THE EFFECTS OF SYMPHYTUM OFFICINALE ON
DIFFERENTIATION
RATES OF MOUSE STEM CELLS
By Nikita Tanguturi
High school sophomore, American Heritage Boca/Delray
ABSTRACT
The purpose of this experiment is to measure the effects of symphytum officinale, also known as comfrey, on the reproduction of mouse mesenchymal stem cells. Symphytum officinaleis also referred to as the common comfrey plant, with many homeopathic uses. This experiment will be using C57BL/6 mouse mesenchymal stem cells from cyagen. They are derived from the bone marrow of the cells, cultured as monolayer, and cryopreserved at the 6thpassage. To test the hypotheses, the stem cells must be differentiated with an Osteogenic Differentiation medium and correct proportions of Symphytum Officinaleextract. After the cells have differentiated, they will be stained with a 1 mL alizarin red S working solution for 3-5 minutes. The experiment has yet to be concluded.
BACKGROUND
What is Symphtum Officinale?
•- It’s referred to as the common comfrey plant, a member of the comfrey family
•- It’s a perennial herb, native to Europe and brought to the Americas by immigrants
•- It can be found in North America now
•- It has a unique rhizomatic root structure which makes it hard to dig up and easy to reproduce
POTENTIAL MEDICAL PROBLEMS
Symphytum Officinale has pyrrolizidine alkaloids
-Unsaturated
-Causes hepatotoxicity and can be tumorogenic
-Also called necine bases
-Deemed unsafe by the FDA for internal use
Can be used externally except for
-Pregnant women
-People with a liver disease
-Broken skin
-Taking drugs that affect the liver
HISTORICAL IMPLICATIONS
Its Latin name, symphytum, refers to ancient uses of the plant.
-Symphis translates to “growing together of bone”
-Phyton translates to “plant”
It has also been cultivated since 400 BC as a healing herb, but there is no definitive proof
-Used as a tea for upset stomachs, ulcers, diarrhea and coughs
-Don’t know effectivity
MESENCHYMAL STEM CELLS
Make several types of cells belonging to skeletal tissues
They are multipotent- produces more than one type
-chondrocytes
-osteoblasts
-Adipocytes
Found in bone marrow
Studies using mouse bone marrow is a good praxis for human innovation
- This project will use C57BL/6, a black lab rat whose genotype is primarily known
HYPOTHETICAL PROCEDURES
1. Prepare the stem cell medium
-MSC Osteogenic Differentiation Basal Medium
2. Prep culture vessels
-0.1% gelatin solution
3. Osteogenic differentiation
-Culture and reseed
-Add medium
-Add each concenttrationof symphytum officinale
4. Differntiate for 2-4 weeks, checking every 3 days
5. Stain analysis
-Fix with formaldehyde
-Stain with alizarin red S
CASE STUDY 1: LONG BONE REPAIR IN CANINES
Chandel
An analysis on long bone canine fractures via radiology
-Post plate insertion surgery
-Given a 30c dose 2x a day for 3 weeks
-Done in Jaipur, India
The conclusion showed
-Reduction in time needed to fully recover
Led the scientists to believe it had osteo-inducing properties
CASE STUDY 2: META-STUDY ANALYSIS
Masoudi
Conducted metastudy of 44 studies
-1992-2016
-search terms were “Symphytum officinale L.”, “therapeutic properties”, “pharmacological effects”
Conclusions
-Had anti-cancer properties
-Can potentially be a carcinoma treatment
-Decreases osteoarthritic pain activity
-Helps by reducing pain and stiffness
CASE STUDY 3: HUMAN FRACTURE TREATMENT
Tsintzas
Analyzed fractured bones in humans
-Given 30CH, once daily for 10 days, and then 200CH, once a week for the next three weeks.
Included 4 isolated cases
-Comminuted fracture of right clavicle, minimally displaced fracture of the distal metaphysis of his right radius, minimally displaced intraarticular fracture of the distal part of the radius, and comminuted fracture of the fifth metatarsal bone
-One example of the four isolated trials found in the study explains how, when a 34 year old man presented with a fractured right clavicle, along with being an avid smoker, was able to return to light training in 4 weeks and foul practice in 8 weeks.
CASE STUDY 4: EFFECTS ON RAT BONE DENSITY
Sakakura
Administered 6C on lab rats
-48 rats total
-Promoted an increase in radiographic bone density around titanium micro-implants placed in their tibia
Radiographic and mechanical analysis.
-More osteogenic growth at early stages of osseointegration and bone formation
IMPLICATIONS
Further Research
In order to be ethical, stem cells are the best way to evaluate the rates of osteogenic production
-Doesn’t rely on the sacrifice of animals but still evaluates the effects of symphytum officinale
-Will be more accurate
-Can never ensure compliance of human subjects
-Doesn’t rely on radiology
Ensures all testing is done from the same baseline, whereas genetic differences in both people and rats can affect results
-No in-vitro studies have been conducted at this time, only in- vivo
CITATIONS
“Eurostemcell.” Mesenchymal Stem Cells: The 'Other' Bone Marrow Stem Cells, 5 Nov. 2015, www.eurostemcell.org/mesenchymal-stem-cells-other-bone-marrow-stem-cells.
Chandel, Swaroop. Role of Symphytum Officinale as an Osteoinducerin Long Bone Fracture Repair in Canine. 2014, krishikosh.egranth.ac.in/bitstream/1/93744/1/32148%20(Chandel,%20Swaroop,%20Surgery,%20NVC,%202014.pdf.
Masoudi, Mansoureh, and MiladSaiedi. Anti-Cancer and Osteoarthritic Pain Activity of Symphytum Officinale. 2017,
“Comfrey: Uses, Side Effects, Interactions, Dosage, and Warning.” WebMD, WebMD, www.webmd.com/vitamins/ai/ingredientmono-295/comfrey.
“Symphytum Officinale.” Wikipedia, Wikimedia Foundation, 9 Aug. 2018, en.wikipedia.org/wiki/Symphytum_officinale.
Tsintzas, Dionysis, and George Vitkhoulkas. “Take a Look at the Recent Articles.” OAText- Open Access Text, www.oatext.com/Fracture-treatment-with-the-aid-of-the-homeopathic-remedy-Symphytum-officinale-A-report-of-four-cases.php.
Society for Science and the Public (2016-17). International Science and Engineering Fair 2016-17: International Rules & Guidelines. Washington, DC: Society for Science and the Public.
Sung JH, Yang HM, Park JB, Choi GS, Joh JW, Kwon CH, Chun JM, Lee SK, Kim SJ. Isolation and characterization of mouse mesenchymal stem cells. Transplant Proc. 2008 Oct;40(8):2649-54. doi: 10.1016/j.transproceed.2008.08.009. PubMed PMID: 18929828.
Sakakura K, Neto R, Belluci M, Wenzel A, Scaf G, et al. (2008) Influence of homeopathic treatment with comfrey on bone density around titanium implants. A digital subtraction radiography study in rats. ClinOral Implants Res 19.