Grant Information
| Knowledge Area | Subject of Investigation | Field of Science | Percent |
|---|---|---|---|
| 206 - Basic Plant Biology | 680 - Other products of the forest | 2000 - Chemistry | 100% |
Asystemhas been designed to allow efficient extraction, characterization, and bioactivity screening of medicinal plants. In the past we have experienced difficulty in formulating bioactive extracts, due primarily to poor solubility or limitations of using toxic solventsin bioassay systems. Our added capability now allows us to conduct solubility enhancement experiments and enable the development of formulations that are compatible for bioactivity investigations in cellular and animal models.
Goal: to identify bioactive compounds from medicinal plants and develop formulations to enable their comprehensive evaluations.
Objective 1. Develop effective extraction methods for plants that have promising bioactivity in improving human health (e.g., prevention and treatment of cancer; weight loss, diabetes; and antimicrobial). Plant extract preparations: We have identified a number of plants for complete extraction.A ginger extract will be prepared. Ginger has been indicated to be chemotherapeutic. We did an extraction study to compare the efficiency in recovering gingerols. The exact solubility of gingerols in water is not known but it is no more than 10 microgram per milliliter, which produced no cytotoxicity in our preliminary study. Gingerols are fat-soluble and solubility enhancement will likely bring the concentration to a bioactive level. Hops is another plant that will be extracted. Hops (Humulus lupulus) has been indicated to be antimicrobial due to beta-acidsand could affect microflora balance of the gastrointestinal tract. Hop resin is an oil and not soluble in water. Any solubility enhancement will likely disperse it for exerting bioactivity. Turmeric (Curcuma longa) root extract will also be prepared. Turmeric root extract contains curcuminoids, which as a class has been reported to show a range of medicinal properties as a antioxidant, anti-inflammatory, antiviral, antibacterial, antifungal, anti-angiogenesis and cancer chemoprevention. Curcuminoidsare only soluble to a concentration of 0.35 microgram per milliliter but to inhibit angiogenesis it is required to be around 15 microgram per milliliter.Chromatographic fingerprinting analyses of bioactive extracts: For each active extract that warrants further investigations, a comprehensive instrumental analysis will be performed. The purpose is to gain insights on possible constituents in the extract without having to perform laborious isolation and identification early in the investigation. The laboratory is equipped with two HPLC systems with photodiode array (PDA), ELS (evaporative light scattering), and MS (mass spectrometry) detection capabilities. The nearby chemistry core facilities provide services on Nuclear Magnetic Resonance (NMR) and sophisticated MS chromatography to aid in the characterization process.Objective 2. Develop formulations that are compatible for in vitro and in vivo evaluations. Due to poor solubility, many of the ethanolic extracts or isolated natural compounds are rarely tested accurately for bioactivity. We will first perform solubility enhancement for these poorly soluble samples with natural solubilizers (steviol glycosides such as rubusoside, stevioside, and/or revaudioside A) and DMSO as the control. The use of DMSO is widespread in sample preparations for in vitro bioactivity testing. Bioactivity screening will begin with our in house cytotoxicity assays that will include an appropriate positive control, for example, curcumin. Human cancer cell lines such as prostate carcinoma (PC3), breast carcinoma (MDA-MB-231), and colon adenocarcinoma (HT-29) cell lines will be used. Normal human cancer cell lines will not be used as the compounds we will enhance are known to cause cytotoxicity to human cancer cells. Our aim is mainly to check if our solubility enhancement has potentially changed these properties or not. These cells are available from the LSU AgCenter's cell culture core laboratory. Cells are maintained at 37°C in a humidified atmosphere with 5% CO2 in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum, HEPES, penicillin-streptomycin, sodium pyruvate, L-glutamine, and non-essential amino acids. In vitro cytotoxicity assays will be conducted using the MTS (3-(4, 5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. HT-29, PC3, and MDA-MB-231 cells are added to 96-well plates at 1 x 104 cells/well, respectively, and allowed to adhere overnight. The cells are then treated with various extracts or natural compounds in triplicate wells and incubated at 37°C for 72 hrs. On day three, a 20 μL aliquot of MTS solution premixed with phenazine methosulfate is added directly to each well. Absorbance is then measured at a wavelength of 490 nm using a Bio-Rad Microplate Reader (Hercules, CA). Percent viability is calculated as cell viability relative to vehicle-treated control (100%). This assay has been used in the lab for generating cytotoxicity data exemplified in our recent publications. ANOVA with repeat measure analysis, if applicable, will be used to analyze the data using SAS software. Different treatment means will be separated using Tukey's test. This is particularly suitable when comparing many means as the best way to avoid type 1 error. If the number of comparisons is limited, a Bonferoni adjustment dividing the alpha level by the number of comparisons would be used to reduce the type 2 error with Tukey by using limited contrasts or using a few comparisons within a LSD mean comparison test.Objective 3. Determine the stability and explore the mechanisms of enhanced formulations. Stability, along with solubility, is an important factor for adequate drug exposure. Each bioactive formulation will be assessed for storage and application stability. The bioactive formulations in powder form will be evaluated for storage stability over time and under normal storage conditions (e.g., room temperature, ambient humidity, ventilation). Storage time for three months is often a benchmark to predict storage stability over longer time. In addition to normal storage conditions, a wider range of storage conditions will include heat and freeze, and dry (e.g., in desiccators) and humid. Water adsorption of the samples will be evaluated bya gravimetric method. Application stability evaluation will include the reconstitutability into physiologically relevant aqueous solutions from powder, chemical and physical stability of the active compounds in solubility-enhanced form over time (e.g., minimal two hours to as long as three days) and during dilutions to application concentrations. At each evaluation point, samples will be taken for instrumental analyses following similar protocols developed for previously completed studies. The complexes formed between a solubilizer and an active compound(s) will be characterized. These include particle size, zeta potential, FT-IR, and differential scanning calorimetry (DSC). Mechanism of solubility enhancement will be explored based on these data.
Target Audience
The use of natural ingredients, especially those that are generally regarded as safe or GRAS, such as essential oils of thyme and clove, for managing plant productions is highly desirable from the angle of environmental friendliness and animal/human safety. Therefore, identifying natural ingredients that have antimicrobial and insecticidal activities and are GRAS became the focus of our investigation. We have tested some of the natural ingredients in controlling plant pests (e.g., bacterial diseases in tomatoes, ball moss on ornamentals). Uses of natural ingredients in pest control and prevention are of great interest in organic agriculture, home vegetable gardening, and urban landscaping. Chemical pesticides use synthetic ingredients that may be more efficacious and long-lasting; however, those good features also could impose adverse effects on human health and the environment because they are more toxic and reside in the environments longer. We have showcased some of the results, and likely, users are receptive to the projects we did. Therefore, users of organic pesticides or naturally derived pesticides are potential audiences to my research results. We collaborated with plant pathologists, entomologists, and horticulturalists. A turmeric extract was developed using various methods of extraction. The extract was analyzed for curcumin content (the chemical marker). The turmeric extract has been reported to provide many health benefits, including anti-inflammation, anti-bacteria, anti-oxidation. Beyond this project, I plan to continue the turmeric study and move it to a targeted audience of nutraceutical developers and the general public, especially the elderly population, for consumption.
Changes / Problems
Nothing Reported
Training & Professional Development
The final year embraced many undergraduate students at the LSU campus who showed genuine interest in nature's ability and tools. The chemical tools inside plant materials are fascinating, and their discovery enhanced our investigations. The research-based on botanical ingredients is disseminated to undergraduate and graduate students on campus. The project was seen as a platform to get students involved. Five undergraduate student research projects were conducted during the final project year. A summer workshop was held to train five undergraduate students on the preparation of extraction and instrumental analysis. Despite the pandemic's negative effect, a growing interest in involvement has been observed in the last two years. Moreover, every undergraduate student research project included a bioassay component, allowing students to appreciate collaborative research leading to practical applications.
Dissemination Streams
We disseminate our research results through publications. One good example is the use of essential oils to control ball moss that decreased the growing vigor of ornamental plants. We disseminated our results to LSU facility services that manage the campus landscape and Baton Rouge Green (a non-profit organization). We also reached out to local nurseries about our use of natural compounds for safely managing plant diseases.
Next Reporting Steps
Nothing Reported
Target Audience
We have focused on validating natural ingredients that have antimicrobial and insecticidal activities. Uses of natural ingredients in pest control and prevention are of great interest in organic agriculture and home vegetable gardening. Chemical pesticides use synthetic ingredients that may be more efficacious and long-lasting, but those good features also impose adverse effects on human health and the environment because they are more toxic and reside in the environments longer. Therefore, users of organnic pesticides or naturally derived pesticides are potential audience for my research results. To get there requires collaborations from plant pathologists, entomologists, horticulturalists, and environmental toxicologists thus they are the interim audience for my research. Other audience include undergraduate students who embrace natural ingredients and may become future users and researchers of these ingredients.
Changes / Problems
Nothing Reported
Training & Professional Development
The research based on botanical ingredients is disseminated to undergraduate and graduate students on campus. It provides inspiring thoughts over future career opportunities in plant protection and human health. Three undergraduate students received some laboratory training in botanical sample preparation, filtration, extraction, and instrumental analysis. More undergraduate training is anticipated for the next year.
Dissemination Streams
We disseminate our research results through publications when we can. However, we were very open to let our research effort in discovering natural active ingredients and developing natural formulations known to others that have issues with the purity and solubility of the active ingredients. A lot of reaching out activities were conducted to find appropriate application areas such as food sanitation, plant bacterial and fungal diseases, and veterinary medicine. Through these disseminations to the research community we were able to identify collaborators, some of whom were interested in forging future joint projects. We also let our analytical capability be known to educate potential collaborators.
Next Reporting Steps
Due to the COVID-19 pandemic, the project's completion has been delayed. In the final extended year we will make major efforts to find collaborators that will teste the formulated natural active ingredients for insecticidal and antimicrobial effectiveness. We have identified some areas to test including but not limited to plant protection from bacterial and fungal diseases, natural insecticide for organic and home gardening, prevention of oral pathogens from infestation, sanitation of veterinary environments, animal specimen preservation, and others to be explored. More undergraduate students will be supported through the ongoing research efforts. <br><br>
<br>What was accomplished under these goals? Plant oils such as essential oils, neem oil, pyrethrum oil have long been used to control or prevent insect and microbial infestations. Essential oils are mostly steam distilled and thought to be pure from a volatility point of view. However, essential oils are in fact extracts and remain a mixture of many components. Most of the antimicorbial properties are a reflection of an overall effect, although a major component of an essential has been discovered that represents that particular essential oil. Clove essential oil, for example, is primarily composed of eugenol. Thyme essential oil is primarily composed of thymol. Tea tree essential oil has a less dominant single compound than eugenol or thymol in relation to its essential oil. We took a further step in testing the single compound (pure single entity) and its residing essential oil (a mixture of entities) for eugenol and thymol. We found eugenol or thymol can single handedly represent the antimicorbial activity of each essential oil. We further analyzed each essential oil and found eugenol was dominant at 78% and thymol was dominant at 60%. These results validated that eugenol and thymol are active ingredients and most like have represented the overall antimicrobial activities. One of the challenges in testing essential oils in standard antimicrobial assays is to disperse the oil in the aqueous medium. There are conventional ways to faciliate the dispersion by using organic solvents such as ethanol or chemical surfactants such as polyethylene glycol 400. These methods not only bring potential toxicity into the test results but also are problematic into the next phase of developmental research. Our research took a unique approach that uses active ingredients from plants to faciliate the dispersion. It was a big step forward and a major accomplishment by developing an all plant-based formulation for current research and for future application research. Next year, we will find collaborations to test some of the fomulations in agricultural fields, human oral health, veterinary sanitation, and others to be explored. I continued to make an effort in supporting the citrus greening disease research by responding to the need in preparing active ingredients for preliminary evaluation. We also sent samples to test the phytotoxicity in apple trees that were subjected to the fireblight disease during blooming at the largest apple growing area of the country in Wenatchee, Washington. I have collaborated with extension faculty member on the use of solubilized product on walnut flight disease. This accomplishment opened the door to collaboration to test the efficacy in controlling or preventing the fireblight disease. In addition to the above applications in agriculture, we continued to collaborate with researchers in the medicinal benefit of green tea materials by providing instrumental analysis. <br><br><b>Publications</b><br>
Target Audience
We have focused on natural ingredients that have antimicrobial and insecticidal activities. Uses of natural ingredients in pest control and prevention are of great interest in organic agriculture and home vegetable gardening. Chemical pesticides use synthetic ingredients that may be more efficacious and long-lasting, but those good features also impose adverse effects to human health and the environment because they are more toxic and reside in the environments longer. Therefore, users of organnic pesticides or naturally derived pesticides are potential audience to my research results. To get there requires collaborations from plant pathologists, entomologists, horticulturalists, and environmental toxicologists thus they are the interim audience to my research.
Changes / Problems
Nothing Reported
Training & Professional Development
The research based on botanical ingredients is disseminated to undergraduate and graduate students on campus. It provides inspiring thoughts over future career opportunities in plant protection and human health. People who worked on this project were trained in botanical sample preparation and learned some insights on how the sample they prepared was tested in bioassays. The best learning experiences involve collaborations of people from different disciplines, which allow the trainees to gain appreciation over the nature of multiple-desciplinary collaboration.
Dissemination Streams
We disseminate our research results through publications when we can. However, we were very open to let our research effort in discovering natural active ingredients and developing natural formulations known to others that have issues with the purity and solubility of the active ingredients. A lot of reaching out activities were conducted to find appropriate application areas such as food sanitation, plant bacterial and fungal diseases, and veterinary medicine. Through these dissemination to the research communities we were able to identify collaborators, some of whom were interested in forging future joint projects.
Next Reporting Steps
Next year will be the last year of the project. We will make major efforts to find collaborators to have the formulated natural active ingredients tested for antimicrobial effectiveness. We have identified some areas to test including but not limited to plant protection from bacterial and fungal diseases, natural insecticide for organic and home gardening, prevention of oral pathogens from infestation, sanitation of veterinary environments, and others to be explored. <br><br>
<br>What was accomplished under these goals? Essential oils have long been used to control or prevent microbial infestation. Essential oils are mostly steam distilled and thought to be pure from volatility point of view. However, essential oils are in fact extracts and remain a mixture of many components. Most of the antimicorbial properties are reflection of overall effects, although a major component of an essential has been discovered that represents that particular essential oil. Clove essential oil, for example, is primarily composed of eugenol. Thyme essential oil is primarily composed of thymol. Tea tree essential oil has a less dominant single compound than eugenol or thymol in relation to its essential oil. We took a further step in testing the single compound (pure single entity) and its residing essential oil (a mixture of entities) for eugenol and thymol. We found eugenol or thymol can single handedly represent the antimicorbial activity of each essential oil. We further analyzed each essential oil and found eugenol was dominant at 78% and thymol was dominant at 60%. These results validated that eugenol and thymol are active ingredients and most likely have represented the overall antimicrobial activities. One of the challenges in testing essential oils in standard antimicrobial assays is to disperse the oil in the aqueous medium. There are conventional ways to faciliate the dispersion by using organic solvents such as ethanol or chemical surfactants such as polyethylene glycol 400. These methods not only bring potential toxicity into the test results but also are problematic into the next phase of developmental research as they are not compatible with the environment. Our research took a unique approach that uses active ingredients from plants to faciliate the dispersion. It was a big step forward and major accomplishment by developing an all plant-based formulation for current research and for future application research. Next year, we will find collaborations to test some of the fomulations in agricultural fields, human oral health, veterinary sanitation, and others to be explored. I continued to make an effort in supporting the citrus greening disease research by responding to the need in preparing active ingredients for preliminary evaluation. We also sent samples to test the phytotoxicity in apple trees that were subjected to the fireblight disease during blooming at the largest apple growing area of the country in Wenatchee, Washington. This accomplishment opened the door to collaboration to test the efficacy in controlling or preventing the fireblight disease. In addition to the above applications in agriculture, we continued to collaborate with researchers in the human medicine area, e.g., ceramide to reverse resistant cancer cells for enhanced chemotherapy. <br><br><b>Publications</b><br>
Target Audience
At the current stage researchers are targeted audience for collaborations intestingantimicrobial formulations. The discovery of bioactive and solubilizing compounds from medicinal plants has prompted the use of natural antimicrobial compounds of botanical origins for potential applications in food sanitation. The work developed in this lab needs to reach out to application scientists such as sprouts sanitation or new formulations for better mosquitos control.
Changes / Problems
Nothing Reported
Training & Professional Development
The people who work on this project have the opportunities to be trained in botanical sample preparation for antimicrobial testing food, plants, and animals. Cross-dissciplinary collaborations servedas a great training groundfor real case studies that involve expertise from both chemistry and biology.
Dissemination Streams
The results have been submitted for publications, some of which have been published or accepted for publications. In addition, there are real efforts made to reachout to other researchers that are interested in using new and natural antimicrobials in various applications such as food sanitation, plant bacterial diseases, and veterinary medicine. During this reporting period we worked with organic sprouts researchers and prepared and submitting a USDA grant applicaiton.Through these dissemination efforts we were able to identify collaborators and forge future joint projects.
Next Reporting Steps
We will continue to test our samples for antimicrobial activities and further strengthen collaborations that have been established. Although new interests in collaborations emerged last year, we will continue to focuson food ingredients that have antimicrobial properties and enable them to be applied in immersion, spray, injection, and other forms of delivery. Antimicrobial applications for postharvest sanitation will be expolored for fruits, vegetables, animials, human dental health, and plant health affected by bacterial agents. Identification and purification of neem oil compounds will also be pursued in the coming year. <br><br>
<br>What was accomplished under these goals? Many essential oils are potential antimicrobial agents. We have been working with several essential oils and validated their antimicrobialproperties against food borne bacteria and companion animals in veterinary settings. Beer hop extract was prepared and standardized. The standardized extract was tested against veterinary bacteria in an effort to find alternative preservatives that are less irritating to the vet animals when applied to infected eyes. Some plant essential oils were also purified and tested. These include the essential oils of tea tree, cinnamon bark, clove, thyme, geranium, and citrus peel.We then performed solubility enhancementand developed water-soluble formulations. Clove oil like other essential oils was not miscible with water and did not disperse well. Using our botanical solubilizers we were able to disperse it better in water and allow antimicrobial testing. We also purified pyrethrum oil and tested its water dispersion against insect pests in laboratory settings. Finally, we employed botanical ingredients to disperse awidely used antibacterial agentin an effort to enhance its absorption into citrus trees to control HLB or citrus greening disease. <br><br><b>Publications</b><br>
Target Audience
Currently the target audience is researchers and developers of antimicrobial applications, as well as those who are interested in natural antimicrobials. The discovery of bioactive compounds from medicinal plants has led to the use or re-use of botanicals that are in human uses. Some formulations employing natural antimicrobials have the potential for organic food production.
Changes / Problems
Nothing Reported
Training & Professional Development
Faculty and studentsworking on this project havebeen trained in botanical sample preparation for antimicrobial testing.
Dissemination Streams
Results have been submitted for publications, some of which have been accepted. In addition, the principal investigator has reached out to other researchers that are interested in using new and natural antimicrobials.
Next Reporting Steps
We will continue to follow up on the natural antimicrobials that have shown promises and better results than the currently available formulations. The focus will be placed on food ingredients that have antimicrobial properties and enable them to be applied in immersion, spray, injection, and other application forms. Antimicrobial applications will also move into postharvest sanitation of fruit and vegetables. Dental health will be another area to work on in the next year. <br><br>
<br>What was accomplished under these goals? We have discovered and enabled the use of some plant extracts for inhibiting bacteria. Sweet leaf extract was found to inhibit mastits-causing bacteria of both gram positive and negative types. Beer hop ethanol extract was also prepared and purified from hop botanicals, which was found to be antibacterial against gram positive, but less so against gram negative bacteria. Plant essential oils were also purified and tested. These include tea tree oil, cinnamon bark, clove leaf, thyme, and limonene. We then performed solubility enhancement for some and developed water-soluble formulations. Tea tree oil like other essential oils was not miscible with water and did not disperse well. Using our botanical solubilizers we were able to turn the oil into a water-soluble formulation. Hop ethanol extract was very potent against gram-positive bacteria but it precipitated when ethanol was replaced with water. Again, by employing botnaical solubilizers hop extract was solubilized into water. Tea tree oil killed all bacteria in 10 minutes of exposure, bioequivalent to the positive control of iodine. Moreover, we were able to scale uplaboratory volume to small scale field tests and allow collaborations into the application phase. <br><br><b>Publications</b><br>