Screening of essential

oils

Larvae
Pupae

Fumigation

Evaluation of essential
oils at high conc.

Contact
toxicity

Evaluation of essential
oils at low conc.

Larvae

Mechanism of action

Pupae

GC-MS
SPME
Stability
Particle size
analysis

Zeta potential
Flash
point
Viscosity

Characterization of oil

Preparation of
formulations
Characterization of
formulation
Field bioassay

Repellency

Adult

Contact
toxicity

Larvae
Pupae

SEM and TEM

analysis of treated
larva
Haemolymph
characterization of
treated adult
Emulsion
Microencapsulation

Gels

Screening of plant essential oils

Literature
reports
Aromacity
Basis for
selection
of
essential
oils

Mentha piperita
Mentha citrata
of
Ease
Eucalyptus
globulus
availability
Cymbopogon citratu
Vetiver zizanoides
Curcuma longa
C. x citratus

Oils were obtained from Kanta Chemicals,

Khari Bowli, New Delhi, India. The essential
oils were stored in plastic bottles at 4C.

Evaluation of essential oils (at

higher concentration)

Repellency assay
Larger
chamber
(606060c
m)
50

REPELLENC
Y
CHAMBER

Smaller
chamber
(202020cm)
Petri plate (90mm)
Oils mixed with 2-3 drops of Tween
containing filter
Concentration of oils
paper
a) 7.8 l/cm2
impregnated with
b) 3.15 l/cm2
oils
Observed for 2 h

Repellency (%) of different essential oils to adult housefly, M.

domestica at different doses (a) 3.15 l/cm2, (b) 7.8 l/cm2
(a)

Other oils showed

slight inc. in
repellency

(b)

Significant inc. in
repellency for M.
citrata from 3.15
l/cm2 to 7.8 l/cm2
Medical and
veterinary
entomology (USA),

Larvicidal bioassay
20 larvae (2nd
instar) taken
on a filter
paper
containing
diet

larvae
observed for
any change in
appearance
and mobility
(48 h)
Larval mortality
assessed by their
withering and
brownish appearance

essential oil
(15.7 l/cm2 ) in
Tween 80
sprinkled on diet

100% larval mortality in 48 h

At 24 h, M. piperita and E. globus showed 94 and
87% larval mortality
Medical and
C. longa was poor performer with 63% larval
mortality
veterinary
in 24 h
entomology (USA),

Pupicidal assay
20 pupae (3 days old) were
taken on a filter paper in a
Petri plate
Concentration of oils-3.15
l/cm2
Observed for 6 days
Percentage inhibition rate:
M. piperita, M. citrata and E. globulusMedical and
veterinary
100%, C. citratus-95%, C. longa-55%

entomology (USA),

Evaluation of essential oils (at lower

concentration)

Oils evaluated: M. piprita, M. citrata, E.

globulus, C. citratus (from the previous
experiment) and Citrussinensis
Concentration
of oils used:
Contact
1. 0.16l/cm2
toxicity
Fumigatio
2. 0.25l/cm2
n
3. 0.50l/cm2
4. 1.01l/cm2
5. 2.01l/cm2

Contact toxicity

Contact toxicity: Larvicidal

M. x piprita
M. citrata
E. globulus
C. citratus
C. x sinensis

LC50 (l/cm2)

LT50 (days)*

0.54
1.39
0.60
0.41
0.71

1.5
1.7
2.0
2.3

Contact toxicity: Pupicidal

Concentrati
on (l/cm2)

M.
piprita

M.
citrata

E.
globulus

C.
citratus

C. x
sinensis

0.16
0.25

54.5

22.7

36.3

36.4

27.3

63.6

31.8

54.5

45.4

36.4

0.50

77.3

40.9

63.6

59.1

54.5

1.01

100

54.5

77.3

68.2

59.1

2.01

100

68.2

90.9

77.3

72.7

Fumigation assay

Paper strip
impregnated with
different conc. of
oils
Fumigation
(48h) done with
3 conc. 40 l/L
50 l/L, and
70 l/L

Conical flask (1 L)

Larvae or
pupae at the
bottom

Fumigation: Larvicidal
M. x piprita
M. citrata
E. globulus
C. citratus
C. x sinensis

* Values in l/L

LC50 (24 h)*

LC50 (48 h)*

62.6
79.5
66.1
69.7
71.2

48.4
61.9
50.1
48.6
52.6

Essential
oils
M. piprita

M. citrata

E. globulus

C. citratus

C. x
sinensis

Concentratio
Number of
Percentage
n
adults
Fumigation:
Pupicidalinhibition rate
emerged
(PIR)
40 l/L

100

50 l/L

100

70 l/L

100

40 l/L

21

25

50 l/L

18

36

70 l/L

12

57

40 l/L

68

50 l/L

89

70 l/L

100

40 l/L

100

50 l/L

100

70 l/L

100

40 l/L

15

46

SEM image analysis

SEM image of housefly larvae by M. x

piperita oil application

The intersegmental region of larvae showed swollen

spinose areas.
Larvae showed proliferation of spinous cells

SEM image of housefly larvae by M.

citrata oil application

Larvae showed proliferation of spinous cells espessially

at the anterior region
Swollen intersegmental region

SEM image of housefly larvae by E.

globulus oil application

Treated larvae showed significant shrinkage at spinose

rings with protruding intersegmental regions
proliferation of spinous cells with bleb formation

SEM image of housefly larvae by C.

citratus oil application

Larvae showed skin shrinkage and distortion with

protruding intersegmental regions.
The larvae also reveals shrinkage of anterior region
with bleb formation

SEM image of housefly larvae by C. x

sinensis oil application

Treated larvae showed dehydrated larval skin

Anterior region of treated larvae was shrunken and
withered

Characterization of
oils

GC-MS analysis of M. x piperita & M. citrata

Peak

Compounds

*KI

1
2

-Pinene
2Bromocyclohexa
nol
-Pinene
(+)-Sabinen
-Phellandrene
-Myrcene
-Terpinen
(+)-Limonene
p-Cineole
-Ocimene
p-Cymene
Isovaleric acid
2-methylbutyl
ester

948
1165

3
4
5
6
7
8
9
10
11
12
13

M.
piperita
1.64
0.08

M. citrata
1.36

943
897
964
958
998
1018
1059
976
1042
1118
1054

1.61
0.82
0.37
1.02
3.49
5.53

1.36
-

0.7
0.16
0.1

0.98
0.15

2.29
1.79
1.23
0.72
0.48
0.19

Peak

Compounds

*KI

14
15

- Terpinolen
1-Octenyl
acetate
3-Octanol
Cis-Linalooloxid
- Terpineol
(+)-Isomenthone
Menthone
Menthofuran
Menthyl acetate
D-Linalool
-Terpineol
Menthol
Linalool acetate
Menthyl acetate
Isopulegol
Caryophyllene
oxide
+/- Nerolidol

1052
1191

16
17
18
19
20
21
22
23
24
25
26
27
28
29
30

M.
piperita
0.23

M. citrata
0.36
0.59

979
1164
1109
1148
1148
1142
1304
1082
1041
1164
1272
1304
1196
1507
1564

0.44
-

0.2
0.43

0.57

25.83
5.04
0.72
0.4
0.1
26.53

24
-

7.35
0.26
0.32
4.26

1.17

3.82
26.69
0.43
0.16
1.36
-

Peak

Compounds

*KI

M.
piperita

14

Caryophyllene

1494

15

(+)-Isomenthol

1127

16

Pulegone

1212

4.11

17

-Farnesene

1440

0.9

0.49

18

-Terpineol

1143

1.42

4.54

19

(+)-Sabinol

1085

0.16

20

p-Allylanisole

1172

21

Germacrene D

1515

22

Citral b/a

1174

4.33

23

2,6-Octadien-1-ol 1352

2.38

24

Piperitone

1158

1.01

25

(+)-Carvone

1190

0.55

1.01

Monoterpene

11.98

13.22

Oxygenated monoterpene

79.56

72.6

Sesquiterpenes

2.51

5.97

Oxygenated Sesquiterpenes

4.83

2.63

Total

98.88

94.42

1.38

M.
citrata
4.37

0.87
0.62

GC-MS/SPME analysis of M. x
piperita & M. citrata
Peak

Compounds

*KI

M. citrata

948

M.
piperita
1.53

-Pinene

-Pinene

943

1.87

0.3

(+)-Sabinen

897

0.75

-Myrcene

958

0.21

Limonene

1018

5.16

0.68
1.17

Cineole

1059

9.24

0.57

-Ocimene

976

0.18

0.69

-Terpinen

998

0.31

p-Cimene

1042

0.61

0.23

10

2-Ethylhexanol

995

0.15

11

Piperitone

1158

0.52

12

Menthone

1148

35.54

0.28

13

Menthofuran

1142

5.9

0.06

Peak

Compounds

*KI

M. citrata

1082

M.
piperita
0.1

14

(+-)-Linalool

15

Menthyl acetate

1304

5.33

0.21

16

Linalool acetate

1272

39.24

17

Caryophyllene

1494

2.16

18

(.+/-.)-Menthol

1164

24.01

3.15

19

(+)-Pulegone

1212

5.44

20

1440

21

(Z)-.beta.Farnesene
-Terpineol

0.12

22

36.14

3.6

1143

0.25

4.09

Germacrene D

1515

0.24

0.18

23

Neryl acetate

1352

1.63

24

Geraniol acetate

1352

0.56

25

cis-Geraniol

1228

1.60

Monoterpene

10.62

Oxygenated monoterpene

86.33

87.47

Sesquiterpenes

2.4

3.9

Total

99.35

94.50

3.13

GC-MS analysis of E. globulus

Peak

Compounds

*KI

Area (%)

-Pinene

948

14.15

-Pinene

943

3.70

-Myrcene

958

1.88

-Phellandrene

969

0.93

1,4-Cineol

1012

4.07

D-Limonene

1018

10.09

1,8-Cineole

1059

33.62

-Ocimene

976

0.20

-Terpinen

998

2.92

10

o-Cymene

1042

3.68

11

- Terpinolen

1052

5.95

12

-Pinene oxide

961

0.39

13

o-Methyl-methylstyrene

1073

0.35

Peak

Compounds

*KI

Area (%)

14

Menthone

1148

0.31

15

Linalool

1082

2.34

16

Linalool acetate

1272

2.35

17

Terpinenol

1137

2.42

18

Fenchol

1138

0.96

19

-Terpinyl acetate

1348

0.21

20

-Terpineol

1158

0.82

21

DL-Menthol

1164

0.34

22

trans-Pinocarveol

1131

0.15

23

Borneol

1088

0.33

24

Citral b/a

1174

1.19

25

-Terpineol

1143

4.72

26

Verbenone

1119

0.10

27

Neryl acetate

1352

0.22

28

p-Tolylmethylcarbinol

1169

0.28

29

Geraniol acetate

1352

0.24

30

trans-Geraniol

1228

0.24

GC-MS/SPME analysis of E.
globulus
Peak
Compounds
*KI
Area (%)
1

-Pinene

948

16.88

-Pinene

943

1.01

Myrcene

958

0.83

1,4-Cineole

1012

0.84

D-Limonene

1018

5.54

p-Cineole

1059

56.54

-Terpinene

998

1.11

p-Cimene

1042

1.76

1023

3.41

10

4-Methyl-3-(1methylethylidene)-1cyclohexene
(+-)-Linalool

11

Linalool acetate

1272

3.42

12

Terpinenol

1137

1.27

13

-Terpineol

1143

3.39

3.36

GC-MS analysis of C. citratus

Peak

Compounds

*KI

Area (%)

-Pinene

948

4.73

-pinene

943

1.56

-Myrcene

958

0.29

-Phellandrene

969

0.10

1,4-Cineol

1012

0.50

D-Limonene

1018

2.24

1, 8-cineole

1059

7.52

-Ocimene

976

0.39

-Terpinen

998

0.68

10

o-Cymene

1042

0.52

11

4-Nonanone

1052

1.32

12

6-Methyl-5-hepten-2one
Citronellal

938

2.27

1125

0.44

13

Peak

Compounds

*KI

Area (%)

14

(+)-Cycloisosativene

1125

0.63

15

Capraldehyde

1204

0.28

16

-Cyclocitral

1175

0.35

17

Linalool

1082

1.52

18

(+)-Verbenol

1136

0.58

19

Terpinenol

1137

0.61

20

Fenchol

1138

0.51

21

-Citral

1174

17.98

22

- Citral

1174

29.02

23

-Terpineol

1143

0.86

24

(-)-Borneol

1138

0.75

25

Germacrene D

1515

1.32

26

Geraniol acetate

1352

4.18

27

-Cadinene

1469

1.13

28

-Muurolene

1435

2.13

29

Geraniol

1228

5.45

30

p-Cymen-8-ol

1197

0.08

GC-MS/SPME analysis of C. citratus

Peak

Compounds

*KI

Area (%)

-Pinene

948

2.67

Camphene

943

0.71

(-)-Limonene

1018

0.86

1,8-Cineole

1059

8.59

trans-.beta.-Ocimene

976

0.14

Terpinolene

1052

0.41

Nonan-4-one

1052

0.58

Methyl-5-heptene-2-one 938

1.42

-Citronellal

1125

0.14

10

(+-)-Linalool

1082

1.32

11

d-Verbenol

1136

1.12

12

Linalool acetate

1272

0.78

13

Caryophyllene

1494

2.33

Peak

Compounds

*KI

Area (%)

14

-Citral

1174

39.35

15

- Citral

1174

25.56

16

-Terpineol

1143

0.64

17

Camphol

1138

0.21

18

Geraniol acetate

1352

4.12

19

Cadina-1(10),4-diene

1469

0.56

20

Germacrene D

1515

1.68

21

Geraniol

1228

5.68

22

Caryophyllene oxide

1507

0.32

GC-MS analysis of C. x sinensis

GC-MS/SPME analysis of C. x sinensis

Mentha oil Nano-encapsule

Preparation for Nano-encapsule

Different parts of PEG6000 were heated (65
C) and supplied with M. piperita essential
oil
After being melt, it is mixed with 5.0, 7.5, and
10.0% of oil to prepare nano-particles of
different oil load.
The melted mixture stirred with glass rod to
ensure even distribution
After being cooled at 25 C, mixture grounded
in a mortar pestel, sieved and stored in air
tight container till further experiments.

Encapsulation efficiency and oil load

The encapsulation efficiency (%) and oil loading (%) of the
prepared nanoparticles were calculated by using the
following formulae:

Nano-particle 5%

7.5%

10%

Where, Wm = weight of nanoparticles just after preparation,

Encapsulation
78.2 after complete 83.4
W0 = weight of nanoparticles
evaporation of M.78.8
piperita oil
efficiency (%)
W1= amount of oil introduced into nanoparticle,
Oil loading (%) 4.5
6.75
9
W2= total amount of polymer used.

Oil release studies

Nano-particle samples (0.1 g) containing different quantities of M.
piperita essential oil was dissolved separately in 10 mL of absolute
ethanol. The mixture of nano-particles and ethanol was heated in a hot
water heater (60 C) until completely dissolved. An aliquot (3 ml) was
removed at appropriate time intervals, filtered and assayed
spectrophotometrically at 313 nm (Perkin-Elmer) for the determination of
cumulative amount of oil release up to 4 h. Each determination was
carried out in triplicate.

Particle size
analysis
Nanoparticle
size analysis
was carried by
particle size
analyzer
(Mastersizer
2000,
Malvern).

Avg. size = 531 nm

Avg. size = 422 nm

Zeta potential
analysis
ZP = -7.12 mV

ZP = -4.58 mV

FTIR spectra
FTIR spectra of nanoparticles
were recorded using KBr pellet
in a Nicholet (model Impact410) spectrophotometer.
FTIR spectra of nanocapsule at
different concentration and shell
material; PEG 6000 (Fig. 1)
showed closely matching
characteristic peaks of C-H
bending vibrations at 841 and
947 cm1, C-O stretching
vibrations at 1060, 1104 and
1145 cm1 and C-N stretching
vibrations at 1359, 1341, 1279
and 1240 cm1. A C-H stretching
vibration at 2882 cm1 and a C=C

XRD
X-ray diffraction (XRD) reveal
information about thecrystal
structure, chemical
composition, and physical
properties of materials and
thin films.
XRDIt is based on
Peak :
theelasticscattering
of X-rays
2(d/inte
nsity) the electron
5%
7.50%
from
clouds of10%
19.45atoms
20.0in the
Peakindividual
1
the
(4.55/82.9 (4.43/94.1 19.69
system.
)
)
(4.5/100)
Peak 2
Peak 3

PEG6000
20.01
(4.43/91.4
)
23.27
23.97
23.51
23.98
(3.82/67.8 (3.71/76.3 (3.78/67.2 (3.70/77.5
)
)
)
)
24.3
23.79
23.56 (3.67/99.9 (3.73/99.9 24.27

DSC
Differential scanning
calorimetry is a
technique used to study
the effect of heat on
polymer, and to determine
thethermal transitionsof
a polymer.
DSC analysis indicate
endothermic process in
the temperature range
between 40-78 C, with an
energetic effect of 328.2
J/g (10%), 25-55 C, 40-85
C, with an energetic
effect of 317.0 J/g (7.5%),
with an energetic effect of

Composition analysis of oil

Chemical composition of M. piperita oil was determined by
GC-MS with a flame ionization detection (FID) detector fitted
with a 60 m 0.25 mm 0.25m WCOT column coated with
diethylene glycol (AB-Innowax 7031428, Japan). The samples
were analysed on Shimadzu instrument fitted with the same
column.
Table 3. Major chemical components of M. piperita essential oil

S. No.
1
2
3
4
5
6
7

Compounds
Menthol
Menthone
Menthyl acetate
p-Cineole
Menthofuran
+/- Nerolidol
Pulegone

M. Piperita oil
26.53
25.83
7.35
5.53
5.04
4.26
4.11

SEM image analysis

Surface characterization of the gold coated nanoparticles
were done by using scanning electron microscope (ZEISS
EVO 50) at analytical condition: EHT 20.00 kV, WD
9.5 mm, Signal A SE1.

Fig. 1. SEM image of Mentha piperita nanoencapsule, (a) 5%, (b) 7.5%, (c) 10%

Repellency assay
The repellency index (RI) was calculated according to
RI = [(NcNt)/Nc ]* 100
where Nc is the percentage in zone b for the control, and Nt
is the percentage in the zone b of the treatment. The RI
means were analyzed by ANOVA. The means were separated
using the Duncan test.

Larvicidal : LAB Assay

Larvicidal : FIELD Assay

Field dimension: 10 m 8 m
Larvae take for each replicates = 800
3 Replication

Mentha oil Emulsified

Concentrate (EC) & Emulsion

Emulsion preparation
Emulsified concentrate (EC) formulation of M. piperita
oil was prepared by taking (w/w) 40% oil, 45%
aeromax, 3% butanol-1 and 12% surfactant.
Two surfactants, CABS-70 and NP-20 were used in the
variable ratio (0:100, 10:90, 20:80, 30:70, 40:60, 50:50,
60:40, 70:30, 80:20, 90: 10, 100:0) to prepare ECs of
three different surfactant concentrations.
Prepared EC were scanned on the basis of their physical
properties (creaming volume , i.e. the appearance of an
oil phase at the surface of the system, sedimentation,
Emulsified
of
color, appearance)
andConcentration
best three, designated
as A, B
concentrate
surfactants
(%)
and C were
selected for
further characterization.
(EC)

CABS-70

NP-20

Characterization of Emulsion
Effect of surfactant concentration
Effect of homogenization
Effect of centrifugation
Effect of temperature
Viscosity analysis
Freeze thaw property
pH analysis
Flash point analysis

Effect of surfactant concentration

le 2. Properties of three emulsified concentrate (EC) formulation of M. piperit

Properties
pH
Relative
Creaming
Volume (%)
Electrical
conductivity
Flash point (C)
Fire point (C)

A
6.6
1.01

B
6.65
0

C
7.54
2.04

8.8
mS/cm
92
102

47.7 S/cm

30.8 S/cm

91
101

90
101

The two of ECs, A and B exhibited slightly acidic pH while EC

C had slightly alkaline pH.
There was no creaming layer observed in EC B whereas EC
C showed a relative creaming value of 2% .
Relatively high flash point of observed in this study, makes it
less volatile and safer to transport and handle.

Effect of homogenization
EC formulations of M. piperita oil were
subjected to three cycle of homogenization,
each 15000 rpm for 30 min and characterized
after each cycle.
Determination of the stability of emulsions
Determination of droplet size
Determination of Zeta potential

Determination of the stability of emulsions

Creaming kinetics of each ECs were evaluated by
preparing 1% emulsion in double distilled water.
100 ml of each emulsion were transferred to a
glass cylinder with stopper and kept for 2 h at
room temperature (21 C 2) and visually
examined.
The height of visible creamy layer (H C) and height
of emulsion (HE) was recorded. Relative creaming
layer was determined as:

Determination of droplet size

Droplet size distribution of the Mentha oil emulsion was
determined by Dynamic Light Scattering (Malvern
instruments).
Sampling was carried out with 0.01% emulsion, freshly
prepared in double distilled water.
Each sample was analyzed twice with each analysis
consisting of three replicates.

Determination of Zeta potential

The zeta potential was measured by a Zetasizer
2000 (Malvern Instruments).
The measurements were carried out with 0.01%
emulsions, in fully automatic mode.
Each sample was analyzed twice, each analysis
consisting of three replicates.

Effect of temperature
Each ECs were filled in cylindrical tubes (height ,
dia. ) and were kept at 6 different temperature
variations; 4 C, 15 C2, 30 C2, 45 C2 and 60
C2.
Observations were made each week for 6 weeks.
For each ECs three replicates were used.
Storage of ECs at different temperature
demonstrated yellowish-reddish-dark reddish
appearance.
Highest relative creaming volume was seen in EC
C at different temperature, while EC A and EC B
showed comparatively thin creaming layer.

Effect of centrifugation
Effect of centrifugation was measured with 10 ml of
1% emulsions at a centrifugal acceleration of
15,000 g for 20 min and 4 C.
Each sample was subjected to 3 centrifugation
cycle, after which their creaming value was
measured.
Emulsion showed thin creaming layer at the upper
surface without any phase separation. EC B
showed relative creaming volume of 2%, after 3 rd
centrifugation cycle, thus maximum stability.
Minimum stability was found in EC C, having
relative creaming volume of 6%, after 3rd
centrifugation cycle.

Viscosity analysis
Viscosity was measured
with a Brookfield
Viscosimeter
(Brookfield, USA).
Viscosity of ECs was
measured at different
shear rate.
Viscosity profile of EC
A, while Fig. 6a & 6b
shows that of EC B and
C, respectively.

Freeze/thaw cycles
Each ECs were filled in cylindrical tubes (height , dia. ) and
stored for 24 h in a freezer at 21 C and then for 12 h at
room temperature (21 C2).
This cycle was repeated six times. After each cycle, 1%
emulsion was prepared and any change observed.
No visible change in appearance was observed in EC A
and B after 6 freeze/thaw cycles while EC C showed
tendency to rupture i.e. dispersion of oil droplets in
emulsion.

Micro-Emulsion

Micro-Emulsion preparation

Particle size determination

Particle size was determined by Dynamic Light
Scattering (Malvern instruments).
Sampling was carried out with 0.01% microemulsion, freshly prepared in double distilled water.
Each sample was analyzed twice with each analysis
consisting of three replicates.
Avg. size=134 nm

Determination of Zeta potential

The zeta potential was measured by a Zetasizer
2000 (Malvern Instruments).
The measurements were carried out with 0.01%
emulsions, in fully automatic mode.
Each sample was analyzed twice, each analysis
consisting of three replicates.
ZP= -13.2 mV

NMR analysis

Viscosity analysis
Viscosity was measured with a Brookfield
Viscosimeter (Brookfield, USA).
Viscosity of ECs was measured at different
shear rate.

Effect of centrifugation
Effect of centrifugation was measured with 10 ml of
1% micro-emulsions at a centrifugal acceleration of
15,000 g for 20 min and 4 C.
Each sample was subjected to 5 centrifugation
cycle, after which their creaming value was
measured.
Micro-emulsion showed no change in visual
appearance after 3 centrifugation cycle but after
that a phase seperation was observed.

Freeze/thaw cycles
Each micro-emulsion (5 ml) were filled in
cylindrical tubes (height , dia. ) and stored for
48 h in a freezer at 21 C and then for 48 h at
room temperature (21 C2).
This cycle was repeated six times.
No visible change in appearance was observed
in micro-emulsion after 6 cycle of freeze-thaw.

pH & Electrical Conductivity

pH was measured by a HI 2212 pH meter
(Hannah instruments, USA) at room
temperature (21 C 2).
Conductivity was measured by HI 9835
conductivity meter (Hannah instruments,
USA) at room temperature (21 C 2).
Value of pH for micro-emulsion was; 3.96,
while conductivity for the same was; 863
S/cm.