Indian Journal of Experimental Biology Vol. 42, October 2004, pp. 981-988

Antitumour activity of some plants from Meghalaya and Mizoram against murine ascites Dalton's lymphoma

G Rosangkima & s B Prasad*

Cell and Tumour Biology Laboratory, Department of Zoology, School of Life Sciences, North-Eastern Hill University, Shillong 793 022, India.

Received 12 November 2003, revised 25 June 2004

Total five plants, three from Mizoram (Dillenia pentagyna, Ageratum conyzoides, Blumea lanceolaria) and two from Meghalaya (Potentilla fulgens, Taxus baccata) were studied for their antitumour activity against murine ascites Dalton's lymphoma (DL) in vivo. Only three plants showed the different magnitude of anti tumour activity. Out of these three plants, the antitumour activity was maximally observed with the methanol extract of the stem bark of D. pentagyna as compared to the aqueous extract of the roots of A. conyzoides and aqueous extract of the root of P. fulgens. An increase in glutathione levels in Dalton's lymphoma cells was observed during tumour growth. Changes in glutathione and protein levels were also investigated in the liver and Dalton's lymphoma cells of tumour-bearing mice following the treatment with the extract of D. pentagyna which showed the highest anti tumour activity as compared to the other two plant extracts. Glutathione in the liver and DL cells of treated tumour-bearing mice was found to be decreased. The protein concentration in liver and DL cells decreased mainly at 96 hr of treatment. It may be concluded that the natural product of D. pentagyna promises to be more active against Dalton's lymphoma than others and the decrease in glutathione level may be one of the important steps in resulting this antitumour effect.

Keywords: Antitumour activity, Dillenia pentagyna, Dalton's lymphoma, Glutathione

IPC Code: Int. CI.7 A61P

Chemotherapy is an effective treatment against cancers either singly or in combination with surgery and/or radiotherapy. In chemotherapy, drugs like cisplatin, carboplatin, cyclophosphamide, doxo­rubicin, melphalan, mitomycin-C, gemcitabine, etc have been used for the treatment of cancers 1,2.

However, therapeutic efficacy of most of them are limited due to the development of various side effects in the host and/or the acquired drug resistance by the cancer cells2

•3

. In an attempt to abate these side effects and better remedy against various malignancies, many plant derivatives have been used with varying success4

• Higher plants, a source of medicinal compounds, have been well-known to play a dominant role in the health care of human beings5.

More than 50% of all modern drugs in clinical use are of natural product origin4

•5

• Many natural products have been recognized to have the ability to induce apoptosis in various tumour cells of human origin6

• A

*Correspondent author: Telephone: 91-0364-2722318'(0); 0364-2550093(R) Fax: 91-0364-2550076/2551634 E-mail: sbpnehu@hotmail.com;sbprasad@nehu.ac.in

variety of plant extracts i.e. turmeric (Curcuma Zonga) and its active constituent, curcumin, roots of tea plant (Camellia sinensis var assamica) and betel leaf have been reported to have potential anti tumour and/or anti-carcinogenic activities4

•7

.12

• Taxol, extracted from the stem bark of Taxus brevifolia is well-known to be effective against many cancers 13. The use of traditional herbal medicines and/or direct use of some parts of plants against various ailments is very common among the tribes of North-Eastern states of Indial4

. Hence, these preliminary reports/information generated interest for the evaluation of some medicinal plants from this region to ascertain their anticancer activity against a murine experimental tumour system.

Meghalaya and Mizoram are small hilly North­Eastern states of India. Meghalaya lies between 25°.00' and 26°.1O'N latitude and 89°.45' and 92°.45'E longitude l5

. Mizoram lies between 21°.58' and 24°.35'N latitude and 92°.15' and 93°.26'E longitude l6

• These states have rich deciduous type of forests with a variety of vegetation. Collection of data through literature, personal interview and

982 INDIAN J EXP BIOL, OCTOBER 2004

questionnaires to different persons, including local herbal practitioners revealed that some plants are used by these people for the treatment of cancer suspected diseases. Among these plants, shrubby cinquefoil herb (Potentilla fulgens Wall., Rosaceae) and common yew tree (Taxus baccata Linn., Taxaceae) from Meghalaya and goat weed herb (Ageratum conyzoides Linn., Asteraceae), Blumea lanceolaria Linn. (Asteraceae) locally called as buarze, and simpoh tree (Dillenia pentagyna Roxb., Dilleniaceae) from Mizoram were noted to be more common in use l7

.

However, the details on the evaluation and establishment of the anti tumour activity of these plants through scientific study have not been investigated. Thus, considering the importance of these plants with the probable anticancer medicinal value, particularly in the life of the people of these states and other people in general, the present study has been undertaken. This may be helpful to derive a comparative therapeutic value of these plants and to establish their antitumour activity particularly against murine Dalton's lymphoma and other cancers ' in general.

Glutathione, an endogenous intracellular thiol­contammg tripeptide (L-y-glutamyl-L-cysteinyl­glycine), is an important cellular antioxidant and has been the focus of interest in cancer chemotherap/8.19. It is involved in many cellular functions, i.e, bioreductive reductions, maintenance of enzyme activity, amino acid transport, protections against oxidati ve stress, radiation and chemotherapy, detoxification of xenobiotics, and drug metabolism2o. After the evaluation of the antitumour activity of different plants, it is also aimed at to determine the effect of most potent plant extract on the changes, if any, in glutathione and protein levels so as to understand their possible involvement in ensuing anti tumour effect.

Materials and Methods Animals and tumour model--Inbred Swiss albino

mice and ascites Dalton's lymphoma were originally obtained from Gauhati University about 15 years back. The inbred mice colony is being maintained under laboratory conditions keeping 5-6 animals in propylene cages using paddy husk as bed at 24°-25°C. The animals were fed with commercially available food pellets diet (Amrut Laboratory, New Delhi) and water ad libitum. Ascites Dalton's lymphoma tumour was maintained in vivo in 10-12 weeks old mice by

serial intraperitoneal (i.p.) transplantation of lOxl07

viable tumour cells/animal in 0.25 ml phosphate­buffered saline (PBS), pH 7.4. Tumour-transplanted mice usually survived for about 19-21 days.

As the regression properties of Dalton's lymphoma using a known anticancer drug like cisplatin has been established earlier21 , this tumour model was used in present studies which may' form a base to understand the confirmatory antitumour activity, if any, of the plant extracts against the same tumour.

Plants and preparation of extracts-The above mentioned five plants and their respective parts were selected according to the traditional practices by the people of these states. The roots of A. conyzoides and P. fulgens, leaves of B. lanceolaria and T. baccata and stem bark of D. pentagyna were collected, washed in distilled water followed by 70% alcohol and shade dried in sterilized container at about 40°C in an oven. The plant tissues were cut into small pieces and ground with a sterile mortar and pestle, and processed further for extraction under cleaned sterilized condition. These steps assure that the extracts were free from microbial contamination.

Aqueous extract of the ground plant tissues were prepared following the method of Amadioha22 with some modifications. Briefly, ground tissue samples were extracted for 2 hr by boiling with distilled water. After removal of insoluble materials by filtration through Whatman No. 1 filter paper, the filtrates were centrifuged at 800 g for 15 min and the clear supernatants were collected. The supernatants were evaporated slowly to dryness at 55°-60°C.

Methanol extract of all the plant tissues were also prepared using a slight modification of the method described by Alasbahi et aP3. The ground plant tissue samples were extracted with absolute methanol for 24 hr at room temperature (20°-25°C). The tissue-solvent mixtures were filtered through Whatman No.1 filter paper. The filtrates were centrifuged at 800 g for 15 min and the supernatants were collected. The supernatants were then evaporated to dryness at 55°-60°C. All the extracts were stored at 5°C until used for anti tumour activity tests.

The plant extracts were tried to dissolve preferentially in double distilled water,; phosphate­buffered saline (PBS), methanol and sodium hydroxide solution. Depending on the solubility criterion the various extracts were dissolved in the

ROSANGKIMA & PRASAD: ANTITUMOUR ACTIVITY OF PLANTS AGAINST MURINE LYMPHOMA 983

solvents as follows: aqueous extract of A. conyzoides and methanol extract of B. lanceolaria were dissolved in phosphate-buffered saline (PBS) and 2% methanol respectively while all other extracts were dissolved in 0.05% sodium hydroxide solution.

Evaluation of antitumour activity-The anti tumour activity of the plant extracts were determined following the method of Sakagami et aF. Various concentrations of the extracts and doses used in the antitumour study have been given in Tables 1 and 2. Tumour transplantation day was designated as day 0 and the early sign of tumour development was visible from the day 3-4 after transplantation. Therefore, plant extracts were administered (ip) to these mice everyday for 5 days following 3rd day of tumour transplantation. In each treatment group, 10 mice were used. Control mice received the same volume of the respective extract vehicle alone i.e. PBS, 2% methanol and 0.05% NaOH.

The deaths, if any, of the hosts were recorded daily and the survival pattern of the hosts was determined for different groups. The anti tumour efficacy of different extracts was reported in percentage of average increase in life span (lLS), and was calculated using the formula (T/Cx100) - 100, where, T and C are the mean survival days of treated and control groups of mice respectively. The survival patterns in the treated groups were compared with the respective controls using paired Student's t-test following 3-4 independent experimental determinations.

Protein and glutathione estimation-D. pelltagYlla, showing the highest anti tumour activity, was selected for further exploration to find its effect on the changes in the level of protein and glutathione in the liver (major site of glutathione and protein metabolism) as well as tumour cells after the treatment. The dose of the stem bark extract of D. pelltagYlla showing maximum antitumour activity was noted to be 20 mg/kg body weight (Table 2) . Therefore, for this part of experiment, same dose of the extract was administered (ip) to the tumour-bearin o mice on the

~ b

10 day of tumour transplantation when the tumour was at the logarithmic phase of growth. After 24, 48, 72 and 96 hr of the treatment, liver and ascites Dalton's lymphoma were collected. Ascites tumour was centrifuged at 2000 g for 10 min at 4°C to separate the Dalton's lymphoma (DL) cells pellet. Protein and glutathione estimations were accomplished following the method of Lowry et Ol.24,

and Sedlak and Lindsay25 respectively.

Results and Discussion In the antitumour studies, ascites Dalton's

lymphoma has been commonly used as an important murine experimental tumour model 21 ,26. Various test parts of the plants, doses of extracts and their effects on the survivability of the hosts in different experimental groups have been described in Tables 1 and 2. The deaths of mice, if any, was recorded daily and the survival pattern of mice in different experimental groups was determined (Tables 1 and 2).

Out of five plants used in the present study, three plants showed comparatively better antitumour activity against ascites Dalton's lymphoma. Aqueous extract of the root of A. conyzoides (100 mg/kg body weight) and P. fulgens (30-100 mg/kg body weight), and methanol extract of stem bark of D. pentagyna (10-30 mg/kg. body weight) significantly prolonged the survival time of host mice (Tables 1 and 2). The comparison of the antitumour effects of these plants depicted in the form of survivability showed that antitumour activity was highest with D. pentagyna (ILS -70%) followed by P. flilgens (lLS -36%) and A. conyzoides (ILS -27%). However, aqueous extract of T. baccata, B. lanceolaria, D. pentagyna and methanol extract of T. baccata, B. lanceolaria, A. conyzoides and P. flilgens did not exhibit significant antitumour activity (Tables 1 and 2).

In all the treatment experiments using different extracts, 50; 100 and 200 mg/kg body weight doses were used. However, in D. pelltagyna (methanol extract) and P. fulgells (aqueous extract), 50 mglkg dose showed higher antitumour activity than that of 100 or 200 mg/kg. Therefore, in these cases, even lower doses were evaluated. In D. pentagyna 30, 20 and 10 mg/kg doses were evaluated because maximum percentage ILS (71 %) was observed with the dose of 20 mg/kg body weight. In P. fulgens the dose lower than 50 mg/kg body weight i.e. 30 mg/kg body weight did not show better percent ILS, therefore, further lower doses were not evaluated. In A. con),zoides, as compared to 100 mg/kg body weight, even 50 mg/kg body weight dose showed almost no antitumour activity, therefore, in this case, doses lower than 50 mg/kg body weight were not evaluated (Tables I and 2) . Thus, it indicates that the di fferent plant extracts have to be used in different doses.

The most potent antitumour activity was observed with the methanol extract of D. pentagyna, whereas its aqueous extract did not show anti tumour activity

984 INDIAN J EXP BIOL, OCTOBER 2004

(Table 2). It appears that the active component of this plant is not extracted in water or it may have been destroyed while extraction with boiling water. On the other hand, methanol extraction may be favourable

for compound(s) extraction, stability and activity. The antitumour activIty representing maximum host survivors at the most effective dose of each plant extract has also been compared (Fig. 1).

0 ;;-

Iii > .~

::J (/)

100 •

I:j:~~"'I .. 80

60 -.-Control -e- Blumea /ancoo/aria

40

.I -I... 1-. I 1-A-t.

°11lLj

At various stages of tumour growth i.e. 5th, loth and

15th day, representing initial, middle and later stages of tumour growth, GSH content in DL cells increased with tumour growth being maximum on the 10th day of tumour growth. A slight decrease over the next 4-5 days was noted when tumour growth was probably declining (Fig. 2). In Ehrlich ascetic tumour cells maximum GSH concentrations was observed by about 7th day of tumour growth, followed by a decrease on Fig. l-Comparative sur.yival patterns of tumour-bearing mice treated with most potent dose of different plant extracts. Control, mice received extract vehicle only. Methanol extracts of leaves of B. lanceolaria (200 mg/kg body weight), stem bark of D. pelltagYlla (20 mg/kg body weight) and leaves of T. baccata (50 mg/kg body weight). Aqueous extracts of roots of A. cOllyzoides (100 mg/kg body weight) and P. illigens (50 mg/kg body weight) were administered daily for 1-5 days to tumour-bearing mice after tumour transplantation.

-A- Dlllenia pentagyna -"'- Ageratum conyzOldes __ Taxus baccala

20 -+- PoIentilla fulgens • 'YT ++

0 1 I

• '" +

0 5 10 15 20 25 30 35 40

Days after transplantation

Table l-Antitumour activity of aqueous extracts of plants against murine ascites Dalton's lymphoma in vivo

Plant Test part Dose" Life span (day) Significance ILS Survival on day 30

(mg/kg) (mean ± SE) (p<)b (%)C (survivalltotal)

B. lanceolaria Leaf* 200 15.2 ± 1.13 NSe

100 17.0 ± 0.65 NS 50 19.0 ± 0.48 NS

o (control)d 19.5 ± 0.42 A. conyzoides Root** 200 19.3 ± 0.57 NS

100 25.3 ± 0.59 <0.001

D. pentagyna Stem bark*

P.flligens Root*

T. baccata Leaf*

50 a (control)

200 100 50

a (control)

200 150 100 50 30

a (control)

200 100 50

o (control)

22.4 ± 0.60 19.8 ± 0.19 18.6±1.01 18.0 ± 0.48 18.2 ± 0.57 19.0 ± 0.89 22.8 ± 1.29 21.8 ± 0.98 27.0 ± 0.33 27.2 ± 0.67 25.4 ± 0.65 19.8±0.42 20.0 ±0.72 19.8 ± 0.36 20.4 ± 0.79 20.0 ± 0.31

a 0.25 ml volume of the extract solution was administered daily on days 3-7 b Statistical analysis was carried out by Student's t test C Activity criteria are passed for ILS(%) ~ 20% d Control animals received the same volume of the extract vehicle eNS: not significant *Extract was dissolved in 0.05% NaOH **Extract was dissolved in PBS

NS

NS NS NS

NS NS

<0.01 <0.01 <0.02

NS NS NS

-22 all a -12 0/10 -3 all 0

Oil a

-3 all a 27 all 0 3 Oil a

all 0

-2 all a -5 Oil a -4 0/10

Oil a

14 Oil a 9 all a 35 Oil a 36 2/10 27 2/10

Oil 0

0 0/10 -1 0/10 2 0/10

0/10

ROSANGKIMA & PRASAD: ANTITUMOUR ACfIVITY OF PLANTS AGAINST MURINE LYMPHOMA 985

the 14th day of tumour growth, which was correlated with a decrease in cell proliferation27

• The increase of aSH in tumour cells could be involved in facilitating

the proliferation and metabolism of tumour cells in the host and may support the finding that aSH controls the onset of tumour cell proliferation by regulating protein kinase C activity and intracellular pH28. As the aSH level in DL cells was observed to increase with tumour growth (Fig. 2), it became an investigating interest to find the changes in aSH, if any, after the extract treatment and for this exploration, the most potent plant i.e. D. pentagyna was used. Plant extract treatment significantly decreased the level of glutathione in liver as well as DL cells (Fig. 3). On the other hand, the treatment caused an increase of protein in liver and DL cells initially during 24- 48 hr but it also decreased significantly later at 72-96 hr of the treatment (Fig. 4). Various reports on the mechanism behind the antitumour activity of various plant extracts indicate that different plant extracts exhibited their anti tumour activities through different mechanism of action in the hose·9, '3,29,3o. In the antitumour effect of tea plant (Camellia sinensis var assamica, Theaceae) root extract, the activity of superoxide dismutase, a free

--- * ~ - 6 ~ C) -(/) 4 Q.) (5 E ...:;, 2 I fI) (9

O~~--~--~---T----~

5 10 15 Days of tumour growth

Fig. 2-Changes in GSH levels in Dalton's lymphoma (DL) cells at different stages of tumour growth ill vivo. Values are the mean ± SE. Student's t test; as compared to value on the day 5, n = 4, *p ~ 0.05.

Table 2-Antitumour activity of methanol extract of the plants against murine ascites Dalton's lymphoma ill vivo

Plant Test part Dose" Life span (day) Significance ILS Survival on day 30

(mg/kg) (mean ± SE) (p<)b (%t (survival/total)

B. lanceolaria Leaf* 200 20.0± 0.66 NS1 5 0/10 100 18.4 ± 0.74 NS -3 0/10 50 19.5 ± 0.98 NS 3 0/10

o (control)" 19.0±0.36 0/10

A. cOllyzoides Root** 200 14.8 ± 1.21 NS -33 0110 100 17.6 ± 0.58 NS -8 0/10 50 19.6 ±0.66 NS 0 0/10

o (control) 19.2 ± 0.94 0/10

D. pelltagYlla Stem 200 13.5 ± 0.66 NS -32 0110 bark** 100 19.6 ± 1.34 NS -2 0/10

50 23.8 ± 0.62 NS 19 0/10 30 32.4 ± 0.31 <0.001 62 8110 20 34.0 ± 0.97 <0.01 71 10/10 10 26.2 ± 0.45 <0.01 31 0110

o (control) 20.0 ± 0.49 0/10

P·fulgells Root** 200 14.4 ± 0.91 <0.05 -27 0/10 100 16.6 ± 0.40 NS -16 0/10 50 18.0 ± 0.92 NS -9 0/10

o (control) 19.7 ± 0.47 0/10

T. baccata Leaf** 200 18.8 ± 0.57 NS -7 0110 100 20.4 ± 0.46 NS 1 0/10 50 21.0 ± 0.63 NS 4 0/10

o (control) 20.2 ± 0.51 0/10

Details of a-e are same as in Table I *Extract was dissolved in 2% methanol **Extract was dissolved in 0.05% NaOH

986 INDIAN J EXP BIOL, OCTOBER 2004

radical scavenger, was found to be increased in the serum of tumour-bearing mice, suggesting the involvement of tea root extract in the enhancement of the defense mechanism9

•

As far as present findings are concerned, the exact mechanism of action of the plant extract against murine ascites Dalton's lymphoma may not be elucidated in detail at present. However, the decrease in aSH level by the extract treatment seems to playa significant role in antitumour activity of the extract of D. pentagyna against ascites Dalton's lymphoma. aSH, a major nonprotein thiol, is involved in

12~--------------------------~

Liver

i?ZIControi ~Treatment (24 h) ISS! Treatment (48 h) ElTreatment (72 h) [[[]]Treatment (96 h)

DL cells

Fig. 3-The pattern of changes in GSH levels in the liver and DL cells after ill vivo treatment with a single dose of methanol extract of the stem bark of D. pelltagYlla (20 mg/kg body weight). Values are the mean ± SE. Student's t test; as compared to respective control, n = 3 - 4, *p:<:; 0.05.

350

300

j 250 Qj ~ 01 200 0, E ~ 150 c ]l ~ 100 c..

50

0

Liver

IZ2l Control ~ Treated (24 hr) ISS:l Treated (48 hr) E3 Treated (72 hr) !lID Treated (96 hr)

*

DL cells

Fig. 4-Changes in protein concentration in the liver and DL cells after ill vivo treatment of tumour-bearing mice with a single dose of methanol extract of the stem bark of D. pelltagYlla (20 mglkg body weight). Values are mean ± SE. Student's t test, as compared to respective control, n = 4. *p:<:; 0.05.

protection against endogenous and exogenous toxic compounds3) and its role in the detoxification of chemotherapeutic agents is widely acknowledged32. It plays a role in protection against tissue damage produced by oxidative stress, radiation and chemotherapy33. Elevation of GSH levels has been shown to increase the resistance of cancer cells to cisplatin34 , while a depletion of GSH levels could potentiate the cytotoxicity of a variety of anti tumour agents32

. In the present study also the GSH depletion caused by the extract treatment may have a role in increasing cell death by enhancing susceptibility of the cells to oxidative stress thereby increasing hosts survivability. Resistance of many cells against oxidative stress is associated with high intracellular levels of aSH 35. In fact, loss of GSH and oxidative damage have been suggested to play a role in apoptotic cell death als036

. This decrease of GSH could possibly be due to inhibition of glutamyl­cysteine synthetase and glutathione synthetase, the enzymes catalyzing glutathione synthesis, and this needs to be examined. The decrease of aSH in DL cells by anticancer drug, cisplatin has also been attributed as one of the key steps during cisplatin­mediated antitumour activity37 and the present findings on the GSH status of liver and DL cells also indicate that the decreased level of aSH in the tissues, specially in tumour cells may play an important role towards the anti tumour efficacy of stem bark extract of D. pentagyna against Dalton's lymphoma.

In contrast to a regular decrease of aSH in liver and DL cells, there was slight increase in protein level initially (48 hr) and it decreased during later period (96 hr) of the treatment (Fig. 4). This may suggest that the extract treatment may not interfere much with the protein synthesizing machinery in the cells at least in the beginning. However, during later periods due to cumulative cytotoxic effects in the tumour cells, and possibly some toxic effect in the liver of the host it may cause a decrease in the protein level. In the cisplatin-mediated anti tumour activity a consistent significant decrease in protein level has been reported in liver as well as DL cells38.

It may be concluded that out of different plants used in present study D. pentagyna have shown effective antitumour properties against murine ascites Dalton's lymphoma. The decrease of GSH level in tumour cells may have a role in mounting oxidative stress and thereby increasing susceptibility to cell death. Further, it needs to characterize its active

ROSANGKIMA & PRASAD: ANTITUMOUR ACTIVITY OF PLANTS AGAINST MURINE LYMPHOMA 987

component, toxIcItIes and elucidate more insight on the mechanism of antitumour activity.

Acknowledgement The authors thank UGC, New Delhi and DST, New

Delhi for financial assistance.

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