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).
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 vigorouslyuprooting 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.
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 acidand 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 mlof 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 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 inconcentration 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 equipmentFGCU has it
Spectrophotometer FGCU has it
Sand 10 gallons scavenge
Ply woodscavenge
Ethyl acetate FGCU has it
Methanol FGCU has it
WaterFGCU has it
AluminaFGCU has it
Salicylic acidChemical supply
Methyl JasmonateChemical supply
Item / Quantity / From / Cost per unit / PriceAloe vera Plants / 30 / cactusstore.com / $3.00 / $90.00
Shipping 20% of plant cost / 90 / cactusstore.com / $0.20 / $18.00
Miraclegrow Potting Mix (16qt) / 2 / Driftwood Garden Center / $7.49 / $14.98
Vermiculite (8qt) / 2 / Driftwood Garden Center / $4.99 / $9.98
21cm Clay pot / 30 / Driftwood Garden Center / $1.99 / $59.70
Kiddie Pool / 1 / Toys R Us / $5.99 / $5.99
Total / $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 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:
Website:
Mentoring Professors:
Dr. Marilyn Cruz-Alvarez
Dr. Joseph Kakareka
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 Society138:107-116
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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