Effects of herbal extracts on C. elegans neuromuscular disorders caused by SOD mutations

Becca MeyerBen OslundKenneth TanCheung Chiu Yin

Introduction

Degeneration of muscle neurons hinders the transmission of signals to muscle cells.

Affects movement.Nature of movement depends on the health of the nervous system.

Introduction

SOD (SOD1 or SOD2) Scavenges free radicals Prevents oxidative stress

SOD mutations/oxidative stress implicated in neurodegenerative diseases

Introduction

Wen Pi Tang TCM prescription antioxidant been shown to delay the onset of ALS

Other TCMs Doubts about effectiveness of treatments

Objectives

To study the effects of various herbal extracts on the mobility of wild type and mutant C. elegans that express a neuromuscular disorder caused by an SOD mutation.

Comparing the protein profiles, movement and lifespan.

Hypothesis

Herbal extracts will be able to alleviate or delay the onset of neuromuscular disease symptoms in mutant type C. elegans compared to wild type C. elegans.

Test organism- C. elegans

Benefits of C. Elegans as a model Common genes and biological traits

Short lifespan▪ Easy to test and see results

Variables

Controlled

• Method of preparation of extracts.

• Species of C. elegans.

• Treatment of C. elegans

• Amount of food available for C. elegans

Independent

• Concentration of extracts

• Type of extract C. elegans are immersed in

• Time of exposure to extracts

Dependent

• Movement of C. elegans

• Protein profile of C. elegans

• Duration of lifespan of C. elegans

Materials Anti-aging prescription

& herbs Wen Pi Tang Ma Huang He Shou Wu Dang Gui Yan Hu Suo

Petri Dishes 250 ml Beakers Measuring Cylinder Centrifuge tube Recorder (Video

Camera) Hot Plate

DroppersDissecting microscopeE.Coli (OP50)Paintbrush hairMaterials for NGM AgarSodium Chloride (NaCl)PeptoneAgar5 mg/ml cholesterol in ethanol (Not autoclaved)1M MgSO4

1 M KPO4 buffer pH 6.0Deionised water

Prescription- Wen Pi Tang

Components of Wen Pi Tang: Rhei rhizoma (Da Huang) Ginseng radix (Ren Shen) Aconiti tuber (Fu Zi) Zingiberis rhizoma (Gan Jiang) Glycyrrhizae radix (Gan Cao)

Wen Pi Tang has been found to delay onset of ALS in transgenic mice.

Herbs

Rhei rhizoma(Da Huang)

All 4 herbs are said to maintain the health of the nervous system.

Ephedra sinica (Ma Huang)

Polygonum multiflorum (He Shou Wu)

Angelica sinesis (Dang Gui)

Rhizoma corydalis (Yan Hu Suo)

Procedure- Preparation of herbal extracts

1. Chop and extract with distilled water.

2. Soak for 20 minutes and boil for 30 minutes.

3. Filter4. Evaporate filtrate in a speed

vacuum without heating.5. Determine antioxidant levels in

extracts using the DPPH assay.

Procedure- Preparation of Nematode Growth Medium Plates

Mix 3 g NaCl, 17 g agar, and 2.5 g

peptone in a 2 litre Erlenmeyer flask. Add

975 ml H2O. Cover mouth of flask with

aluminum foil. Autoclave for 50 min.

Cool flask in 55°C water bath for 15

min.

Add 1 ml 1 M CaCl2, 1 ml 5 mg/ml cholesterol in

ethanol, 1 ml 1 M MgSO4 and 25 ml 1 M KPO4

buffer. Swirl to mix well.

Fill plates 2/3 full of NGM agar.

Leave plates at room temperature for 2-3 days

before use to allow for detection of contaminants,

and to allow excess moisture to evaporate. Plates stored in an air-tight container at room temperature will be usable for

several weeks.

Procedure- Seeding NGM Plates with E. Coli

Apply approximately 0.05 ml of E. coli (OP50) liquid culture to small or medium NGM plates or 0.1 ml to large NGM plates using a pipette.

The drop can be spread using the pipette tip or a glass rod.

Allow the E. coli (OP50) lawn to grow overnight at room temperature or at 37°C for 8 hours.

Cool plates to room temperature before adding worms.

Procedure- Observation and data collection

Remove a cube of agar containing C.

elegans and place it onto the edge of

another Petri dish.

Inoculate a few drops of OP50 E. Coli onto

the agar plate, creating a trail from

the cube to the other side of the Petri Dish.

Locate one worm on the surface and mark the original position

Prod the worm with a hair (from a

paintbrush) to cause it to move

Record the time taken for it to move to another location

Measure the distance moved

Videotape the whole process for analysis

of movement

Procedure- Protein Profiling Pt 1

•Homogenize the C. elegans in a homogenizer.

•Mix the mixture obtained with SDS and an anionic detergent.

•Heat the mixture to 60 degrees Celsius.

•Rinse the wells of the get with a running buffer

•Prepare the anode buffer with Tris-Cl, distilled deionized water.

•Prepare the cathode buffer with SDS, Tris, Tricine and distilled deionized water.

•Cover the gel in the lower positive electrode chamber of the electrophoresis apparatus with the anode buffer.

•Cover the gel in the negative electrode chamber of the electrophoresis apparatus with the cathode buffer.

•Add the denatured protein mixture with a pipette into the wells in the gel.

•Connect the electrophoresis apparatus to a power source and turn it on.

Procedure- Protein Profiling Pt 2 (Staining)

1. Stain gel in 10 volumes of Coommassie Blue. Stain for 1-2 hours with agitation.

2. Destain gel in with 50% methanol and 10% acetic acid for 1-2 hours. 

3. Destain gel again using 7% methanol and 10% acetic acid.

Literature Review and initial proposal Prior to 31st Oct

Discussion and collaboration 29 Oct – 6 Nov

Finalisation of Proposal 9 Nov

Preliminary Experimentation (Determining the concentration and time interval etc.)

Nov-Jan (AOS)

18 Jan – 18 Mar (HCI)

*Depending on whether results of preliminary experimentation are favourable

Preliminary Round of Projects Day Competition (HCI)

March

Experimentation 1 April – 1 Jul (HCI)

Jan-May (AOS)AOS Symposium June

Semi-Finals (HCI) July

Final Experimentation (HCI) 1 – 31 JulCompilation of Results 1 – 7 Aug

Finals Judging Aug

Writing of research paper 1 – 14 Sep

References

Carroll, P. M., & FitzGerald, K. J. (2003). Model organisms in drug discovery. San Francisco, CA: John Wiley and Sons.

Glenn, C. F., Chow, D. K., David, L., & Cooke, C. A. (2004). Behavioral Deficits During Early Stages of Aging in Caenorhabditis elegans Result From Locomotory Deficits Possibly Linked to Muscle Frailty. Journal of Gerontology: BIOLOGICAL SCIENCES, 59A(12), 1251-1260.

Gold Paul E. Cahill, Larry Wenk Gary L. (2002) Ginkgo biloba: A cognitive enhancer? Psychological Science in Public Interest pp2-11 Blackwell Publishing

Hou, Joseph P.. and Jin, Youyu. (2005). Miraculous Tonic Herbs. The healing power of Chinese herbs and medicinal recipes. pp. 135-136. The Haworth Press

References

Huang, C., Xiong, C., & Kornfeld, K. (2004). Measurements of age-related changes of physiological processes that predict lifespan of Caenorhabditis elegans. PNAS, 101(21), 8084-8089.

LeDoux, M. (2005). Animal Models of Movement Disorders. Amsterdam: Elsevier.

Suzuki, M., Goto, T., Tsuji, T., & Ohtake, H. (2005). A Dynamic Body Model of the Nematode C. elegans with Neural Oscillators. Journal of Robotics and Mechatronics, 17(3), 318-319.

Taylor, David (1996) Herbal Medicine at Crossroads. Environmental Health Perspectives pp 924-928. Brogan and Partners

THE END