Experimental studies on Influence of different doses of Vermicompost on essential oil yield of Ocimum tenuiflorum var. CIM-Ayu

 

A. Ranganadha Reddy1*, R. Bharath Kumar1, T. C. Venkateswarulu1, M. Indira1, D. Ramyakrishna1, M. Deepthi1, K. P. Sastry2

1School of Biotechnology, Vignan University, Vadlamudi, Guntur 522 213, Andhra Pradesh, India

2CIMAP Uppal, Hyderabad 500 039, Andhra Pradesh, India.

Corresponding Author E-mail : rangaaluri@gmail.com

 

ABSTRACT:

Vermicompost is a naturally occurring organic biofertilizer and it contains 1.5 – 2.5 % Nitrogen, 0.9 – 1.7 % Phosphorus, 1.5 – 2.4 % Potash, 0.5 – 1.0 % Calcium, 0.2 – 0.3 % Magnesium, 0.4 - 0.5 % Sulphur. Ocimum tenuiflorum yields aromatic oil containing eugenol (71.3%). The present study focused on the influence of different doses of vermicompost on the essential oil yield of Ocimum tenuiflorum var. CIM-Ayu. In the experiment, six different doses of vermicompost (0- 3.0 tons/ha) was applied and during the course of investigation the essential oil from the herbs was collected at two different stages (i.e. at 45, 90 days after planting) and it was noticed that the essential content did not follow any particular pattern. Similarly, the chemical constituents of the oil were not influenced by the treatments. It was also observed that reduction in plant height and number of tillers/clump that was manifested in reduced herb yield was compensated by a higher oil concentration in the leaves. The highest content of Eugenol, was reported in addition to  Methyl Eugenol, Beta Elemene and Caryophyllene.

 

KEYWORDS: Ocimum tenuiflorum, Vermicompost, Essential oil, Eugenol, Methyl Eugenol, Beta Elemene and Caryophyllene.


 

INTRODUCTION:

Ocimum tenuiflorum (Krishna Tulasi) belonging to the family Lamiaceae (labiate) [1] is a medicinal [2] and aromatic crop that is thought to have originated in north central India and now grows native throughout the eastern world tropics, which yields essential oil and aroma chemicals and find diverse uses in the perfumery and cosmetic industries as well as in indigenous systems of medicine.[3][4] Out of 160 species of Ocimum, recorded in India, Ocimum tenuiflorum Linn is cultivated in India on a commercial scale. It is grown in various parts of the country viz. in west Bengal, Maharashtra, Uttar Pradesh, Madhya Pradesh, Bihar, Jammu, Assam etc.[5]

 

Essential oil of Ocimum tenuiflorum contained higher amount of linalool (39.39-55.26%) and moderate amounts of methyl chavicol (0-6.66%), nerol (0.48-8.0%), geraniol (0.26-1.75%) and citral (4.26-6.79%).[6] Recent analysis of the chemical constituents in the essential oils of Ocimum tenuiflorum revealed the presence of α-humulene (0.71%), germacrene D (2.11-4.91%), β-elemene (1.59-6.35%), methyl eugenol (36.47-76.27%) and β-caryophyllene (8.71-56.63%).[7] The essential oils, mainly used in food industries, perfumery, and also possess anti-bacterial and insecticidal properties. It inhibits the in vitro growth of Mycobacterium tuberculosis and Micrococcus pyrogenes variety aureus. It has marked insecticidal activity against mosquitoes. Vermi compost improve herbs quality foliage yield, essential oil content, and oil yield Vermi compost contains Nitrogen, Phosphorous, Potassium, Calcium, Sulphur and Magnesium- Conserved Nutrients from Nature. Vermicompost is the product obtained by composting organic residues using earth worms.[8] Earth worms like Edudrilus eugeniae, Eistenia fetida and Perionyx erxcauatus have been identified as the organisms to process the biodegradable organic material.[9] The chemical composition of basil oil is reported as -pinene 0.1-0.4%, camphene 0.02-0.1%, ßpinene 0.07-0.8%, myrcene 0.12-0.8%, limonene 2.0-9.3%, cis-ocimene 0.1-0.6%, p-cymene 0.05-0.15%, cis-3-hexenol 0.02-0.08%, fenchyl acetate 0.1-0.5%,camphor 0.37-0.75%, linalool 40-54%, fenchyl alcohol 2-9%, methyl chavicol 23-26%, -terpineol 0.8-1.9%, citronellol 0.65-3.7%, geraniol 0.03-0.30%, methyl cinnamate 0.05-0.34% and eugenol 5-12%.[10]

 

Table-1: Physico-chemical properties of the sweet basil oil [11]

Assay of GLC:

Comparable to standard [methyl chavicol (71% to 75%) Linalout (18% to 24%) traces 4-10%]

Appearance:

Pale yellow liquid

Refractive index:

1.453 to 1.655

Saponification value:

2.0000 to 8.0000

Specific Gravity:

0.9320 to o.9830

Optical Rotatim:

- 4 to -8

Specific Gravity:

0.9320

 

MATERIALS AND METHODS:

Plant Material:

Fully grown slips of size 4-6” were planted in field at 60x60 cm spacing in 3x4 m plots as per the lay out plan.

Ocimum tenuiflorum var. CIM-Ayu were collected from the experimental garden of CIMAP, Hyderabad and other chemical are analytical grade, NSP chemicals.

 

Methods:

Experiment I: Different doses of Vermicompost were applied in the following composition   on herb Ocimum tenuiflorum var. CIM-Ayu”

 

Table-2: Different levels of vermicompost applications

S NO

Treatment

Vermicompost tons/ha

Vermicompost Kg/plot

1.

T1

Control

Control

2.

T2

3.00

7.20

3.

T3

1.50

3.60

4.

T4

1.00

2.40

5.

T5

0.75

1.80

6

T6

0.50

1.45

Note: The treatment composition of vermicompost was taken as 3.0, 1.5, 1.0, 0.75, 0.50 tons/ha.

 

Table-3:  Shows  the  replication  of  treatments  with Vermicompost  correspondingly  the    information  has  been  provided  in  parenthesis.

T1 Control

R1T1  (Control)

R2T1  (Control)

R3T1  (Control)

R4T1  (Control)

R1T2 7.20 Kg VC

R2T2 7.20 Kg VC

R3T2 7.20 Kg VC

R4T2 7.20 Kg VC

R1T3 3.60 Kg VC

R2T3 3.60 Kg VC

R3T3 3.60 Kg VC

R4T3 3.60 Kg VC

R1T4 2.40 Kg VC

R2T4 2.40 Kg VC

R3T4 2.40 Kg VC

R4T4 2.40 Kg VC

R1T5 1.80 Kg VC

R2T5 1.80 Kg VC

R3T5 1.80 Kg VC

R4T5 1.80 Kg VC

R1T6 1.45Kg VC

R2T6 1.45Kg VC

R3T6 1.45Kg VC

R4T6 1.45Kg VC

(VC: Vermicompost, T: treatment, R: replication)

 

Experiment –I

Treatment imposition:

The crop was planted in garden on 10.10.2010. Vermicompost  as per treatments was applied 10 days after planting. 

 

Maintenance:

The crop was kept weed free and regularly irrigated.

 

Observations on essential oil yield:

Observations were taken at different intervals starting from 10 days after vermicompost application. In each treatment five plants were removed at random from the plants and washed under tap water. The plants were partitioned   in to lamina, petiole and stem. Fresh and dry weights of individual components were taken separately. Later observations were recorded. Details about the observations recorded are presented here (see Table-4):

 

Table-4: Table showing the different stages of fresh plant samples was collected at regular intervals of time i.e., (30, 60, 90, 120 days)

S.No

Regular intervals

Code

1

30

Stage I

2

60

Stage II

3

90

Stage III

4

120

Stage IV

 

Oil content and quality:

At   45, 90 days after planting leaf samples were collected and separated in to small branches. 250 g leaves were weighed and using a Clevenger apparatus the oil content was estimated. .

 

Statistical Analysis:

The data generated in this study were statistically analyzed following the procedure described by Mandal and Numbiar (1999)[11][16]. The results are presented and discussed at 5 % probability level uniformly. The non significant results are denoted as N.S and * indicates the significant results. In the statistical analysis, F'-Test was performed with the difference between the highest and


lowest average values. If the difference is more than C.D value, then it is significant and if it is less than C.D value, then it is non significant.

 

Gas Chromatography parameters:

Lemon grass short method:

GC analysis was carried out using Varian CP-3800 with Galaxie chromatography data system fitted with flame ionization detector (FID) and an electronic integrator. Separation of the compounds was achieved employing a Varian CP-Sil 5CB capillary column (ID: 50 m X 0.25 mm; film thickness 0.25 µm) with 5% dimethyl polysiloxane. Nitrogen was the carrier gas at 0.5 ml/min constant flow rate. The column temperature program was: 1200C (2 min) to 2400C (6 min) at 80C/min ramp rate. The injector and detector temperature were 2500C and 3000C, respectively. Samples (0.2 µL) were injected with a 20:80:20 split ratio. Retention indices were generated with a standard solution of n-alkanes (C6-C15). Peak areas and retention times were measured by an electronic integrator. The relative amounts of individual compounds were computed from GC peak areas without FID response factor correction. [13][14][15]

 

RESULTS AND DISCUSSION:               

Essential Oil characteristics: During the course of investigation the essential oil from the herbs was collected at three different stages and it was noticed that the essential content did not follow any particular pattern (Table-5 and Figure-1). Similarly, the chemical constituents of the oil were not influenced by the treatments (Table-6, 8, and10).

 

Table-5: Influence of different levels of Vermicompost on the Oil content (%) at different stages.

Treatment

days after planting

I

II

T1

0.21

0.24

T2

0.23

0.28

T3

0.25

0.30

T4

0.25

0.34

T5

0.25

0.30

T6

0.21

0.31

F'- Test

*

*

C.D.(P=0.05)

0.027

0.036

C.V.%

8.18

8.575

Quantity of essential oil volume yielding in different stages at 45,90 days after vermicompost application. It has been observed that reduction in plant height and number of leaves that was manifested in reduced herb yield was compensated by a higher oil concentration in the leaves.

Stge-II

 
 


Figure-1: Influence of different levels of Vermicompost on the essential Oil content (% ) of Ocimum tenuiflorum var. CIM-Ayu at different days after planting

 

An increase in essential oil content was noticed upto 90 days in all the treatments. The essential oil content noticed at observation three was less because the observation was taken during growth period.  It was observed that the essential oil content did not follow any particular pattern. 

 

Chemical constituents of the oil:

Influence of different levels of vermicompost on the chemical constituents of essential oil. Stage I– Chemical constituents of the oil after every 45 days 

 

It has been also observed that, the oil content and chemical composition of oil did not change with regard to influence of the age of the crop.( after every 45 days)


 

 


Compounds

T1

T2

T3

T4

T5

T6

Methyl Eugenol

6.027

0.177

0.159

0.698

0.917

0.161

Linalool

0.302

0.195

0.324

0.537

0.391

0.266

Limonene

0.175

0.356

0.586

1.368

0.328

0.289

Methyl Chavicol

0.733

0.540

0.611

0.816

0.691

0.514

Beta Elemene

6.748

13.251

11.682

8.867

11.783

11.861

Caryophyllene

7.591

10.994

14.805

13.006

11.829

12.197

Eugenol

45.024

70.080

66.441

65.614

65.334

69.580

 



Figure-2: GC Analysis for Vermicompost application of Plant samples after 45 days


 


In the above graph, the highest content of the Eugenol , Limonene, Linalool, Methyl Chavicol, Methyl Eugenol, Beta Elemene and Caryophyllene was present in the essential oil component. It has been represented where as in X-axis Retention Time (RT), min.and Y-axis Height of the peak (µV) (refer to   Figure-2).


 

Table -7: Chemical Composition of oil after 45 days of Vermicompost application

S.No

Name

Time [Min]

Quantity [% Area]

Height [µV]

Area [µV.Min]

Area % [%]

1

Unknown

0.03

0.03

69.7

1.8

0.027

2

Unknown

5.73

0.03

61.8

2.2

0.034

3

Unknown

5.82

0.49

937.6

32

0.492

4

Unknown

5.9

0.01

11.2

0.7

0.01

5

Unknown

6.87

0.33

441.7

21.5

0.331

6

Unknown

7.24

0.02

54.4

1.5

0.023

7

Unknown

7.29

0

13.6

0.2

0.002

8

Unknown

7.65

0.02

31.2

1.2

0.018

9

Unknown

7.72

1.17

2541.8

76.2

1.172

10

Unknown

7.81

0.07

126.2

4.3

0.066

11

Unknown

7.89

0.47

994.8

30.6

0.471

12

Unknown

8.02

0.38

663.9

24.9

0.384

13

Unknown

8.14

0.37

787.2

24.4

0.375

14

Unknown

8.46

0.05

89.6

3.2

0.049

15

Limonene

8.63

1.37

2041.3

88.9

1.368

16

Unknown

8.74

0.3

594.1

19.6

0.302

17

Unknown

8.98

0.03

47.6

2.1

0.033

18

Linalool

9.19

0.54

1028.6

34.9

0.537

19

Unknown

9.25

0.07

146.4

4.6

0.07

20

Unknown

9.35

0.02

22.4

1.3

0.02

21

Unknown

9.99

0.04

63.7

2.3

0.035

22

Unknown

10.21

0.02

26.5

1.2

0.018

23

Methyl chavicol

10.32

0.82

1545.1

53.1

0.816

24

Unknown

10.51

0.49

817.6

32.1

0.494

25

Unknown

11.05

0.04

65.3

2.5

0.038

26

Unknown

11.42

0.07

145.5

4.8

0.074

27

Unknown

11.95

0.14

150.8

8.9

0.136

28

Unknown

12.57

0.16

191.6

10.6

0.163

29

Eugenol

12.77

65.61

122785.4

4263.3

65.614

30

Methyl eugenol

13.26

0.7

1341.4

45.4

0.698

31

Unknown

13.65

0.55

1042.6

35.9

0.552

32

Beta elemene

13.76

8.87

16650.1

576.1

8.867

33

Caryophyllene

14.35

13.01

24559.7

845.1

13.006

34

Unknown

14.5

0.01

14.2

0.8

0.012

35

Unknown

14.83

1.14

1440.4

74

1.14

36

Unknown

15.04

0.12

150.5

7.8

0.12

37

Unknown

15.17

0.07

52.3

4.6

0.071

38

Unknown

15.29

0.21

356.2

13.5

0.208

39

Unknown

15.4

0.27

418.2

17.4

0.268

40

Unknown

15.67

0.02

54.2

1.5

0.023

41

Unknown

15.74

0.03

46.1

1.7

0.026

42

Unknown

15.79

0.02

40.2

1.2

0.019

43

Unknown

16.61

0.12

195.5

7.8

0.12

44

Unknown

16.77

0.16

75.8

10.3

0.158

45

Unknown

16.99

0.06

50.2

4

0.062

46

Unknown

17.53

0.05

72

3.2

0.05

47

Unknown

17.68

0.16

98.6

10.1

0.156

48

Unknown

20.75

1.27

821.9

82.6

1.271

Total

100

183976.5

6497.6

100


The chemical composition contents was present in above mentioned table shows that the out of 48 unknown samples the only seven components (viz. Eugenol ,Limonene, Linalool, Methyl Chavicol, Methyl Eugenol, Beta Elemene and Caryophyllene) were shown the considerable enhancement in terms of quantity after adding the required quantity (2.40 kg/Plot) vermicompost application.( see Table-7)


 

Stage II -Chemical constituents of the oil after every 90 days 

Table-8: Quantity of oil after every 90 days of  Vermicompost application.

Compounds

T1

T2

T3

T4

T5

T6

Linalool

0.363

0.418

0.424

0.341

0.302

0.330

Limonene

0.665

0.673

1.410

0.555

0.474

0.490

Methyl Chavicol

0.720

0.576

0.695

0.568

0.463

0.533

Beta Elemene

11.031

13.394

8.110

9.282

11.748

10.521

Caryophyllene

18.322

17.822

19.967

15.205

11.288

18.012

Methyl Eugenol

1.049

0.098

28.126

8.307

0.127

0.099

Eugenol

61.163

60.665

33.558

61.145

71.509

65.250

 

Figure-3: GC Analysis for Vermicompost application of Plant samples after 90 days 

 


It has been also observed that, the oil content and chemical composition of oil did not change with regard to  influence of the age of the crop.( after every 45 days.)

 

In the above graph, the highest content of the Eugenol ,Limonene, Linalool, Methyl Chavicol, Methyl Eugenol, Beta Elemene and Caryophyllene was present in the essential oil omponent. It has been represented where as in X-axis Retention Time (RT), min.and Y-axis Height of the peak (µV).( refer to Figure-3)


 

Table-9:Chemical Composition of oil after 90 days of Vermicompost application

S.No

Name

Time [Min]

Quantity [% Area]

Height [µV]

Area [µV.Min]

Area % [%]

1

Unknown

5.87

0.25

2994.8

113.4

0.249

2

Unknown

6.08

0.01

127.8

5.9

0.013

3

Unknown

6.67

0.01

111.2

4.5

0.01

4

Unknown

6.87

0.16

1581.4

72.9

0.16

5

Unknown

7.16

0.01

110.2

3.9

0.009

6

Unknown

7.24

0.01

149.5

5.1

0.011

7

Unknown

7.59

0.01

138.8

4.3

0.009

8

Unknown

7.72

1.17

16570.4

534.8

1.174

9

Unknown

7.81

0.05

636.4

21.6

0.047

10

Unknown

7.88

0.35

4934.8

161

0.353

11

Unknown

8.02

0.38

4684

171

0.375

12

Unknown

8.14

0.28

3866.1

125.4

0.275

13

Unknown

8.46

0.07

973.7

32.9

0.072

14

Limonene

8.63

1.41

10661.1

642.6

1.41

15

Unknown

8.74

0.76

10224

344.1

0.755

16

Unknown

8.86

0.03

192.4

11.5

0.025

17

Unknown

8.98

0.03

258.7

13.1

0.029

18

Linalool

9.19

0.42

5086.8

193.1

0.424

19

Unknown

9.25

0.1

1361.4

47.2

0.104

20

Unknown

9.33

0.02

138.4

7.2

0.016

21

Unknown

9.67

0.01

126

4.2

0.009

22

Unknown

9.77

0.02

187.6

8.1

0.018

23

Unknown

9.99

0.07

710

33

0.072

24

Unknown

10.21

0.03

259.2

11.9

0.026

25

Methyl chavicol

10.32

0.69

8964.5

316.5

0.695

26

Unknown

10.43

0.03

314.1

13.8

0.03

27

Unknown

10.51

0.36

2862

164.5

0.361

28

Unknown

11.42

0.02

240.1

9.2

0.02

29

Unknown

11.95

0.02

247.2

8.5

0.019

30

Unknown

12.02

0.01

72.1

3.2

0.007

31

Unknown

12.15

0.02

305.8

10.6

0.023

32

Unknown

12.24

0.01

103.5

5.6

0.012

33

Eugenol

12.79

33.56

351512.5

15289.6

33.558

34

Unknown

13.03

0.06

147

27.8

0.061

35

Methyl eugenol

13.29

28.13

316007.4

12814.6

28.126

36

Unknown

13.58

0.02

140.9

8.6

0.019

37

Unknown

13.65

0.49

6419.8

224.1

0.492

38

Beta elemene

13.76

8.11

103937.9

3695.2

8.11

39

Unknown

14.12

0.03

369.4

15.6

0.034

40

Caryophyllene

14.36

19.97

249302.5

9097.2

19.967

41

Unknown

14.58

0.15

685.1

67.9

0.149

42

Unknown

14.83

1.11

13976.6

504.4

1.107

43

Unknown

15.05

0.04

389.9

16.7

0.037

44

Unknown

15.12

0.01

90.9

3.7

0.008

45

Unknown

15.17

0.03

190.8

12

0.026

46

Unknown

15.29

0.1

1159.2

46.2

0.101

47

Unknown

15.4

0.11

1349.4

50

0.11

48

Unknown

15.45

0.04

375.3

16.3

0.036

49

Unknown

15.58

0.03

248.8

14.7

0.032

50

Unknown

15.67

0.05

648.1

24.5

0.054

51

Unknown

15.74

0.05

401

21.1

0.046

52

Unknown

15.79

0.03

333.7

12.8

0.028

53

Unknown

15.85

0.04

274.6

18.6

0.041

54

Unknown

16

0.02

195.7

9.9

0.022

55

Unknown

16.38

0.01

77.4

2.7

0.006

56

Unknown

16.55

0.03

356.1

12.7

0.028

57

Unknown

16.61

0.25

3003.7

113.1

0.248

58

Unknown

16.76

0.1

839.3

45.9

0.101

59

Unknown

16.94

0.05

279.1

23.8

0.052

60

Unknown

17.26

0.05

372.4

24.1

0.053

61

Unknown

17.53

0.07

445.6

33.9

0.074

62

Unknown

17.64

0.02

135.1

7.8

0.017

63

Unknown

17.93

0.01

108.5

6

0.013

64

Unknown

18.21

0.03

262.1

12.8

0.028

65

Unknown

18.47

0.09

652.2

39.1

0.086

66

Unknown

19.37

0.3

673.1

135.5

0.297

67

Unknown

19.85

0.02

96.7

7.9

0.017

Total

100

1134652

45561.4

100


 

The chemical composition contents was present in above mentioned table shows that the out of 67 unknown samples the only seven components (viz. Eugenol ,Limonene, Linalool, Methyl Chavicol, Methyl Eugenol, Beta Elemene and Caryophyllene) were shown the considerable enhancement in terms of quantity after adding the required quantity (2.40 kg/Plot) vermicompost application.( refer Table-9)

 

CONCLUSIONS:

The influence of vermicompost on the essential oil yield of ocimum at two different stages (i.e. 45 and 90 days) was studied by using standardization method of oil analysis it was observed that the essential oil content did not follow any particular pattern.  The eugenol content of the oil was higher due to vermicompost application.

 

REFERENCES:

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Received on 12.07.2016          Modified on 27.07.2016

Accepted on 10.08.2016        © RJPT All right reserved

Research J. Pharm. and Tech 2016; 9(12):2087-2093.

DOI: 10.5958/0974-360X.2016.00425.X