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Department of Pharmaceutical Sciences, Kumaun University, Nainital

CHAPTER: 3

PHYTOCHEMICAL

ANALYSIS

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Phytochemical Analysis

Department of Pharmaceutical Sciences, Kumaun University, Nainital Page 48

BOTANICAL IDENTITY

The plant sample for authentication was collected from Bhimtal (Nainital),

Uttarakhand, India. The sample was identified by Dr. Tariq Husain, Head

Biodiversity and Angiosperm Taxonomy, National Botanical Research Institute

(NBRI), Lucknow, India as Citrus medica L. of family Rutaceae with accession

no. 97840. The specimen was deposited in the same institute for future reference.

COLLECTION OF THE PLANT MATERIALS

The parts of Citrus medica Linn. selected for present study were root, stem bark,

leaves, flowers, fruit, fruit peel and seeds. The different parts of the plant were

collected when they were expected to have maximum amount of active

constituents. All samples were collected locally from Bhimtal (Nainital).

Leaves: The usual time for the collection of leaves is when the flowers are just

beginning to expand or the flowering is just arriving at its height. At this time the

leaves are in the healthiest state and contain an optimum of the products of the

plants metabolism and, therefore, suited to exert the most desirable therapeutic

action (Wallis, 1997; Evans, 2002). The leaves were collected in March-April,

early in the morning in dry weather. The collected leaves were dried under shade

at room temperature, coarsely powdered and stored in well closed container.

Bark: The time of collection of bark is usually spring or early summer, when the

sap is rising in the stem and the cambium is active, therefore, more easily torn

than at other seasons. So the stem bark was collected in March-April (Wallis,

1997).

Root: Roots are usually collected when their tissues are fully stored with reserve

foods, it being assumed that medicinal constituents will be also most abundant at

this season (Wallis, 1997). For present study roots were collected in autumn

season (November-January), washed with running water to remove soil.

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Both root and bark samples were sun dried, powdered and packed separately in

tightly closed containers. Small pieces of root and root hair were used for the

extraction of essential oil.

Flowers: Flowers were collected when fully expanded in the month of March-

April early in the morning, in dry weather (Wallis, 1997). Fresh flowers were

exhausted for extraction of volatile oil.

Fruits: Fully mature fruits were available from November-January. Fresh peels

were utilized for the extraction of volatile oil while shade dried peels for

preparation of extracts.

Juice: After removing peels ripe fruits were squeezed to collect juice and stored

at 4-6ºC until used.

Pulps: Juiceless pulps were dried under sun, powdered and stored in well closed

container.

Seeds: Seeds separated from fully mature fruits were dried under sun and stored

for further use.

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PREPARATION OF PLANT EXTRACTS

a) Ethanolic extract of root, leaf, bark, peel and pulp

Extracts of bark and pulp were prepared by soaking 100 gm of powdered samples

in 250 ml of 70% ethanol while 100 gm of root, leaf and peel samples were

soaked in 500 ml of 70% ethanol for 96 h. Each mixture was stirred every 24 h

using sterile glass rod. At the end of extraction each extract was passed through

Whatman filter paper no. 1 (Whatman, UK). The filtrates were concentrated on a

rotary evaporator under vacuum at 40ºC, dried and weighed. Percentage yield of

crude extract of each sample was determined in terms of g/100 g of sample (Aqil

& Ahmad, 2003). Dried extracts were stored at 4-6ºC until used and marked as

EE(root), EE(leaf), EE(bark), EE(peel) and EE(pulp) for ethanolic extract of root,

leaf, bark, peel and pulp, respectively (Fig 3.1). Percentage yield and colour of

extracts are mentioned in Table 3.1.

100 g powdered drug

Macerated in 70% ethanol (v/v) for 96 h at room temperature

Stirred every 24 h

Filtered through Whatman filter paper and marc pressed

Filtrate concentrated on a rotavap

at 40ºC and dried

Percentage yield of dried extract

of each sample was determined in terms of g/100 g sample

Extracts stored at 4-6ºC until used

and marked as EE(root), EE(leaf), EE(bark), EE(peel) and EE(pulp)

Fig 3.1: The scheme for extraction of Citrus medica L. parts.

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Department of Pharmaceutical Sciences, Kumaun University, Nainital 1

These extracts were used for the evaluation of antioxidant and antimicrobial

activities. Ethanolic root extract and fruit juice were also screened for

antiurolithiatic activity.

Table 3.1: Percentage yield and colour of C. medica L. extracts.

S.No. Ethanolic extract (EE) % Yield Colour

1 Root 7.43 Brown

2 Leaf 9.60 Blackish green

3 Bark 5.80 Yellowish brown

4 Peel 24.60 Yellowish brown

5 Pulp 23.26 Chocolate coloured

b) Root extracts for anthelmintic activity

Powdered root sample was extracted by continuous hot extraction method using

Soxhlet extractor (Houghton & Raman, 1998) in ethanol (70% v/v) at 50ºC for 3

h. Solvent extract was concentrated under vacuum and dried (Fig 3.2).

Root powder

Fig 3.2: The scheme for extraction of Citrus medica L. root.

Residue

(Discard)

Solvent extract

Concentrated on a rotavap

Dried in oven at 40 ºC

EtOH extract-root

Soxhlet extraction in

EtOH (70%) at 50ºC for 3 h

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Root extract obtained by hot extraction method (Fig 3.2) was further fractionated

in chloroform and acetone (Fig 3.3). The fractions obtained were also screened for

anthelmintic activity.

Dried root extract in 70% ethanol (EtOH extract-root)

(as prepared in Fig 3.2)

Fig 3.3: The scheme for fractionation of Citrus medica L. root extract.

c) Seed extract for anthelmintic activity

The collected seeds were washed thoroughly under running tap water, dried under

sun and coarsely powdered. The petroleum ether extract of course powder was

prepared by Soxhlet extraction at 40ºC for 3-4 h and then the solvent was

recovered at 40ºC under reduced pressure. The extract was oily, pale yellow

coloured liquid and stored at room temperature till used in the experiment.

Fractionated with chloroform

(5 aliquots, each 20 ml)

Filtered

Filtrate Residue

Concentrated on a rotavap & dried Fractionated with acetone

(5 aliquots, each 20 ml)

Chloroform fraction Filtered

Filtrate Residue

Concentrated on a rotavap & dried

Acetone fraction

Dried

Ethanolic fraction Estelar



d) Distilled water of fruit

A fresh ripe fruit of citron was cut into small pieces with peel and taken in a

round bottom flask. Some juice was already squeezed and filled in the same flask.

The sample taken was distilled and the distillate was collected. In literature

distilled water of the fruit is reported as a sedative (Kirtikar & Basu, 1993), so this

distilled water of fruit was screened for sedative/CNS activity.

e) Decoction of fruit

For preparation of decoction fresh ripe fruits were collected, weighed and cut into

small pieces with peel; nothing have been removed. About five times of its weight

distilled water was added and heated for 2-3 h in gentle heat to reduce volume to

approximately 1/4th, then strained and used fresh for evaluation of analgesic

activity (Bhatia, 2001).

PRELIMINARY PHYTOCHEMICAL INVESTIGATION OF PLANT

EXTRACTS

Selected extracts were screened for the presence of various groups of compounds

as mentioned below.

Detection of alkaloids

Small portion of the solvent free extract was stirred with few drops of dilute HCl

and filtered. The filtrate was then tested for following tests:

a) Mayer’s test: Few drops of Mayer’s reagent were added with 2-3 ml of

filtrate. It gives a cream coloured precipitate.

b) Dragendorff’s test: Few drops of Dragendorff’s reagent were added with 2-3

ml of filtrate. It gives an orange brown precipitate.

c) Hager’s Test: Few drops of Hager’s reagent were added with 2-3 ml of

filtrate. It gives yellow precipitate.

d) Wagner’s test: Few drops of Wagner’s reagent were added with 2-3 ml of

extract. It gives a reddish brown precipitate.

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Detection of carbohydrates

Small quantity of the extract was dissolved in distilled water and filtered. The

filtrate was then subjected to Molisch’s test (general test); Benedict’s and

Fehling’s test for reducing sugars.

a) Molisch’s test: 2-3 ml of aqueous extract was mixed with few drops of α-

naphthol solution in alcohol. It was shaken and concentrate H2SO4 was added

from the side of the test tube. It shows a violet ring at the junction of two liquids.

b) Benedict’s test: 2 ml of Benedict’s reagent was mixed with 2 ml of test

solution in a test tube. The mixture was heated in boiling water bath for 5 min.

The solution appeared green, yellow or red depending on the amount of reducing

sugar present in test solution.

c) Fehling’s test: 1 ml of Fehling’s A and 1 ml of Fehling’s B solution were

mixed, then boiled for 1 min. Equal volume of test solution was added and heated

in boiling water bath for 5-10 min. Appearance of first yellow and then brick red

precipitate indicates the presence of reducing sugars.

Detection of glycosides

Small portion of the extract was hydrolysed with dilute HCl for few hours on

water bath and was subjected to Legal’s test and Keller-Kiliani test for cardiac

glycosides; and Borntrager’s test for anthraquinone glycosides.

a) Legal’s test (for cardenolides): 1 ml pyridine and 1 ml sodium nitroprusside

was mixed with 2-3 ml of the extract. A pink colour appears.

b) Keller-Kiliani test (for deoxysugars): Glacial acetic acid, one drop of 5%

FeCl3 solution and concentrate H2SO4 were added to 2-3 ml of the extract.

Reddish brown color appears at the junction of two layers and upper layer turns

bluish green.

c) Borntrager’s test: Dilute H2SO4 was added to 3 ml of extract, boiled and

filtered. To the cold filtrate, equal volume of chloroform was added, shaked well

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and organic layer was separated. On addition of ammonia, ammoniacal layer turns

pink.

d) Modified Borntrager’s test for C-glycosides: To 5 ml extract, 5 ml of FeCl3

(5%) and 5 ml dilute HCl were added and heated in boiling water bath for 5

minutes. Benzene was added in cooled solution and organic layer was separated

after shaking. Equal volume of dilute ammonia was added in organic layer.

Ammoniacal layer shows pinkish red colour.

e) Test for coumarin glycosides: Alcoholic extract when made alkaline, shows

blue or green fluorescence.

Detection of triterpenoids and sterols

Triterpenes and sterols were detected by performing following tests:

a) Liebermann-Burchard test: 2 ml of the extract was added with chloroform

and mixed, to this mixture 1-2 ml acetic anhydride was added and 2 drops of

concentrate H2SO4 was added from the side of test tube. First red, then blue and

finally green color appears.

b) To the chloroform extract acetic anhydride was added. Then concentrate

H2SO4 was added from the side wall of test tube, upper layer turns green and

indicates presence of steroids.

Detection of resins

Extract was dissolved in acetone and this solution was added in distilled water;

turbidity indicates presence of resins.

Detection of phenolic compounds and tannins

Small quantity of extract was dissolved in alcohol and subjected to following tests

for detecting presence of phenolic compounds and tannins.

a) Dilute FeCl3 solution (5% w/v) test: 2-3 ml of test solution with a few drops

of ferric chloride shows bluish-black colour.

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b) Gelatin solution: Reaction of extract with 1% solution of gelatin containing

10% sodium chloride gives white precipitate.

Detection of proteins and amino acids

A small quantity of test solution was dissolved in few ml of water and subjected

to the following tests.

a) Ninhydrin test (general test for amino acids): 3 ml of test solution was mixed

with 3 drops of 5% v/v of Ninhydrin reagent. Heating on a water bath gives blue

coloration.

b) Millon’s test (for proteins): 3 ml test solution was mixed with 5 ml Millon’s

reagent and it gives white precipitate. Heating of this solution on a water bath

gives red precipitate.

c) Biuret test (for proteins): To 3 ml test solution, 4% w/v NaOH and few drops

of 1% w/v copper sulphate solution were added. A blue color was observed.

(Anonymous, 2001; Evans, 2002; Kokate, 2006; Khandelwal, 2010).

RESULTS

The results of preliminary phytochemical screening of extracts are shown in Table

3.2.

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Table 3.2: Preliminary phytochemical screening of different extracts of C. medica

L.

S.No Chemical compounds ChE

(F)

EE

(F)

EtOH

(root)

EE

(peel)

EE

(pulp)

Fruit

deco

1 Alkaloids + + + + + +

2 Carbohydrates +

+

+

+

+

+

3 Glycosides (G)

Cardiac G. Cardenolides

Deoxysugars Anthraquinone G.

Coumarin G.

+

+

-

-

+

-

-

-

+

+

-

-

-

-

-

nt

-

-

-

nt

-

-

-

nt

4 Triterpenoids & steroids

Steroids

+

+

+

nt

+

nt

-

-

-

-

-

-

5 Resins + - - nt nt nt

6 Phenolic compounds &

Tannins

+ + + + + +

7 Proteins & amino acids -

-

-

+

-

-

ChE(F)- chloroform extract fractionated, EE(F)- ethanolic extract fractionated, EtOH

(root)- ethanolic extract of root by Soxhlet extractor, EE (peel)- ethanolic extract of peel,

EE (pulp)- ethanolic extract of pulp, G- glycosides. (+)- presence of the

phytoconstituent, (-)- absence of the phytoconstituent, (nt)- not tested.

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EXTRACTION AND CHEMICAL ANALYSIS OF ESSENTIAL OILS

Plant material

Leaves, peels, flowers and roots of C. medica L. were collected from Bhimtal to

extract essential oils. To observe effect of seasonal variation on essential oil

composition of C. medica leaf oil; two samples of leaves were collected, one in

March-April (spring season) and another sample on August-September (rainy

season); oils were extracted by steam distillation method and coaded as LS04 and

LS09, respectively. Variation on essential oil composition may also occur due to

growth of plants in different places having variations in altitude and other climatic

conditions. To study such variations leaf samples were collected from Almora

(1646 m), Berinag (1720 m) and Bhimtal (1370 m) in October and oils were

obtained by hydrodistillation method and coaded as L1-11, L2-11 and L3-11,

respectively. Essential oil from fruit peels of citron was extracted by steam

distillation method and coaded as P301. Flower and root oils were obtained by

hydrodistillation method and coaded as F304 and CMR1, respectively.

Chemical analysis of extracted oils was performed by GC/GC-MS; % yield and

colour of oils was recorded.

Extraction of essential oils

The collected plant materials were weighed before extraction. The essential oil

from larger samples was extracted by steam distillation method and from smaller

samples by hydrodistillation method. In steam distillation method, the essential oil

from fresh plant material was obtained by using a copper still fitted with a spiral

glass condenser. The distillates were saturated with NaCl and the oil was

extracted with hexane. The extracts were dried over anhydrous sodium sulphate

and the solvent was removed by using rotavap at 25ºC under reduced pressure. In

hydrodistillation method, Clevanger apparatus was used to extract essential oil

from fresh plant material and the extracted oil was dried over anhydrous sodium

sulphate.

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GC and GC-MS analyses

GC analysis : Varian 3700 and Hewlett Packard 6980 gas

chromatograph system fitted with FI detector, a

HP-5MS capillary column (30 m X 0.25 mm, film

thickness 0.25 µm) and SRI model 203 Peak

Simple chromatography data system was

used. Helium gas was used as a carrier gas.

GC-MS analysis : AGILENT 5973 Network Mass Selective Detector

interfaced with AGILENT 6856 GC system. An

HP-5MS capillary column (30 m x 0.25 mm, film

thickness 0.25 µm) was used. Helium gas was used

as a carrier gas. The Mass spectra were taken under

EI condition at 70 eV.

For the gas chromatographic analysis of oil samples, the initial oven temperature

was maintained at 50ºC for first 10 minutes and then programmed at the rate of

3ºC/min to 250ºC after which it was again maintained at 250ºC for the final 5

minutes. The composition data were taken from area percentage data without the

use of correction factors and rounded off to the first decimal place.

For calculation of Retention indices (RI) of various components a co-injection

was made of a mixture of oil and n-alkanes (C8 - C23). The Retention indices of

various constituents were calculated by using the following equation:

log Rt(unknown) – log Rt(Cn)

RI = 100 N +

log Rt(CN) – log Rt (Cn)

Where:

Rt(unknown) = Retention time of the compound

Rt(CN) = Retention time of higher alkane

Rt(Cn) = Retention time of lower alkane

N = Carbon No. of lower alkane

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For GC-MS analysis of oil sample the oven temperature was programmed at 50ºC

for first 10 minutes followed by 3ºC/min to final temperature 230ºC and

isothermal at 230ºC for the last 10 minutes.

RESULTS

The percentage yield of essential oils collected from different parts of the plant

was 0.28-0.5% in leaf, 0.4-0.5% in flower, 0.12-0.15% in peel and 0.6-0.75% in

root. Colour and percentage yield of essential oils are mentioned in Table 3.3.

Table 3.3: Percentage yield and colour of different oil samples of C. medica L.

S. No. Source of essential oil Yield (%) Colour

1. Leaf 0.28-0.50 Colourless

2. Flower 0.40-0.50 Colourless

3. Peel 0.12-0.15 colourless to pale yellow

4. Root 0.60-0.75 greenish

The major constituents identified in the leaf oil samples were limonene (18.4-

65.6%), linalool (19.1-50.5%), neral (2.4-6.2%), geranial (3.8-7.7%) and

citronellol (trace-4.6%). The leaf oil sample collected in spring season was rich in

linalool (50.4%) and limonene (18.6%) while the oil collected in rainy season

contains more percentage of limonene (56.6%) and less amount of linalool

(19.1%). However, it was observed that remarkable variation occur in the

essential oil composition of samples collected from Almora, Berinag and Bhimtal.

After completing the identification and comparing these oil compositions with

one another, it was found that the limonene content was 28.1%, 18.4% and

37.3%, respectively while linalool content was 37.5%, 50.5% and 35.2% in

Almora, Berinag and Bhimtal samples, respectively.

The identified components present in aerial parts of the plant were more or less

similar while composition of root oil was different. The identified components

with their percentage in oil from different aerial plant parts were; α-pinene (nil-

trace), sabinene (nil-trace), β-pinene (nil-trace), 6-methyl-5-hepten-2-one (nil-

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0.5%), β-myrcene (nil-1.2%), limonene (15.2-76.8%), lavender lactone (nil-

1.5%), β-(Z)-ocimene (nil-2.2%), cis-linalool oxide (trace-3.2%), trans-linalool

oxide (trace-2%), p-mentha-2,4(8)-diene (nil-trace), linalool (1.6-55.6%), Z-

isocitral (nil-1.4%), E-isocitral (nil-0.6%), citronellol (trace-3.2%), neral (2.4-

4.3%), carvone (nil-0.4%), linalool acetate (nil-1%), geranial (2.4-5.2%),

limonene-10-ol (nil-2.8%) and phytol acetate (nil-1.5%); see Tables from 3.5-3.7.

Table 3.4: Chemical composition of leaf essential oil of C. medica L. collected

from different places.

Compound RIobserved Percentage in the oil

L1-11 L2-11 L3-11

α-pinene 939 -- -- trace

sabinene 976 -- -- trace

β-pinene 979 -- -- trace

6-methyl 5-hepten-2-one 983 0.5 1.1 0.5

β-myrcene 988 0.5 0.3 trace

limonene 1026 28.1 18.4 37.3

β-(Z)-ocimene 1037 -- -- 0.6

cis-linalool oxide 1069 0.3 0.3 trace

trans-linalool oxide 1085 0.5 0.7 trace

linalool 1098 37.5 50.5 35.2

citronellol 1225 2.4 4.6 2.5

neral 1234 6.2 6.2 4.1

linalool acetate 1249 1.6 2.8 2.0

geranial 1268 7.5 7.7 3.8

Total compounds identified

Total percentage of identified compounds

10

85.1

10

93.3

14

86

RI- retention indices, L1-11- Almora sample, L2-11- Berinag sample, L3-11- Bhimtal

sample.

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Fig 3.4: GC profile of leaf essential oil of C. medica L. (L2-11).

Fig 3.5: GC profile of leaf essential oil of C. medica L. (LS04).

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Table 3.5: Chemical composition of leaf essential oil of C. medica L. collected at

different seasons.

Compound RIobserved Percentage in the oil

LS04 LS09

α-pinene 937 trace --

β-pinene 980 trace --

6-methyl-5-hepten-2-one 983 -- 0.5

β-myrcene 988 -- 1.0

limonene 1024 18.6 56.6

lavender lactone 1035 trace --

β-(Z)-ocimene 1037 -- 2.2

cis-linalool oxide 1068 3.2 trace

trans-linalool oxide 1085 1.5 trace

p-mentha-2,4(8)-diene 1087 trace --

linalool 1098 50.4 19.1

Z-isocitral 1160 1.4 --

E-isocitral 1175 0.6 --

citronellol 1225 3.2 trace

neral 1234 2.4 4.3

carvone 1243 0.4 --

linalool acetate 1249 -- trace

geranial 1268 5.2 4.8

limonene-10-ol 1292 1.2 --



15

88.1

11

88.5

RI- retention indices, LS04- leaf oil collected in spring season, LS09- leaf oil collected

in rainy season.

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Table 3.6: Chemical composition of peel oil of C. medica L.

Compound RIobserved Percentage in the peel oil (P301)

α-pinene 939 trace

sabinene 976 trace

β-pinene 979 trace

β-myrcene 988 1.2

limonene 1026 76.8

β-(Z)-ocimene 1037 trace

linalool 1098 1.6

citronellol 1225 3.0

neral 1234 2.8

linalool acetate 1249 1.0

geranial 1268 3.4

phytol acetate 2221 1.5



12

91.3

RI- retention indices.

In the root oil sample total 10 components were identified representing 52.6% of

total oil composition. The identified components with their percentage were;

limonene (2%), cis-and trans-linalool oxide (trace), linalool (1.5%), geijerene

(39%), carvone (trace), isogeijerene (0.6%), 2Z-hexanyl valerate (0.3%), 2E-

hexanyl valerate (3.4%) and myrtenal acetate (5.8%); see Table 3.8.

The results of the total identified components of essential oils obtained from

different samples along with their percentage composition and observed Retention

Indices are summarized in Tables 3.4-3.8.

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Table 3.7: Chemical composition of flower oil of C. medica L.

Compound RIobserved Percentage in the flower oil (F304)

limonene 1024 15.2

lavender lactone 1035 1.5

cis-linalool oxide 1068 2.4

trans-linalool oxide 1085 2.0

p-mentha-2,4(8)-diene 1087 trace

linalool 1098 55.6

Z-isocitral 1160 0.6

E-isocitral 1175 0.3

citronellol 1225 0.2

neral 1234 3.0

carvone 1243 0.3

geranial 1268 2.4

limonene-10-ol 1292 2.8



13

86.3


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Fig 3.6: GC profile of peel essential oil of C. medica L. (P301).

Fig 3.7: GC profile of flower essential oil of C. medica L. (F304).

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Fig 3.8: GC profile of root essential oil of C. medica L. (CMR1)

Table 3.8: Chemical composition of root oil of C. medica L.

Compound RIobserved Percentage in the root oil (CMR1)

limonene 1026 2.0

cis-linalool oxide 1069 trace

trans-linalool oxide 1085 trace

linalool 1098 1.5

geijerene 1139 39.0

carvone 1239 trace

isogeijerene 1247 0.6

2Z-hexanyl valerate 1283 0.3

2E-hexanyl valerate 1287 3.4

myrtenal acetate 1326 5.8


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DISCUSSION

C. medica L. grown in hilly regions of Uttarakhand was not investigated earlier

for its essential oil composition of different parts like root and flower and

variation in leaf oil composition with period of collection and with

altitude/climatic conditions. Leaf, peel, flower and root oils were analysed by GC

and GC-MS.

It is reported that limonene is the major component (27.8-43.2%) in leaf oil of

different varieties of C. medica L. like ethrog, sarcodactylis, corsican, diamante,

rhobs-el-arsa and poncire commun (Lota et al., 1999). In the present study, the

limonene content (18.4-37.3%) in leaf oil was found comparable to other

varieties. Neral (11.9-16.3%) and geranial (17-23.4%) were the next major

constituents in other varieties of C. medica L. as mentioned earlier (Lota et al.,

1999) while in the present study neral (4.1-6.2%) and geranial (3.8-7.7%) were

present in comparatively less amount. The next major constituent observed in the

present sample of C. medica leaf oil was linalool (35.2-50.5%) while less content

of linalool (1.1-1.6%) was reported in other varieties of C. medica L. (Lota et al.,

1999), see Table 3.4, comparisons were made on leaf oils obtained by

hydrodistillation method. All leaf oils of citron analysed in this study exhibited a

linalool-limonene composition which differed from other varieties of C. medica

L. leaf oils having limonene-geranial-neral composition (Lota et al., 1999).

Seasonal variation greatly affects the composition of leaf essential oil with respect

to limonene-linalool content. On comparing the percentage variation it was

observed that the content of linalool (50.4%) was more than the limonene (18.6%)

in spring season sample and this composition was reversed in sample collected in

rainy season with more limonene (56.6%) and less linalool (19.1%) content.

A detail study on leaf oils obtained from Almora, Berinag and Bhimtal samples

showed a remarkable difference especially with respect to major components in

their percentage composition. The leaf oil sample from Bhimtal showed presence

of limonene (37.3%), linalool (35.2%), neral (4.1%), geranial (3.8%), citronellol

(2.5%), and linalool acetate (2%) as major constituents. The essential oil from

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Almora sample contained linalool (37.5%), limonene (28.1%), geranial (7.5%),

neral (6.2%), citronellol (2.4%) and linalool acetate (1.6%) as main constituents.

The essential oil from Berinag sample have shown almost same composition but

percentage variation was observed with high content of linalool (50.5%) followed

by limonene (18.4%), geranial (7.7%), neral (6.2%), citronellol (4.6%), linalool

acetate (2.8%) and 6-methyl-5-hepten-2-one (1.1%).

The results indicated that with increase in altitude limonene content was

decreased as 37.3% in Bhimtal (1370 m) sample, 28.1% in Almora (1646 m)

sample and 18.4% in Berinag (1720 m) sample. Linalool, neral and geranial

contents were increased with increase in altitude from 35.2-50.5%, 4.1-6.2% and

3.8-7.7%, respectively. With altitude, other climatic conditions may also affect

the composition of essential oils.

Peel oil composition of five varieties viz., ethrog, sarcodactylis, corsican,

diamante and rhobs-el-arsa differed only quantitatively. The major component

was always limonene (46.9-93.6%) with other components like γ-terpinene (trace-

31.3%), geranial (0.8-14.4%) and neral (0.4-7.6%). (Lota et al., 1999). The

‘rhobs-el-arsa’ peel oil exhibited a high amount of limonene (93.6%) and the

percentage of other components were very low (≤ 1.5%), geranial and neral were

accounted for only 0.8% and 0.4%, respectively. The ‘diamante’ oil also exhibited

a high content of limonene (70.4%) and also contained appreciable proportions of

geranial and neral, 14.4% and 7.6%, respectively. The peel oils of the corsican,

sarcodactylis and ethrog varieties belonged to the limonene/γ-terpinene

chemotype with limonene (46.9-51.9%) and γ-terpinene (26.2-31.3%) as major

constituents, while contents of geranial and neral ranged from 2.6-5.4% and 1.3-

2.8%, respectively (Lota et al., 1999).

The peel oil composition of present sample also exhibited limonene (76%) as the

major constituent and was also characterized by the presence of geranial (3.4%),

citronellol (3%), neral (2.8%), linalool (1.6%), phytol acetate (1.5%), β-myrcene

(1.2%), linalool acetate (1%) and other components in trace amount. To the best

of our knowledge, earlier no work was done on the essential oil composition of

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citron flower and root samples grown in Kumaun region of Uttarakhand. Essential

oil of flowers like other aerial parts was also dominated by linalool (55.6%) and

limonene (15.2%). Root oil of citron exhibited high content of geijerene (39%).

Geijerene and its precursor pregeijerene, released by citrus roots were identified

as attractants for the plant parasitic nematodes and also to entomopathogenic

nematodes, which act against citrus root feeding insects. So these compounds

were regarded as safe and effective insect control agents to protect roots of citrus

and other important plants (Ali et al., 2010).

Some of these essential oil compositions were helpful to understand the biological

activity of their constituents and are mentioned in concerned chapters.

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REFERENCES

Adams RP. (1995). Identification of Essential Oil Components by Gas

Chromatography/Mass Spectroscospy. Illinois, USA: Allured Publishing Corp.

Ali JG, Alborn HT, Stelinski LL. (2010). Subterranean herbivore-induced

volatiles released by Citrus roots upon feeding by Diaprepens abbreviatus

recruit entomopathogenic nematodes. J Chem Ecol, 36, 361-68.

Anonymous. (2001). The Ayurvedic Pharmacopoeia of India, part I, vol III. New

Delhi, India: Govt of India.

Aqil F, Ahmad I. (2003). Broad spectrum antibacterial and antifungal properties

of certain traditionally used medicinally plants. World J Microbiol Biotechnol,

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Bhatia SC. (2001). Perfumes and Cosmetics, 1st ed, vol II. New Delhi, India: CBS

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Kingdom: Saunders, p. 64, 193.

Houghton PJ, Raman A. (1998). Laboratory Handbook for the Fractionation of

Natural Extracts, 1st ed. London, UK: Chapman & Hall, p. 29.

Khandelwal KR. (2010). Practical Pharmacognosy, 20th ed. Pune, India: Nirali

Prakashan, p. 25.1-25.7.

Kirtikar KR, Basu BD. (1993). Indian Medicinal Plants, vol I. Dehra Dun, India :

Bishan Pal Singh Mahendra Pal Singh, p. 486.

Kokate CK. (2006). Practical Pharmacognosy, 4th ed. Delhi, India: Vallabh

Prakashan, p. 108-11.

Lota ML, Serra Dde R, Tomi F, Bessiere JM, Joseph C. (1999). Chemical

composition of peel and leaf essential oils of Citrus medica L. and C.

limonimedica Lush. Flavour Fragr J, 14, 161-166.

Wallis TE. (1997). Text Book of Pharmacognosy, 5th ed. Delhi, India: CBS

Publishers and Distributors, p. 69, 104, 160, 353.

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