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Isolation of anti-leukemia compounds from Citrus reticulata
Life Scienrrs, Vol. 58, No. l.5, pp. 1265’-1276,1!296 Copyright0 19% ELMvicrScience Inc. Printed in the USA. Au rights resewed am-3205/96 5l.5.00 t .oo
ELSEVIER
PII SOO24-3205(96)OOOS8-4
ISOLATION OF ANTI-LEUKEMIA COMPOUNDS FROM CITRUS RETICULA TA Nai Ki Makl, Yee Ling Wong-Leung2, Shuk Chong Cha.nl# , Jianming Wenl *, Kwok Nam Leung3, and Ming Chui Fung4 lDepartment of Biology, 2Department of Chemistry, Hong Kong Baptist University, 3Department of Biochemistry, and 4Department of Biology, Chinese University of Hong Kong (Received in final form February 7, 1996)
Summary
In vitro effects of medicinal plant extracts from the pericarpium of Citrus reticulata (cv Jiao Gan) (PCRJ) on the growth and differentiation of a recently characterized murine myeloid leukemic cell clone WEHI 3B (JCS) were investigated. Extracts of PCRJ not only inhibited the proliferation of JCS cells in a dose dependent manner, but also induced differentiation of JCS cells into macrophages and granulocytes. Morphological differentiation of PCRJ treated JCS cells was associated with an increase in phagocytic activity of the cells. Furthermore, both in vitro clonogenicity and in vivo growth of PCRJ treated JCS leukemic cells in syngeneic BALB/c mice were significantly reduced. The survival rate of mice receiving PCRJ Using ‘H-NMR, 13C-NMR, and treated JCS tumour cells was also increased. GCMS, two active components isolated from PCRJ were identified as nobiletin and tangeretin. Key Words: leukemia, tangeretin, nobiletin, cytokines, citrus fruits
The commercial importance of citrus plants is the use of their fruits as sources of food Fruits from citrus species have also been widely used as a source juices for human consumption. of traditional medicines. Many active components of medicinal values were isolated from citrus These included anti-allergic agents, anti-oxidants, anti-tumour agents, and immunospecies. modulating agents (l-4). It has been demonstrated that D-limonene and the oils isolated from citrus fruits can inhibit the formation of pulmonary adenoma and the occurrence of forestomach tumours induced by carcinogens such as 4-(methylnitrosamino)-I-(3-pyridyl)-1-butanone (NNK) (3). Compounds with in vitro anti-tumour activity against squamous cell carcinoma, and human We have recently promyelocytic leukemia cells have also been detected in citrus fruits (5, 6). The disease that the reported a subclone of murine myeloid leukemic WEHI 3B (JCS) cells (7). WEHI 3B cell causes when injected into mice appears to resemble human acute myeloid leukemia Correspondence : Dr. N.K. Mak, Hong Kong Baptist University, Hong Kong. Tel: 852-2339-7059; E-mail : [email protected] Present Address : #Department of Biology, Chinese University of Hong Kong and *Department Pathology, School of Basic Medicine, Sun Yat-Sen University of Medical Sciences, China.
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(8). The JCS leukemic cells had been shown to be induced to differentiate into macrophages with the characteristics of mature macrophages by tumour necrosis factor-alpha (TNF-a) or by a combination of TNF-a and interleukin-4 (IL-4) (9). In this study, compounds with anti-tumour and differentiation inducing activities on the JCS cells were isolated and identified from the pericarpium of Citrus reticulata.
Methods Animals and Cell cultures : Inbred male BALB/c mice were used in this study. The myeloid leukemic cell lineWEH1 3B (JCS) was maintained in RPM1 1640 medium supplemented with 10% fetal calf serum (FCS) (Gibco), 2 mM glutamine, and antibiotics PSN (50 U/ml penicillin G, 50 u g/ml streptomycin, and 100 &ml neomycin). Cells were incubated at 37°C in a humidified 5% CO2 incubator. Extraction and isolation : Dried peels of Citrus reticulata (cv. Jiao Gan) were initially blended to become powder in a homogenizer. Crude PCRJ extract (PCRJ-C) was prepared by extracting PCRJ powder with distilled water (1:s w/w) at 40°C for 2.5 hrs. Aqueous fraction was collected and the residue was extracted repeatedly with 5 volume of distilled water for 2 times. The resultant supernatants were pooled and the suspended particles were then removed by centrimgation (5OOg, 30 minutes). Organic fraction of PCRJ (PCRJ-E) was prepared by extracting PCRJ-C repeatedly with diethyl ether (1: 1 v/v). After extraction, residual ether was removed using rotary evaporator. PCRJ-A was the aqueous fraction after extraction of PCRJ-C with ether. D 1 fraction was the precipitate prepared by repeatedly extracting PCRJ-E with water (3 : 1v/v) D 1 fraction was further chromatographed using thin layer chromatography (silica gel 60) with benzene : acetone (6: 1) as mobile phase to give Dl-FI to Dl -F5 fractions. The purity of the isolated components was checked with HPLC. Chemical and structural characterization of plant extract: Chemical composition of PCRJ extracts was analysed using Biuret test for protein determination, Wagner’s test and Mayer’s test for alkaloid, Fehing’s test for the presence of reducing sugar, lead acetate test for flavanoids, and FeCl3 methods for tannin and phenolic compounds (10,ll). The ‘H-NMR and 13C-NMR spectra of isolated compounds were obtained on Jeol F.X Spectrometer (JNM-Zx270) using tetramethylsilane as an internal standard. Mass spectrum was analysed on a HP 589011 Gas Chromatography using HP 5972 series Mass Selective Detector. The melting points of purified compounds were determined on a Melt Temp II melting point apparatus. UV- and IR- spectra were measured using Hitachi 150-20 and Hitachi 270-30 Spectrometer, respectively. Proliferation assay: Proliferative responses were determined by incubating 5x1 O3 JCS cells in 200 ul of appropriately diluted plant extracts in the wells of 96 well flat-bottom microtiter plates at 37” C in a humidified atmosphere containing 5% C02. The cultures were incubated for 48 hours. During the last 6 hours of incubation, the cultures were pulsed with 0.5 uCi of 3H-methylthymidine (3H-TdR, specific activity 41 Ci/mmole, Amersham). The incorporated radioactivity was determined using a Beckman liquid scintillation counter. Cell morphology: Cell morphology was examined using cytocentrimge preparations as described previously (7). The number of cells at immature (blast), intermediate, and mature stages were determined. A total of six hundred cells were scored on each slide and the results were expressed as means * S.D.
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Phagocytosis: Phagocytic activity was determined by a modification of the method of yeast phagocytosis assay as described previously (7). Briefly, 1~10~ opsonized gluteraldehydeinactivated yeast cells (Sacchuromyces cerevisiae) were added to cultures of PCRJ treated JCS cells in 96-well plates. Phagocytic activity of PCRJ treated cells was determined 16 hours after incubation at 37°C. A total of 500 cells were counted for each sample. Colony Assay: Clonogenicity of PCRJ treated JCS cells was determined as described previously (9). Briefly, semisolid agar cell cultures were set up in the wells of leukocyte migration plates (Sterilin). Cultures, 0.4m1, containing 0.33% agar (Sigma), 400 untreated or PCRJ treated cells, RPMI-1640 medium, 20% FCS, 2mM glutamine, and antibiotics PSN, were incubated at 37°C in a humidified 5% CO2 incubator. Colonies consisting of more than 50 cells were counted 7 days after incubation. In viuo tumorigenicity assay: The zn VIVOgrowth of tumour cells was monitored using the method as described previously (9,12). Briefly, 1~10~ PCRJ treated viable JCS cells were injected intraperitoneally into BAL,B/c mice in groups of five. Tumour cells recoverable from the peritoneal For the monitoring of the cavity of mice were counted at day-14 post-tumour inoculation. survival rate of tumour-bearing mice, groups of twelve mice were injected with treated or The survival rate of tumour bearing mice was untreated tumour cells as described above. monitored over a period of 70 days.
Results Inhibition of leukemic cell growth : The anti-proliferative activity of extracts prepared from PCRJ on the myeloid leukemic JCS cells was studied by incubating the cells with various fractions of PCRJ for 48 hours. Results in Figure 1 showed that fractions of PCRJ at the range of 1 - 400~ g/ml inhibited the growth of JCS leukemic cells in a dose dependent manner. The inhibitory effect of PCRJ-E was greater than PCRJ-A. The inhibitory activity of PCRJ was slightly increased when the crude PCRJ-C extract was prepared at an extraction temperature of 100°C (data not shown).
4
100
-
80
-
‘,
3 cd
a ms ‘% ?$ ‘+= ;;j ‘2 9
s
60-
-
4020
-
PCRB-C
-
PCRB-A
A
PCRB-E
0 0
100
200 Cont.
of PCRJ
300 (ugim
400 1)
Fig. 1 Anti-proliferative
activity of PCRJ fractions on WEHI 3B (JCS)
Anti-leukemia Compounds in Citrus Fruits
1272
Vol. 58, No. 15, 1996
Induction of JCS cell differentiation : JCS cells were treated with various concentrations of PCRJ-C, PCRJ-G and PCRJ-E for 3 days. Morphological changes in PCRJ treated leukemic cells were monitored by examination of cytospin preparations. Results in Table 1 showed that all of the PCRJ-C, PCRJ-A, and PCRJ-E treated JCS cells undergo a significant morphological change 3 days after treatment. Both monocytic (macrophages) and granulocytic differentiation (PMN, polymorphonuclear cells) could be observed. PCRJ-C induced a higher proportion of granulocytic differentiation of JCS cells than PCRJ-A and PCRJ-E. The kinetics of JCS cell differentiation had also been examined. Results showed that the JCS cells started to differentiate at 17 to 24 hours after exposure to PCRJ-C, and the number of differentiated cells reached a maximum at 48 to 72 hours after the treatment (data not shown). To further examine the biological activity of PCRJ treated JCS cells, yeast phagocytosis assay was used. Results in Fig. 2 showed that the phagocytic activity of PCRJ-E treated JCS cells was increased in a dose dependent manner.
Table Induction of in vitro differentiation
1
of JCS leukemia cells by various PCRJ fractions.
Blast
% of cells Intermediate
(mean f S.D) Macrophages
PMN
Control
90.1+1.2
5.3*0.6
3.5*0.7
0
PCRJ-c
531 265 132
17.3k4.0 11.3k4.4 62.7*2.3
37.040.9 36.6*4.4 29.W3.4
23.8*1.8 50.7=tl.S 7.Q2.4
19.0*1.5 1.4hO.4 0
PCRJ-A
625 312
36.3&l .2 85.7*3.8
34.0+1.8 12.7k3.0
25.4*1.6 0
4.4kO.l 0
PCRJ fractions
Cont. @g/ml)
30.6&l, 1 18.9*0.4 1.0*0.9 49.6kO.9 179 33.1h4.2 28.Q2.8 0.8*0.4 35.9h4.0 89 71.4*2.2 17.3kl.4 44 11.3+1.2 0 JCS cells (4xlO3/ml) were cultured with various concentrations of PCRJ fractions for 3 days at 37°C. Cytospin preparations were made and cells were stained for morphological identification. PCRJ-E
In viva tumourigenicity of PCRJ treated JCS cells : The in vivo tumourigenicity of PCRJ treated JCS cells was studied by monitoring the growth of tumour cells in syngeneic BALB/c mice. Partially purified PCRJ (PCRJ-E) was used in this study. Results in Figure 3A showed that treatment of JCS with PCRJ-E significantly reduced their capacity of growth (over 99.8% The mean survival time was also increased from 24 days reduction) in syngeneic BALBlc mice. in control mice to 54 days in mice inoculated with PCRJ-E (650 &ml) treated JCS cells (Fig. 3B). The in vitro growth property of PCRJ treated JCS cells was also studied using colony assay method. Three days of PCRJ treated JCS cells were cultured in semi-solid soft agar medium and examined for the formation of colonies. Results showed that the in vitro formation of colonies from PCRJ treated groups was significantly lower than the control group ( > 90 % reduction of colony formation) (data not shown).
Vol. 58, No. 15, 1996
u) %0
1273
Anti-leukemia Compounds in Citrus Fruits
40 30
m
PCRJ-E
(44
uglml)
m
PCRJ-E
(88
uglmi)
.i
Fig. 2 Phagocytic activity of PCRJ-E treated JCS cells
Characterization of isolated active components : In view of the facts that most of the activities were found in the PCRJ-E fraction, this fraction was then selected for fkther analysis. Results from chemical analysis indicated that PCRJ-E contained a significant amount of flavonoids (data not shown). The Dl fraction of PCRJ-E was then fractionated by TLC and HPLC. A total of 5 fractions were observed in both TLC and HPLC. Figure 4 indicates the HPLC profile of Dl fraction. All Dl-Fl to Dl-F5 show anti-proliferative and differentiation inducing activities to various extent. Two of the most active fractions (Dl-F2 and Dl-F3) were isolated for kther structural analysis. The melting point of Dl-F2 and Dl-F3 was 152-154°C and 135.5’C, By comparing their melting points, ‘H-NMR and 13C-NMR spectral data of these respectively. two fractions with previosuly reported values (13,14), Dl-F2 and Dl-F3 were identified as tangeretin (4’5,6,7,8-pentamethoxyflavone) and nobiletin (3’,4’,5,6,7,8-hexamethoxyflavone), respectively (data not shown)
r
4
108 u)
z
0
Control
m
PCRJ-E (650 u@I,)
n
B
A 0
PCRJ-E (650 ughl) PCRJ-E (325 ughl) Control
B
0 ;
10'
E a z 106 b “E
3 105 104
-
I
0 Time
Fig. 3 In vivo growth (A) and in vivo tumourigenicity
10
20 after
30 tumour
40
50
inoculation
60
70
(days)
(B) of PCRJ treated KS cells
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Compounds
in Citrus Fruits
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Discussion Proliferation and differentiation of haematopoietic stem cells are under control by many factors including haematopoietic growth factors such as interleukin-3 (IL-3) granulocyte/macrophage-colony stimulating factor (GM-CSF), granulocyte-colony stimulating factor (G-CSF), macrophage-colony stimulating factor (M-CSF), erythropoietin, and stem cell factor (15). Abnormahties in the normal development of these progenitors result in various types of haematopoietic diseases including leukemia. We have recently demonstrated that the growth and differentiation of a myelomonocytic leukemia cell clone WEHI 3B (JCS) was controlled by cytokine TNF-a and IL-4 (7,9). TNF-a induces monocytic differentiation of JCS cells into mature macrophages. Along the direction of searching for anti-tumour compounds for the treatment of leukemia, we have been examining the extracts prepared from natural products for their antileukemia activity. In this study, the anti-leukemia activity of extracts prepared from PCRJ was investigated. Results from this study indicated that PCRJ-C exhibited not only growth inhibitory effects on JCS cells, but could also induce JCS cells to differentiate into macrophages and PMN The differentiation of PCRJ treated JCS cells was also associated with an increase in phagocytic activity and a reduction in vitro clonogenicity and in vivo tumorigenicity of the cells. These properties were similar to the terminal differentiation of JCS cells induced by cytokines TNF-c( and IL-4 (9).
0
HPLC profile of Dl fraction. Microsorb-MV Ranin Ci8 (5pm) column was used with acetonitrile-water (4:6 v/v) as the eluent. Flow rate was 1mlJmin. The eluates were monitored with a photodiode-array detector (338nm).
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Anti-leukemia Compounds in Citrus Fruits
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Since PCRJ-E showed potent anti-proliferative and differentiation-inducing activity against JCS cells, attempts were made to f&her fractionate the active components from PCRJ-E. Two of the active compounds were identified as tangeretin and nobiletin. These two kinds of compounds are both methoxylated flavones. The presence of differentiation inducing compounds for other myeloid leukemic cell lines (e.g. Ml cells) in the hot methanolic extracts of C. reticulutu has recently been reported (6). The active components were also highly methoxylated flavones. In our extraction protocol, although the PCRJ-C preparation inhibited the growth of Ml cells, the differentiation inducing activity of these PCRJ preparations on Ml cells was not observed (data not shown). These results suggest that methoxylated flavonoids may be a source of anti-leukemia compounds for various forms of myeloid leukemia. Induction of terminal differentiation of leukemic cells has been used as a method for the treatment of leukemia. Inducers of different chemical nature have been examined and some of them such as G-CSF, retinoic acids have been used for clinical trial of certain forms of myelomonocytic leukemia (16,17). The drawback of using cytokines (e.g. G-CSF, M-CSF) as inducers for the treatment of leukemia is partly due to the fact that leukemic cell clones with different sensitivity and response to cytokines exist in leukemic animals including human. Isolation and identification of new potent anti-leukemia drugs with anti-proliferative and differentiationinducing activity toward myeloid leukemia cells would become important in chemotheraputic treatment of leukemia. Results from this investigation clearly showed that both of the it7 viva growth and tumourigenicity of PCRJ treated JCS leukemic cells in syngeneic inbred mice were greatly reduced. All these data indicate that extracts of PCRJ contain potent anti-leukemia substances. The biological activity and the nature of other active ingredients in PCRJ are currently under investigation.
Acknowledgement We wish to thank Miss Chu Wan Yiu for her excellent technical help. by the Faculty Development Grant of Hong Kong Baptist University Research Grant.
This work was supported and the RGC Earmarked
References 1 2. 3. 4. 5. 6. 7. 8. 9.
H. MATSUDA, M. YANO, M KUBO, M. IINUMA, M. OYAMA and M. MIZUNO, Yakugaku Zasshi, 111 193-198 (1991). Y.T. CHEN, R.L. ZHENG, Z.J. JIA and Y. JU, Free Radical Biol. Med., 9 19-21 (1990). L.W. WATTENBERG and J.B. COCCIA, Carcinogenesis, 12 115-l 18 (1991) E. JR MIDDLETON, G. DRZEWIECKI and J. TATUM, Planta Med., 53 325-328 (1987). C. KANDASWAMI, E. PERKINS, D.S. SOLONIUK, G DRZEWICKI and E.JR MIDDLETON, Cancer Letters, 56, 147- 152 (199 1). S. SUGIYAMA, K. UMEHARA, M. KUROYANAGI, A. UENO and T. TAKI, Chem. Pharm. Bull., 41 714-719 (1993). N.K. MAK, M.C. FUNG, K.N. LEUNG, and A.J. HAPEL, Cell. Immunol., 150 1-14 (1993). C. GAMBA-VITALO, M.D. CARMAN and A.C. SARTORELLI, Exp. Hematol., 11 I30137 (1989). K.N. LEUNG, N.K. MAK, M.C. FUNG and A.J. HAPEL, Immunology, u 65-72 (1994)
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10. R.M.C. DAWSON, D.C. ELLIOT, W.H. ELLIOT and K.M. JONES, Data for Biochemical Research. 3rd edition, Oxford University Press (1987). 11. R.M. SILVERSTEIN, G.C. BASSLER and T.C. MORRILL, Seectrometric Identification Organic Compounds. 5th ed., John Wiley & Son Inc. (1991). 12. N.K. MAK, K.N. LEUNG, M.C. FUNG and A.J. HAPEL, A.J, Immunobiol., _@ 1-12 (1994) 13, K. MACHIDA and K. OSAWA, Chem. Pharm. Bull. 37(4) 1092- 1094 (1989). 14. M. BNUMA, S. MATSUURA and K. KUSUDA, Chem. Pharm. Bull. 28(3) 106-716 (1980) 15. CM. HEYWORTH, S.T. VALLANCE, A.D. WHETTON and T.M. DEXTER J. Cell Sci. Supplment. 13 57 - 74 (1990) 16. K.L. WHITE, S.J. WILEY, T. FROST, J. J. MCKENDRICK, R.P. HERMANN, M. SELDON, A. ENNO, R. BELL, I. BUNCE, K. TAYLOR, M. MORGAN, L. EDWARDS and F.C. FIRKIN, Aust. NZ J. Med., 22 449-454 (1992) 17. M.B.VAN DER WEYDEN, D.J. CURTIS, and J. SZER, Aust. NZ. J. Med., 22 446-448 (1992)
ELSEVIER
PII SOO24-3205(96)OOOS8-4
ISOLATION OF ANTI-LEUKEMIA COMPOUNDS FROM CITRUS RETICULA TA Nai Ki Makl, Yee Ling Wong-Leung2, Shuk Chong Cha.nl# , Jianming Wenl *, Kwok Nam Leung3, and Ming Chui Fung4 lDepartment of Biology, 2Department of Chemistry, Hong Kong Baptist University, 3Department of Biochemistry, and 4Department of Biology, Chinese University of Hong Kong (Received in final form February 7, 1996)
Summary
In vitro effects of medicinal plant extracts from the pericarpium of Citrus reticulata (cv Jiao Gan) (PCRJ) on the growth and differentiation of a recently characterized murine myeloid leukemic cell clone WEHI 3B (JCS) were investigated. Extracts of PCRJ not only inhibited the proliferation of JCS cells in a dose dependent manner, but also induced differentiation of JCS cells into macrophages and granulocytes. Morphological differentiation of PCRJ treated JCS cells was associated with an increase in phagocytic activity of the cells. Furthermore, both in vitro clonogenicity and in vivo growth of PCRJ treated JCS leukemic cells in syngeneic BALB/c mice were significantly reduced. The survival rate of mice receiving PCRJ Using ‘H-NMR, 13C-NMR, and treated JCS tumour cells was also increased. GCMS, two active components isolated from PCRJ were identified as nobiletin and tangeretin. Key Words: leukemia, tangeretin, nobiletin, cytokines, citrus fruits
The commercial importance of citrus plants is the use of their fruits as sources of food Fruits from citrus species have also been widely used as a source juices for human consumption. of traditional medicines. Many active components of medicinal values were isolated from citrus These included anti-allergic agents, anti-oxidants, anti-tumour agents, and immunospecies. modulating agents (l-4). It has been demonstrated that D-limonene and the oils isolated from citrus fruits can inhibit the formation of pulmonary adenoma and the occurrence of forestomach tumours induced by carcinogens such as 4-(methylnitrosamino)-I-(3-pyridyl)-1-butanone (NNK) (3). Compounds with in vitro anti-tumour activity against squamous cell carcinoma, and human We have recently promyelocytic leukemia cells have also been detected in citrus fruits (5, 6). The disease that the reported a subclone of murine myeloid leukemic WEHI 3B (JCS) cells (7). WEHI 3B cell causes when injected into mice appears to resemble human acute myeloid leukemia Correspondence : Dr. N.K. Mak, Hong Kong Baptist University, Hong Kong. Tel: 852-2339-7059; E-mail : [email protected] Present Address : #Department of Biology, Chinese University of Hong Kong and *Department Pathology, School of Basic Medicine, Sun Yat-Sen University of Medical Sciences, China.
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(8). The JCS leukemic cells had been shown to be induced to differentiate into macrophages with the characteristics of mature macrophages by tumour necrosis factor-alpha (TNF-a) or by a combination of TNF-a and interleukin-4 (IL-4) (9). In this study, compounds with anti-tumour and differentiation inducing activities on the JCS cells were isolated and identified from the pericarpium of Citrus reticulata.
Methods Animals and Cell cultures : Inbred male BALB/c mice were used in this study. The myeloid leukemic cell lineWEH1 3B (JCS) was maintained in RPM1 1640 medium supplemented with 10% fetal calf serum (FCS) (Gibco), 2 mM glutamine, and antibiotics PSN (50 U/ml penicillin G, 50 u g/ml streptomycin, and 100 &ml neomycin). Cells were incubated at 37°C in a humidified 5% CO2 incubator. Extraction and isolation : Dried peels of Citrus reticulata (cv. Jiao Gan) were initially blended to become powder in a homogenizer. Crude PCRJ extract (PCRJ-C) was prepared by extracting PCRJ powder with distilled water (1:s w/w) at 40°C for 2.5 hrs. Aqueous fraction was collected and the residue was extracted repeatedly with 5 volume of distilled water for 2 times. The resultant supernatants were pooled and the suspended particles were then removed by centrimgation (5OOg, 30 minutes). Organic fraction of PCRJ (PCRJ-E) was prepared by extracting PCRJ-C repeatedly with diethyl ether (1: 1 v/v). After extraction, residual ether was removed using rotary evaporator. PCRJ-A was the aqueous fraction after extraction of PCRJ-C with ether. D 1 fraction was the precipitate prepared by repeatedly extracting PCRJ-E with water (3 : 1v/v) D 1 fraction was further chromatographed using thin layer chromatography (silica gel 60) with benzene : acetone (6: 1) as mobile phase to give Dl-FI to Dl -F5 fractions. The purity of the isolated components was checked with HPLC. Chemical and structural characterization of plant extract: Chemical composition of PCRJ extracts was analysed using Biuret test for protein determination, Wagner’s test and Mayer’s test for alkaloid, Fehing’s test for the presence of reducing sugar, lead acetate test for flavanoids, and FeCl3 methods for tannin and phenolic compounds (10,ll). The ‘H-NMR and 13C-NMR spectra of isolated compounds were obtained on Jeol F.X Spectrometer (JNM-Zx270) using tetramethylsilane as an internal standard. Mass spectrum was analysed on a HP 589011 Gas Chromatography using HP 5972 series Mass Selective Detector. The melting points of purified compounds were determined on a Melt Temp II melting point apparatus. UV- and IR- spectra were measured using Hitachi 150-20 and Hitachi 270-30 Spectrometer, respectively. Proliferation assay: Proliferative responses were determined by incubating 5x1 O3 JCS cells in 200 ul of appropriately diluted plant extracts in the wells of 96 well flat-bottom microtiter plates at 37” C in a humidified atmosphere containing 5% C02. The cultures were incubated for 48 hours. During the last 6 hours of incubation, the cultures were pulsed with 0.5 uCi of 3H-methylthymidine (3H-TdR, specific activity 41 Ci/mmole, Amersham). The incorporated radioactivity was determined using a Beckman liquid scintillation counter. Cell morphology: Cell morphology was examined using cytocentrimge preparations as described previously (7). The number of cells at immature (blast), intermediate, and mature stages were determined. A total of six hundred cells were scored on each slide and the results were expressed as means * S.D.
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Phagocytosis: Phagocytic activity was determined by a modification of the method of yeast phagocytosis assay as described previously (7). Briefly, 1~10~ opsonized gluteraldehydeinactivated yeast cells (Sacchuromyces cerevisiae) were added to cultures of PCRJ treated JCS cells in 96-well plates. Phagocytic activity of PCRJ treated cells was determined 16 hours after incubation at 37°C. A total of 500 cells were counted for each sample. Colony Assay: Clonogenicity of PCRJ treated JCS cells was determined as described previously (9). Briefly, semisolid agar cell cultures were set up in the wells of leukocyte migration plates (Sterilin). Cultures, 0.4m1, containing 0.33% agar (Sigma), 400 untreated or PCRJ treated cells, RPMI-1640 medium, 20% FCS, 2mM glutamine, and antibiotics PSN, were incubated at 37°C in a humidified 5% CO2 incubator. Colonies consisting of more than 50 cells were counted 7 days after incubation. In viuo tumorigenicity assay: The zn VIVOgrowth of tumour cells was monitored using the method as described previously (9,12). Briefly, 1~10~ PCRJ treated viable JCS cells were injected intraperitoneally into BAL,B/c mice in groups of five. Tumour cells recoverable from the peritoneal For the monitoring of the cavity of mice were counted at day-14 post-tumour inoculation. survival rate of tumour-bearing mice, groups of twelve mice were injected with treated or The survival rate of tumour bearing mice was untreated tumour cells as described above. monitored over a period of 70 days.
Results Inhibition of leukemic cell growth : The anti-proliferative activity of extracts prepared from PCRJ on the myeloid leukemic JCS cells was studied by incubating the cells with various fractions of PCRJ for 48 hours. Results in Figure 1 showed that fractions of PCRJ at the range of 1 - 400~ g/ml inhibited the growth of JCS leukemic cells in a dose dependent manner. The inhibitory effect of PCRJ-E was greater than PCRJ-A. The inhibitory activity of PCRJ was slightly increased when the crude PCRJ-C extract was prepared at an extraction temperature of 100°C (data not shown).
4
100
-
80
-
‘,
3 cd
a ms ‘% ?$ ‘+= ;;j ‘2 9
s
60-
-
4020
-
PCRB-C
-
PCRB-A
A
PCRB-E
0 0
100
200 Cont.
of PCRJ
300 (ugim
400 1)
Fig. 1 Anti-proliferative
activity of PCRJ fractions on WEHI 3B (JCS)
Anti-leukemia Compounds in Citrus Fruits
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Vol. 58, No. 15, 1996
Induction of JCS cell differentiation : JCS cells were treated with various concentrations of PCRJ-C, PCRJ-G and PCRJ-E for 3 days. Morphological changes in PCRJ treated leukemic cells were monitored by examination of cytospin preparations. Results in Table 1 showed that all of the PCRJ-C, PCRJ-A, and PCRJ-E treated JCS cells undergo a significant morphological change 3 days after treatment. Both monocytic (macrophages) and granulocytic differentiation (PMN, polymorphonuclear cells) could be observed. PCRJ-C induced a higher proportion of granulocytic differentiation of JCS cells than PCRJ-A and PCRJ-E. The kinetics of JCS cell differentiation had also been examined. Results showed that the JCS cells started to differentiate at 17 to 24 hours after exposure to PCRJ-C, and the number of differentiated cells reached a maximum at 48 to 72 hours after the treatment (data not shown). To further examine the biological activity of PCRJ treated JCS cells, yeast phagocytosis assay was used. Results in Fig. 2 showed that the phagocytic activity of PCRJ-E treated JCS cells was increased in a dose dependent manner.
Table Induction of in vitro differentiation
1
of JCS leukemia cells by various PCRJ fractions.
Blast
% of cells Intermediate
(mean f S.D) Macrophages
PMN
Control
90.1+1.2
5.3*0.6
3.5*0.7
0
PCRJ-c
531 265 132
17.3k4.0 11.3k4.4 62.7*2.3
37.040.9 36.6*4.4 29.W3.4
23.8*1.8 50.7=tl.S 7.Q2.4
19.0*1.5 1.4hO.4 0
PCRJ-A
625 312
36.3&l .2 85.7*3.8
34.0+1.8 12.7k3.0
25.4*1.6 0
4.4kO.l 0
PCRJ fractions
Cont. @g/ml)
30.6&l, 1 18.9*0.4 1.0*0.9 49.6kO.9 179 33.1h4.2 28.Q2.8 0.8*0.4 35.9h4.0 89 71.4*2.2 17.3kl.4 44 11.3+1.2 0 JCS cells (4xlO3/ml) were cultured with various concentrations of PCRJ fractions for 3 days at 37°C. Cytospin preparations were made and cells were stained for morphological identification. PCRJ-E
In viva tumourigenicity of PCRJ treated JCS cells : The in vivo tumourigenicity of PCRJ treated JCS cells was studied by monitoring the growth of tumour cells in syngeneic BALB/c mice. Partially purified PCRJ (PCRJ-E) was used in this study. Results in Figure 3A showed that treatment of JCS with PCRJ-E significantly reduced their capacity of growth (over 99.8% The mean survival time was also increased from 24 days reduction) in syngeneic BALBlc mice. in control mice to 54 days in mice inoculated with PCRJ-E (650 &ml) treated JCS cells (Fig. 3B). The in vitro growth property of PCRJ treated JCS cells was also studied using colony assay method. Three days of PCRJ treated JCS cells were cultured in semi-solid soft agar medium and examined for the formation of colonies. Results showed that the in vitro formation of colonies from PCRJ treated groups was significantly lower than the control group ( > 90 % reduction of colony formation) (data not shown).
Vol. 58, No. 15, 1996
u) %0
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Anti-leukemia Compounds in Citrus Fruits
40 30
m
PCRJ-E
(44
uglml)
m
PCRJ-E
(88
uglmi)
.i
Fig. 2 Phagocytic activity of PCRJ-E treated JCS cells
Characterization of isolated active components : In view of the facts that most of the activities were found in the PCRJ-E fraction, this fraction was then selected for fkther analysis. Results from chemical analysis indicated that PCRJ-E contained a significant amount of flavonoids (data not shown). The Dl fraction of PCRJ-E was then fractionated by TLC and HPLC. A total of 5 fractions were observed in both TLC and HPLC. Figure 4 indicates the HPLC profile of Dl fraction. All Dl-Fl to Dl-F5 show anti-proliferative and differentiation inducing activities to various extent. Two of the most active fractions (Dl-F2 and Dl-F3) were isolated for kther structural analysis. The melting point of Dl-F2 and Dl-F3 was 152-154°C and 135.5’C, By comparing their melting points, ‘H-NMR and 13C-NMR spectral data of these respectively. two fractions with previosuly reported values (13,14), Dl-F2 and Dl-F3 were identified as tangeretin (4’5,6,7,8-pentamethoxyflavone) and nobiletin (3’,4’,5,6,7,8-hexamethoxyflavone), respectively (data not shown)
r
4
108 u)
z
0
Control
m
PCRJ-E (650 u@I,)
n
B
A 0
PCRJ-E (650 ughl) PCRJ-E (325 ughl) Control
B
0 ;
10'
E a z 106 b “E
3 105 104
-
I
0 Time
Fig. 3 In vivo growth (A) and in vivo tumourigenicity
10
20 after
30 tumour
40
50
inoculation
60
70
(days)
(B) of PCRJ treated KS cells
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Discussion Proliferation and differentiation of haematopoietic stem cells are under control by many factors including haematopoietic growth factors such as interleukin-3 (IL-3) granulocyte/macrophage-colony stimulating factor (GM-CSF), granulocyte-colony stimulating factor (G-CSF), macrophage-colony stimulating factor (M-CSF), erythropoietin, and stem cell factor (15). Abnormahties in the normal development of these progenitors result in various types of haematopoietic diseases including leukemia. We have recently demonstrated that the growth and differentiation of a myelomonocytic leukemia cell clone WEHI 3B (JCS) was controlled by cytokine TNF-a and IL-4 (7,9). TNF-a induces monocytic differentiation of JCS cells into mature macrophages. Along the direction of searching for anti-tumour compounds for the treatment of leukemia, we have been examining the extracts prepared from natural products for their antileukemia activity. In this study, the anti-leukemia activity of extracts prepared from PCRJ was investigated. Results from this study indicated that PCRJ-C exhibited not only growth inhibitory effects on JCS cells, but could also induce JCS cells to differentiate into macrophages and PMN The differentiation of PCRJ treated JCS cells was also associated with an increase in phagocytic activity and a reduction in vitro clonogenicity and in vivo tumorigenicity of the cells. These properties were similar to the terminal differentiation of JCS cells induced by cytokines TNF-c( and IL-4 (9).
0
HPLC profile of Dl fraction. Microsorb-MV Ranin Ci8 (5pm) column was used with acetonitrile-water (4:6 v/v) as the eluent. Flow rate was 1mlJmin. The eluates were monitored with a photodiode-array detector (338nm).
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Since PCRJ-E showed potent anti-proliferative and differentiation-inducing activity against JCS cells, attempts were made to f&her fractionate the active components from PCRJ-E. Two of the active compounds were identified as tangeretin and nobiletin. These two kinds of compounds are both methoxylated flavones. The presence of differentiation inducing compounds for other myeloid leukemic cell lines (e.g. Ml cells) in the hot methanolic extracts of C. reticulutu has recently been reported (6). The active components were also highly methoxylated flavones. In our extraction protocol, although the PCRJ-C preparation inhibited the growth of Ml cells, the differentiation inducing activity of these PCRJ preparations on Ml cells was not observed (data not shown). These results suggest that methoxylated flavonoids may be a source of anti-leukemia compounds for various forms of myeloid leukemia. Induction of terminal differentiation of leukemic cells has been used as a method for the treatment of leukemia. Inducers of different chemical nature have been examined and some of them such as G-CSF, retinoic acids have been used for clinical trial of certain forms of myelomonocytic leukemia (16,17). The drawback of using cytokines (e.g. G-CSF, M-CSF) as inducers for the treatment of leukemia is partly due to the fact that leukemic cell clones with different sensitivity and response to cytokines exist in leukemic animals including human. Isolation and identification of new potent anti-leukemia drugs with anti-proliferative and differentiationinducing activity toward myeloid leukemia cells would become important in chemotheraputic treatment of leukemia. Results from this investigation clearly showed that both of the it7 viva growth and tumourigenicity of PCRJ treated JCS leukemic cells in syngeneic inbred mice were greatly reduced. All these data indicate that extracts of PCRJ contain potent anti-leukemia substances. The biological activity and the nature of other active ingredients in PCRJ are currently under investigation.
Acknowledgement We wish to thank Miss Chu Wan Yiu for her excellent technical help. by the Faculty Development Grant of Hong Kong Baptist University Research Grant.
This work was supported and the RGC Earmarked
References 1 2. 3. 4. 5. 6. 7. 8. 9.
H. MATSUDA, M. YANO, M KUBO, M. IINUMA, M. OYAMA and M. MIZUNO, Yakugaku Zasshi, 111 193-198 (1991). Y.T. CHEN, R.L. ZHENG, Z.J. JIA and Y. JU, Free Radical Biol. Med., 9 19-21 (1990). L.W. WATTENBERG and J.B. COCCIA, Carcinogenesis, 12 115-l 18 (1991) E. JR MIDDLETON, G. DRZEWIECKI and J. TATUM, Planta Med., 53 325-328 (1987). C. KANDASWAMI, E. PERKINS, D.S. SOLONIUK, G DRZEWICKI and E.JR MIDDLETON, Cancer Letters, 56, 147- 152 (199 1). S. SUGIYAMA, K. UMEHARA, M. KUROYANAGI, A. UENO and T. TAKI, Chem. Pharm. Bull., 41 714-719 (1993). N.K. MAK, M.C. FUNG, K.N. LEUNG, and A.J. HAPEL, Cell. Immunol., 150 1-14 (1993). C. GAMBA-VITALO, M.D. CARMAN and A.C. SARTORELLI, Exp. Hematol., 11 I30137 (1989). K.N. LEUNG, N.K. MAK, M.C. FUNG and A.J. HAPEL, Immunology, u 65-72 (1994)
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10. R.M.C. DAWSON, D.C. ELLIOT, W.H. ELLIOT and K.M. JONES, Data for Biochemical Research. 3rd edition, Oxford University Press (1987). 11. R.M. SILVERSTEIN, G.C. BASSLER and T.C. MORRILL, Seectrometric Identification Organic Compounds. 5th ed., John Wiley & Son Inc. (1991). 12. N.K. MAK, K.N. LEUNG, M.C. FUNG and A.J. HAPEL, A.J, Immunobiol., _@ 1-12 (1994) 13, K. MACHIDA and K. OSAWA, Chem. Pharm. Bull. 37(4) 1092- 1094 (1989). 14. M. BNUMA, S. MATSUURA and K. KUSUDA, Chem. Pharm. Bull. 28(3) 106-716 (1980) 15. CM. HEYWORTH, S.T. VALLANCE, A.D. WHETTON and T.M. DEXTER J. Cell Sci. Supplment. 13 57 - 74 (1990) 16. K.L. WHITE, S.J. WILEY, T. FROST, J. J. MCKENDRICK, R.P. HERMANN, M. SELDON, A. ENNO, R. BELL, I. BUNCE, K. TAYLOR, M. MORGAN, L. EDWARDS and F.C. FIRKIN, Aust. NZ J. Med., 22 449-454 (1992) 17. M.B.VAN DER WEYDEN, D.J. CURTIS, and J. SZER, Aust. NZ. J. Med., 22 446-448 (1992)