Isolating Mesembrine Alkaloids form Sceletium tortuosum for Potential Dosage Studies to Determine Candidacy for Inhibition of Serotonin

Reuptake Pumps to Treat DepressionDavid Santeliz, Student Researcher, University of Redlands, CA, 92373

Research Advisor Dr. David SchrumAbstractMany plants in the world have been key in deriving chemical compounds for medicinal use; Sceletium tortuosum, a plant indigenous to South Africa, is no exception for its desirable properties in the application of depression. Unlike most benzodiazepines (BZD) and other antidepressants, selective serotonin reuptake inhibitors (SSRIs) are safer and have been shown to have less side-effects than their BZD counterparts.  Sceletium tortuosum is a succulent shrub that when dried and fermented possesses biologically active alkaloid compounds that may be extracted using various acid digestions and organic extractions to collect the drugs of interest.  These four psychoactive alkaloids are isomers of Mesembrine with very similar chemical structures, making separating them an analytical challenge.  The goals of this project were to see if we could isolate and characterize these four compounds of interest.  Acid digestions and organic extractions utilizing dichloromethane were performed on commercially obtained Sceletium tortuosum samples.  To isolate each compound TLC separations were conducted using a dichloromethane/ methanol/ ammonium hydroxide solvent system.  Gas Chromatography-Mass spectrometry (GC/MS) analyses were also performed with the samples to correctly identify each of the four Mesembrine analogs as well as to determine their % by weight from the commercial sources.  The optimum TLC solvent system was determined to be 93% dichloromethane/7% methanol/0.5% ammonium hydroxide based on experimentally obtained Rf factors.  GC-MS data obtained was also used to further identify each of the four alkaloid compounds as compared to mass spectra found in the literature.  Future work will entail scaling up the extraction process to obtain larger quantities of the four alkaloids to be used in potential dosage studies.  These dosage studies will help to understand whether a single alkaloid or all play a part in eliciting the desired psychoactive properties in treating depression. 

Introduction

Methodology

Acknowledgements/ReferencesAcknowledgements: Student predecessors: Robert Hendry, and Matt Brown, Research Advisior: Dr. David Schrum, Collaborator: Dr. John Krstenansky and the Keck Graduate Institute, Dr. Wachs, University of Redlands Chemistry Department, Summer Research Program along with fellow Donors

References: 1. E.A. Shikanga et. al. Validated RP-UHPLC PDA and GC–MS Methods for the Analysis of Psychoactive

Alkaloids in Sceletium tortuosum. South African Journal of Botany. 2012, 82, 99–107 2. National Institute of Mental Health. Mental Health Information: Depression.

http://www.nimh.nih.gov/health/topics/depression/index.shtml#part_145394 (Accessed April 3, 2015).

1. Extraction• Raw fermented S. tortuosum (kanna) was weighed added into a mixing bowl.• A 1: 2 ratio of kanna to 0.05M sulfuric acid was used to suspend raw plant in acid solution. • A paint strainer was used to filter raw poultice of plant• Filtrate was re-filtered via Celite and vacuum filtration.• Transparent-brown filtrate was collected in an Erlenmeyer flask. • Filtrate was washed using several washes of dichloromethane (DCM) in a separatory funnel, number of washes

dependent on volume of filtrate.• Organic layers where collected and mixtures kept in Erlenmeyer flasks. • Organic filtrates were rotary evaporated and oils collected in round bottom flasks.• Oils were suspended in acetonitrile and IR, and GC-MS analysis taken of oils.

2. Separation• Thin layer chromatography (TLC): Separation efficiency was determined to maximize optimal solvent concentration

for separation in massive scale separation. Analyte is UV active therefore separation was identified under UV light as dark spots on TLC.

• Solvent System: DCM: Methanol: Ammonium Hydroxide

3. Identification• Gas chromatography-mass spectroscopy (GC-MS) to identify alkaloid compound with respect to reference values

(Shikanga et al, 2012)

1. TLC Separation• Separation- TLC: Our data vs Literature RF factors.

Results

Conclusion & Future Work1. TLC data presented 3 spots for JK131203-05 further validated as 3 separate alkaloids via GC-MS. Alkaloids were

identified as mainly mesembrine and partial concentrations of mesembrenone. The ideal solvent ratios to separate these compounds was 9.5 DCM: 0.5 MeOH: 0.05 concentrated NH4OH.

2. TLC data presented 4 spots under UV for JK131203-08 further validated as 4 separate alkaloids via GC-MS. Alkaloids were identified as mainly mesembrenol, and smaller concentrations of Mesembrine, mesembranol, and mesembrenone. The ideal solvent ratios to separate each compound on TLC was 9.3 DCM: 0.7 MeOH: 0.05 NH4OH.

3. Recently determined solvent concentrations will be essential to use in mass scale separation of organic filtrates from large quantities of plant material to isolate each alkaloid in separate tubes for single dosage studies. Using concentrations on Automated Column Chromatography for mass scale alkaloid separation.

4. To verify whether desired alkaloid is present NMR and IR will be used for identification techniques post extraction. 5. High performance liquid chromatography (HPLC) will be used for post-dosage studies to determine the levels of

alkaloid present in brain serum of mice and understand what alkaloid are metabolized in the body. Levels of alkaloid give insight into the alkaloid compounds that interact with the brain. With this blood analysis the mystery behind whether a single alkaloid if not all are responsible for eliciting the therapeutic properties in the brain regarding anxiety and depression.

1. Depression • A Central Nervous System (CNS) Disorder.• Effects approximately 6.7% of US adults, average age 32 and females being 70% more

prone than males2.• Symptoms: Broad spectrum of psychological and emotional changes• Causes: Decrease production therefore activity of Serotonin.

2. Sceletium tortuosum (S. tortuosum):• A very ancient people, the Koi-San of South Africa, used this succulent white daisy-

producing plant to help alleviate a broad spectrum of ailments. Just recently was it introduced to the western world of medicine, this kanna as known to the villagers is Scelentium tortuosum belonging to the family Mesembreanthemacea1.

3. Compounds of Interest:• Further research on its psychoactive compounds lead to the discovery of four alkaloid

molecules: mesembrine, mesembrenone, mesembrenol, and mesembranol.

1. These properties bound to radio-ligands have been proven to show inhibitory properties towards serotonin reuptake pumps in the pleasure centers of the brain and anti-inflammatory properties towards PDE4b enzymes in the upper respiratory tract.

2. These effects posses medicinal value in the field of auto-immune diseases and neurological disorders primarily clinical anxiety and depression.

3. Each compound is close in molecular structure, therefore the challenge to separate each structure stems from this molecular property.

4. With no avail to separate these compounds it is imperative to improve separation techniques to understand individual interaction of each alkaloid with the receptors

5. It is vaguely understood whether each alkaloid elicits its own effect or whether the therapeutic properties stems from the presence of all four alkaloids in the brain. The needs to improve separation methods in order to isolate these alkaloids is imperative if one is to get to the source of the plant’s psychoactive properties.

Results Identification

2. Identification

• Identification- GC-MS: Our data vs Literature

E.A. Shikanga Et. al. Validated RP-UHPLC PDA and GC–MS methods for the analysis of psychoactive alkaloids in Sceletium tortuosum. South African Journal of Botany. 2012 82 99–107 (accessed January 2015)

CNSforum. Antidepressant Drugs Mechanismshttps://www.cnsforum.com/educationalresources/imagebank/antidepressants/drug_ssri_norm (accessed April 3,2015)

Hardvard. Health: Reward Center of Brain http://www.health.harvard.edu/media/content/images/newsletter/M0711a-1.jpg ( accessed April 3, 2015)

Winkipedia. Mesemrbine. http://en.wikipedia.org/wiki/Mesembrine. (accessed June 13, 2015).

Chemistry Connections. UV TLC. http://chemconnections.org/organic/chem226/Labs/TLC/TLC-uv-vis-plate-08.gif (Accessed June 2015)

UC Davis Chem Wiki. Gas Chromatography. http://chemwiki.ucdavis.edu/Analytical_Chemistry/Instrumental_Analysis/Chromatography/Gas_Chromatography ( accessed Feb 3, 2015).

Mass Spectroscopy Data

Gas Chromatography Data

Kanna oil eluent with 4 alkaloid compounds GC peaks from JK131203-08 column chromatography fraction.

JK131203-05 9.5:0.5:0.055% MeOH

JK131203-05 9.7:0.3:0.053% MeOH

RF factors w/5% MeOH:0.91 0.78 0.44, 0.88 0.74 0.56

RF factors w/3% MeOH: 0.81 0.73 0.67 0.37, 0.82 0.73 0.65 0.38

Collaborator’s RF:0.44 0.53 0.64

JK131203-08 9.2:0.8:0.058% MeOH

JK131203-08 9.3:0.7:0.057% MeOH

RF factors w/8% MeOH:0.77 0.67 0.57,0.74 0.67 0.56

RF factors w/7% MeOH: 0.92 0.81 0.69 0.55, 0.95 0.82 0.70 0.56

Collaborator’s RF:0.27 (major)

Solvent system:

DCM: MeOH: NH4OH

Major peaks pertinent to each alkaloid:

Mesembrenone: 287 m/z

Mesembrine: 289 m/z

Mesembrenol: 219 m/z

Mesembranol: 290 m/z

Peak 1: Mesembrenol (219, 77, 70, 42)

Peak 2: Mesembranol (290, 219, 70, 42)

Peak 3: Mesembrine (288, 289, 218, 96,42)

Peak 4: Mesembrenone (287,70,42)