0 1 9 2 - 0 5 6 1 / 9 1 $3.00 + .00 Pergamon Press plc. ;c) 1991 International Society for lmmunopharmacology.
Int. J. lmmunopharmac., Vol. 13, No. 5, pp. 5 4 9 - 5 5 4 , 199I. Printed in Great Britain.
I M M U N O M O P H A R M A C O L O G I C A L EFFECTS OF P O L Y S A C C H A R I D E S F R O M A C A N T H O P A N A X SENTICOSUS ON E X P E R I M E N T A L ANIMALS M. L. SHEN,* S. K. ZHAI, ~ H. L. CHEN, + Y. D. Luo, + G. R. Tu ~ and D. W. Ou *H *Department of Pathology, University of Illinois at Chicago, Chicago, U.S.A.; *Shanghai Institute of Materia Medica, Chinese Academy of Sciences; *Shanghai First Tubercolosis Hospital; and ~Institute of Chinese Materia Medica, Beijing, China
(Received 19 September 1990 and in final form 11 January 1991)
Abstract -- Polysaccharides PES isolated from a common Oriental herb Acanthopanax senticosus were found to have a wide spectrum of immunomodulatory activities on experimental animals. The potential usefulness of these polygaccharides is suggested in the observations that PES inhibited transplanted tumor growths and ameliorated toxicities of the toxic substances in experimental animals. Of most interest is the observations that they suppressed human TB propagation in mice and guinea pigs, as evaluated by lymph node responses and OT skin tests in the guinea pig model, and the quantitation of the TB in the lungs in the mouse model.
Acanthopanax senticosus is a well-known herbal tonic. Its therapeutic activities were proven in recent years both in patients and animals (Brekham & Dardymov, 1969; Miyanomae & Frindel, 1988; Asano, Takahashi, Miyashita, Matsuzaka, Muramatsu, Kuboyama, Kugo & Imai, 1986). Since the herb is a mixture of constituents with a wide variety of activities, isolation and purification of the active constituent is necessary in order to increase the expected effects and to understand the mechanisms of action of this specific effect so that better utilization with scientific bases can be obtained. Several eleutherosides had been isolated from A. senticosus by Russian scientists and proven to possess adaptogenic activities, a term defined as " a state of nonspecifically increased resistance" of the organism (Kaloeva, 1986; Bohn, Nebe & Birr, 1987). The Shanghai Institute of Materia Medica successfully isolated the polysaccharides (PES) from the herb and found them to be immunostimulatory (Xu, Feng, Pan, Ye, Zhai & Shen, 1980). Since then, polysaccharides with different molecular weights were isolated by other laboratories (Wagner, Proksch, Riess-Maurer, Vollmar, Odenthal, Stuppner, Jurcic, Le Turdu & Heur, 1984; Fang, Proksch & Wagner, 1985) and, in addition to the IIAuthor to whom correspondence should be addressed. 549
immunostimulatory activities, were found also to possess hypoglycemic effects (Hikino, Takahashi, Otaka & Konno, 1986). In this paper, the effects of PES on the recipient's resistance against various factors, as well as its stimulatory effects on the mouse immune system, will be presented.
EXPERIMENTAL PROCEDURES
Preparation o f polysaccharides. The isolation procedure of the polysaccharides (PES) from the plant A. senticosus was presented in previous reports (Xu et al., 1980). The pyrogen-free polysaccharide used throughout this study was prepared under sterile conditions at a concentration of 2 m g / m l of H20 by the Xin-Yee Drug Company, Shanghai, China. Briefly, the water extract was added to the ethanol to a final concentration of 30%. The formed precipitate was removed, and the supernatant was added to ethanol again to a final concentration of 80%. The precipitate formed at this ethanol concentration was dissolved in HzO, dialysed, and purified by passing through an activated carbon
550
M. L. SHENet al.
column. The eluate from the column was concentrated and adjusted to 80% ethanol. The precipitate formed (PES) was washed with acetone and dried. The yield was 0.5%.
Animals. Eight- to ten-week-old C57BL or JCR female mice weighing 1 8 - 2 2 g, and 3-month-old Hartley female guinea pigs weighing 3 8 0 - 4 7 0 g were supplied by the Biological Testing Branch of the National Cancer Institute and The Animal House of the Chinese Academy of Sciences (Shanghai, China). Animals were divided into groups according to their body weight. Saline or glucose was given to the control group. All reagents, unless indicated, were injected intraperitoneally.
Reagents. Mitogens concanavalin A (Con A) and lipopolysaccharides (LPS), cyclophosphamide, thioacetamide, horseradish peroxidase conjugated goat anti-mouse immunoglobulin, and O-phenyldiamine dihydrochloride were obtained from the Sigma Chemical Co. Thymidine (methyl-3H, 2 Ci/mmol, 1 mCi/ml) was purchased from the New England Nuclear Co. Old tuberculin (OT) was prepared by the Shanghai Biological Product Company. Guinea pig complement was purchased from Pel Freeze Bioproduct Co. (Rogers, Arkansas, U.S.A.) Sarcoma 180 (ATCC TIB 66) and mouse strain E Ehrlich carcinoma (ATCC CCL 77) were purchased from the American Type Culture Collection.
Assay for IgM antibody plaque-forming cells to SRBC (PFC). Glucose or PES was given to the C57BL mice at 100 mg/kg for 4 days, 10 mice/ group. One-fifth of a milliliter of 25% SRBC was given 10 min after the first injection of drugs. On the fifth day, the splenic cell suspension was prepared in complete medium (RPMI-1640 medium supplemented with 10 mM of glutamine, 50 ~g of penicillin, 50 ~g of streptomycin, and 5% of fetal calf serum). Cells, 106, were mixed with 0.1 ml of 25% SRBC, 0.1 ml of guinea pig complement, and complete medium with a final volume of 0.75 ml. The well-mixed suspension was added to a chamber, made of two glass slides separated by two narrow strips of cover-glass, and sealed with wax. The chamber was incubated at 37°C for 45 min. The number of plaques formed was counted and the number of PFC/106 lymphocytes and PFC/whole spleens were calculated.
Assay for the anti-BSA antibody by the ELISA method. C57BL female mice were divided into two groups. One-fifth of a milligram of BSA was injected into each mouse. Glucose or PES at 100 mg/kg was given every day for 5 days. On the sixth day, mouse sera were collected and frozen until use. The BSAcoated assay plates were prepared by adding 0.2 ml of 1 mg BSA/ml 0.05 M sodium carbonate buffer (pH 9.6) and 0.02% NaN3 into each well of a 96-well plate. The plates were incubated at room temperature for 3 h, then stored at 4°C until use. Before each assay, each plate was washed three times with PBS containing 0.05% Tween 20 and 0.02% NAN,. Then 100 IA of tested mouse serum of different dilutions supplemented with 20 ~l of rabbit serum and 1:350 peroxidase conjugated goat antimouse immunoglobulin were added in succession. The plate was incubated at 37°C for 1 h, and washed three times with PBS containing NAN3. Finally, 100 tA of the freshly prepared substrate (0.04% of Ophenyldiamine dihydrochloride in 0.1 M of citrate phosphate buffer, pH 5, and 0.012% of H202) was added. After incubating at 37°C for 30 min, the reaction was stopped by the addition of 25 ~1 of 12.5% H2SO4. The color of the reaction product was determined by a Dynatech MR 500 Microelisa Autoreader at 490 nm. Results were compared as concentration units using a standard curve prepared from pooled mouse serum collected from BSA immunized mice and stored at - 7 0 ° C .
Assay for phagocytosis. Glucose or PES was given at 100 mg/kg for 6 days to C57BL mice, five mice/ group. On the seventh day, the mice were injected i.p. with 0.5 ml of 1% SRBC suspension. Thirty minutes later, intraperitoneal cells were washed out with saline. The ceils were cytospinned onto slides and stained. The percentage of macrophages that ingested SRBC (phagocytic ratio) and the number of SRBC in the macrophage (phagocytic index) were calculated by counting 200 macrophages under a microscope. Assay for lymphocyte transformation. C57BL mouse spleen cell suspension was prepared in Hank's solution supplemented with 5% fetal calf serum. After centrifugation, the cell pellet was treated with 0.85% NH4C1 to lyse red blood cells. After being washed twice with RPMI-1640 medium, the live cells were counted in the presence of 0.1% trypan blue dye and resuspended in a complete medium at 2.5 × 106 live cells/ml. To the 96-well culture plate, 100 ~1 of the cell suspension, mitogens at sub-
551
Polysaccharides from A. senticosus optimal concentrations (LPS 5/~g/ml, Con A 1 /ag/ml), and PES of different concentrations were added in triplicate. The final volume was 200/al. After incubation for 68 h in a 5o70 CO2 incubator, 10 tA of 3H-TdR (1 /aCi) were added to each well. The plate was incubated for another 6 h and the cells were collected by a harvester. The c o u n t s / m i n in each well were determined.
Assay for anti-toxic effects. Twenty J C R mice were divided into two groups. Glucose or PES was injected at 100 m g / k g for 7 days. Cyclophosphamide at 10 m g / k g , or thioacetamide at 100 m g / k g , was injected on the fifth day. On the eight day, blood was collected from the orbital vein. The a m o u n t of white blood cells from the cyclophosphamide-injected mice was counted. For thioacetamide-injected mice, serum g l u t a m i c - p y r u v i c transaminase was assayed by A b b o t t ' s Spectrum Analyser. Assay for the inhibition of in vivo tumor growths. Glucose or PES was injected into 20 J C R mice (10 each) at 200 m g / k g for 10 days. After the second injection, 2 × 106 of Sarcoma 180 tumor cells or Ehrlich carcinoma cells were inoculated subaxillarily into all 20 mice. On the eleventh day, the mice were sacrificed and the t u m o r weights were evaluated. Assay for the effects on the tubercle bacillus infected animals. Three weeks before the experiment, tubercle bacillus (TB) of the human strain, H37RV, provided by the Shanghai Biological Products C o m p a n y , was inoculated and incubated in L o w e n s t e i n - J e n s e n ' s medium ( L - J ) at 37°C. After 3 weeks, colonies were removed f r o m the medium and 0.5 m g / m l of the colony suspension was injected into guinea pigs at the inguinal site. The next day, the animals were divided into two groups. N o r m a l saline or PES (40 m g / k g ) was injected every other day. On the eighteenth day, guinea pigs were challenged with 0.1 ml of 1:20 O T hypodermically. On the twenty-first day, animals were examined for their skin reaction and then autopsied for examination of the lymph nodes. In the mouse experiments, J C R mice were divided into four groups. Saline and PES were given intramuscularly to two groups while the other two groups were given intraperitoneal injections. The PES dosage was 40 m g / k g / d a y . Mice were given the drugs for 2 days, followed by intravenous injections of 0.1 mg of H37RV. Drugs were given continuously for another 20 days and animals were sacrificed on
Table 1. Effects of PES on the PFC of mouse spleen cell, Group Glucose PES
PFC/106 spleen cells 650 _+ 98 1293 + 208**
PFC × 103/spleen 182 +_ 17 511 _+ 78**
Glucose or PES was given to the C57BL mice at 100 mg/kg for 4 days, 10 mice/group. 0.2 ml of 25°7o SRBC were given 10 min after the first injection of drugs. The PFC assay was performed on the fifth day. The values represent the mean _+ S.D. of each group. Significant difference from the control (glucose), **P
Int. J. lmmunopharmac., Vol. 13, No. 5, pp. 5 4 9 - 5 5 4 , 199I. Printed in Great Britain.
I M M U N O M O P H A R M A C O L O G I C A L EFFECTS OF P O L Y S A C C H A R I D E S F R O M A C A N T H O P A N A X SENTICOSUS ON E X P E R I M E N T A L ANIMALS M. L. SHEN,* S. K. ZHAI, ~ H. L. CHEN, + Y. D. Luo, + G. R. Tu ~ and D. W. Ou *H *Department of Pathology, University of Illinois at Chicago, Chicago, U.S.A.; *Shanghai Institute of Materia Medica, Chinese Academy of Sciences; *Shanghai First Tubercolosis Hospital; and ~Institute of Chinese Materia Medica, Beijing, China
(Received 19 September 1990 and in final form 11 January 1991)
Abstract -- Polysaccharides PES isolated from a common Oriental herb Acanthopanax senticosus were found to have a wide spectrum of immunomodulatory activities on experimental animals. The potential usefulness of these polygaccharides is suggested in the observations that PES inhibited transplanted tumor growths and ameliorated toxicities of the toxic substances in experimental animals. Of most interest is the observations that they suppressed human TB propagation in mice and guinea pigs, as evaluated by lymph node responses and OT skin tests in the guinea pig model, and the quantitation of the TB in the lungs in the mouse model.
Acanthopanax senticosus is a well-known herbal tonic. Its therapeutic activities were proven in recent years both in patients and animals (Brekham & Dardymov, 1969; Miyanomae & Frindel, 1988; Asano, Takahashi, Miyashita, Matsuzaka, Muramatsu, Kuboyama, Kugo & Imai, 1986). Since the herb is a mixture of constituents with a wide variety of activities, isolation and purification of the active constituent is necessary in order to increase the expected effects and to understand the mechanisms of action of this specific effect so that better utilization with scientific bases can be obtained. Several eleutherosides had been isolated from A. senticosus by Russian scientists and proven to possess adaptogenic activities, a term defined as " a state of nonspecifically increased resistance" of the organism (Kaloeva, 1986; Bohn, Nebe & Birr, 1987). The Shanghai Institute of Materia Medica successfully isolated the polysaccharides (PES) from the herb and found them to be immunostimulatory (Xu, Feng, Pan, Ye, Zhai & Shen, 1980). Since then, polysaccharides with different molecular weights were isolated by other laboratories (Wagner, Proksch, Riess-Maurer, Vollmar, Odenthal, Stuppner, Jurcic, Le Turdu & Heur, 1984; Fang, Proksch & Wagner, 1985) and, in addition to the IIAuthor to whom correspondence should be addressed. 549
immunostimulatory activities, were found also to possess hypoglycemic effects (Hikino, Takahashi, Otaka & Konno, 1986). In this paper, the effects of PES on the recipient's resistance against various factors, as well as its stimulatory effects on the mouse immune system, will be presented.
EXPERIMENTAL PROCEDURES
Preparation o f polysaccharides. The isolation procedure of the polysaccharides (PES) from the plant A. senticosus was presented in previous reports (Xu et al., 1980). The pyrogen-free polysaccharide used throughout this study was prepared under sterile conditions at a concentration of 2 m g / m l of H20 by the Xin-Yee Drug Company, Shanghai, China. Briefly, the water extract was added to the ethanol to a final concentration of 30%. The formed precipitate was removed, and the supernatant was added to ethanol again to a final concentration of 80%. The precipitate formed at this ethanol concentration was dissolved in HzO, dialysed, and purified by passing through an activated carbon
550
M. L. SHENet al.
column. The eluate from the column was concentrated and adjusted to 80% ethanol. The precipitate formed (PES) was washed with acetone and dried. The yield was 0.5%.
Animals. Eight- to ten-week-old C57BL or JCR female mice weighing 1 8 - 2 2 g, and 3-month-old Hartley female guinea pigs weighing 3 8 0 - 4 7 0 g were supplied by the Biological Testing Branch of the National Cancer Institute and The Animal House of the Chinese Academy of Sciences (Shanghai, China). Animals were divided into groups according to their body weight. Saline or glucose was given to the control group. All reagents, unless indicated, were injected intraperitoneally.
Reagents. Mitogens concanavalin A (Con A) and lipopolysaccharides (LPS), cyclophosphamide, thioacetamide, horseradish peroxidase conjugated goat anti-mouse immunoglobulin, and O-phenyldiamine dihydrochloride were obtained from the Sigma Chemical Co. Thymidine (methyl-3H, 2 Ci/mmol, 1 mCi/ml) was purchased from the New England Nuclear Co. Old tuberculin (OT) was prepared by the Shanghai Biological Product Company. Guinea pig complement was purchased from Pel Freeze Bioproduct Co. (Rogers, Arkansas, U.S.A.) Sarcoma 180 (ATCC TIB 66) and mouse strain E Ehrlich carcinoma (ATCC CCL 77) were purchased from the American Type Culture Collection.
Assay for IgM antibody plaque-forming cells to SRBC (PFC). Glucose or PES was given to the C57BL mice at 100 mg/kg for 4 days, 10 mice/ group. One-fifth of a milliliter of 25% SRBC was given 10 min after the first injection of drugs. On the fifth day, the splenic cell suspension was prepared in complete medium (RPMI-1640 medium supplemented with 10 mM of glutamine, 50 ~g of penicillin, 50 ~g of streptomycin, and 5% of fetal calf serum). Cells, 106, were mixed with 0.1 ml of 25% SRBC, 0.1 ml of guinea pig complement, and complete medium with a final volume of 0.75 ml. The well-mixed suspension was added to a chamber, made of two glass slides separated by two narrow strips of cover-glass, and sealed with wax. The chamber was incubated at 37°C for 45 min. The number of plaques formed was counted and the number of PFC/106 lymphocytes and PFC/whole spleens were calculated.
Assay for the anti-BSA antibody by the ELISA method. C57BL female mice were divided into two groups. One-fifth of a milligram of BSA was injected into each mouse. Glucose or PES at 100 mg/kg was given every day for 5 days. On the sixth day, mouse sera were collected and frozen until use. The BSAcoated assay plates were prepared by adding 0.2 ml of 1 mg BSA/ml 0.05 M sodium carbonate buffer (pH 9.6) and 0.02% NaN3 into each well of a 96-well plate. The plates were incubated at room temperature for 3 h, then stored at 4°C until use. Before each assay, each plate was washed three times with PBS containing 0.05% Tween 20 and 0.02% NAN,. Then 100 IA of tested mouse serum of different dilutions supplemented with 20 ~l of rabbit serum and 1:350 peroxidase conjugated goat antimouse immunoglobulin were added in succession. The plate was incubated at 37°C for 1 h, and washed three times with PBS containing NAN3. Finally, 100 tA of the freshly prepared substrate (0.04% of Ophenyldiamine dihydrochloride in 0.1 M of citrate phosphate buffer, pH 5, and 0.012% of H202) was added. After incubating at 37°C for 30 min, the reaction was stopped by the addition of 25 ~1 of 12.5% H2SO4. The color of the reaction product was determined by a Dynatech MR 500 Microelisa Autoreader at 490 nm. Results were compared as concentration units using a standard curve prepared from pooled mouse serum collected from BSA immunized mice and stored at - 7 0 ° C .
Assay for phagocytosis. Glucose or PES was given at 100 mg/kg for 6 days to C57BL mice, five mice/ group. On the seventh day, the mice were injected i.p. with 0.5 ml of 1% SRBC suspension. Thirty minutes later, intraperitoneal cells were washed out with saline. The ceils were cytospinned onto slides and stained. The percentage of macrophages that ingested SRBC (phagocytic ratio) and the number of SRBC in the macrophage (phagocytic index) were calculated by counting 200 macrophages under a microscope. Assay for lymphocyte transformation. C57BL mouse spleen cell suspension was prepared in Hank's solution supplemented with 5% fetal calf serum. After centrifugation, the cell pellet was treated with 0.85% NH4C1 to lyse red blood cells. After being washed twice with RPMI-1640 medium, the live cells were counted in the presence of 0.1% trypan blue dye and resuspended in a complete medium at 2.5 × 106 live cells/ml. To the 96-well culture plate, 100 ~1 of the cell suspension, mitogens at sub-
551
Polysaccharides from A. senticosus optimal concentrations (LPS 5/~g/ml, Con A 1 /ag/ml), and PES of different concentrations were added in triplicate. The final volume was 200/al. After incubation for 68 h in a 5o70 CO2 incubator, 10 tA of 3H-TdR (1 /aCi) were added to each well. The plate was incubated for another 6 h and the cells were collected by a harvester. The c o u n t s / m i n in each well were determined.
Assay for anti-toxic effects. Twenty J C R mice were divided into two groups. Glucose or PES was injected at 100 m g / k g for 7 days. Cyclophosphamide at 10 m g / k g , or thioacetamide at 100 m g / k g , was injected on the fifth day. On the eight day, blood was collected from the orbital vein. The a m o u n t of white blood cells from the cyclophosphamide-injected mice was counted. For thioacetamide-injected mice, serum g l u t a m i c - p y r u v i c transaminase was assayed by A b b o t t ' s Spectrum Analyser. Assay for the inhibition of in vivo tumor growths. Glucose or PES was injected into 20 J C R mice (10 each) at 200 m g / k g for 10 days. After the second injection, 2 × 106 of Sarcoma 180 tumor cells or Ehrlich carcinoma cells were inoculated subaxillarily into all 20 mice. On the eleventh day, the mice were sacrificed and the t u m o r weights were evaluated. Assay for the effects on the tubercle bacillus infected animals. Three weeks before the experiment, tubercle bacillus (TB) of the human strain, H37RV, provided by the Shanghai Biological Products C o m p a n y , was inoculated and incubated in L o w e n s t e i n - J e n s e n ' s medium ( L - J ) at 37°C. After 3 weeks, colonies were removed f r o m the medium and 0.5 m g / m l of the colony suspension was injected into guinea pigs at the inguinal site. The next day, the animals were divided into two groups. N o r m a l saline or PES (40 m g / k g ) was injected every other day. On the eighteenth day, guinea pigs were challenged with 0.1 ml of 1:20 O T hypodermically. On the twenty-first day, animals were examined for their skin reaction and then autopsied for examination of the lymph nodes. In the mouse experiments, J C R mice were divided into four groups. Saline and PES were given intramuscularly to two groups while the other two groups were given intraperitoneal injections. The PES dosage was 40 m g / k g / d a y . Mice were given the drugs for 2 days, followed by intravenous injections of 0.1 mg of H37RV. Drugs were given continuously for another 20 days and animals were sacrificed on
Table 1. Effects of PES on the PFC of mouse spleen cell, Group Glucose PES
PFC/106 spleen cells 650 _+ 98 1293 + 208**
PFC × 103/spleen 182 +_ 17 511 _+ 78**
Glucose or PES was given to the C57BL mice at 100 mg/kg for 4 days, 10 mice/group. 0.2 ml of 25°7o SRBC were given 10 min after the first injection of drugs. The PFC assay was performed on the fifth day. The values represent the mean _+ S.D. of each group. Significant difference from the control (glucose), **P
