CHEMICAL INVESTIGATION, ANTIMICROBIAL AND ANALGESIC

ACTIVITY OF SOLIDAGO CANADENSIS, SENECIO RUFINERVIS

AND LEUCAS HYSSOPIFOLIA

Supervisor Research Scholar

Devendra Mishra
Prof. Ganga Bisht
Head, Deptt. of Chemistry
Kumaun University, Nainital.
 Objectives

 To find out the essential oil composition of the plants selected

for study.
 Isolation and characterization of the major components of
essential oil/extracts.
 To determine Analgesic and Antimicrobial activity of
essential oil/ fractions of the plant extract.
 CONTENTS

CHAPTER 1 Introduction

CHAPTER 2 Experimental Methods

CHAPTER 3 Phytochemical Investigation

CHAPTER 4 Biological Activities

i) Analgesic activity

ii) Antimicrobial activity

PLANTS SELECTED FOR STUDY

1) Senecio rufinervis D.C. (ASTERACEAE)

Senecio rufinervis is a tall aromatic herb. The plant has

occurrence in Uttarkhand and Nepal at the altitude of 1800-

3000m. It has been screened for the presence of

sesquiterpenoids rich essential oil by Thakuri et al, 2006.

 
 2) Solidago Canadensis Linn. (ASTERACEAE)

Solidago canadensis is an herbaceous perennial plant native to the

United States. Polyacetylenes, diterpenes, triterpenes (Lu Tiansheng

et al, 1993) and flavonoids (Budzianowski et al, 1990) were isolated

from S. canadensis. Essential oil composition and antimicrobial

activity of S. canadensis have also been reported (Kalemba et al,

1990, Weyerstahl et al, 1993 & Amtmann, M. 2009).

 3) Leucas hyssopifolia Benth. (LAMIACEAE)

L. hyssopifolia Benth. (Labiatae), is an annual herb found

throughout India as a weed in cultivated fields, wastelands

and roadsides. Flavonoid glycosides were reported from the

aerial parts of Leucas lavandulaefolia (Chandrashekhar et al,

2005). Alkaloids, steroids and tritepenoids were isolated

from L. aspera, L. stricta and L. cephalotes (Kamat et al,

1994 ). The aerial parts of Leucas neufliseana were reported

to contain diterpenes and flavones (Khalil et al, 1996).

 Table 1. Essential oil composition of Senecio rufinervis D.C.

Extraction Essential oil

 Steam distillation.

 Characterization of essential oil by GC/GC-MS analysis.

Table 1. Essential oil composition of Senecio rufinervis D.C.

S.No. Compounds R.T Root oil % Leaf oil % Mode of Identification

α-Pinene 5.62 - 3.24 a,b
1
Sabinene 6.5 - 0.25 a,b
2
β- Pinene 6.8 - 12.23 a,b
3
4 α-Phellandrene 7.61 - 0.87 a,b
Limonene 8.2 - 0.1 a,b
5
P-Cymene 8.26 - 4.15 a,b
6
Borneol 13.17 2.15 - a,b
7
α-Copaene 21.27 8.14 - a,b
8
α-Cubebene 22.11 2.06 - a,b
9
α-Longipinene 22.5 6.46 - a,b
10
Iscocomene 22.82 1.68 - a,b
11
β- Cubebene 24.04 - - a,b
12
 (Z)-Caryophyllene 24.16 2.08 - a,b
13
(E)-Caryophyllene 25.12 0.32 - a,b
14
α -Murrolene 25.34 1.1 - a,b
15
α-Gurjunene 25.52 2.1 - a,b
16
α-Humulene 25.58 - 1.6 a,b
17
Seychllene 26.41 1.1 - a,b
18
Germacrene D 26.64 24.95 40.19 a,b,c
19
β-Selinene 26.73 0.93 - a,b
20
Cis-β-Guaine 27.7 0.57 - a,b
21
γ -Murrolene 28 - 2.38 a,b
22
δ-Cadinene 28.33 - 2.8 a,b
23
Trans-β-Guaine 29.61 17 - a,b
24
25 Germacrene D-4-ol 32.84 - 2.66 a,b
 
  Total 70.64 70.47  

a= Mass spectra, b= R.T, c=1H, 13C-NMR

 TABLE 2. Essential oil composition of Solidago canadensis Linn.

Mode of
S.No. Compound R.T Leaf Oil % Root Oil % Identification
1 α-pinene 5.62 0.41 0.28 a,b
2 Sabinene 6.67 0.29 0.2 a,b
3 Camphene 6.78 0.23 - a,b
4 β- Pinene 6.8 0.22 0.72 a,b
5 p-Cymene 8.27 0.28 a,b
6 Limolene 8.4 4.23 4.34 a,b
7 1,4-Cineole 8.76 - 2.9 a,b
8 1,8-Cineole 14.12 - - a,b
9 (Z)-β-Ocimene 14.7 - 1.09 a,b
10 trans- Carveol 15.52 - 2.17 a,b
11 Thymol methyl ether 16.48 - 1.5 a,b
12 Carvacrol methyl ether 16.84 - 1.99 a,b
13 Bornyl acetate 17.16 3.37 2.17 a,b,c
14 Thymol 18.98 - 20.25 a,b,c
15 Carvacrol 19.37 - 5.51 a,b
16 δ-Elemene 22.16 2.43 1.3 a,b
 17 α-Cubebene 22.33 2.42 - a,b

18 Isoledene 23.48 2.42 1.02 a,b

19 β-Elemene 24.15 - 5.51 a,b

20 Germacrene-D 26.64 64.06 6.26 a,b,c

21 γ -Muurolene 28.32 0.42 1.3 a,b

22 γ -Curcumene 29.02 2.18 - a,b

23 β -Copaen-4-α-ol 29.45 - 3.5 a,b

24 Cubeban-11-ol 30.32 - 3.32 a,b

25 Junenol 30.49 - 2.13 a,b

26 Cubenol<1,10-diepi-> 30.63 - 3.04 a,b

27 β -Cedren-9-one 31.51 - 2.11 a,b

28 Eremoligenol 32.25 - 1.35 a,b

29 Muurola-4,10(14)-dien-1-β-ol 32.36 - 1.5 a,b

30 epi-α-Cadinol 32.81 1.38 1.8 a,b

31 α -Muurol 33.3 0.83 0.53 a,b

32 Muurolene<14-hydroxy-α-> 34.45 - 1.01 a,b

  Total   84.89 79.08  

a= Mass spectra, b= R.T, c=1H, 13C-NMR.

 Flow Sheet

Solidago canadensis Linn. leaf oil (3.0 g)

CC(Et2O: n-hexane) Silica gel = 100g ( 230-400 mesh)

5% Et2O in 10% Et2O in 15% Et2O in 20% Et2O in

n-hexane
n-hexane n-hexane n-hexane n-hexane

Fr. (1-7) Fr. (8-10) Fr. (11-14) Fr. (15-19) Fr. (20-25)

D2 D3

(140 mg) (12 mg)

Scheme 3.0 Isolation of compounds from Solidago canadensis Linn.

 Flow Sheet

Solidago canadensis Linn. root oil (3.0 g)

CC(Et2O: n-hexane) Silica gel = 100g ( 230-400 mesh)

5% Et2O in 10% Et2O in 20% Et2O in

n-hexane
n-hexane n-hexane n-hexane

Fr. (1-7) Fr. (8-14) Fr. (11-17) Fr. (18-22)

Hydrocarbon mixture

D4
50mg

Scheme 3.1 Isolation of compound from Solidago canadensis Linn.

 Identification of compound D2

Physicochemical data of D2

Yield: 160 mg, viscous liquid.

EIMS: 204 (M+), 189, 161 (100%), 133, 119, 105, 91, 79, 77, 67, 65 and 43.

IR KBr νmax cm-1: 2927, 2871, 1689, 1463, 1383, 980, 734.

1
H -NMR (300 MHZ, CDCl3) δ ppm: δ 0.89 (6H, d, J=6.6 HZ, 2Me-12,13), 1.52 (3H, s, Me-14),

4.80 (2H, dd, J=13.2 HZ, H-15), 4.80 (2H, dd, J=13.2 HZ,16.5 HZ, H’-15), 5.24 (2H, m), 5.81 (1H, s).

13
C-NMR (75 MHZ, CDCl3) δ ppm: δ 129.8 (d, C-1), 29.7 (t, C-2), 34.5 (t, C-3), 148.8 (s, C-4), 135.5

(d, C-5), 133.2 (d, C-6), 52.9 (d, C-7), 26.5 (t, C-8), 40.7 (t, C-9), 133.7 (s, C-10), 32.7 (d, C-11), 19.7

(q, C-12), 20.9 (q, C-13), 15.8 (q, C-14), 109.0(t, C-15).
 14
1 9
2 8
10

3 7
13
5
4 6 11

15 12

Germacrene D
 Identification of compound D3

Physicochemical data of D3

Yield: 10mg, viscous liquid.

EIMS: 196 (M+), 154(12.43), 136(47.66), 121(72.37), 108(22.71), 107(21.11), 95(81.68),

93(100.00), 92(41.35), 91(51.42), 80(33.63), 79(34.06), 77(20.79), 67(35.58), 43(10.94).

IR KBr νmax cm-1: 2926, 2857, 1723, 1461, 1377, 1216, 1034, 764.

1
H -NMR (300 MHZ, CDCl3) δ ppm: 0.90 (6H, s, Me- 9/10), 1.28 (3H, s, Me-7), 2.06 (3H,s, Me-12), 4.89

(2H,m, H-1).

13
C-NMR (75 MHZ, CDCl3) δ ppm: 79.9(d, C-1), 48.2(s, C-2), 37.5(t, C-3), 27.5(t, C-4), 45.5(d, C-5), 28.7(t,

C-6), 21.1(q, C-7), 48.2(s, C-8), 19.1(q, C-9), 15.9(q, C-10), 170.7(s, C-11), 19.1 (q, C-3).
 7

9
2 O
3 1
11 O
8
6
4
5 10 12

Bornyl acetate
 Identification of compound D4

Physicochemical data of D4

Yield: 50 mg, crystalline solid.

EIMS: 150 (M+), 135 (100%), 115, 107, 105, 91, 77.

IR KBr νmax cm-1: 3425, 2963, 2871, 1708, 1623, 1584, 1348, 831, 770.

1
H -NMR (300 MHZ, CDCl3) δ ppm: δ 1.15-1.17 (d, 6H), 2.19 (s, 3H),

3.06-3.10 (sept, 1H), 4.56 (1H, ArOH), 6.50 (s,1H), 6.64-6.66 (d, 1H), 6.98-7.01 (d, 1H).

13
C-NMR (75 MHZ, CDCl3) δ ppm: δ152.9 (s, C-13), 136.5 (s, C-4), 131.8 (s, C-1),

126.3 (d, C-2), 121.7 (d, C-5), 116.1 (d, C-6), 26.8 (d, C-8), 22.7 (q, C-9), 22.7 (q, C10), 20.9 (q, C-7).
 HO
8 1 2

3
7 10
6

9 5 4
Thymol
 Flow
Sheet

Leucas Roots
(300 g)

100%
24 hours
(60-80 C) Pet ether
o

Etheral Extract Fraction 1

Defatted Plant
material

100% ethyl 24 hours

acetate

Ethyl Acetate extract

Fraction 2

Scheme 3.2 Soxhlet extraction of Leucas hyssopifolia Benth.

1 f-2 f-3 f-4 f-5 f-6

Chl C C C C C
oro hl hl hl hl hl
for or or f-7
or or or
m: of of of of of
Et or or or or or
Pre Pre m Pre Pre
OA m
p.c p. p. p.m m m
:E :E
tlc(95 tlc tO tlc D5 tlc:E D6 :E D7 :E
tO tO tO tO
:5) A A A A A
c c c c c
(9 (8 (7 (5 (7
0: 0: 0: 0: 0:
1 2 3 5 3
0) 0) 0) 0) 0)
 Identification of compound D5

Physicochemical data

Yield: 50mg, white powder.

EIMS: 284 (M+), 241, 185, 171, 143, 129, 115, 101, 87, 73, 60, 55.

IR KBr νmax cm-1: 3463, 2977, 2922, 2865, 2367, 1964, 1708, 1381, 1122, 760.

H -NMR (300 MHZ, CDCl3) δ ppm: 0.90 (t, 2H), 1.65 (m, 2H), 1.61 (m, 26H),
1

1.58 (m, 2H), 3.52 (q, 3H), 11.0 (s, 1H).

13
C-NMR (300 MHZ, CDCl3) δ ppm: 14.07 (C-1), 22.67 (C-2), 31.91 (C-3), 29.43 (C-4, C-14),

29.65 (C-5 to C-13), 29.08 (C-15), 24.74 (C-16), 33.88 (C-17), 178.17 (C-18).
 O

3 5 7 9 11 13 15 17
1
HO
2 4 6 8 10 12 14 16 18

Stearic acid
Identification of compound D6

Physicochemical data

Yield: 35mg, oily liquid.

EIMS: 282 (M+), 264, 222, 111, 97, 83, 69, 55.

IR KBr νmax cm-1: 30.18, 2927, 2856, 2365, 1709, 1460, 1216, 932, 764.

1
H -NMR (300 MHZ, CDCl3) δ ppm: 1.27 (q,14H), 1.51 (m, 2H), 1.31 (m, 6H), 1.63(q,4H),

0.97 (t, 2H), 5.35 (q, 2H), 2.30 (t, 2H), 9.77 (s, 1H).

13
C-NMR (300 MHZ, CDCl3) δ ppm : 180 (C-1), 36.6 (C-2), 24.9 (C-3), 29.4 (C-4), 29.6 (C-

5), 29.7 (C-6, C-13, C-14), 31.4 (C-7, C-12), 27.9 (C-8, C-11), 130.2 (C-9, C-10), 29.4 (C-15),

31.8 (C-16), 22.6 (C-17), 13.9(C-18).

 O
17 2
18
1
15 4 OH
16 3
13 6
14 5
11 8
12 7
10 9

Oleic acid
 Identification of compound D7

Physicochemical data

Yield: 30mg, white powder

EIMS: 256 (M+), 213, 129, 73, 60, 57, 43.

IR KBr νmax cm-1: 2954, 2917, 2849, 2676, 1703, 1471, 1187, 940, 782.

1
H -NMR (300 MHZ, CDCl3) δ ppm: 0.87 (t, 3H), 1.25 (m, 22H), 1.31 (2H),

1.69 (t, 2H), 2.34 (t, 2H), 11.00 (s, 1H).

13
C-NMR (300 MHZ, CDCl3) δ ppm: 20.96 (C-16), 27.40 ( C-15), 29.67 (C-3), 50.23 (C-4),

53.38 (C-5), 69.63 (C-13), 70.32 (C-14), 77.60 (C-6, C-12), 35.8 (C-2), 170.30 (C-1).
 O

3 5 7 9 11 13 15
1
HO 2 16
4 6 8 10 12 14

Palmitic acid
 Identification of compound D8

Physicochemical data

Yield: 30mg, greenish powder.

IR KBr νmax cm-1: 3400, 3000, 2900, 1700, 1470-1380, 1250, 1100, 830.

H -NMR (300 MHZ, CDCl3) δ ppm: δH5.28 (1H, t, J = 3.6 Hz, H-12), 3.21 (1H, dd, J = 10.2/4.4 Hz, H-3),
1

2.18 (1H, d, J =11.7 Hz, H-18), 1.19 (1H, m, Ha-22), 2.00 (1H, dd, J = 13.0/4.0 Hz, Hb-22), 1.25 (3H, s, Me-23),

0.98 (3H, s, Me-24), 0.77 (3H, s, Me-25), 1.08 (3H, s, Me-26), 1.14 (3H, s, Me-27), 0.93 (3H, d, J = 6.5 Hz, Me-

29), 0.91 (3H, d, J = 5.9 Hz, Me-30).

13
C-NMR (300 MHZ, CDCl3) δ ppm: δC39.2 (C-1), 27.5 (C-2), 78.5 (C-3), 38.7(C-4), 55.5 (C-5), 18.3 (C-6),

33.1 (C-7), 39.6 (C-8), 47.8 (C-9), 36.9 (C-10), 16.6 (C-11), 125.7 (C-12), 138.4(C-13), 41.7 (C-14), 29.5 (C-

15), 24.1 (C-16), 47.7 (C-17), 53.1 (C-18), 39.2 (C-19), 39.2 (C-20), 30.5 (C-21),36.9 (C-22), 28.0 (C-23), 15.2

(C-24), 14.8 (C-25), 16.4 (C-26), 23.1 (C-27), 180.4 (C-28), 22.9 (C-29), 22.8(C-30).
 28
20
21
19
H
12
13 22
11 18 OH
25 17
1 9 14 30
16
2 O
10 8 15
H
3 27
5 7
HO 4
6
23 24 Ursolic acid
 ANALGESIC ACTIVITIES

Writhing reflex test

Hot plate method

 100
*
90 *
80 * *
70
60
% inhibition

50
40
30
20
10
0
D8 (25) D8 (50) D8 (75) Pentazocine

Fig. 2. Effect of S. rufinervis essential oil (D8 25, 50 and 75 mg/kg i.p) in acetic
acid induced writhing. Pentazocine (10 mg/kg i.p) was used as standard. (*)
show significance at P<0.05 versus vehicle treated group.
 Table 4. Effect of S. rufinervis essential oil (D8 25, 50 and 75 mg/kg i.p) in hot plate test in mice.

Latency (s)
Group (mg/kg)
Post drug time (h)

Predrug 0.25 0.5 1

D8 (25) 7±0.8 8.8±1.1 8±1.0 7.6±0.9

D8 (50) 6±1.74 12.6±4* 9±0.9* 6.66±0.88

D8 (75) 4.9±0.4 8.8±0.4* 7.8±0.6* 7.6±1.2

Pentazocine (10) 4.9±0.59 8±0.6* 8±1.0* 7.8±0.58*

Pentazocine (10 mg/kg i.p) was used as standard. (*) show significance at P<0.05 versus pre drug reaction time.
Fig. 1. Effect of S. canadensis essential oil (D7 25, 50 and 75 mg/kg i.p) in acetic acid induced writhing.
Pentazocine (10 mg/kg i.p) was used as standard. (*) show significance at P<0.05 versus vehicle treated group.
Table – 1: Effect of S. canadensis Essential oil (D7 25, 50 and 75 mg/kg i.p) in Hot Plate test in mice

Latency (s)

Group (mg/kg) Pre drug Post drug time

0.5 h

D7 (25) 4.94±0.486 4.92±0.497

D7 (50) 4.942±0.164 6.16±0.59*

D7 (75) 4.924±0.424 8.42±0.55*

Pentazocine (10) 4.996±0.59 7.9±0.411*

Pentazocine (10 mg/kg i.p) was used as standard. (*) show significance at
P<0.05 versus pre drug reaction time.
 Vehicle D9 (25 mg/kg) D9 (50 mg/kg)
30 D9 (75 mg/kg) Pentazocine

25
Number of writhes

20

15 *a

10 * *a

5
*
0
1
Treatment

Fig. 4. Effect of ethyl acetate extract of Leucas hyssopifolia (D9) on the nociception induced by intraperitoneal injection of
acetic acid. Mean±SEM. Pentazocine (10 mg/kg i.p) was used as positive control. *, significantly different from control i.e.
vehicle treated group, a significantly different from D9 (50 mg/kg).
(p<0.05, ANOVA followed by Tukey’s test)
Table 5. Effect of ethyl acetate fraction of L.hyssopifolia (D9 25, 50 and 75 mg/kg i.p) in hot plate test in mice.

Latency (s)

Pre drug Post drug

Group (mg/kg) 0.25 h 0.5 h 1h
Vehicle 4.46±1.6 4.48±1.7 4.5±1.4 4.5±1.4
D9 (25) 5.19±0.4 6.2±0.2 8.65±0.48*# 7.0±0.9
D9 (50) 4.98±0.2 5.6±0.4 9.12±0.27*# 6.4±0.88
D9 (75) 4.7±0.4 5.2±0.4 7.48±0.78*# 7.4±1.2
Pentazocine (10) 4.9±0.59 8.6±0.6* # 8.9±1.0* # 7.8±0.58* #
 ANTIMICROBIAL ACTIVITIES

Disc diffusion method.

Table 1. Antimicrobial activity of Solidago canadensis Linn. essential oil

Microorganisms Zone of inhibition (mm)

Leaves (μg/ml) Roots (μg/ml) Control (μg)

1000 500 250 125 1000 500 250 125 AM(25) CP(25) NY(150)

S. aureus - - - - 10 8 6 - 42 20 -

S. faecalis 9 8 7 6 12 10 6 - 21 - -

B. subtilis 11 10 9 8 8 6 - - 20 26 -

S. typhi 8 6 - - 8 7 - - - 28 -

E. coli - - - - 11 9 8 - 21 20 -

P. aeruginosa - - - - 10 7 6 - 8 - -

C.albicans - - - - 8 6 - - - - 10

A. niger 7 - - - 6 - - - - - 11

AM (25 μg ): Amoxycillin (25 μg), CP (25 μg): Chloramphenicol (25 μg), NY(150): Nystatin (150 μg).
 Table 2. Antimicrobial activities of essential oils of S. rufinervis

Micro- Zone of inhibition (mm)

organisms

Leaves (μg/ml) Roots (μg/ml) Control (μg)

2000 1000 500 250 2000 1000 500 250 AM(25) CP(25) NY(150)

S. aureus 7 6 - - 6 - - - 42 20 -

S. faecalis 9 7 6 - 8 6 - - 21 - -

B. subtilis 10 8 6 - 11 9 7 - 20 26 -

S. typhi 6 - - - - - - - - 28 -

E. coli 10 9 7 - 10 8 6 - 21 20 -

P.aeruginosa 11 7 6 - 10 7 - - 8 - -

C.albicans - - - - - - - - - - 10

A. niger 7. - - 6 - - - - - 11

AM(25): Amoxycillin (25 μg), CP(25): Chloramphenicol (25 μg), NY(150): Nystatin (150 μg).
 Table 4. Antimicrobial activities of ethyl acetate fraction of Leucas hyssopifolia

Microorganisms Zone of inhibition (mm)

EAF (μg/ml) Control (μg)

2000 1000 500 250 AM(25) CP(25) NY(150)

S. aureus 7 6 6 - 42 20 -

P. vulgaris 7 6 5 - 21 - -

B. subtilis 9 7 6 - 20 26 -

B. mycoides 7 6 5 - - 28 -

E. coli - - - - 21 20 -

P. aeruginosa 6 5 - - 8 - -

C.albicans - - - - - - 10

A. niger 7. - - - - - 11

AM(25): Amoxycillin (25 μg), CP(25): Chloramphenicol (25 μg), NY(150): Nystatin (150 μg).