Download Materials and Methods Throughout this project the focus was on

Materials and Methods
Throughout this project the focus was on discovering different medicinal properties of
Trifolium pratense, and Pueraria lobata. Each plant was tested for antioxidants,
polyphenols, antibiotic properties, and anti tumorous properties.
Collection of specimens
Most of the specimens of Trifolium pratense (red clover) was collected in Transylvania
County at 1232 Asheville Highway, Brevard, NC 28712. The specimens were collected by
plucking then out of the ground by the base of the stem. They were placed in a bucket and
dried for about two weeks, or until the flowers were dark brown and crispy.
Pueraria lobata [common name] was purchased from Mountain Rose Herbs (confirmation
#5288689), city, state.
Preparation of Extracts
The dried flower heads of the red clover were pinched from the stem and ground to a fine
powder in a standard coffee grinder. The powder was stored in a clean container at room
temperature. for safe keeping. In order To make the extracts from Pueraria lobata and
Trifolium pratense, a 1 part (by weight) plant powder to 40 part (by volume)70% ethanol
alcohol ratio was used. In the particular extract that was used for the majority of the
testing, In most cases, 10g plant powder and 400g 70% ethanol alcohol was used. These
were weighed using a scale. The 10 g of plant powder and 400 g ethanol were then placed
in a large beaker and put in a large beaker, placed in an xxxxx sonicator [give name and
model #] for 30 min, in order to fully mix it. A sonicator is a machine using ultrasonic
waves in water to mix extracts and other combinations. Once the specimen was sonicated,
and then filtered into a new beaker with the use of through Wattman filter paper #2 , in
order to remove the plant pieces. To remove the 70% ethanol, the mixture filtrate was then
placed on in a rotovap, to remove the 70% ethanol. A Rotovap is a rotary evaporator. It is
used to dry down extracts by using pressure to cause them to boil. Since the rotovap does
not use heat it reduces the risk of losing vital chemicals due to overheating. The Rotovap
spins the beaker containing the extract and increases the pressure causing the liquid to
boil. Our extract was placed on the rotovap rotary evaporator [brand name & model #]
with the pressure set at 58 [units], and was slowly brought down to 21 [units ]. The water
bath was 40 degrees C, and the coil temperature was set to 0 degrees C. The filtrate was
maintained in placed on the rotovap until the extract was sufficiently dried down.
After the specimen was dried down, 60 ml of hexane, 90 ml of ethanol, and 30 ml of
methanol were added to the residue extract in order to separate the it to a greater extent
[???????]. A separatory funnel was used to separate it [it?????] into two parts, phases, one
made of consisting of extracted compounds in hexane and extract and the other made of
consisting of extracted compounds in methanol plus 70% ethanol. The hexane mix was put
into a different beaker with the use of a pipette. A gooey residue was left in the methanol
ethanol section, which was pipetted out and stored in a separate beaker.
TLC
Thin layer chromatography was used to isolate isoflavones from three different extracts.
The three extracts tested were made of Trifolium pratense and Pueraria lobata. The original
extracts were made and then the Trifolium pratense extract was separated using a 30mL
methanol, 90mL 70% ethanol and 60 mL hexane mixture. The hexane was separated from
the methanol and ethanol creating two different extracts of one plant.
Then we drew the origin 1.5 cm above the bottom of the plate. The origin is the original line
on the TLC plate that the extracts are placed on before insertion into the chamber. The
Trifolium pratense extract made with hexanes, Trifolium pratense extract made with
methanol and ethanol, Pueraria lobata extract and two pure isoflavones, genistein and
genistin, were dotted in lines about one cm long and one cm apart onto the origin.
Then the plates were inserted into a chamber with a mixture of 100 parts ethyl acetate, 20
parts methanol and 10 parts water. Then the plates were allowed to sit for around five
minutes until the liquid was a few cms from the top of the plate. The liquid traveling up the
plate separated the chemicals and isoflavones in the extracts.
Antioxidant Test
Once TLC had been completed multiple plates were sprayed with an antioxidant test spray
made with .4 mM DPPH in methanol. This test showed which chemicals on the plate were
antioxidants by bleaching the antioxidants and turning the rest of the plate purple. After
the plates were sprayed they were allowed to sit for around 10 minutes, and then
examined for results.
Polyphenol Test
The plates that hadn’t been used in the antioxidant test were taken and sprayed with a
polyphenol mixture made with 2% iron chloride in ethanol. Once sprayed, the plates were
dried using a hair dryer on medium heat for 10 minutes. The polyphenols turned a dark
gray or black depending on their concentration. The non-polyphenols turned an orange
color. This helps to give an idea of the Rf value of the polyphenols.
Potato Disk Bioassay:
Materials:
1 beaker
1 liter graduated cylinder
2 1 gallon glass bowls
2 knives
2.5 cm long screw
2.5 cm screw
3 cm copper pipe tip
Agrobacterium tumefacien
Autoclave
Bleach
Blowtorch
Capped test tubes
Centrifuge
Distilled water
Filter cap flasks
Gloves
Hood
Large loops
LB agar plates
LB broth
Metal plate
Micropipet (20-200 μL)
Micropipet tips
PBS
Pipets
Pipet Helper
Plant extracts
Russet potatoes
Sterilized 6-well plates
Sink water
Sterilized distilled water
Optional:
Tweezers
Under the hood, the Agrobacterium strain 348 was put on two LB agar plates using a
loop. It was sealed using parafilm. Then it sat out for two days at room temperature. After
agrobacterium colonies had grown a large loop of the colonies were put into a sterile filter
top flask with 5 ml of LB broth.
The filter top flask was then incubated at 28°C for two days. After two days the LB
broth was cloudy showing that the bacterium had grown. The LB broth was then put into a
15mL Conical Centrifuge Tube and centrifuged for fifteen minutes.
Then a russet potato was washed and peeled. One liter 10% bleach and distilled
water solution was put into one of the glass bowls. It was left in the solution for two
minutes. Then gloves were put on and the potato was put the other glass bowl with just
distilled water for ten minutes.
The centrifuge tube was taken out and the LB broth was poured into a beaker with
dawn liquid soap. The centrifuge tube containing the Agrobacterium pellet was then put in
a rack and 5ml of PBS solution was mixed in with it.
The metal plate was then sterilized with the blowtorch. Then gloves were put on
and the potato was taken out of the distilled water and put on the plate. A sterilized knife
was then used to cut the potato in .5 cm thick slices. A sterilized 3 cm copper pipe tip that
had been filed in on the inside was then used to cut a disk from the center of the potato
slice. A 2.5 cm screw head was used with a 3.5 cm washer to help get the copper pipe to cut
the potato.
The copper pipe was then taken to a 6-well plate and the long 2.5 cm screw head
was used to put the potato into the well. This was done for all of the slices of the potato.
Then using a 20-200μL micropipet, 200μL of the PBS Agrobacterium mixture was
put on most of the potato discs. The number of plain controls depended on how many disks
had been made and put into wells. After the 200μL had been put onto some of the disks the
DMSO plant extract solution was used. 200μL of each extract was put onto the potato discs.
In this certain experiment each extract was put on two discs with Agrobacterium and one
with just a plain potato disc.
At the end when all of the potato discs had whatever they were supposed to have, .5
ml of sterilized distilled water was added to each well. Then the wells were checked on
everyday to make sure that there was enough sterile distilled water in each well every day
for 21 days.
At the end of 21 days the discs were stained with 500μL of 5% I2 and left for five
minutes. Then the discs were observed under a dissecting microscope and tumors were
counted on all of the discs. Then numbers were written down and made into a graph and
chart.