121

Cancer Letters,49 (1990) El--126

Elsevier Scientific Publishers

Antitumour

Ireland Ltd.

principles from 1x0~a ja vanica

S.C.

Nair and

K.R.

Panikkar

Amala

Cancer Research Centre,

Amala Nagar, Trichur 680 553, Kerala (India)

(Received 20 December 1988) (Accepted 14 September 1989)

Summary The antitumour agent from Ixora jauanica jlowers shows broad activity against transplantable solid tumours (DLA) in mice by inhibiting the growth of tumour and arresting the growth of already formed tumours; with lesser activity against Ascites tumours. In vitro cytotoxic studies showed 50% cytotoxicity to Dalton’s lymphoma (DLA) and Ehrlich Ascites tumour cells at a concentration of 12 c(9 and 65 pg, respectively, with no activity against normal lymphocytes but prejerentiaf actiuity for lymphocytes derived from leukemia patients (ALL) (CML), and K 562 suspension cell culture. Tritiated thymidine incorporation studies indicated the mechanism of action of the agent at the site of DNA synthesis. The purified fractions contained Ferulic acid, Pyrocatacheuic acid and cajjeic acid. jauanica; lxoru Keywords: antitumor; Dalton’s lymphoma; Ehrlisch carcinoma.

tinal antiseptic and as astringent [Z] . Externally the flower parts are used to heal sores and chronic ulcers. The roots were found to contain aromatic oil, tannins, fatty acids and a white crystalline substance. Flowers contain a colouring and astringent principle of the nature of an organic acid, a wax, a yellow colouring matter related to quercetin. We have been able to detect antibacterial properties in the leaves and flowers, against bacteria cultured from clinical specimens. The anticancer properties of this plant has not been examined by modern techniques. Screening of plants have been carried out against various cell lines like Dalton’s lymphoma ascites cells, HeLa, CHO (Chinese Hamster Ovary cells) P388, L1210 leukemia, B-16 melanoma in our laboratories [3,4]. Preliminary experiments indicated the presence of antitumour principles in the flowers and leaves of Ixora jauanica. We are presenting here the cytotoxic properties of the flowers and leaves of Zxoru jauanica using in vivo and in vitro experiments against cell lines of Dalton’s lymphoma, K-562 and lymphocytes .

Introduction lxora jauanica is a garden plant and its flowers constitute one of the ingredients of a preparation used in the treatment of cancer by the Indian system of medicine [l]. A survey of Indian medicinal plants were carried out and several plants are found to possess anticancer activity as judged by their cytotoxicity and animal experiments against various cancer cell lines. The flowers and leaves of Ixora jauanica are used as a sedative stomichic tonic, intes-

Materials and methods Ixora jauanica flowers used in this study were available locally and collected after identification with the aid of treatises on regional flora and comparison with the herbarium sheets of authentic species. These were washed, dried in the shade, powdered using a mechanical grinder and passed through a No. 400 sieve. The extraction with various solvents

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of different polarity indicated maximum activity with 50% ethanol. The extraction was carried out using 5 g of the dry powder with 100 ml of 50% ethanol and kept overnight at room temperature (23OC). This was filtered and the residue was extracted twice subsequently with the same solvent. The supernatants were pooled and evaporated to dryness in a water bath and redissolved in 30 ml of 50% ethanol. This extract was diluted with PBS pH 7.4 to a concentratioh of 5 mg/ml and used for in vitro cytotoxic studies. All values reported here are expressed in terms of the original flower powder. Lymphocytes were prepared using FicollHypaque method from either healthy donors or patients with leukemia. Dalton’s lymphoma cells grown in the peritoneal cavity of mice were aspirated and washed in normal saline. These cells were used either for cytotoxicity experiments or for injection into fresh mice. Purification

The concentrated crude extract was then subjected to extraction in ethyl acetate. The ethyl acetate fraction was purified by descending paper chromatography on Whatman No. 1 filter paper into a homogenous compound with butanol/acetic acid/water (6 : 1 : 2) as the solvent system. The active spot was identified as the substances having yellow colour moving immediately below the solvent front. This was cut and eluted with methanol and evaporated to dryness (40 mg) and this powder was suspended in PBS for use in cytotoxic studies. The cytotoxicity studies were carried out using Dalton’s lymphoma ascites tumour cells grown in the peritoneal cavity of Swiss albino mice. The cells drawn from the animals were separated and washed with normal saline. Various concentrations of the drug extract was incubated with 1 x lo6 cells in PBS for 3 h at 37 OC. The percentage of dead cells were determined using the Trypan Blue exclusion method and the minimum concentration required for 50% cytotoxicity was noted [3]. Lymphocytes were isolated from the peripheral blood of healthy normal persons

and patients with leukemia (acute lymphocytic leukemia and chronic myeloid leukemia) and used for cytotoxic studies in a similar way [5,6] using 1 X 106 cells. Animal experiments Animal experiments were carried out using male Swiss albino mice in 3 groups of 8 animals. Dalton’s lymphoma ascites cells were drawn asceptically washed with PBS and 1 X 106 cells in PBS buffer were injected i.p. to all 3 groups. One group of mice were given 1 ml of the extract containing 4 mg equivalent to the original powder (i.p.) 24 h after the transplantation of the tumour, for 10 days. The second group of mice were given the drug similarly for 10 days from the 11th day onwards. The third group was used as a control and were given the same volume of PBS without any drug. All animals were given standard mouse diet (Lipton India) and water ad libitum. The development of tumours and increase in life span of the treated animals was compared with the untreated control mice. The same experiment was also carried out with the crude sample. Another experiment using 3 groups of 8 Swiss albino mice was carried out to assess the effect on solid tumour formation. Dalton’s lymphoma ascites tumour cells grown previously in mice were asceptically aspirated and after proper washing 1 x lo6 cells were injected into the hind leg of the animals of all the groups S.C. After 24 h of tumour transplantation, the experimental mice were administered S.C. with 2.5 mg in 0.1 ml PBS equivalent to the original powder at the. site of the tumour for 10 days. A similar course of treatment was given to the second group from the 1 lth day onwards for 10 days and the third group were considered as the control. Tumour development was noted and the tumour volume measured at suitable intervals with the help of vernier calipers using the following formula f7] : 4/3 nrft$ Inhibition ojDNA incorporation studies The mechanism of action of this drug was

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studied by using Dalton’s lymphoma cells by its role in the inhibition of DNA synthesis. Tritiated thymidine was obtained from Bhabha Atomic Research Centre, Bombay. Incorporation of tritiated thymidine with DNA was done by incubating Dalton’s lymphoma ascites tumour cells (4 x lo6 cells) with 2 &i of [3H]thymidine in the presence of various concentrations of the extract. The cells were added along with thymidine and incubated for 4 h at 37 OC in PBS (pH 7.4). After the incubation the DNA was precipitated with 0.8 M perchloric acid at 4OC and redissolved in 0.5 M sodium hydroxide. The radioactivity was counted using a dioxan based cocktail in a liquid scintillation counter LKB 1209 Rackbeta

161.

Results The purified flower extract gave 50% cytotoxicity to DLA cells at a concentration of 121 pg. This indicated a ZO-fold purification of the original crude extract. Comparison of this cytotoxic effect in Ehrlich ascites and Dalton’s lymphoma ascites indicated a better activity since Ehrlich with Dalton’s lymphomas required 65 pg for 50% cytotoxicity which is approximately 5 times the concentration required for the Dalton’s lymphoma cells. Normal lymphocytes were found to be Table 1. Concentration extract bg)

600 300 100 50 20 10

In vitro cytotoxicity of

loot

t \

7 \\

mm

z

T 50 -_-_-.? 2 $_ 30

____

i

t

i

I

10 &

1 5

10

15

Concentration

20

-x---

(pg)

Fig. 1.

In vitro cytotoxicity of the purified flower extract on lymphocytes obtained from leukemia patients by the Ficol-Hypaque method - a concentration of 7 pg/ml and 13 pg/ml equivalent to the purified flower extract was required for 50% cytotoxicity to lymphocytes derived from the peripheral blood of patients with acute lymphocytic leukemia (ALL) (0 -0) and chronic respectively. Blood myeloid leukemia (CML) (0 -0) samples were collected prior to the commencement of chemotherapy.

insensitive to the purified flower extract while lymphocytes from leukemic patients were acted upon by the extract at a low concentration of 20 c(g (Fig. 1, Table 1). It was found that a concentration of 7 pg/ml and 13 pg/ml equivalent of the purified flower extract was required for 50% cytotoxicity to lymphocytes

of the purified flower extract on lymphocytes. Percentage

of dead cells

Normal lymphocytes

Lymphocytes from (ALL) patients

Lymphocytes from (CML) patients

12 2 0 0 0 0

100 100 100 100 100 74

100 100 100 100 100 20

Normal lymphocytes were isolated from the while leukemic lymphocytes was drawn from leukemia (ALL) and a 52-year-old female Department. Blood samples were collected 3 separate studies.

peripheral blood of healthy human subjects by the Ficol-Hypaque method the peripheral blood of a 19-year-old female patient with acute lymphocytic patient with chronic myeloid leukemia (CML) admitted to our Oncology prior to the commencement of chemotherapy. Values are an average of

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Cytotoxicity Table 2. K 562 cells in culture. Concentration of the PFE

of the purified flower extract on

Number of live cells in millions after incubation with extract

kg)

25 pg 50 pg Control

Oh

24h

48h

72h

1 1 1

12.7 8.5 23

20.5 15.5 47

65 41 85

Two different concentrations of the purified flower extract was incubated with K562 cells in 10 ml RPM1 medium. The extract was not added to the controls. Values expressed are an average of 5 separate experiments.

derived from the peripheral blood of patients with acute lymphocytic leukemia (ALL) and chronic myeloid leukemia (CML) respectively. Studies on solid tumours in mice showed that the experimental mice subjected to the administration of flower extract failed to develop tumours. The second group that was administered with the purified flower extract 10 days after transplantation of tumour cells developed tumours initially but further growth of tumours was arrested without any decrease in the tumour volume indicating tumour static property of the extract. A substantial increase in the life span, 66.85% in the case of treated mice 24 h after tumour transplantation and 58.40% in case of mice treated 10 days after Table 3.

Tumour reduction

1

2

10

_

15

:

20

;

25

:

30

DAYS. Fig. 2. Solid tumour reduction in Swiss albino mice. 0 -0, Control mice that were left untreated; 0 -0, treated mice administered 2.5 mg/O.l ml PBS equivalent of the purified flower extract(s.c.), for 10 days 24 h after transplantation of tumour cells; A -A, Treated mice administered 2.5 mg/O.l ml PBS equivalent of the purified flower extract (s.c.), 10 days after transplantation of tumour cells, for 10 days.

tumour transplantation, was noted (Fig. 2, Table 3). The results of the ascites tumour studies indicated that the experimental mice did not develop ascites tumour for the first 20 days. After the 25th day a slight development was noted. The control animals developed ascites tumour and the treated animals showed an ILS of 50.20% (Table 4). Administration of the extract 10 days after tumour transplantation did not resist the growth of tumour as in the case of solid tumour experiment since the results were almost similar to that of controls.

in Swiss albino mice; DLA; solid tumour.

Average no. of mice survived after transplantation of tumour

Control (E) Treated (El) Treated (E2)

;

5

Average life

30 Days

45 Days

60 Days

8/8 8/8 8/8

O/8 7/8 6/8

O/8 2/8 l/8

(days)

I.L.S. = T - C x 100% C

34.33 57.28 54.38

66.85 58.40

Increase in life span (ILS) of treated mice (El) administered with 2.5 mg/O. 1 ml PBS equivalent of the purified extract (flower), 24 h after tumour transplantation and treated mice (E2), administered 2.5 mg/O. 1 ml PBS equivalent of the extract 10 days after tumour transplantation, subcutaneously for 10 days, the control mice (E) given the same volume of PBS without drug. Each set used 8 mice for the experiments.

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Table 4.

Ascites DLA tumour. Average number of mice survived after tumour transplantation

Control (n) Treated (n)

25 Days

35 Days

45 Days

60 Days

3/8 8/8

O/8 8/8

O/8 3/8

O/8 O/8

Average life (days)

I.L.S. = T - C x 100% C

24.88 37.37

50.20

Increase in life span (ILS) of treated mice (m) administered with 4 mg/ml PBS equivalent of the purified flower extract (i.p.) for 10 days, 24 h after tumour transplantation with respect to control (n) given the same volume of BPS without drug. n = Number of mice used in each set (8)

The effect of the purified extract on DNA synthesis was studied by incorporating tritiated thymidine into Dalton’s lymphoma ascites cells. The results showed a concentration dependent decrease in thymidine incorporation. The purified extract showed maximum effect on the thymidine incorporation into cellular DNA as 50% inhibition in incorporation was achieved at a concentration of 7 pg/ml. This was found to be lower than the amount required for 50% cytotoxicity to DLA cells in vitro suggesting the mechanism of action of the drug is at the site of DNA synthesis and not only at the cellular membrane level (Fig. 3). The in vitro cytotoxic studies with K562 cultured cells in RPM1 medium, showed an activity resulting in the inhibition of the cell growth in the first 24 and 48 h even though the growth

h

100 90

5

10

15

Concentration

20

25

J,

(pg)

Fig. 3. Effect of the purified flower extract on inhibition of DNA synthesis - 50% inhibition in incorporation of [3H]thymidine into cellular DNA of DLA cells was obtained at a concentration of 7 &ml.

has tended to come to normal by approximately 72 h (Table 2). The purified samples were investigated for the identification of the chemical nature and we were able to find three compounds Pyrocatacheuic acid, ferulic acid and caffeic acid in the extract. The identification was carried out by chemical and spectroscopy methods. Discussion

The in vitro cytotoxic experiments showed activity both in the crude and purified flower extracts and it was purified only 20-fold as per cytotoxic studies. These drugs had no action on normal lymphocytes while leukemic lymphocytes were acted upon. The preferential action may be due to the specificity of the drug towards lymphocytes of the leukemic subjects probably at the membrane level. This is a positive finding as the drug can selectively distinguish the cancerous lymphocyte from the normal. The flower parts are also being used for healing wounds normally resistant to drugs. A similar cytotoxic principle was isolated in our laboratory from a Nigella sat&urn, another medicine used in the healing of wounds normally resistant to wound healing drugs. The in vitro cytotoxic studies with differnt cell lines is only a degree of variation which are due to the specific nature of the cell line. Thus we find Ehrlich ascites cells require four times

126

the concentration necessary for Dalton’s lymphoma cells. We have isolated another drug from Nigella satiuum which is found to be more effective towards Ehrlich ascites than Dalton’s lymphoma which also suggest that the action of the drug is specific to cells. The experiment with K 562 cell lines indicate initial action which is being overcome probably due to the increase in number of cells, thereby decreasing the effective concentration of the drug. The growth rate could have been arrested had we maintained the same ratio of the drug at all times. The effect of the drug on the prevention of tumour development in the ascites and solid tumours suggest that the effectiveness of the drug as an agent which prevents the development of transplanted tumours even though established tumours could not be reduced, but further development could be arrested in the case of solid tumours. This is not so in the ascites form sites of tumour probably due to the proliferation and development being in the peritoneal cavity. The mechanism of action of the drug could be at the DNA level since the thymidine incorporation studies reveal inhibitions in the DNA incorporation proportional to the drug concentration. We do not know the exact mechanism which may inhibit any of the enzymes in the replication site of DNA synthesis stage. The in vitro cytotoxic activity on various cell lines and lymphocytes may be due to the action of the drug on the cell membrane as a first step before its specific activity. The effective prevention of growth in K 562 cells initially may also be due to the action on the DNA but in the later stages the concentration of the drug was low compared with the cell number and the consequent inability to inhibit the further growth. The nature of the active ingredient is not

known, but we were able to isolate 3 compounds present in the purified extract viz., Pyrocatacheuic acid, ferulic acid and caffeic acid. These 3 compounds are present in the drug in the free form or most probably in the combined form. A better extraction of the active principle is obtained with 50% ethanol and therefore the active principles are most likely present as derivatives of these compounds in combination with carbohydrates or proteins or some basic substances. The finding of these experiments reveal the potential anticancer properties of the lxora javanica flowers. Acknowledgement We are thankful to Prof. A.G. Ramachandran Nair, Prof. of Chemistry Pondicherry University, Pondicherry, India for the identification of the active principle. References Panikkar, K.R., Bhanumathy, P. and Raghunath, P.N., (1986) Anticancer activity of an ayurvedic oil preparation. Ancient SC). Life, VI, 107. Nadkami, K.M. (1976) Indian Materia Medica. Popular Prakashan, Bombay, p. 698. Kuttan, R., Bhanumathy, P., Niramala, K. and George, M.C. (1985) Potential anticancer activity of tumeric (Curcuma longa) Cancer Lett., 20, 197-202. Kuttan, G., Vasudevan, D.M. and Kuttan, R. (1988) Isolation and identification of tumour reducing component from Mistletoe extract (Iscador) Cancer Lett., 41,3. Nath 1. and Hanjans, N.S. (1983) Separation of human lymphocytes and prepartion of Rosette forming cells from human peripheral G.P. Talwar, p. 275. Unnikrishnan, M.C. and Kuttan, R. (1988) Cytotoxicity of extracts of spices (isolated lymphocytes) to culture cells. Nutr. Cancer, 11,4. De sombrc, D. (1974) Solid tumour growth (measurements) Cancer Res., 34, 1971,

Antitumour principles from Ixora javanica.

The antitumour agent from Ixora javanica flowers shows broad activity against transplantable solid tumours (DLA) in mice by inhibiting the growth of t...
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