Phytochemical study and In-Vitro Cytotoxic Activity of Cinnamomum tammala Linn. Against Ehrlich Ascites Carcinoma (EAC) and Dalton’s Ascitic Lymphoma (DAL) Cell Lines

 

 

M S Saluja1, B Sangameswaran2, A Sharma1 and C Dubey1

1Department of Pharmacy, Suresh Gyan Vihar University, Jaipur, Rajasthan

2Department of Pharmacognosy, SRM University, Chennai, Tamilnadu.

 

 

ABSTRACT:

Present study was performed to explore the cytotoxic activity of various concentration (10- 200ug/ml) of Acetonic and 50% ethanolic Extract from the leaves of Cinnamomum tammala linn Against EAC and DAL cell lines using Trypan Blue method and to find out the phytoconsituents presented in leaves. Results found that both of extracts exhibited significant cytotoxity activity at 200ug/ml, but higher cytotoxic activity was found in acetonic extract.

 

KEYWORDS: Cytotoxic Activity, Cinnamomum tammala Linn. Ehrlich Ascites Carcinoma (EAC) and Dalton’s Ascitic Lymphoma (DAL)

 

INTRODUCTION:

Cinnamomum tammala commonly known as Tejpata belongimg to family Lauraceae. It is a moderate sized evergreen tree attaining a height of 8 m, and a girth of 150 cm. Its bark produces mucilage. Leaves lanceolate, glabrous; alternately placed, opposite and short stalked. 3-nerved from the base(1,2).

 

It is found in tropical and sub-tropical Himalayas, Khasi and Jaintia hills and in eastern Bengal, India.

 

In the essential oil from the leaves, mostly  monoterpenoides were found: Linalool (50%) is the  major compound, whereas a-pinene, p-cymene, ß-pinene and limonene range around   5 to 10% each. Phenylpropanoids appear only in traces: Newer work reports 1% cinnamic aldehyde and no eugenol, whereas older literature speaks of traces of both compounds (3).

 

The leaves are used extensively in northern India as a spice - Tejpat. In Kashmir they are used as a substitute for paan (betel leaves). The essential oil is used in perfuming soap and in medicine. Four essential oils of C. tamala screened for fungicidal activity against F. moniliforme [Gibberella fujikuroi], a postharvest fungal pathogen of cereal crops were effective in inhibiting fungal growth. Activity of the four oils increased with concentration. C. tamala essential oil exhibited fungi toxicity against A. flavus and A. parasiticus at 3000 ppm and 1000 ppm, respectively(4). Leaves of C. tamala are used in colic and diarrhoeal preparations. C. tamala leaf extracts produce a hypoglycemic effect in experimental rats(5). Hydrodistilled essential oils of C. tamala screened for their anti-fungal activity against Trichophyton mentagrophytes and Microsporum microsporum audounil causing ring worm diseases in animals and humans exhibited fungicidal or fungistatic toxicity.(4) Plant parts are used in many Ayurvedic preparations e.g. sudarshan, choorna and chanderprabhavati. Other products: The leaf extracts are used as clarifiers in dyeing procedures with myrobalans or kamala(6)

 

It is useful in the treatment of    circulation, muscles and joints complications and relieves arthritis, inflamed joints, muscular pains, rheumatism and sprains. Benefits the digestive system and helps improve appetite. Helps control infections.(7)

 

Table No. 1. Preliminary phytochemical screening of extract of leaves of Cinnamomum tamala

S. No.

Constituents

Tests

Acetone extract

Ethanolic extract

1.

CARBOHYDRATES

 

Molisch’s test

-

+

Fehling’s test

 

-

+

2.

GLYCOSIDES

 

 

Legal’s test

-

-

Borntrager’s test

-

-

Baljet test

 

-

-

3.

FIXED OIL AND FATS

 

Spot test

+

+

Saponification test

 

+

+

4.

PROTEINS AND AMINO ACIDS

 

 

Millon’s test

-

+

Ninhydrin test

-

+

Biuret test

 

-

+

5.

SAPONINS

Foam test

 

-

+

6.

PHENOLIC COMP. AND TANNINS

 

FeCl3 test

+

+

Lead acetate test

 

+

+

7.

PHYTOSTEROLS

 

Salkowski test

+

+

Libermann-bucchard test

 

+

+

8.

ALKALOIDS

 

Dragendorff’s test

+

+

Mayer’s test

 

+

+

9.

GUMS AND MUCILAGE

 

Froth test

-

-

Alcoholic test

 

-

-

10.

FLAVONOIDS

 

 

Lead acetate test

+

+

Con. H2SO4 test

+

+

FeCl3 test

+

+

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Where, +  = Presence, −  = Absence

 

Table No 2.  In-Vitro Cytotoxicity activity of Acetonic Extract

Sample

Cells   1 x 106

Concentration µg/ml

S. No.

No. Cells

% Death

Live

Dead

Acetonic Extract

Dalton’s ascitic lymphoma

200

1

01

99

98%

03

97

100

2

03

97

97.5%

02

98

50

3

05

95

96%

03

97

20

4

79

21

26%

69

31

10

5

94

06

7%

92

08

Control 10 µg

6

98

02

2%

98

02

 

Ehrlich ascitic carcinoma

200

1

03

97

98%

01

99

100

2

03

97

96%

05

95

50

3

08

92

94%

04

96

20

4

78

22

25%

72

28

10

5

98

02

2.5%

97

03

Control  10 µg.

6

98

02

2%

98

02

 

MATERIAL AND METHODS:

Collection and Authentification of the Plant:

The leaves of Cinnamoamum tamala (Family -Liliaceae) were collected from Garden of National Botanical Research Institute, Lucknow, India in month of July 2009. The plant material was authenticated by Dr. Sayeeda Khatoon, chemotaxonomist and the voucher specimens (NAB 180023) were deposited in the departmental herbarium for future reference.

 

 

Preparation of Crude Drug For Extraction :( 8)

The authenticated leaves were used for the preparation of the extract. The leaves was collected and dried under shade and then coarsely powdered with the help of mechanical grinder. The powdered was passed through sieve no. 40 and stored in an airtight container for extraction.

 

Preparation of extracts of Cinnamoamum tamala:

The powdered plant material (1000g) was first macerated with petroleum ether to remove fatty substances and filtered by using Whatman filter paper no. 1.

 

 

Table No. 3:- In-vitro Cytotoxicity activity of Alcoholic Extract

Sample

Cells 1 x 106

Concentration µg/ml

S. No.

No. Cells

% Death

Live

Dead

Alcoholic Extract

Dalton’s ascitic lymphoma

200

1

62

38

40%

58

42

100

2

62

38

35%

68

32

50

3

72

28

29%

70

30

20

4

88

12

10.5%

91

09

10

5

94

06

5%

96

09

Control 10 µg

6

96

04

3.5%

97

03

 

Ehrlich ascitic carcinoma

200

1

62

38

34%

70

30

100

2

60

40

33%

74

26

50

3

78

22

27.5%

67

33

20

4

87

13

10%

93

07

10

5

92

08

5.5%

97

03

Control  10 µg.

6

96

04

3.5%

97

03

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

The filtrate is dried under reduced pressure by using rotavapour (Buchi, USA) and then dried in lyophilizer (Labconco, USA). The marc left after Pet ether extraction was dried and then extracted with acetone for 3 days (3 X 5L). The extract was separated by filtration and concentrated on rotavapour (Buchi, USA) and then dried in lyophilizer (Labconco, USA) under reduced pressure and low temperature to obtain of solid residue.   The marc left was dried and then further exhaustively extracted with of ethanol for 3 days (3 X 5L). The extract was separated by filtration and concentrated on rotavapour (Buchi, USA) and then dried in lyophilizer (Labconco, USA) under reduced pressure and low temperature to obtain solid residue.

 

Ethyl alcohol was used as a solvent for alcoholic extract whereas acetone for acetonic extract.

 

Extracts of Cinnamoamum tamala was subjected to qualitative tests for the identification of various active constituents viz. carbohydrate, glycoside, alkaloid, amino acids, flavanoids, fixed oil, tannins, gum and mucilage, phytosterols etc. The phytoconstituents were identified by chemical tests, which showed the presence of various constituents in the different extracts8. The results are shown in Table No.1.

 

In Vitro Cytotoxic Activity: (9)

For In Vitro cytotoxic activity, Ehrlich Ascites Carcinoma (EAC) and Dalton’s Ascitic Lymphoma (DAL) were used, which were originally obtained from Cell Culture Department of Central Drug Research Institute, Lucknow. The activity was found using trypan blue cytotoxic assay.

 

 

Trypan blue method

·           Given sample (plant extract) i.e., acetone extract or aqueous extract dissolved in suitable solvent.(water/ethanol).

·           Different concentration of the sample solution pipette out.

·           The volume in all the tubes is made up to 800µl with PBS.(Phosphate buffer saline).

·           100 µl DLA/EAC (1 million cells/ml) were added to all the test tube.

·           A control having solvent alone is also prepared (20 µl).

·           Incubated at 37oC for three hours.

·           Add 100 µl of trypan blue to all test tube.

·           Count the no. of cells using the Haemocytometer with the help of compound microscope.

·           No .of dead cells/total 100 cells counted gives the percentage cytotoxicity.

 

RESULTS:

The phytochemical evaluation shows the presence of flavonoid, phenolic compounds, tannins, glycosides, saponins, and carbohydrate in the petroleum ether and ethanolic extact as showed in Table no 1. The results of In Vitro Cytotoxic test were shown in Table 2, 3. The Acetonic Extract shows remarkable cytotoxic activity against the tested cells Dalton’s ascetic lymphoma (DLA) and Ehrlich Ascites Carcinoma (EAC). Alcoholic extracts also showed cytotoxic activity against the tested cell line. The extract shows more activity against Dalton’s ascetic lymphoma when compared with Ehrlich ascitic carcinoma.

At 200 µg concentration it shows 98% (DLA), 98% (EAC) of activity for Acetonic Extract, 40% (DLA), 34% (EAC) of activity for Alcoholic extract.

 

 

CONCLUSION:

The phytochemical study found the presence of alkaloids and flavanoids, which are responsible for its cytotoxic activity.

The whole study concluded that the Acetonic Extract shows remarkable cytotoxic activity against the tested cells Dalton’s ascetic lymphoma (DLA) and Ehrlich Ascites Carcinoma (EAC). Alcoholic extracts also showed cytotoxic activity against the tested cell line. The extract shows more activity against Dalton’s ascetic lymphoma when compared with Ehrlich ascitic carcinoma.

At 200 µg concentration it shows 98% (DLA), 98% (EAC) of activity for Acetonic Extract, 40% (DLA), 34% (EAC) of activity for Alcoholic extract. This holds great promise for future research in human beings. The anticancer properties of Cinnamomum tamala will provide useful information in the possible application in cancer prevention and cancer therapy.

 

 

ACKNOWLEDGMENT:

The authors are grateful to Dr. Ch. V Rao, M Pharm, Phd, Scientist E2, NBRI, Lucknow for their valuable guidance during research work.

 

REFRENCES:

1.      Bradu BL and Sobti SN. Cinnamomum tamala in NW Himalayas. Evaluation of various chemical types for perfumery value. Indian Perfumer, 1988; 32(4), 334-340.

2.      Buss AD and Waigh RD. Natural products as leads for new pharmaceuticals. In Berger’s medicinal chemistry and drug discovery. 1995; 5th edi.,Vol. I, 983-1033.

3.      Gupta AK. Quality Standards of Indian medicinal plans, 2003; 3: 146-157.

4.      Foye WO Lemke TL Williams DA. Principles of medicinal chemistry 5th edition Williams and  Wilkins, USA , 1995; p.21

5.      Chandra A, Mahdi A, Ahmad A, Singh R K. Indian herbs result in hypoglycemic responses in streptozotocin-induced diabetic rats, Nutrition Research, 2007; 27 :161– 168,.

6.      Kirtikar and Basu (1981)Indian medicinal plants,1981; 3: 2146-2147

7.      Zhong Shu, Y., Recent natural products based drug development: A pharmaceutical industry perspective. Journal of natural products, 1998; 61, 1053-1071.

8.      Khandelwal, K.R., Practical Pharmacognosy technique and experiments. 2nd ed.Pune : Nirali Prakashan, 200; 149-56.

9.      B. Rajkapoor et al. Antitumour activity of Bauhinia variegata on Dalton’s ascitic lymphoma, Journal of Ethnopharmacology. 2003; 83: 107–109.

 

 

 

Received on 12.10.2009

Accepted on 14.12.2009     

© A&V Publication all right reserved

Research Journal of Pharmacognosy  and Phytochemistry. 2(1): Jan.-Feb. 2010, 37-40