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The anti-proliferation effect of Cordyceps militaris on human 2_2_

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The anti-proliferation effect of Cordyceps militaris on human 2_2_ Powered By Docstoc
					The anti-proliferation effect
of Cordyceps militaris on
human mucoepidermoid
pulmonary carcinoma
Overview of the presentation

1.   Background information
2.   Methodology and Results
3.   Implications
4.   Future perspectives
Background
information
Cancer statistics in H.K.
• Cancer: no. 1 killer in HK
• In 2003, 31.6% of death was due to
  cancers in HK
• It is expected that 1 in 3 men & 1 in 4
  women would develop cancer in HK
  (Department of Health)
The Global Picture
• USA: 50% men and 1/3 of women expected
  to get cancer (American Cancer Society, 2006)
• Developed countries: ~ 1/4 deaths due to
  cancers
• Developing countries: cancer has become a
  major health problem
• Global cancer rates could increase by 50%
  to 15 million new cases in 2020 (WHO, 2003)
Different treatments &
drawbacks
• Surgical resection: not applicable to
  inoperable tumors, traumatic surgery
• Chemotherapy: cytotoxic drugs destroy
  normal cells, has little contribution to
  survival (Morgan et al., 2004)
• Radiotherapy: harm the normal cells as
  well
Cordyceps
•   Record dates back to Tang Dynasty
•   A genus of Ascomycetes fungi
•   Host-specific
•   Two of the well-known active
    ingredients: adenosine, cordycepin
Medicinal value of Cordyceps
• Used to treat kidney, lung, heart and
  liver ailments, male and female sexual
  dysfunction, fatigue, cancers, etc.
• Considered particularly beneficial to
  kidneys and lungs in Traditional
  Chinese Medicine (TCM)
The structure of cordycepin
• Altered nucleoside
• 3’deoxyadenosine
• Inserted between DNA,
  halting DNA replication



                            (Holliday et al.)
(Holliday et al.)
Cordyceps militaris
• Also known as North
  Chinese caterpillar
  fungus
• Infect insect larvae
  Bombyx mori (silkworm)
• From which cordycepin
  was first isolated (Won et al.)
Methodology
& Results
Cultivation of mycelia
• Preparation of sterilized
  rabbit food medium
• Inoculation of mycelia with
  aseptic technique
• Shaking at 27°C for 5 days
• Allowed to stand still &
  grow
Extraction method
• 4 types of extracts
    –   Aqueous mycelia
    –   Aqueous fruiting body
    –   50% ethanol mycelia
    –   50% ethanol fruiting body
•   Mycelia made brittle with liquid N2
•   Grinded with mortars and pestles
•   +50% EtOH/ H2O, sonicated for 1 hr
•   Lyopholisation of the supernatant
High Performance Liquid
Chromatography (HPLC)
• liquid mobile phase
  mechanically pumped
  through a column that
  contains a stationary
  phase
• Reversed phase, with
  progressively non-
  polar mobile phase
• Retention time different for different
  molecules
• Peak area directly proportional to the
  concentration
• Standards (adenosine, cordycepin)
  added to the samples  confirmation
• UV absorbance at 215nm
Chromatograms
Figure 1. Chromatogram of adenosine (0.1mg/ml) Figure 2. Chromatogram of cordycepin (0.1mg/ml)
           DAD1 A, Sig=215,4 Ref=360,4 (ISABELLA\06021406.D)           DAD1 A, Sig=215,4 Ref=360,4 (ISABELLA\06021401.D
    m AU
                                   adenosine                   m AU                                    cordycepin


    800                                                        800




    600                                                        600
                           2.457




                                                                                                       3.559
    400                                                        400




                                                                                                                        14.429
    200                                                        200




                                                                                         11.411
                                                                                        2.149
                                                                           0.676
               0.676




      0                                                            0


           0           2             4         6               8       0           10   2         12           4   14            6   16
Figure 3. The chromatograms of the 4 extracts
        DAD1 A, Sig=215,4 Ref=360,4 (ISABELLA\06021421.D)
                                          DAD1 A, Sig=215,4 Ref=360,4 (ISABELLA\
m AU                                                          m AU

             H2O fruiting body                                                   50% EtOH fruiting body




                                                                                                                              3.581
                                                 3.537
 800                                                             800




 600                                                             600




                1.047
            DAD1 A, Sig=215,4 Ref=360,4 (ISABELLA\06021416.D) Ref=360,4 (ISAB
                                            DAD1 A, Sig=215,4




                                                             4.330




                                                                                                               1.077
m AU
 400                                                         m AU
                   1.263
                                                               400




                                                                                 5.630
                                 2.272




                                                                                                                                            4.412



                                                                                                                                                                5.681
                                                                                                         7.180
                              2.018




                                                                                               7.494 2.317
                                         3.111




                                                                                                      2.077
                          1.471
                          1.741




                                                                        5.168




                                                                                                                           3.166
                                                                                                  1.797
 200                                                             200




                                                                                                 6.819
                                                                     4.772




                                                                                                                                               5.235
                                                                                                6.271




                                                                                                                                             10.010
                                                                                                                       8.515
                                                                                                                       8.021
                                                                                                                       8.257


                                                                                                                                             9.346
                                                                                                                                             9.826
                                                                                                                                            4.864
                                                                                             1.515
 800




                                                                                                                                           4.654
                                                              800
    0                                                                 0



        0                       2                        4                   0           6                2            8              4              10

 600                                                          600

              H2O mycelia                                                        50% EtOH mycelia
                  1.054




                                                                                               1.078
 400                                                          400




                                                                                                                 7.236
                                     2.373




 200
                                                                         4.627
                                                              200




                                                                                                               2.469
                        1.304



                                  2.687




                                                                                                            6.428
                                  2.005




                                                                                                           1.494




                                                                                                           8.185
                                                                                  5.655


                                                                                                           6.622




                                                                                                                                                    9.157
                                                                                  5.281




                                                                                                                                                            9.676
                                 2.972
                                                    3.461




                                                                                                                                                               4.704
                                                                                                        2.750
                                                                                                        2.078
                                                                                                        1.707



                                                                                                       3.044
                                                                                                                                          3.547
    0                                                                0


        0                           2                        4              0                   6               2             8                     4           10
HPLC Results (Triplicate)
Table 1. Percentage of cordycepin in the crude extracts of Cordyceps
Types of extract                       % of cordycepin

Aqueous fruiting                       1.96 ± 0.006
body extract
50% EtOH fruiting                      2.45 ± 0.006
body extract
Aqueous mycelia                        0.05 ± 0.004
extract
50% EtOH mycelia                       0.06 ± 0.003
extract
Table 2. Percentage of adenosine in the crude extracts of Cordyceps

Types of extract                             % of adenosine
Aqueous fruiting body                        0.66 ± 0.003
extract
50% EtOH fruiting body                       0.61 ± 0.005
extract
Aqueous mycelia extract 0.23 ± 0.013

50% EtOH mycelia                             0.19 ± 0.029
extract
Cell culture
• Cell line thawing – human
  mucoepidermoid pulmonary
  carcinoma (NCI-H292)
• Aseptic technique
• Subculture every 3 days and cell
  number counting
• Log phase – constant doubling time
   cell proliferation assay
Cell proliferation assay
• 3-(4,5-dimethylthiazol-2-yl)-
  2,5-diphenyltetrazolium bromide
  (MTT) assay
• 48-hour treatment




               (http://www.phs.osaka-u.ac.jp/homepage/b012/cyto/cyto.html)
The IC50 (50% inhibitory
concentration) values

Table 3. The IC50 values of aqueous and 50% ethanol extracts of fruiting body

Aqueous extract of
                                     103.9 (± 2.9) μg/ml
fruiting body

50% EtOH extract
                                     49.1 (± 1.8) μg/ml
of fruiting body
    The inhibitory effect of the
    extracts on NCI-H292
Figure 4. The inhibitory effect of aqueous and 50% ethanol fruiting body extracts on the proliferation of NCI-H292




                                                                                     p < 0.01, n=12, triplicate
Figure 4. The inhibitory effect 50% ethanol mycelia extracts on the proliferation of NCI-H292




               *    * *              *        *        *




                                                                         p < 0.01, n=12, triplicate
Implications
• Anti-tumour effect:
  – fruiting body >>> mycelia
  – 50% EtOH fruiting body >>> aqueous
• Content of cordycepin correlates with
  the anti-tumour effect
• Some other components may also be
  responsible for the anti-tumour effect
• Cordyceps militaris may be a better
  alternative
Future perspectives
• The mechanism behind with
  protein profile analysis
• Normal cell line for toxicity study
• Animal models – investigate the
  effects at the organism level
• Cultivated Cordyceps – more
  available, easier to be
  standardized
Acknowledgements
• Prof. NGAI Sai Ming Ice
• Miss TSAI Sau Na Helen
• Other EG08 labmates
References
1.   http://www.cancer.org/docroot/CRI/content/CRI_2_4_1x_Wha
     t_Is_Cancer.asp?rnav=crihttp://www.info.gov.hk/dh/publicat/ar
     0304/pdf/a11.pdf
2.   http://www.info.gov.hk/dh/publicat/ar0304/pdf/a08.pdf
3.   http://www.phs.osaka-u.ac.jp/homepage/b012/cyto/cyto.html
4.   http://www.protocol-
     online.org/prot/Cell_Biology/Cell_Growth___Cytotoxicity/MTT
     _Cell_Proliferation_Assay/index.html
5.   http://www.who.int/mediacentre/news/releases/2003/pr27/en/
6.   Ahn, Y.J., Park, S.J., Lee, S.G., Shin, S.C., Choi, D.H. 2000.
     Cordycepin: selective growth inhibitor derived from liquid
     culture of Cordyceps militaris against Clostridium spp. Journal
     of Agricultural and Food Chemistry. 48(7):2744-8.
7.    Buenz, E.J., Bauer, B.A., Osmundson, T.W., Motley, T.J.
      2005. The traditional Chinese medicine Cordyceps sinensis
      and its effects on apoptotic homeostasis. Journal of
      Ethnopharmacology. 96(1-2):19-29.
8.    Holliday, J., Cleaver, M. 2004. On the Trail of the Yak –
      Ancient Cordyceps in the Modern World. MS in press.
9.    Morgan, G., Ward, R., Barton, M. 2004.
      The contribution of cytotoxic chemotherapy to 5-year survival
      in adult malignancies. Clinical Oncology. 16(8):549-60.
10.   Won, S.Y., Park, E.H. 2005. Anti-inflammatory and related
      pharmacological activities of cultured mycelia and fruiting
      bodies of Cordyceps militaris. Journal of Ethnopharmacology.
      96(3): 555-561.
What is cancer?
• Proto-oncogene  oncogene
• Tumour suppressor gene inactivated
• Mutated genes with products
  participating in DNA repair
• Uncontrolled cell proliferation 
  angiogenesis  metastasis
Therapeutic Applications
•   Pulmonary, renal, liver, heart diseases
•   Hyposexuality
•   Hyperlipidemia
•   Hyperglycemia
•   Immune disorders
•   Adjunct in cancer therapy

				
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