highest percentage of oil. Eugenol and chavibetol

were the major components in the varieties aluva and nadankodi, while only very small

quantities of chavibetol were present in the varieties thulasikodi and salem. Constituents

like sabinene, pinene, limonene, cinenole etc. also were identified. These components

varied greatly in different varieties. The percentage of eugenol was the maximum in P betle

var. salem and the highest percentage of chavibetol was in P betle var. aluva. P betle var.

salem contained the least amount of chavibetol.

All the varieties of P nigrum and two varieties of P betle (aluva and nadankod~)

were mixed chemotypes whereas, i? betle varieties salem and thulasikodi were eugenol

chemotypes. Studies on the chemical constituents of essential oil of Piper showed that

pepper oil is a complex mixture of hydrocarbons, monoterpenes and sesquiterpenes. The

main difference observed between P betle and i? nigrum was that caryophyllene was

present only in the varieties of P nigrum whereas, eugenol and chavibetol were present

only in P betle. This supports the taxonomic grouping of these taxa under different spe-

cies. The rest of the components were common in both the species, thus showing their

intrageneric relationship. Considering the economic importance of this family, it has been

thought that, a systematic study with regard to the chemical constituents of essential oil

of the genus Piper will help in evaluating the present taxonomic grouping and in selecting

the different species for any improvement programme.