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Anti-inflammatory and immunomodulatory activities of the extracts from the inflorescence of Chrysanthemum indicum Linné
Journal of Ethnopharmacology 101 (2005) 334–337
Short communication
Anti-inflammatory and immunomodulatory activities of the extracts from the inflorescence of Chrysanthemum indicum Linn´e Wenming Cheng a,b , Jun Li b,∗ , Tianpa You a , Chengmu Hu b a
Department of Chemistry, University of Science and Technology of China, Hefei 230026, China b School of Pharmacy, Anhui Medical University, Hefei 230032, China Received 19 May 2004; received in revised form 10 February 2005; accepted 12 April 2005 Available online 18 July 2005
Abstract Chrysanthemum indicum Linn´e (CI) has a long history for the treatment of inflammation, hypertension and respiratory diseases in China. The purpose of the present study was to investigate the anti-inflammatory and immunomodulatory properties of the inflorescence or bud of CI extracts. The ethanol extract of CI (CIEE) was fractionated to a petroleum ether soluble fraction (CIPF), an ethyl acetate soluble fraction (CIEF), a butanol soluble fraction (CIBF) and a water soluble fraction (CIWF) successively. CIBF (150 mg/kg, p.o.) caused a significant inhibition on the auricle edema in mice. CIBF (150, 300 mg/kg, p.o.) not only significantly increased the delayed-type hypersensitivity (DTH) reaction induced by 2,4-dinitro-fluorobenzene (DNFB) but also significantly enhanced antibody generation by splenic cells of mice and IgG and IgM levels in mice sera in response to sheep red blood cells (SRBC) in cyclophosphamide (CP)-induced mice. Furthermore, CIBF (150, 300 mg/kg, p.o.) obviously potentiated the function of the mononuclear phagocytic system in CP-induced mice. The above results reveal that CIBF possesses anti-inflammatory, humoral and cellular immunomodulatory and mononuclear phagocytic activities, probably due to the presence of flavonoids. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Chrysanthemum indicum; Anti-inflammation; Immunomodulation; Mononuclear phagocytosis
1. Introduction Chrysanthemum indicum Linn´e (Compositae) is a wild herb, which was widely spread, productive and of high quality in Anhui province of China. The inflorescence or bud of Chrysanthemum indicum has a long history using as a Chinese traditional medicine mainly for the treatment of inflammation, hypertension and respiratory diseases. Previous studies have reported that Chrysanthemum indicum possesses the anti-bacteria, anti-virus, anti-oxidant, antiinflammatory and immunomodulatory properties (Wang et al., 2000). Phytochemical profile of this plant has shown the presence of flavonoids, terpenoids and phenolic compounds (Yoshikawa et al., 1999). However, the anti-inflammatory
∗
Corresponding author. Tel.: +86 551 5161116; fax: +86 551 5161116. E-mail address: [email protected] (J. Li).
0378-8741/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2005.04.035
and immunomodulatory functions of active components from this plant remain to be investigated. In the present study, we screened the anti-inflammatory components from the dried bud of Chrysanthemum indicum and then explored the influence of active components on cellular immunity, humoral immunity and the phagocytic function of mononuclear phagocytic system.
2. Material and methods 2.1. Animals Kunming mice, BALB/c mice (weighing 18–22 g, n = 10 per group) and purebred Guinea pigs (weighing 250–350 g, n = 5), were purchased from Animal center, Anhui Medical University, Hefei, China. All animals were housed for 1 week before experiment. All animal procedures were followed in
W. Cheng et al. / Journal of Ethnopharmacology 101 (2005) 334–337
accordance with the approved protocol for use of experimental animals set by the standing committee on animal care at Anhui Medical University. 2.2. Plant material, extraction and chemical analysis The bud of Chrysanthemum indicum was obtained from the local market and authenticated based on its microscopic and macroscopic characteristics. A voucher specimen (CI-1) has been deposited at School of Pharmacy, Anhui Medical University, Hefei, China. The dried bud of Chrysanthemum indicum (8 kg) was macerated with 95% ethanol at room temperature for 2 weeks. The ethanol extract (CIEE) was suspended in water, then extracted by petroleum ether, ethyl acetate and butanol successively, yielding a petroleum ether soluble fraction (CIPF), an ethyl acetate soluble fraction (CIEF), a butanol soluble fraction (CIBF) and a water soluble fraction (CIWF), about 3.4, 2.6, 4.0 and 5.0% of the dried plant material, respectively. The fraction with obvious anti-inflammatory activity was analysed on the thin layer chromatography (TLC) plate of RP-18 F254s . A mixture of methanol and water (60:40) was used as mobile phase. The flavonoids was detected at 365 nm as yellow fluorescent spots with the solution of 1% AlCl3 in ethanol. The tested fraction was compared with reference substances, which were luteolin, luteolin-7-glucoside and acacetin-7-rhamnoglucoside. The flavonoid content of the active fraction was also determined (Lin, 2002). 2.3. Antigen and complement 2,4-Dinitrofluorobenzene (DNFB) was purchased from Sigma Chemical Co., USA. Sheep red blood cells (SRBC) were collected from a healthy adult sheep in a local breed mill. SRBC in Alsever’s solution were washed three times with sterile saline. The blood cells collected from five purebred Guinea pigs were challenged with SRBC by 10:1 (v/v), and then centrifugated at 2000 rpm for 10 min. The pellet used as complement was preserved at −70 ◦ C.
335
2.5. DNFB-induced delayed-type hypersensitivity (DTH) reaction in cyclophosphamide (CP)-induced mice BALB/c Mice of either sex were divided into five groups of 10 each. CIBF (75, 150, 300 mg/kg, p.o.) was administered for 5 days. On day 3, CP (50 mg/kg, i.p.) was administered and continued for 2 days. On day 5, the mice were sensitized by applying 50 l of 1% DNFB in mixed acetone–olive oil (4:1, v/v) to the skin of shaved abdomen. On day 10, they were challenged for contact hypersensitivity with 50 l of 1% DNFB by applying it to both sides of the left earlap of mice. Forty-eight hours later, the mice were executed. Both earlaps of mice were cut down and weighed as previously described (Wei et al., 2001). 2.6. Antibody generation by the splenic cells of mice and IgG and IgM levels in mice sera in response to SRBC in CP-induced mice Two batches of mice of either sex were divided into five groups of 10 each, respectively. CIBF (75, 150, 300 mg/kg, p.o.) was administered on day 1 till the day of the experiment. On day 3, both batches of mice were immunized with 0.2 ml of 1 × 108 SRBC. CP (50 mg/kg, i.p.) was administered on day 3 and continued for 2 days. On day 7, blood from one batch of mice was collected. Simultaneously, the spleens of mice were culled and the splenic cell suspension of 5 × 106 ml−1 was prepared in Gey’s solution. Another batch of mice were immunized again on day 7. On day 14, blood sample was collected and divided to two halves. One-half was prepared to determine IgM level. Another half was pretreated with 2-mercaptoehanol (incubated at 37 ◦ C for 0.5 h to block IgM) and sheep anti-mouse (SAM) IgG in order to determine IgG level. An aliquot (0.5 ml) of the splenic cell suspension or mice sera was diluted by 800-fold with saline and challenged with 0.5 ml of 10% (v/v) SRBC suspension, complement and saline subsequently. Both the antibody generation by the splenic cells of mice and IgM or IgG levels in mice sera were determined as previously described (Shen et al., 2001). The values of absorbance indicating the level of IgM, IgG or total Ig in mice sera multiplied by dilution factor are converted into HCIgM , HCIgG and HCIg , respectively.
2.4. Anti-inflammatory testing The anti-inflammatory property of Chrysanthemum indicum bud was investigated using the model of auricle edema induced by dimethylbenzene in mice. Male mice were divided into seven groups of 10 each. Reference drug (Naproxen, 50 mg/kg), tested extracts (150 mg/kg) and control vehicle were given intragastrically for 5 days. A 0.05 ml of dimethylbenzene was spread on both sides of the left earlap of mice 1 h after the final administration of drugs. Two hours later, the mice were executed. Both earlaps of mice were cut down and weighed as previously reported (Ding et al., 2001).
2.7. Macrophage phagocytosis by carbon clearance method in CP-induced mice Mice of either sex were divided into five groups of 10 each. CIBF (75, 150, 300 mg/kg, p.o.) was administered for 5 days prior to the injection of carbon particles (0.1 ml/10 g). CP (50 mg/kg, i.p.) was administered on day 3 and continued for 2 days. On day 5, mice were injected with 0.1 ml of carbon suspension through the tail vein (Biozzi et al., 1953). Blood samples were collected from the orbital plexuses of individual mice immediately at 2 min (t1 ) and 10 min (t2 ) after injection of carbon suspension, lysed with 4 ml 0.1%
336
W. Cheng et al. / Journal of Ethnopharmacology 101 (2005) 334–337
Table 1 Anti-inflammatory activity of the extracts from Chrysanthemum indicum on auricle edema in mice
Table 2 Effect of CIBF on antibody generation by the splenic cells of mice in response to SRBC and DNFB-induced DTH reactivity in CP-induced mice
Drugs
Dose (mg/kg, p.o.)
Tumidity (mg)
Inhibition (%)
Drugs
Dose (mg/kg, p.o.)
Absorption value
Tumidity (mg)
Control CIEE CIPF CIEF CIBF CIWF Naproxen
– 150 150 150 150 150 50
24.3 ± 5.1 22.5 ± 3.9 20.9 ± 4.3 20.4 ± 4.1 15.8 ± 3.3a 21.6 ± 4.9 19.1 ± 4.0a
– 7.4 14.0 16.0 35.0 11.1 21.4
Control CP control CIBF + CP CIBF + CP CIBF + CP
– 50 75 150 300
0.25 ± 0.02 0.11 ± 0.01a 0.15 ± 0.03 0.17 ± 0.03b 0.18 ± 0.01b
2.2 ± 0.5 1.6 ± 0.4a 1.8 ± 0.5 2.2 ± 0.3b 2.4 ± 0.7b
Mean ± S.E.M. (n = 10). a P < 0.01, statistically significant as compared to control. b P < 0.01, statistically significant as compared to CP control.
Mean ± S.E.M. (n = 10). a P < 0.05, statistically significant as compared to control.
Na2 CO3 . The absorbances were measured spectrophotometrically at 670 nm. Then the liver and spleen of individual mice were culled and weighed. The rate of carbon clearance κ and phagocytic index α are calculated as follows: κ = (log A1 − log A2 )/(t2 − t1 ), α = κ1/3 × body weight/(liver weight + spleen weight) (Xu et al., 1982). 2.8. Statistical analysis The results are presented as mean ± S.E.M. Statistical significance between the groups was analysed by Student’s t-test. P-value less than 0.05 was considered to be statistically significant.
3. Results 3.1. Chemical analysis CIBF appeared the same way as luteolin, luteolin-7glucoside and buddleoglucoside on TLC plate. The total flavonoid content of CIBF was about 53%, which indicates that CIBF mainly consists of the above flavonoids. 3.2. Anti-inflammatory activity CIBF (150 mg/kg, p.o.) was found to significantly inhibit auricle edema induced by dimethylbenzene in mice, with a higher inhibition rate (35.0%) than that of naproxen (50 mg/kg, p.o., 21.4%) (Table 1).
3.3. Antibody generation by the splenic cells of mice in response to SRBC and DNFB-induced DTH reactivity in CP-induced mice CIBF (150, 300 mg/kg, p.o.) not only produced a significant, dose-related increase on the antibody generation by the splenic cells of mice in response to SRBC, but also obviously increased DTH reactivity induced by DNFB in CP-induced mice (Table 2). 3.4. IgG and IgM levels in mice sera in response to SRBC and macrophage phagocytosis in CP-induced mice CIBF (150, 300 mg/kg, p.o.) significantly, dosedependently enhanced serum IgG and IgM levels in response to SRBC in CP-induced mice (Table 3). Accordingly, a significant, dose-related potentiation of the macrophage phagocytic activity was observed as evident by the rate of carbon clearance and phagocytic index (Table 4).
4. Discussion The dimethylbenzene induced auricle edema in mice is the preliminary and simple model for searching for potential antiinflammatory constituents. The crude extract of Chrysanthemum indicum has been found to be effective on the model and potentiate phagocytic activity of the macrophage in mice (Wang et al., 2000). The present study not only proved the anti-inflammatory activity of the extract of Chrysanthemum indicum, but also showed that CIBF was more active than other fractions.
Table 3 Effect of CIBF on IgG and IgM levels in mice sera in response to SRBC in CP-induced mice Drugs
Dose (mg/kg, p.o.)
HCIgM
HCIg
HCIgG
Control CP control CIBF + CP CIBF + CP CIBF + CP
– 50 75 150 300
684.5 ± 18.9 294.4 ± 18.2a 324.2 ± 13.4 362.4 ± 16.4b 395.4 ± 21.5b
788.0 ± 68.4 462.6 ± 47.7a 613.2 ± 57.1 701.2 ± 87.2b 731.2 ± 66.9b
224.0 83.1 123.7 171.7 243.2
Mean ± S. E M. (n = 10). a P < 0.01, statistically significant as compared to control. b P < 0.01, statistically significant as compared to CP control.
± ± ± ± ±
60.7 22.5a 15.0 21.0b 60.0b
W. Cheng et al. / Journal of Ethnopharmacology 101 (2005) 334–337
337
Table 4 Effect of CIBF on macrophage phagocytic activity in CP-induced mice
Acknowledgements
Drugs
Dose (mg/kg, p.o.)
Rate of carbon clearance (κ)
Phagocytic index (α)
Control CP control CIBF + CP CIBF + CP CIBF + CP
– 50 75 150 300
0.086 ± 0.021 0.042 ± 0.010a 0.053 ± 0.013 0.061 ± 0.012b 0.065 ± 0.014b
7.83 ± 1.40 5.78 ± 0.55a 6.12 ± 0.73 6.91 ± 0.82b 7.25 ± 0.99b
Financial support for the project ‘Primary Study of National Grave Basic Research Programme’ (No. 2002CCC02900) by the Ministry of Science and Technology of China is gratefully acknowledged.
Mean ± S.E.M. (n = 10). a P < 0.01, statistically significant as compared to control. b P < 0.01, statistically significant as compared to CP control.
CP, an immunosuppressive agent, can inhibit cell reproduction (Shao et al., 2000), especially after the challenge of the antigen. DNFB, a chemical sensitizer, which in combination with skin proteins acquires antigenicity (Wei et al., 2001), was used to elicit contact hypersensitivity reaction in mice. CIBF (150, 300 mg/kg, p.o.) significantly enhanced antibody generation by the splenic cells of mice in response to SRBC and DNFB-induced DTH reactivity in CP-induced mice. This suggests that CIBF not only boosts the production of antibody by lymphocytes, but also causes activation of the T cells, which subsequently proliferate and release cytokines, leading to anti-inflammatory and immunomodulatory activation. CIBF (150, 300 mg/kg, p.o.) obviously enhanced serum IgG and IgM levels in response to SRBC in CP-induced mice. The result reveals that CIBF improves humoral immunity probably through the reproduction and differentiation of B cells into antibody-secreting plasma cells. Macrophages can process and present antigen to B cells. CIBF (150, 300 mg/kg, p.o.) caused a significant, dose-related potentiation of the macrophage phagocytic activity as shown in Table 4. The result indicates the stimulatory effect of CIBF on the cells of the mononuclear phagocytic system. Through chemical analysis, CIBF mainly consists of three flavonoids, luteolin, luteolin-7-glucoside and acacetin7-rhamnoglucoside, which are probably responsible for its anti-inflammatory and immunomodulatory activities. Therefore, CIBF could be an effective and useful candidate in the development of anti-inflammatory and immunomodulatory drug.
References Biozzi, G., Benacerraf, B., Halpern, B.N., 1953. Quantitative study of the granulopectic activity of the reticulo-endothelial system I: the effect of ingredient present in India ink and of substances affecting blood clotting in vivo on fate of carbon particles admininstration intravenously in rats, mice and rabbits. British Journal of Experimental Pathology 34, 426–457. Ding, C.H., Xu, S.Y., Xiong, Z.Y., Mei, Q., Dai, L.M., Yu, C.W., 2001. Methods of the experimental study on the anti-inflammatory and antipyretic drugs. In: Xu, S.Y., Bian, R.L., Chen, X. (Eds.), Experimental Methods of Pharmacology. People’s Medical Publishing House, Beijing, China, p. 911. Lin, C.R., 2002. The determination of flavonoid content of flowers of Chrysanthemum indicum. Guihaia 22, 467–468. Shao, L., Yu, Q.H., Xu, J.H., Li, X., Zhang, Y.Y., Zhang, H., 2000. The effect of houttuyninum on immunologic function in mice treated with cyclophosphamide. Journal of Shenyang Pharmaceutical University 17, 133–135. Shen, Y.X., Wei, W., Xu, S.Y., 2001. Determination on humoral-mediated immunological function. In: Xu, S.Y., Bian, R.L., Chen, X. (Eds.), Experimental Methods of Pharmacology. People’s Medical Publishing House, Beijing, China, pp. 1433–1438. Wang, Z.G., Ren, A.N., Xu, L., Sun, X.J., Hua, X.B., 2000. The experimental study on the immunological and anti-inflammatory activities of Chrysanthemum indicum. Chinese Journal of Traditional Medical Science and Technology 2, 92–93. Wei, W., Shen, Y.X., Xu, S.Y., 2001. The determination of function of lymphocytes. In: Xu, S.Y., Bian, R.L., Chen, X. (Eds.), Experimental Methods of Pharmacology. People’s Medical Publishing House, Beijing, pp. 1429–1430. Xu, S.Y., Bian, R.L., Chen, X., 1982. Experimental Methods of Pharmacology. People’s Medical Publishing House, Beijing, pp. 934– 938. Yoshikawa, M., Morikawa, T., Murakami, T., Toguchida, I., Harima, S., Matsuda, H., 1999. Medicinal flowers. I. Aldose reductase inhibitors and three new eudesmane-type sesquiterpenes, kikkanols A, B, and C, from the flowers of Chrysanthemum indicum L. Chemical and Pharmaceutical Bulletin 3, 340–345.
Short communication
Anti-inflammatory and immunomodulatory activities of the extracts from the inflorescence of Chrysanthemum indicum Linn´e Wenming Cheng a,b , Jun Li b,∗ , Tianpa You a , Chengmu Hu b a
Department of Chemistry, University of Science and Technology of China, Hefei 230026, China b School of Pharmacy, Anhui Medical University, Hefei 230032, China Received 19 May 2004; received in revised form 10 February 2005; accepted 12 April 2005 Available online 18 July 2005
Abstract Chrysanthemum indicum Linn´e (CI) has a long history for the treatment of inflammation, hypertension and respiratory diseases in China. The purpose of the present study was to investigate the anti-inflammatory and immunomodulatory properties of the inflorescence or bud of CI extracts. The ethanol extract of CI (CIEE) was fractionated to a petroleum ether soluble fraction (CIPF), an ethyl acetate soluble fraction (CIEF), a butanol soluble fraction (CIBF) and a water soluble fraction (CIWF) successively. CIBF (150 mg/kg, p.o.) caused a significant inhibition on the auricle edema in mice. CIBF (150, 300 mg/kg, p.o.) not only significantly increased the delayed-type hypersensitivity (DTH) reaction induced by 2,4-dinitro-fluorobenzene (DNFB) but also significantly enhanced antibody generation by splenic cells of mice and IgG and IgM levels in mice sera in response to sheep red blood cells (SRBC) in cyclophosphamide (CP)-induced mice. Furthermore, CIBF (150, 300 mg/kg, p.o.) obviously potentiated the function of the mononuclear phagocytic system in CP-induced mice. The above results reveal that CIBF possesses anti-inflammatory, humoral and cellular immunomodulatory and mononuclear phagocytic activities, probably due to the presence of flavonoids. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Chrysanthemum indicum; Anti-inflammation; Immunomodulation; Mononuclear phagocytosis
1. Introduction Chrysanthemum indicum Linn´e (Compositae) is a wild herb, which was widely spread, productive and of high quality in Anhui province of China. The inflorescence or bud of Chrysanthemum indicum has a long history using as a Chinese traditional medicine mainly for the treatment of inflammation, hypertension and respiratory diseases. Previous studies have reported that Chrysanthemum indicum possesses the anti-bacteria, anti-virus, anti-oxidant, antiinflammatory and immunomodulatory properties (Wang et al., 2000). Phytochemical profile of this plant has shown the presence of flavonoids, terpenoids and phenolic compounds (Yoshikawa et al., 1999). However, the anti-inflammatory
∗
Corresponding author. Tel.: +86 551 5161116; fax: +86 551 5161116. E-mail address: [email protected] (J. Li).
0378-8741/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2005.04.035
and immunomodulatory functions of active components from this plant remain to be investigated. In the present study, we screened the anti-inflammatory components from the dried bud of Chrysanthemum indicum and then explored the influence of active components on cellular immunity, humoral immunity and the phagocytic function of mononuclear phagocytic system.
2. Material and methods 2.1. Animals Kunming mice, BALB/c mice (weighing 18–22 g, n = 10 per group) and purebred Guinea pigs (weighing 250–350 g, n = 5), were purchased from Animal center, Anhui Medical University, Hefei, China. All animals were housed for 1 week before experiment. All animal procedures were followed in
W. Cheng et al. / Journal of Ethnopharmacology 101 (2005) 334–337
accordance with the approved protocol for use of experimental animals set by the standing committee on animal care at Anhui Medical University. 2.2. Plant material, extraction and chemical analysis The bud of Chrysanthemum indicum was obtained from the local market and authenticated based on its microscopic and macroscopic characteristics. A voucher specimen (CI-1) has been deposited at School of Pharmacy, Anhui Medical University, Hefei, China. The dried bud of Chrysanthemum indicum (8 kg) was macerated with 95% ethanol at room temperature for 2 weeks. The ethanol extract (CIEE) was suspended in water, then extracted by petroleum ether, ethyl acetate and butanol successively, yielding a petroleum ether soluble fraction (CIPF), an ethyl acetate soluble fraction (CIEF), a butanol soluble fraction (CIBF) and a water soluble fraction (CIWF), about 3.4, 2.6, 4.0 and 5.0% of the dried plant material, respectively. The fraction with obvious anti-inflammatory activity was analysed on the thin layer chromatography (TLC) plate of RP-18 F254s . A mixture of methanol and water (60:40) was used as mobile phase. The flavonoids was detected at 365 nm as yellow fluorescent spots with the solution of 1% AlCl3 in ethanol. The tested fraction was compared with reference substances, which were luteolin, luteolin-7-glucoside and acacetin-7-rhamnoglucoside. The flavonoid content of the active fraction was also determined (Lin, 2002). 2.3. Antigen and complement 2,4-Dinitrofluorobenzene (DNFB) was purchased from Sigma Chemical Co., USA. Sheep red blood cells (SRBC) were collected from a healthy adult sheep in a local breed mill. SRBC in Alsever’s solution were washed three times with sterile saline. The blood cells collected from five purebred Guinea pigs were challenged with SRBC by 10:1 (v/v), and then centrifugated at 2000 rpm for 10 min. The pellet used as complement was preserved at −70 ◦ C.
335
2.5. DNFB-induced delayed-type hypersensitivity (DTH) reaction in cyclophosphamide (CP)-induced mice BALB/c Mice of either sex were divided into five groups of 10 each. CIBF (75, 150, 300 mg/kg, p.o.) was administered for 5 days. On day 3, CP (50 mg/kg, i.p.) was administered and continued for 2 days. On day 5, the mice were sensitized by applying 50 l of 1% DNFB in mixed acetone–olive oil (4:1, v/v) to the skin of shaved abdomen. On day 10, they were challenged for contact hypersensitivity with 50 l of 1% DNFB by applying it to both sides of the left earlap of mice. Forty-eight hours later, the mice were executed. Both earlaps of mice were cut down and weighed as previously described (Wei et al., 2001). 2.6. Antibody generation by the splenic cells of mice and IgG and IgM levels in mice sera in response to SRBC in CP-induced mice Two batches of mice of either sex were divided into five groups of 10 each, respectively. CIBF (75, 150, 300 mg/kg, p.o.) was administered on day 1 till the day of the experiment. On day 3, both batches of mice were immunized with 0.2 ml of 1 × 108 SRBC. CP (50 mg/kg, i.p.) was administered on day 3 and continued for 2 days. On day 7, blood from one batch of mice was collected. Simultaneously, the spleens of mice were culled and the splenic cell suspension of 5 × 106 ml−1 was prepared in Gey’s solution. Another batch of mice were immunized again on day 7. On day 14, blood sample was collected and divided to two halves. One-half was prepared to determine IgM level. Another half was pretreated with 2-mercaptoehanol (incubated at 37 ◦ C for 0.5 h to block IgM) and sheep anti-mouse (SAM) IgG in order to determine IgG level. An aliquot (0.5 ml) of the splenic cell suspension or mice sera was diluted by 800-fold with saline and challenged with 0.5 ml of 10% (v/v) SRBC suspension, complement and saline subsequently. Both the antibody generation by the splenic cells of mice and IgM or IgG levels in mice sera were determined as previously described (Shen et al., 2001). The values of absorbance indicating the level of IgM, IgG or total Ig in mice sera multiplied by dilution factor are converted into HCIgM , HCIgG and HCIg , respectively.
2.4. Anti-inflammatory testing The anti-inflammatory property of Chrysanthemum indicum bud was investigated using the model of auricle edema induced by dimethylbenzene in mice. Male mice were divided into seven groups of 10 each. Reference drug (Naproxen, 50 mg/kg), tested extracts (150 mg/kg) and control vehicle were given intragastrically for 5 days. A 0.05 ml of dimethylbenzene was spread on both sides of the left earlap of mice 1 h after the final administration of drugs. Two hours later, the mice were executed. Both earlaps of mice were cut down and weighed as previously reported (Ding et al., 2001).
2.7. Macrophage phagocytosis by carbon clearance method in CP-induced mice Mice of either sex were divided into five groups of 10 each. CIBF (75, 150, 300 mg/kg, p.o.) was administered for 5 days prior to the injection of carbon particles (0.1 ml/10 g). CP (50 mg/kg, i.p.) was administered on day 3 and continued for 2 days. On day 5, mice were injected with 0.1 ml of carbon suspension through the tail vein (Biozzi et al., 1953). Blood samples were collected from the orbital plexuses of individual mice immediately at 2 min (t1 ) and 10 min (t2 ) after injection of carbon suspension, lysed with 4 ml 0.1%
336
W. Cheng et al. / Journal of Ethnopharmacology 101 (2005) 334–337
Table 1 Anti-inflammatory activity of the extracts from Chrysanthemum indicum on auricle edema in mice
Table 2 Effect of CIBF on antibody generation by the splenic cells of mice in response to SRBC and DNFB-induced DTH reactivity in CP-induced mice
Drugs
Dose (mg/kg, p.o.)
Tumidity (mg)
Inhibition (%)
Drugs
Dose (mg/kg, p.o.)
Absorption value
Tumidity (mg)
Control CIEE CIPF CIEF CIBF CIWF Naproxen
– 150 150 150 150 150 50
24.3 ± 5.1 22.5 ± 3.9 20.9 ± 4.3 20.4 ± 4.1 15.8 ± 3.3a 21.6 ± 4.9 19.1 ± 4.0a
– 7.4 14.0 16.0 35.0 11.1 21.4
Control CP control CIBF + CP CIBF + CP CIBF + CP
– 50 75 150 300
0.25 ± 0.02 0.11 ± 0.01a 0.15 ± 0.03 0.17 ± 0.03b 0.18 ± 0.01b
2.2 ± 0.5 1.6 ± 0.4a 1.8 ± 0.5 2.2 ± 0.3b 2.4 ± 0.7b
Mean ± S.E.M. (n = 10). a P < 0.01, statistically significant as compared to control. b P < 0.01, statistically significant as compared to CP control.
Mean ± S.E.M. (n = 10). a P < 0.05, statistically significant as compared to control.
Na2 CO3 . The absorbances were measured spectrophotometrically at 670 nm. Then the liver and spleen of individual mice were culled and weighed. The rate of carbon clearance κ and phagocytic index α are calculated as follows: κ = (log A1 − log A2 )/(t2 − t1 ), α = κ1/3 × body weight/(liver weight + spleen weight) (Xu et al., 1982). 2.8. Statistical analysis The results are presented as mean ± S.E.M. Statistical significance between the groups was analysed by Student’s t-test. P-value less than 0.05 was considered to be statistically significant.
3. Results 3.1. Chemical analysis CIBF appeared the same way as luteolin, luteolin-7glucoside and buddleoglucoside on TLC plate. The total flavonoid content of CIBF was about 53%, which indicates that CIBF mainly consists of the above flavonoids. 3.2. Anti-inflammatory activity CIBF (150 mg/kg, p.o.) was found to significantly inhibit auricle edema induced by dimethylbenzene in mice, with a higher inhibition rate (35.0%) than that of naproxen (50 mg/kg, p.o., 21.4%) (Table 1).
3.3. Antibody generation by the splenic cells of mice in response to SRBC and DNFB-induced DTH reactivity in CP-induced mice CIBF (150, 300 mg/kg, p.o.) not only produced a significant, dose-related increase on the antibody generation by the splenic cells of mice in response to SRBC, but also obviously increased DTH reactivity induced by DNFB in CP-induced mice (Table 2). 3.4. IgG and IgM levels in mice sera in response to SRBC and macrophage phagocytosis in CP-induced mice CIBF (150, 300 mg/kg, p.o.) significantly, dosedependently enhanced serum IgG and IgM levels in response to SRBC in CP-induced mice (Table 3). Accordingly, a significant, dose-related potentiation of the macrophage phagocytic activity was observed as evident by the rate of carbon clearance and phagocytic index (Table 4).
4. Discussion The dimethylbenzene induced auricle edema in mice is the preliminary and simple model for searching for potential antiinflammatory constituents. The crude extract of Chrysanthemum indicum has been found to be effective on the model and potentiate phagocytic activity of the macrophage in mice (Wang et al., 2000). The present study not only proved the anti-inflammatory activity of the extract of Chrysanthemum indicum, but also showed that CIBF was more active than other fractions.
Table 3 Effect of CIBF on IgG and IgM levels in mice sera in response to SRBC in CP-induced mice Drugs
Dose (mg/kg, p.o.)
HCIgM
HCIg
HCIgG
Control CP control CIBF + CP CIBF + CP CIBF + CP
– 50 75 150 300
684.5 ± 18.9 294.4 ± 18.2a 324.2 ± 13.4 362.4 ± 16.4b 395.4 ± 21.5b
788.0 ± 68.4 462.6 ± 47.7a 613.2 ± 57.1 701.2 ± 87.2b 731.2 ± 66.9b
224.0 83.1 123.7 171.7 243.2
Mean ± S. E M. (n = 10). a P < 0.01, statistically significant as compared to control. b P < 0.01, statistically significant as compared to CP control.
± ± ± ± ±
60.7 22.5a 15.0 21.0b 60.0b
W. Cheng et al. / Journal of Ethnopharmacology 101 (2005) 334–337
337
Table 4 Effect of CIBF on macrophage phagocytic activity in CP-induced mice
Acknowledgements
Drugs
Dose (mg/kg, p.o.)
Rate of carbon clearance (κ)
Phagocytic index (α)
Control CP control CIBF + CP CIBF + CP CIBF + CP
– 50 75 150 300
0.086 ± 0.021 0.042 ± 0.010a 0.053 ± 0.013 0.061 ± 0.012b 0.065 ± 0.014b
7.83 ± 1.40 5.78 ± 0.55a 6.12 ± 0.73 6.91 ± 0.82b 7.25 ± 0.99b
Financial support for the project ‘Primary Study of National Grave Basic Research Programme’ (No. 2002CCC02900) by the Ministry of Science and Technology of China is gratefully acknowledged.
Mean ± S.E.M. (n = 10). a P < 0.01, statistically significant as compared to control. b P < 0.01, statistically significant as compared to CP control.
CP, an immunosuppressive agent, can inhibit cell reproduction (Shao et al., 2000), especially after the challenge of the antigen. DNFB, a chemical sensitizer, which in combination with skin proteins acquires antigenicity (Wei et al., 2001), was used to elicit contact hypersensitivity reaction in mice. CIBF (150, 300 mg/kg, p.o.) significantly enhanced antibody generation by the splenic cells of mice in response to SRBC and DNFB-induced DTH reactivity in CP-induced mice. This suggests that CIBF not only boosts the production of antibody by lymphocytes, but also causes activation of the T cells, which subsequently proliferate and release cytokines, leading to anti-inflammatory and immunomodulatory activation. CIBF (150, 300 mg/kg, p.o.) obviously enhanced serum IgG and IgM levels in response to SRBC in CP-induced mice. The result reveals that CIBF improves humoral immunity probably through the reproduction and differentiation of B cells into antibody-secreting plasma cells. Macrophages can process and present antigen to B cells. CIBF (150, 300 mg/kg, p.o.) caused a significant, dose-related potentiation of the macrophage phagocytic activity as shown in Table 4. The result indicates the stimulatory effect of CIBF on the cells of the mononuclear phagocytic system. Through chemical analysis, CIBF mainly consists of three flavonoids, luteolin, luteolin-7-glucoside and acacetin7-rhamnoglucoside, which are probably responsible for its anti-inflammatory and immunomodulatory activities. Therefore, CIBF could be an effective and useful candidate in the development of anti-inflammatory and immunomodulatory drug.
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