16

2.1 INTRODUCTION
Mentha, a genus of labiatae family, includes 20 species spread all over the world few of
them are peppermint, spearmint, wild mint, corn mint, bergamot, of which spearmint is
the most common of all (Paranjpe, 2001). It has pungent taste with post digestive effects
and has hot potency (Syed and Sharma, 2002). Different species of mint have similar
constituents and all are important from dietary point of view (Gopalan et al., 1971). Mint
descends from the Latin word menthe, which is rooted in the greek word minthe
mentioned in the Greek mythology as minthe, a nymph who was transformed in to a mint
plant (Quattrocchi and Umberto et al., 1947).
Water disitillate of spearmint relieves hiccup and flatulence as well as giddiness of
indigestion. The ancients used spearmint to scent their bath and as a restorative (Sivarajan
and Balachandran, 1994). The distilled oil of mint is used to flavor toothpaste,
confectionery, chewing gum and also used to perfume soaps. Mentha constituents have
antifungal, antiviral, antimicrobial, insecticidal, antioxidant, anti amoebic, antihemolytic,
anti allergenic, CNS depressant and anthelmintic activities (Sharma, 1993, Rastogi and
Mehrota, 1998).
Various phenolic acids, flavonoids, terpenoids and other volatile compounds from
different extracts of mint have been reported. Phenolic acids and their derivatives are
widely distributed in plants (Olennikov and Tankhaev, 2010, Voirin and Bayet, 1994).
The bottom pitch waste a thick; fatty acid smelling material is inexpensive and finds use
as an adulterant to perfumed incense stick.
17

The present work comprises of removal of long chain fatty acids by saponification,
isolation of constituents using hydrodistillation and solvents of different polarity and
characterization using GC-MS and GC-FID.
Mentha piperita (Corn Mint) is a species of mint native to the temperate regions of
Europe and Western and Central Asia, eastern to the Himalaya and eastern Siberia.

Mentha piperita

Mentha piperita flowers
Figure 4. M. piperita plant
18

2.1.1 Reported chemical constituents of Mentha piperita
The essential oil of Mentha piperita (yield up to 2.5% in the dried leaves) mostly contains
l-menthol (ca. 50%), menthones (10-30%), menthyl esters (up to 10%) and some
monoterpene derivatives viz., menthofuran. Traces of jasmone (0.1%) improve the oil
quality remarkably. Menthol and menthyl acetate are responsible for the pungent and
refreshing odour; they are mostly found in older leaves and are preferentially formed
during long sunlight periods. On the other hand pulegone is not preferred in perfumery
industries as it does not possess delightful fragrance (Wallis et al., 2005).
OH

O

O

O

l-menthol l-menthone Menthofuran Pulegone
Figure 5. Reported constituents of M. piperita
2.2 AIM OF WORK
Mentha piperita is a comercially important medicinal plant yielding essential oil i.e. used
widely by cosmetic, liquor, confectionary, pharmaceutical and related industries. The oil
is generally extracted by steam distillation. Mentha after steam distillation gives two
types of waste products i.e. high boiling hydrosol and bottom pitch which sells at very
low prices for the preparation of incense sticks. The bottom pitch waste is a thick; fatty
acid smelling material and is inexpensive. The present work comprises of removal of
long chain fatty acids by saponification, isolation of constituents using hydrodistillation,
solvents of different polarity and characterization using GC-MS and GC-FID.
19

2.3 RESULTS AND DISCUSSION
The present investigation has shown that important product like 3-octanol, menthols are
hooked to long chain fatty acids as esters and also serve as fixative to other non hooked
terpenoids. GC-MS data of bottom pitch after saponification showed maximun
concentration of menthols, whereas high boiling hydrosol showed menthone in
appreciable quantities. Moreover, dichloromethane extract of bottom pitch oil contains
viridifloral in small amounts along with other constituents as shown in table 2-7. The
saponified material of bottom pitch was characterized by GC. The GC data showed (fig.
6) presence of oleic and palmitic acid in appreciable amount (50-60%) which have
extensive commercial acceptance in pharmaceutical industries as an excipient
(Smolinske, 1992; French et al., 2002). The fatty acids were characterized by comparing
the GC data with the marker compounds.
Sr. No. Compounds Identified R.I.* % w/w
1 l-menthone 1603.857 25.25
2 Cadinene 1732.943 0.98
3 -cadinol 2114.671 2.74
4 Caryophylline oxide 2180.577 0.94
Table 2. GC-MS data of hydro distilled high boiling hydrosol oil
Sr. No. Compounds Identified R.I.* % w/w
1 Limonene 1239.623 4.43
2 p-Methyl cumyl alcohol 1245.764 10.62
3 Menthol 1469.959 33.92
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Table 3. GC-MS data of hydro distilled bottom pitch oil
Sr. No. Compounds Identified R.I.* % w/w
1 Menthone 1467.91 2.64
2 2-isopropyl-4-menthyl hex-2-enal 2115.062 1.23
3 3-octanol 1527.904 2.46
Table 4. GC-MS data of solvent extracted high boiling hydrosol constituents in
dichloromethane
Sr. No. Compounds Identified R.I.* % w/w
1 Nerolidol 1994.651 1.02
2 Cadinol 2113.233 2.19
3 Duvatriendiol 2235.377 0.58
4 2-(4-hydroxybutyl)cyclohexanol 2314.457 5.96
5 Viridiflorol 2445.199 1.74
Table 5. GC-MS data of solvent extracted bottom pitch constituents in dichloromethane
Sr. No. Compounds Identified R.I.* % w/w
1 1-menthone 1805.962 2.52
2 p-menthane -1,3-diol 2059.01 5.40
3 Piperidine,1-(1-oxo-3-phenyl-2-
propenyl)
2331.492 12.81
21

Table 6. GC-MS data of solvent extracted high boiling hydrosol constituents in
ethylacetate
Sr. No. Compounds Identified R.I.* % w/w
1 l-menthone 2235.377 0.58
2 2-(4-hydroxy butyl) cyclohexanol 2445.199 1.74
Table 7. GC-MS data of solvent extracted bottom pitch constituents in ethyl acetate
R. I. = Retention indices

Figure 6. GC chromatogram of saponified material showing presence of oleic and
palmitic acids.


22

2.4 EXPERIMENTAL
Plant source The waste product of Mentha piperita i. e. Bottom pitch and High boiling
hydrosol was provided by Hindustan Mint and Agro products private ltd. Chandausi.
Uttar Pradesh. India
2.4.1 Isolation procedure
2.4.1.1 Isolation of free fatty acids from bottom pitch
Bottom pitch (30 ml) was subjected to saponification with 5% methanolic KOH under
reflux for 3 h, the saponified material was derivatized to methyl esters using
HCl/methanol under reflux for 6 h. The mixture thus obtained was worked up, extracted
with ether, dried and finally analyzed using GC-FID.
2.4.1.2 Isolation of essential oil from bottom pitch
Bottom pitch after saponification was subjected for 3 h to hydrodistillation using a
Clevenger apparatus (yield: 0.5/30ml). The essential oil obtained was dried, filtered and
stored at 4

C and characterized using GC-MS.

2.4.1.3 Isolation of essential oil from high boiling hydrosol
High boiling hydrosol (30 ml) was subjected to 3 h hydrodistillation using a Clevenger
apparatus (yield: 0.5/30ml). The essential oil obtained was dried, filtered & stored at 4

C
until tested and analysed.
2.4.1.4 Isolation of essential oil from bottom pitch residue by solvent extraction
The hydrodistilled bottom pitch residue was dried, extracted successively with petroleum
ether 60-80 (to remove fats and waxes), dichloromethane and finally with ethyl acetate.
The obtained extracts were concentrated in vacuo at 40C using a rotary evaporator (yield
was 0.1-0.15 ml of each extract).
23

2.4.1.5 Isolation of essential oil from high boiling hydrosol residue by solvent
extraction
The same procedure as described for the bottom pitch residue was adopted for the
isolation of essential oil from high boiling hydrosol.
The complete detail of the procedure applied for the extraction is shown in the flow chart
fig. 7 for High boiling hydrosol and fig. 8 for Bottom pitch.









Figure 7. Procedure followed for the isolation of constituents from high boiling hydrosol



High boiling hydrosol (30 ml)
hydrodistillation
residue
oil (0.5 ml)
petroleum ether
Fats and waxes residue
dichloromethane
oil (0.2 ml)
residue
ethyl acetate
oil (0.15 ml)
residue
24

Bottom pitch (30 ml)
saponified residue
5% KOH
diethyl ether
ether layer
residue
aqueos layer
acidified with HCl
extracted with ether
fatty acids
methyl ester
GC
hydrodistillation
residue
oil (0.5 ml)
petroleum ether
fats and waxes
residue
dichloromethane
oil (0.15 ml) residue
oil (0.1 ml)
residue
ethylacetate

Figure 8. Procedure followed for the isolation of constituents from bottom pitch

25

2.5 Spectral analysis
2.5.1 GC-MS analysis
The GC-MS (70 eV) data were generated on MS-QP2010 series Shimadzu, Tokyo,
Japan equipped with FID, AOC 20i auto-sampler and BP-20 capillary column 30 m x
0.25 mm x 0.25 m (polyethylene glycol, TPA treated). The oil sample (10 l) was
diluted (up to 2 ml) with dichloromethane (HPLC grade), one l of this was injected for
the analysis. The Helium gas was employed as carrier to maintain flow rate of 1.2
ml/min. During the analysis following conditions were maintained; split ratio 1:50; mass
scan 50-800; oven temperature programmed from 40

C to 220

C at the rate of 4

C/min,
held isothermally at 40

C and 220

C for 5 min each. Ion source temperature 200

C;
interface temperature 250

C; injector temperature was kept constant at 220

C.
2.5.2 GC-FID analysis
The chromatographic system adopted to find out fatty acids as their methyl esters
consisted of NUCON 5675 GC, India with flame ionization detector, manual injector and
Stationary phase ECTM-WAX column, 30 m x 0.25 mm x 0.25 m (coated with
polyimide). The oil sample (10 l) was diluted (up to 2 ml) with dichloromethane (HPLC
grade), one l of this was injected for the analysis. The gas pressure was maintained at
56:10:10 psi for NO
2
, H
2
, O
2
respectively. During the analysis following conditions were
maintained split ratio 1:50; oven temperature programmed from 40

C to 240

C

at the rate
of 5

C/min, held isothermally at 240

o
C

for 10 min each. Injector temperature was kept
constant at 230
o
C