Chapter 10
Antitumor Activity of Emblica officinalis Gaertn Fruit Extract 1
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Pratima Sur , D. K. Ganguly , Y. Hara , and Y. Matsuo 1
Division of Pharmacology and Experimental Therapeutics, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Jadavpur, Calcutta 700 032, India Food Research Laboratory, Matsui Norin Company Ltd., 223-1 Miyabara, Fujieda City, 426 Japan Hayashibara Biochemical Laboratory, Fujisaki Cell Center, Okayama 702, Japan
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The antitumor activity of Emblica officinalis Gaertn (Phyllanthus emblica Linn) fruit extract (EFE) was assessed against different human leukemic cell lines, ML-2, U-937, and K-562. Significant cell growth inhibition was observed in all the cell lines. Induction of differentiation, assessed by indirect immunofluorescence, Nitroblue tetrazolium reduction, morphology was observed in EFE treated cells. Balb/C mice, pretreated with the EFE extract, inoculated with Ehrlich Ascites Carcinoma (EAC) cells showed significant cell growth inhibition. The in vivo effect was accompanied by the anti-inflammatory property of EFE. In Indian traditional medicine, the fruit of Emblica officinalis Gaertn has been used extensively as the main ingredient, which has been known to prevent colds, coughs, and enhances immunity (1). So far, the fruit is known as one of the best sources of natural Vitamin C. Such Vitamin C has been found to be more readily assimilated than the synthetic Vitamin C. The fruit is also known to contain a significant amount of pectin (1), a complex polysaccharide containing galactoside residues, which is known to possess anticancer properties (2). The antioxidant, and strong reducing properties of Vitamin C are known to be responsible for its use as a free radical scavenger, indicating its chemotherapeutic potential (3). Many anti-inflammatory drugs are reported to prolong survival of patients with cancer (4). The antiinflammatory property of this fruit is also mentioned in Indian traditional medicine. Considering all previous facts, the present study was undertaken using EFE extract. The in vitro antitumor property against human leukemic cell lines, preliminary in vivo antitumor property against Ehrlich Ascites Carcinoma (EAC), and the antiinflammatory property of EFE were all evaluated in this study. Plant Material. The fruit of Emblica officinalis was collected during the winter season (November-December) in West Bengal, India. Voucher specimens of the
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©1998 American Chemical Society
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dried fruit and extract were deposited at the Division of Pharmacology and Experimental Therapeutics, Indian Institute of Chemical Biology, Calcutta, India. Preparation of Emblica officinalis Fruit Extract (EFE). The fruit is green when young, and gradually turns to pale yellow when it is mature. The EFE was prepared in the following manner: Mature fruit minced into pieces Seeds removed and discarded
Dried at room temperature Soaked in 50% Ethanol for 7 days at room temp, ^filtered Brown filtrate
I Rotary^vaporated Brown Residue ^Dissolved in double distilled water Millipore filter Sterile Extract kept at 5°C Emblica Fruit Extract (EFE) Cells. Human leukemic cell lines ML-2, U-937, and K-562 cells were obtained from the Fujisaki Cell Center, Okayama, Japan. Cells were maintained routinely in a RPMI 1640 medium, and supplemented with 10% fetal calf serum (heat inactivated). Cultures were maintained at 37°C in a 95% humidified atmosphere containing 5% CO2 in air. For preliminary in vivo studies, Ehrlich Ascites Carcinoma (EAC) cells were obtained from the Chittaranjan National Cancer Research Center, Calcutta, India. E A C cells were maintained in Balb/C mice by weekly intraperitoneal inoculation with 10 cells per mouse. Sprague Dawley rats (125-150gm) were used to study the anti-inflammatory effect. 6
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In Vitro Cell Growth Inhibition. Cells at a concentration of about 0.5 χ 10 /ml were used to start the experiment in a 12-well sterile plastic plate. EFE, at desired concentrations, were added on day Ό , and incubated for 5 days at 37°C. Viable cells, as judged by the Trypan blue dye exclusion test, were counted every day (5). 1
106 Cell growth inhibitions, after treatment, were indicated by doubling times, using the formula: Doubling Time = Time(hour)/No. of Doubling Where, No. of Doubling = [log.final cell no. - log.initial cell no.] /0.302 Tritiated Thymidine, Uridine and Leucine Incorporation. Effect of EFE (50, 100, 200 μg/ml) on the synthesis of DNA, R N A , and protein synthesis were observed with EFE treated ML-2 cells after 24 and 72 hour time intervals. The method used was essentially the same as reported earlier (5). Cell Cycle Analysis. Cell cycle analysis was performed with EFE treated ML-2 cells as described earlier (5). Treated ML-2 cells, after 24 and 72 hours, were washed with phosphate buffered saline, and fixed in 70% ethanol. The fixed cells were washed and incubated with RNase (40 μg/ml) for 30 min at 37°C. Cells were again washed and stained with propidium iodide (50 μg/ml) for 30 min. at 0°C. The stained cells were analyzed on a coulter profile flow cytometer. Indirect Immunofluorescence. Cells, after 72 hours of treatment, were washed in 0.9% saline, and incubated for 30 min. at 37°C in heat inactivated, pooled, A B serum to prevent nonspecific FC binding (6). Modulation of the expression of specific cell surface antigens was observed by monoclonal antibodies obtained from Nichirei (Tokyo, Japan), Coultronics and Seralab (UK). Cells were incubated for another 30 min. at 37°C with a second monoclonal antibody containing FITC conjugated (ab') goat anti-mouse antiserum. The cells were washed and suspended in 0.9% saline, and analyzed by a coulter profile flow cytometer (Coulter Electronics, Hialeah, F l ) , as done earlier (7). 2
Morphology. Morphologic differentiation was observed on Giemsa-stained slide preparations, with 72 hour EFE treated ML-2 cells. Nitroblue tetrazolium (NBT) reduction assay. The N B T reduction assay was performed, as described previously (7). EFE treated ML-2 cells were washed and suspended (1 χ 10 cells) in 0.2 ml of RPMI 1640 meduim, containing 5% FCS, 0.1% N B T , and 30 mg of TPA. After 30 minutes of incubation at 37°C, the percentage of cells containing blue black deposits, were counted. 5
In vivo effect of EFE on the growth of EAC in mice. In vivo effects with EFE, against E A C , were observed following three different methodologies: A Animals (n=7) were pretreated with 100 mg/kg EFE for 3 days. After a 24 hour gap, the animals were inoculated with 10 E A C cells intraperitoneally. On the 4 day of inoculation, the animals were sacrificed. Total 5
th
107 intraperitoneal E A C cells were then counted, and compared with the control (without treatment) group. 5
B. Animals (n=7) were inoculated with 10 E A C cells/mouse on day zero. Treatment with EFE (100 mg/kg) started from day 1, and continued to day 3. On day 4, the animals were sacrificed. Intraperitoneal cell counts were then made, and compared with the control. C. Animals (n=7) were pretreated for 3 days as described in A . After a 24 hour period, they were inoculated, and treated as described in B. On day 4, they were sacrificed. Intraperitoneal E A C cells were then counted, and compared with the control. Antiinflammatory activity (Carrageenan-induced Oedema). A 1% solution (0.1 ml) of carrageenan, in 0.9% saline, was injected in male rats beneath the plantar aponeurosis of the right hand paw, and divided into four groups (6 in each). Group 1 served as the control, group 2 received phenylbutazone (100 mg/kg, ip) as standard. Groups 3 and 4 received 50 mg/kg, ip and 100 mg/kg, ip of EFE respectively. Drugs were administered 1 hour before the injection of carrageenan . Foot volume was measured plethesmographically after 4 hours of carrageenan administration. The difference between the control and the treated groups indicated the degree of oedema developed, and the percentage of inhibition was thereby calculated. For the in vivo experiments, significance tests were done using the Student's t-test. Results Differential cell growth inhibitions, as indicated from the doubling times, were observed in EFE treated cell lines (Table I). The prolonged doubling time of treated cells with respect to the control, indicate inhibition of cell growth. Negative doubling time was due to continuous cell death after treatment. The E F E treated ML-2 cells showed the maximum sensitivity to EFE. To make a comparitive study, the time required for different cell lines to inhibit 50% cell growth, with a specific dose of EFE (200 μg/ml of cell suspension), was made (Table II). It was found that the ML-2 cell line was the most sensitive, requiring 32 hours to inhibit 50% cell growth. For the K-562 cell line, the time required was 63.0 hours. The M L - 2 cell line was selected for further study because of its higher sensitivity against EFE. Cell growth inhibition of the ML-2 cells was corroborated by the inhibition of H-Thymidine uptake after treatment, with different doses of EFE (Table III). With 100 μg EFE/ml of ML-2 cell suspension, 65% inhibition of H-Thymidine uptake could be observed in 72 hours. However, uptake of H-Uridine or H Leucine was not inhibited in EFE treated ML-2 cells (data not shown). The D N A histogram patterns of EFE treated ML-2 cells were found to be perterbed after the 72 hour treatment (Table IV). Accumulation of cells in the 'G2-M' compartment was prominent with a depletion of 'S' phase cells, resulting in the inhibition of cell growth. 3
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108 Table I. Doubling Time in Log Phase of Different Tumor Cells under the Effect of EFE Tumor Cell Lines (Hr) Doubling Time EFE Concentration 100
0
(Mg/ml) 200
400
1.
ML-2
30.5
38.7
64.8
-58.9
2.
U-937
36.2
39.5
50.2
98.3
3.
K-562
29.6
30.7
43.6
100
Table II. Time Required for 50% Cell Growth Inhibition with EFE Cell Line
Iso
(Hr)
100 μg/ml
200 μg/ml
ML-2
44.0
32.0
U-937
48.0
40.0
K-562
92.0
63.0
Specific antibodies expressed on macrophage, monocyte, and granulocyte surfaces were observed on the ML-2 cells treated with EFE (100μg/ml) for 72 hours (Table V). Such induction of differentiation, evidenced by indirect immunoflorescence, was supported by enhanced NBT reducing activity (Table VI), and morphological changes (Figure 1). The morphology of different doses of EFE (72 hr) treated cells, showed enhanced cytoplasm to nuclear ratio, and ruffled surfaces. Morphologically mature cells were observed with all the doses of EFE used. Preliminary in vivo cell growth inhibitions are shown in Table VII. Pretreatment with the EFE, followed by i.p. E A C tumor inoculation, showed a 50% tumor growth when compared with the control (protocol-Α). When tumor bearing mice were treated with EFE, as described in protocol-B, they produced a 30% inhibition of the tumors. When the protocols were coupled together, the cell growth inhibition was found to be 82%, showing an additive effect. It is important to note that experiments carried out with extracts of EFE, prepared from fruits from different
109 areas of West Bengal, India, showed similar results as those described above. The appreciable antiinflammatory activity of EFE was found when compared with the control. The treatment with 100 mg/kg of phenylbutazone showed 50% inhibition. 40% and 62.3% (pO.001) inhibitions of oedema were observed with 50 mg/kg and 100 mg/kg EFE treatments, respectively.
Table III. Inhibition of DNA Synthesis Incorporation in ML-2
'Ή-Thymidine 24 Hr Treatment EFE
48 Hr Treatment c.p.m.
μ^ηιΐ
O(Control) 50 100 200
18,466(~) 15,350(17) 15,542(16) 10,742(42)
Cell 72 Hr Treatment
cells
20,503(~) 14,783(28) 10,529(48.7) 9,270(54.8
23,921(») 11,728(51) 8,370(65) 7,250(70)
The results shown represent the average value of the three independent experiments (deviations were within 3-5%). The values in parenthesis indicate percent inhibition.
Table IV. Cell Cycle Analysis DNA Histogram of ML-2 Cells Treated with EFE
Intensity Distribution of Stain Fluorescence at Different Stages of Cell Growth 72 Hr 48 Hr EFE S G -M mg/ml Go-Gi G -M S Go-Gi 2
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O(Control)
39.9
17.5
42.5
36.25
12.9
50.8
50
34.7
13.0
52.2
33.44
23.45
43.1
100
34.5
17.8
47.7
38.14
20.35
41.5
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Discussion. The purpose of the present study was to investigate the antitumor property of EFE. In vitro inhibition of cell growth in ML-2 cells, by EFE, was accompanied by inhibition of D N A synthesis and perturbation of the cell cycle. Growth and maturation are normally synchronized in hematopoitic stem cells. A blocked cellular maturation plays an important role in cancer. Thus, therapy leading to the differentiation is often used to induce stem cells to mature (8,9). Enhanced expression of myeloid differentiation antigens CD14, CD15, CDub, etc. are normally expressed on the surface of monocytes, macrophages and activated granulocytes, and were found to be enhanced on the surface of EFE treated ML-2 cells, indicating differentiation. It has been observed (10,11) that antineoplastic agents, such as inducers of differentiation, cause sustained inhibition of DNA synthesis, while protein or RNA synthesis remains unchanged. Similar observations were found with EFE treated ML-2 cells.
Table V. Indirect Immunofluorescence Reactivity of ML-2 Cells with Antibodies on Monocyte, Macrophage and Granulocyte Surface Antigens after a 72 Hr. Treatment Antibody
Cluster Designation
Mean
Fluorescence
Intensity(MFI)
Saline Control
50 Mg/ml EFE
100 Mg/ml EFE
1.MY9
CD33
1.592
1.254
1.376
2. M Y 4
CD
1 4
0.298
0.312
0.347Î
3. MO
CDi,
0.288
0.294
0.346Î
4. Leu-15
CDub
0.279
0.280
0.317Î
5. MCS-1
CD
0.300
0.298
0.366f
0.284
0.279
0.315
6. Saline
I 5
Ill
υ
ο
υ
SI
I s 15 g "ID (Ν A • Ο j Ο
^ /—s Ο w
ο n •S ο
s e
υ>
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Table VI. Nitroblue Tetrazolium Reduction NBT-Reducing Activity After EFE Treatment in ML-2 Cells Experiment(n=5)
Percent 24 Hr
Positive Cell ±S.D. 48 Hr
72 Hr
Control
0.52 ± 0.0
0.78 ± 0.09
1.2 ± 0 . 7
EFE 50 μ£/ηι1
1.0 ±0.5
5.811.2
7.5 ± 0 . 6
EFE 100 μg/ml
5.3 ±2.1
11.9 ±2.3
12.0 ±2.1
EFE 200 μ§/πι1
6.05 ±3.1
12.0 ±2.8
14.5 ± 1 . 8
Vitamin C in the extract might be partially damaged inspite of every care, due to exposure during the drying of the fruits, and the preparation of EFE. The amount of Vitamin C present in the extract, after drying, possibly acted as a scavenger of free radicals and prevented tumor growth. Pectins, which are complex polysaccharides, and known to inhibit azomethane, induced colon carcinogenesis (12), adenocarcinoma growth, and embolization of tumor ceils (13), were reported to be present in the fruit (1). Such properties of Vitamin C and pectins might be responsible for the antitumor property of the fruit extract. Further in vitro studies with EFE are in progress with different fractions of EFE. Table VII. In Vivo Cell Growth Inhibition Total Number of Viable Intaperitoneal E A C cell /mouse (xlO )
Percent Cell Growth Inhibition
Pretreatment
0.72 ± 0.08*
50.0
Post Treatment
1.00 ±0.2*
30.0
0.25 ±0.18*
82.5
1.43 ±0.14
—
8
Treatment
7
A. B.
C. Pretreatment + Post Treatment D.
Control
*In vivo treatment in Balb/C mice with 100 mg/kg EFE, following methodologies as described in the text. *p
