Effect of Acacia catechu and Rotula aquatica on the DNA: Implications for cancer therapy

 

Swati Patil*1, S R Naik2, V Joshi3, CI Jolly1 and S Narayanan4

1Department of Pharmacognosy and Phytochemistry, Principal K M Kundnani College of Pharmacy, Cuffe Parade, Mumbai-400 005, India.

2Principal, Singhgad’s Institute of Pharmaceutical Sciences, Kusgaon (BK), Lonavala

3Department of Genetics, BARC, Mumbai.

4Orchid Research Laboratories Ltd. 476, Chennai 600119 India.

 

ABSTRACT

Ethnobotanical search has revealed the use of water extracts of bark of Acacia catechu and Rotula aquatica in the treatment of cancer. The aqueous extracts of both above mentioned plants were evaluated for antimitotic activity using the meristamatic cells of Allium cepa roots. The results showed inhibition of prophase stages in cell division. The A.catechu and R. aquatica treated roots of A.cepa were treated with tritiated thymidine.  DNA was extracted from these root tips. The total DNA was then counted in a scintillation counter. The results were compared with a positive control Methotrexate, a known anticancer drug. Roots treated with Plain water were used as negative control. The total DNA count from the treated roots and that from Methotrexate treated roots was less than that of Plain water. The extracted DNA was subjected to gel electrophoresis. DNA from roots treated with extracts and from Methotrexate treated roots showed fragmentation where as that from the Plain water was intact.

 

KEY WORDS: Acacia catechu, Rotula aquatica, Allium cepa,   Antimitotic activity, DNA isolation.

 

INTRODUCTION

Rotula aquatica (lour) family Boraginaceae, synonymously called as Rhabdia Iycoides is a small shrub found in rocky and sandy beds of streams often submerged in water1.

 

Acacia catechu family Leguminaceae synonymously called as Katha is a medium sized tree found throughout India2.

 

The antimitotic activity of the aqueous extracts of above mentioned plants was evaluated using the meristamatic tissue of A.cepa roots. The meristamatic tissue of A.cepa roots has cells undergoing continuous division. This division is similar to the division of human cancer cells. A plant cell is known to be more resistant than a human cell. Hence if an extract is able to arrest mitotic division in a plant cell, it may also be effective against a human cell3. The studies were not only aimed at evaluating the antimitotic activity but also to understand the molecular basis of mechanisms of the aqueous extract of A.catechu and R.aquatica.

 

Cancer is a genetic disease caused by intrinsic errors in DNA replication or induced by exposure to a carcinogen4. DNA is an essential component of the chromosome and there is a definite relation between the chromosome complement of the cell and its DNA content. Apart from a few exceptions all cells in a given species contain a constant number of chromosomes. Thus we may expect that all diploid cells in tissues of any one species would contain the same amount of DNA but that the amount would vary from one species to another5. These observations will have important implications. In the first place if the total amount of DNA is estimated in a sample of that tissue, it is a simple matter to calculate the number of nuclei and hence the numbers of cells present.

TABLE I: Antimitotic activity of A.catechu and R.aquatica extracts

Sl.

Different solutions used for treatment

* Non dividing cells (%)

% Dividing cells

Mitotic Index

P

M

A

T

Avg

SD

SEM

1

Plain water

10

65

12

8

5

90

3.215

1.856

 

Aqueous extract of A. catechu

2

0.3mg/ml

25

65

5

3

2

75

4.509

2.603

3

1mg/ml

35

55

5

3

2

65

4.163

2.404

4

6mg/ml

45

50

3

2

-

55

1.155

0.667

5

10mg/ml

90

7

2

1

-

10

2.000

1.155

 

Methotrexate

6

0.004mg/ml

50

21

11

10

8

50

0.577

0.333

7

0.020mg/ml

60

15

10

8

7

40

5.000

2.887

8

0.040mg/ml

73

12

8

4

3

30

5.000

2.887

9

0.10mg/ml

85

7

5

3

-

10

2.000

1.155

 

Aqueous extract of R. aquatica

1

0.3 mg/ml

25

68

3

3

1

75

2.08

1.20

2

1    mg/ml

35

58

5

1

1

65

1.00

0.58

3

6    mg/ml

58

39

2

1

-

42

0.58

0.33

4

10  mg/ml

88

9

2

1

-

12

1.53

0.88

P: Prophase; M : Metaphase;  A : Anaphase; T: Telophase, * Average of three readings.

 

 

Such a calculation is of value in the studies on cell growth. It has been proved that the amount of DNA per nucleus is constant and is unaffected by any nutritional changes6.

 

Cells need thymidine for DNA synthesis. Thymidine is unique for DNA synthesis. The precursor is deoxyribose uracil monophosphate, which is converted to deoxyribose thymidine monophosphate by enzyme thymidilate synthetase. This enzyme catalyses oxidation of tetrahydrofolate to dihydrofolate, from which the cells receive the methyl group of thymidine, required for DNA synthesis7. When exogenous thymidine or thymine, is provided and since there are no salvage enzymes, it is readily taken by the DNA of the cells. This technique can be used for radioactive labeling of DNA. Thus if (3H) thymidine or 14C thymidine is put in the growth medium, newly replicated DNA will contain 3H and 14C respectively8,9. The roots of A.cepa were dipped in a solution of triated thymidine, the dividing cells showed uptake of thymidine10. After the roots were treated with the aqueous extracts of both the plants, the cell division was arrested. If such cells were to be placed in a solution of triated thymidine they would not take up Thymidine as the cell division has ceased. This means that both the extracts act on DNA of the cells, thereby preventing further cell division. The total DNA was thus isolated after treatment to study the effect of these extracts. The total DNA count of the roots treated with both the extracts and Methotrexate would be much less than that of untreated roots. Running Gel Electrophoresis of the extracted DNA can check DNA fragmentation. The intact DNA molecule is very heavy and is retained in the well. If the DNA molecule is affected which results in fragments of the molecule, it travels at different speeds and have different Rf values.

 

MATERIAL AND METHODS:

The dried bark of A. catechu and entire plant of R. aquatica was collected from Kutch district in Gujarat and authenticated at Blatter Herbarium, St. Xavier's College, Mumbai. A. cepa bulbs (red variety) were purchased from local market and stored for the entire study. Carmine stain, Bromophenol blue, Ethidium Bromide, Gel agarose, Tris HCl, EDTA was procured from M/s Sigma laboratories. All the other solvents used for extraction were of AR grade. Solvents were distilled before use for greater purity.

 

Preparation of extracts:

Total Aqueous Extract:

The dried bark of A. catechu and entire plant of R. aquatica was powdered (24 mesh) and was extracted with hot water by boiling for 30 minutes. The extract obtained was concentrated and dried under controlled temperature of 60°C.

 

Antimitotic activity:

Allium cepa bulbs were sprouted in tap water for 48 h at room temperature. The ones, which developed good roots, were used for the experiment. These roots were treated with above prepared extracts of 10 mg/ml concentration. Water was used as medium / vehicle for dilution. The solutions were vortexed for uniform dispersion. The different concentrations used are mentioned in Table1. A blank with tap water was used as control. Methotrexate was used as a standard control. After 3 h of treatment, the root tips were fixed in a fixing solution of acetic acid and alcohol. Squash preparations were made by staining with acetocarmine stain. The mitotic index was calculated using the Following Formula11.

 

Mitotic Index = Number of dividing cells / Total number of cells X 100.

The Aqueous extract of concentration 10mg/ml showed least mitotic activity i.e. highest activity amongst all. The experiment was repeated thrice with four different concentrations of the aqueous extract. The cell divisions were differentiated and number of cells in each phase of cell division i.e. either Prophase, Metaphase, Anaphase, or Telophase were recorded (Table I).

 

Extraction of DNA from the meristamatic root tip:

Onion bulbs were allowed to sprout as before at room temperature. Four sets of experiments, each containing three bulbs were taken for the study. Each set of bulbs were treated with aqueous extract of A.catechu (10mg/ml), R. aquatica (10mg/ml), Methotrexate (0.1 mg/ml) and plain water respectively. Root tips were excised at 0m, 5m, 15m, 30m, 1 h, and 2 h for each set of experiment. These were named as sample collected at 0,5,15 m, 1 and 2h respectively. After each treatment before sample collection the roots were dipped in a solution of tritiated thymidine (0.01 ml/ml). After thymidine treatment 250mg of the root sample was weighed and taken for extraction of DNA. The Following procedure was used for extraction of DNA from the weighed sample12.

 

 

TABLE II: Effect of A.catechu and R.aquatica extracts and methotrexate on Total DNA Count

Sl.

No

Sample

Collection

(Mins)

Plain water

A. catechu

R. aquatica

Methotrexate

Without TCA

With TCA

Without TCA

With TCA

Without TCA

With TCA

Without TCA

With TCA

A

B

A

B

A

B

A

B

A

B

A

B

A

B

A

B

1

0

1521

100%

1054

100%

1129

100%

1100

100%

448

100%

408

100%

448

100%

408

100%

2

15

1426

94%

1036

98%

457

40%

332

30%

420

94%

390

96%

396

88%

342

84%

3

30

1408

93%

926

88%

401

36%

279

25%

336

75%

330

81%

280

63%

144

35%

4

60

1352

89%

908

86%

306

27%

190

17%

203

45%

187

46%

181

40%

108

26%

5

120

1326

87%

824

78%

254

22%

177

16%

51

11%

33

8%

160

36%

104

25%

 

 

250mg of weighed root tissue was shred with forceps. Freezed the tissue in liquid Nitrogen and grind to a fine powder in a mortar and pestle. Transferred this to a 50ml centrifuge.

 

Added 15ml of extraction buffer and 2ml of 10% sodium Dodecyl sulphate. Mixed by vigorous shaking and then incubated at 65°C for 10mins. Added 5ml of 5M Potassium acetate. Mixed vigorously and incubated on ice for 20mins. Centrifuged at 25000X for 20mins in cold.

 

Poured the supernatant through a muslin cloth into a clean centrifuge tube containing 10ml of Isopropanol. Mixed and incubated the tubes at 20°C for 30mins. Centrifuge at 2000X for 15mins in cold to pellet DNA. This procedure of extraction was carried out for every sample collected from four sets at various times.

 

Estimation of total DNA isolated from the root tips:

100 ul of the supernatant before adding to Isopropanol in the above procedure was spotted on two Whattmann discs for each sample. One disc of every sample spotted was washed with 10% Tri-chloro acetic acid and dried with acetone. These discs were then placed in a vial containing 4%BBOT in toluene. The blank readings of these vials were taken before adding the discs. The blank readings were zero. The Radio-active DNA was counted in a liquid Scintillation counter (Table II).

 

Gel Electrophoresis of the isolated DNA.:

The DNA pellet obtained from the above procedure is dissolved in T.E. Buffer (1 ug/ml).Agarose Gel Electrophoresis was carried out using Type II agarose in horizontal slab gels. 3-5mm gels were made 13. DNA samples dissolved in T.E. Buffer were mixed with the gel loading buffer and applied to the gel using an automatic micropippeter. Gels were run at 4V/cm for 3hrs and visualised using a UV lamp at 366nm. Photographs were taken using a 35mm Asahi pentax MX camera with the usual filters. Gels were placed on a low wavelength UV Transilluminator (254nm) and photographed with 100ASA black and white film.

 

RESULTS:

Table I shows that the total aqueous extract shows significant antimitotic activity which is comparable to that of methotrexate. The mitotic index decreases with concentration of the extract. The phases were differentiated in each case and it is seen that with increase in concentration of the extract the number of non-dividing cells increase. The number of cells entering Prophase is decreasing with increasing in concentration of the extracts. Since the cells do not enter Prophase the further stages of cell division also decreases with increase in concentration. The total DNA count from the treated roots is less than the total count of the non treated ones. The results are similar to that of Methotrexate, a known anticancer drug which was used as a standard (positive control). The 120 mins sample show less amount of DNA than the earlier collected samples (table II).

 

DISCUSSION:

The result that the aqueous extracts of A. catechu and R. aquatica showed least mitotic index i.e. highest activity. Both the extracts show inhibition of prophase stage, where DNA duplication occurs. Thus it can be concluded that the extracts may be acting by inhibiting DNA synthesis. Since the results are comparable to that of Methotrexate, a known anticancer drug which acts by inhibiting folic acid required for cell division and thus arresting cell division. The extracts also may be acting through the same mechanism by inhibiting folic acid synthesis and arresting cell division.

 

The amount of DNA estimated in the root tip of the treated samples showed decrease in the quantity with increase in time of treatment. The amount of DNA from the root tip samples treated with A.catechu and with R.aquatica showed decrease in its amount with increase in time of treatment. These results were similar to that of Methotrexate where a decrease in quantity of DNA was observed with increase in time of treatment.

 

These observations show that the amount of DNA in each nucleus, or indirectly the number of nuclei is decreasing with increase in time of treatment.

 

Since the root tips are dipped in radioactive thymidine after treatment with aqueous extracts, it may also be possible that both these extracts may be acting on the cell membrane, thus reducing the uptake of thymidine by these cells. But since similar results are seen with Methotrexate and it is already seen that these extracts prevent cells from entering prophase, they may be acting by inhibiting folic acid synthesis.

 

Gel electrophoresis show that the amount of DNA in all lanes shows fragmentation. The zero min. samples were vortexed as the whole DNA molecule without fragmentation is a heavy and will not travel out of the well. The DNA from treated samples was not vortexed. They show fragmentation. It may thus be concluded that the extracts have effect on the DNA molecule resulting in its fragmentation and thus arrest of cell division.

 

REFERENCES:

1.       Sharma OP. Chemistry and technology of manufacturing of Katha and Cutch, International book distributors, Meerut, 1984; pp.1-30.

2.       Wealth of India - Raw materials, Publications and Information Directorate, CSIR, New Delhi, 1972(1); pp. 80.

3.       Abhang RY, Joglekar PP, Kulkarni P H. Preliminary study on the effect of             T. Cordifolia  on mitosis. Ancient Science. 1991; 1 and 2:7-8.

4.       Yang CS, Lee M J, Chen L. Human salivary tea catechin levels and catechin esterase activities: implication in human cancer prevention studies, Cancer Epidemiol Biomarkers Prev. 1999; 8(1): 83-89.

5.       Mirsky AE, Ris H. Variable and constant components of chromosomes. Nature. 1949 (163); pp. 166 - 8.

6.       Raven RW, Cancer. Butterworth and Co. Ltd. London, 1957(1); pp. 274-280

7.       O'Connor L, David CS, Huang S. Apoptosis and Cell Division. Current Opinion in Cell Biology. 2000; 12: 257-263

8.       Lehninger LA, Nelson A. Principles of Biochemistry, CBS Publishers and Distributors, Delhi, 1993; pp.725 - 818,

9.       Freifelder D. Molecular Biology, Jones and Bartlett Publishers Inc., Boston, 2nd ed: pp.123-133

10.     Hazra A, Tripathi SK. Folic acid revisited. Ind J of Pharm. 2001 ;( 33): 322-342.

11.     Williams GO, Omoh LE, Mitotic Effects of aqueous leaf extract of Cymbopogon citratus in Allium cepa root tips. Cytobios. 1996; (87):161-168.

12.     Chirikjian J. Biotechnology Theory and Techniques, Jones and Bartlett Publishers, London, 1995(I): pp. 51-59.

13.     Micklos DA, Freyer GA. DNA Science. J Cell Sci. 1974; 15(3): 264-267.

 

 

Received on 13.05.2009

Accepted on 10.06.2009     

© A&V Publication all right reserved

Research J. Pharmacology and Pharmacodynamics  2009; 1(1): 31-34