Differences in Aloin Concentration in Aloe Vera (Aloe
barbadensis Miller) caused by stresses
Jason Carloni
Abstract:
Aloe vera has been used medicinally throughout history by
many different cultures. Many compounds have been found in
the exudates of the aloe plant that have been used
medically by humans and used as a defense mechanism by the
plant. Stress has been known to cause increases in the
production of secondary metabolites that are used for plant
defense. I plan to stress some of the Aloe plants, extract
the exudates, and use TLC, a photo spectrometer and
possibly an HPLC/MS to compare the concentrations of Aloin
in the stressed and non-stressed plants. The differences in
Aloin concentration will indicate the stresses that may
cause increased chemical production in Aloe plants. These
methods may then be used to understand the dosage of
medicinal plants.
Introduction:
Aloe vera (Aloe barbadensis Miller) is a member of the
lilacae family. It is native to tropical and southern
Africa. It has been cultivated for its thick exudates that
contain many active compounds with known therapeutic
properties. The extracts have been found to reduce
oxygenase activity (Hart 1985) and relieve inflammation
(Reynolds 1999).
In other aloe species of Aloe the active phenolic
compounds were most concentrated in the peripheral regions
of the plant leaves (Chauser-Volfson 2002) (Gutterman
2000). The top third of the leaf and the leaf edges have
the highest concentration because those parts are most
susceptible to consumption by herbivores (Chauser-Volfson
2002).
Plants when stressed will often produce more secondary
metabolites as a defense mechanism to better protect itself
from a subsequent attack (Tang 1995). Salicylic acid and
jasmonic acid are some of the compounds in the stress
signal pathway. Jasmonic acid is synthesized when hormones
are released from a wounded leaf (Taiz 2002). Application
of jasmonate to tobacco plants (Nicotiana attenuate) was
found to increase phenolic compounds and alkaloids in the
leaves (Keinänen 2001). Salicylic acid is not a mobile
signal in the pathway but it is found in high
concentrations in the infected part of the leaf and it is
associated with systemic acquired resistance (Taiz 2002).
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Research Objective
The aim of this study is to see if we can increase the
amount of aloin in the leaves by stressing the Aloe vera
plant. The different stresses will consist of water stress,
root stress, and simulated herbivore attack. I will also
try to activate the stress signal pathway by topical
application of jasmonic acid or salicylic acid. These
stresses are expected to cause an increase in aloin in the
leaf exudates.
Methods
Preparation of the plants
Young Aloe vera plants will be ordered from Wards
Scientific Supply. The aloe plants will be transplanted
from their three inch cups to one gallon flower pots. The
pots will be placed in a level baby pool to allow
consistent irrigation. The plants should be allowed to grow
in normal (non-stressed) conditions for at least two
months. The soil should consist of 1:1:1 of soil,
vermiculite and sand (Chukwujekwu 2002) and they should be
under full sunlight (Paez 2000). This will allow the plants
develop in similar conditions and eliminate variability in
growth and chemical composition.
Stress
After the plants have been stabilized we randomly
designate the plants to a particular stress and then begin
to add stresses. The stresses should not cause the plants
to die but it may reduce the rate of growth.
Water stress can be caused simply by denying the
plants water. To do this we will not water the plants for
two weeks.
Root stress will be caused by vigorously uprooting the
plant and replanting it into the same soil. To do this I
will use a fork to break up the roots by sticking it into
the soil at an angle to reach under the plant. Then twist
the fork and break up the roots and soil so the plant can
be lifted out, photographed, and replanted. We will collect
a leaf sample one week later.
The herbivore attacks will be simulated by poking the
surface of the plant with forks several times. The three
largest leaves will be selected to undergo this attack. The
attack will consist of two forks making contact in the
center of the leaf. This will be done three times on each
of the selected leaves. We will collect leaf samples on
week later.
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I will also try to stimulate the plants systemic
acquired resistance by applying a solution of salicylic
acid and water to the leaves. Each of the four largest
leaves will be sprayed with 1ml of a 50ug/ml salicylic acid
solution. This will be applied five days before sample
collection.
Jasmonic acid will also be used to stimulate a defense
response. The four largest leaves will be sprayed with a
solution of 50ug/ml jasmonic acid and distilled water five
days befor collection. Approximately 50μg of methyl
jasmonate per leaf will be applied by spray bottle
(Keinänen 2001).
Some of the plants will have offspring budding from
it. We will collect a sample from the new plant and compare
the compounds produced by offspring of stressed plants with
the offspring of non-stressed plants.
Harvest and Collection of Compounds
After the stress period the leaves will be harvested.
The newest leaf grater than 7.0 cm will be sliced off and
frozen in liquid nitrogen. They will then be stored in a
super cool freezer until we are ready to do the chemical
analysis.
Preparation for analysis will consist of the leaf
being cut into 5 mm segments. 5.0 g of the segments will
then be mixed with 10.0 ml of methanol to cover all the
pieces. They must set for two hours while the chemical
compounds in the exudates and some from the gel dissolve
into the methanol. The plant matter can then be removed and
the methanol can be filtered and evaporated off. Only the
compounds from the aloe are left and ready for analysis.
Testing
The resulting aloe solution will be separated through
column chromatography using a solution of Ethyacetate:
methanol: water (100:16.5:13.5) and the barbaloin has an Rf
value of .31-.35 (Chauser-Volfson 2002) (Gutterman 2000).
The desired portion of aloin will be mixed with an exact
portion of methanol and analyzed for the absorption of
light at Λ360-362nm with a photo spectrophotometer
(Chauser-Volfson 2002) (Gutterman 2000). Then we can use
calibration curves to determine the concentration of
barbaloin in each sample. This will be done with each plant
in each stress group as well as a control group. There
should be five plants in each group totaling 30 plants and
samples.
The HPLC/MS will provide us with accurate information
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identifying all of the compounds that are in solution. It
will also provide us with the relative concentration of the
chemicals.
A student’s t-test will be used to find out if there
is a significant difference in concentration of compounds
between the stressed and the non-stressed Aloe. There will
also be a comparison amongst the different forms of stress.
Time frame
It will take about two weeks to order the plants and
plant them, and then I will allow two months for the plants
to stabilize and grow. Then stresses will be applied for
one week then I will extract samples. It is still unknown
how long it will take to process all the samples. It should
take another week to put all the data together and analyze.
The whole project should take 18 weeks I plan to begin in
September 2004 and end in December 2004.
Materials Needed
30 Aloe vera plants
$3.00 each from cactusstore.com
Column chromatography equipment
FGCU has it
Spectrophotometer
FGCU has it
Sand 10 gallons
scavenge
Ply wood
scavenge
Ethyl acetate
FGCU has it
Methanol
FGCU has it
Water
FGCU has it
Alumina
FGCU has it
Salicylic acid
Methyl Jasmonate
Item
Aloe vera Plants
Shipping 20% of plant cost
Miraclegrow Potting Mix (16qt)
Vermiculite (8qt)
21cm Clay pot
Kiddie Pool
Total
Chemical supply
Chemical supply
Quantity
30
90
2
2
30
1
From
cactusstore.com
cactusstore.com
Driftwood Garden Center
Driftwood Garden Center
Driftwood Garden Center
Toys R Us
Cost per unit
$3.00
$0.20
$7.49
$4.99
$1.99
$5.99
Price
$90.00
$18.00
$14.98
$9.98
$59.70
$5.99
$198.65
How this will benefit mankind
Many cosmetic manufacturers have been using aloe
extracts to supplement their products so they may have an
interest in increasing the yield of active compounds. The
Aloe maybe used as a marketing strategy but better Aloe
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will make it a better product.
These methods of stressing plants can be used to
increase the amount of active compounds produced by other
plants. Then more potent medicinal plants can be grown.
Drug prices have been ever increasing and people
without medical insurance cannot afford medicine. Since
many drugs have been developed from compounds produced by
plants, there is a source for affordable medicine by using
the plants only if we can understand the factors involved
with the potency of the plant and dosage. Other than
medicine men of lost cultures, few people understand the
dosages and potencies of herbal medicines. I hope that
these experiments will enlighten us to the optimal growth
and administration of natural medicine.
Further studies conducted in this manner could also
enlighten us to the different compounds produced by
different plants from different types of stress. This
project can be continued with much valuable information to
be obtained.
Biographical sketch
I received an Associates degree in Pre-Veterinary
Medicine form Broward Community College in 2001. In the
summer of 2001 participated in an Anthropology field school
in Mexico in which I studied the medicine of the Mayans. I
continued my education at Florida Gulf Coast University and
joined another Anthropology field school in the summer of
2004 in Honduras where I studied a medicinal drink of the
Garifuna. I plan to graduate in April of 2005 with a
Bachelor of Arts degree in Biology and a minor in
Anthropology. My course work in plant physiology and
organic chemistry provide me with the understandings and
techniques required to perform this proposed experiment.
Contact Information
Jason Carloni
1195 Main St.
Ft. Myers Beach, Fl. 33931
Cell: (239)560-8929
E-mail:jjcarlon@eagle.fgcu.edu
Website: http://student.fgcu.edu/jjcarlon
Mentoring Professors:
Dr. Marilyn Cruz-Alvarez
Dr. Joseph Kakareka
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References:
Chauser-Volfson E, Shen Z, Hu Z, Gutterman Y (2002) Anatomical
structure and distribution of secondary metabolites as a
peripheral defense strategy in Aloe hereroensis leaves. Botanical
Journal of the Linnean Society 138:107-116
Chukwujekwu, J. C., Fennell, C. W., van Staden, J., (2002) Optimization
of the tissue culture protocol for the endangered Aloe
polyphylla. South African Journal of Botany 68:424-429
Taiz, L., Zeiger, E. Plant Physiology 3rd Ed. Sinauer Associates, Inc.,
Publishers, Sutherland, Massachusetts (2002).
Shen Z, Chauser-Volfson E, Hu Z, Gutterman Y (2001) Leaf age, position
and anatomical influences on the distribution of the secondary
metabolites, homonataloin and three isomers of aloeresin in Aloe
hereroensis (Aloaceae) leaves. South African Journal of Botany
67:312-319
Keinänen, M., Oldham, N. J., Baldwin, I. T. (2001) Rapid HPLC Screening
of Jasmonate-Induced Increases in Tobacco Alkaloids, Phenolics,
and Diterpene Glycosides in Nicotiana attenuata. Journal of
Agriculture and Food Chemistry 49:3553-3558
Gutterman, Y., Chauser-Volfson, E., (2000) The distribution of the
phenolic metabolites carbaloin, aloeresin and aloenin as a
peripheral defense strategy in the succulent leaf parts of Aloe
arborescens. Biochemical Systematics and Ecology 28:825-838
Paez, A., Gebre, G. M., Gonzalez, M. E., Tschaplinski, T. J. (2000)
Growth, soluble carbohydrates, and aloin concentration of Aloe
vera plants exposed to three irradiance levels. Environmental and
Experimental Botany 44:133-139
Reynolds, T., Dweck, A. C., (1999) Aloe vera leaf gel: a review update.
Journal of Ethnopharmacology, 68:3-37
Tang, C-S., Cai, W-F., Kohl, K., Nishimoto, R. K. (1995) Plant Stress
and Allelopathy Allelopathy; organisms, processes, and
applications. 142-157
Genet, W. B. M., Schooten, C. A. M. van, (1992) Water requirement of
Aloe vera in a dry Caribbean climate. Irrigation Science 13:81-85
Hart, L. A., van Enckevort, H., van Dijk, H., Zaat, R., de Silva, K. T.
D., Labadie, R. P. (1985) Two functionally and chemically
distinct immunomodulatory compounds in the gel of Aloe vera.
Journal of Ethnopharmacology, 23:61-71
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