DISCLAIMER
All extractions I do are for mere educational purposes to learn about phytochemistry. No alkaloids were completely isolated, and the solutions containing them were disposed of after seeing their fluorescence. This website DOES NOT encourage alkaloid consumption or extraction, and STRONGLY ADVISES each viewer to consult their local restrictions before doing any experiment.
Personal Introduction
Ever since my grandfather gave me a mushroom book when I was around five years old, I have been very, very, intensely passionate about nature. What started with learning how to differentiate a Boletus from an Agaricus mushroom has grown into knowing a plethora of scientific names and travelling hundreds of kilometres to see an endangered plant. Parallel to this, ever since I realised I had hands, I have been joining atoms with my molecular model kit and inventing molecules too big to fit on an A4 sheet of paper. Here, I share with you one of the many testaments to my passion as a witness to the most fundamental characteristic of science: living in a perpetual cycle of melting and recasting to be at peace with each new truth unveiled from nature.
Extracting a molecule from the seeds of a desert-dwelling plant that’s both fluorescent and psychoactive? Is it a tale? NO! It’s reality.
Today, you will learn a bit more about the incredible romance between plants and chemistry alongside my journey as a paparazzi that adores documenting every one of their secret encounters. Embrace yourself for me to take you on this (quite literally) intoxicating love story. You may want to skip the extraction part, as it is tedious and dull (though it is written to maintain scientific rigour), and proceed to the“The Moment of Truth…” heading.
Peganum harmala, also known as Syrian Rue, is a herbaceous perennial plant from the family Nitrariaceae, native to the Mediterranean region and extending eastward to Central Asia. It inhabits saline, dry, and disturbed areas, even those that have been subject to eutrophication. It has been used to make traditional medicines, dyes, and incense, among others.
Like the caapi vine, Banisteriopsis caapi, Syrian rue contains a relatively high concentration of alkaloids from the β-carboline class, including harmine, harmaline, and harmalol. Some of these alkaloids are Monoamine Oxidase Inhibitors (MAOI) and therefore prevent our body’s enzymes (Monoamine Oxidases) from degrading hallucinogens like N,N-Dimethyltryptamine (DMT), making its effects stronger.
Last year, I was paging through articles on Google Scholar to find a research topic for a school task. I was left utterly dumbfounded when I discovered that harmaline and harmine are fluorescent, and what’s more, that I could easily extract these compounds myself in the lab. My school wasn’t impressed. Little did they know that a few months later, I would still do the extraction anyway.
And here begins the hunt…
Hunt
Is there anything more magical than the existence, the spontaneously arisen existence, of life? Stellar and supernova nucleosynthesis (the fusion of lighter nuclei to yield heavier nuclei) gave rise to almost all of Earth’s elements, including the soil’s nutrients, the air’s carbon, and water’s oxygen and hydrogen. Nuclear fusion (of hydrogen atoms to give helium) in the Sun powers almost all of life on Earth, which definitely includes ours. Plants, like artists of the atom, utilise enzymes to combine the Sun’s energy and Earth’s elements, creating a vast array of chemical masterpieces, such as our beloved harmaline and harmine.
Consequently, I wanted to do everything from scratch to really admire the beauty of evolution. First, I visited a scrubland area in southern Madrid, where the plant had been spotted on iNaturalist. I went in shorts (clever me), offering ticks a free buffet and thistles a chance to paint my legs red, all to find not a single plant. With a sliver of hope, I travelled almost 200km to a town named Molina de Aragón in Guadalajara, Spain, where there was an iNaturalist observation of this plant, hoping to get seeds… and guess what?
Not a plant. Again, all my effort scrambling through chest-high vegetation (not pictured) was absolutely futile.
School was ending soon, and hence, my access to the laboratory. I urgently needed the seeds to carry out the extraction. I had no other choice but to buy them online.
Extraction
Grinding and defatting
The first step is grinding the seeds. This was done with a mortar and pestle. Then, they were defatted using pure n-hexane, CH3(CH2)4CH3, as it is a very apolar molecule due to the small electronegativity difference between carbon and hydrogen.
The seed and n-hexane mixture was stirred and then filtered. The filtrate containing the n-hexane was discarded, and the seeds were left to dry.
Acid-base extraction
The now-defatted seeds were placed in a dilute solution of acetic acid (CH3COOH), the main acid in vinegar, to increase the solubility of the alkaloids in water. The reaction mechanism is shown below in my own drawing of harmine’s protonation. The nitrogen in harmaline and harmine’s pyrrole accepts a proton (H+) with its lone pair of electrons, becoming positively charged and increasing the molecule’s solubility in water, a polar solvent.
The mixture was then filtered. The residue (seeds) was discarded, and the filtrate was collected. This part contained all the desired alkaloids in their salt form. Now, using the opposite process, by increasing the pH to around 9 with sodium carbonate (Na2CO3), the acidic salts are converted to our desired harmine and harmaline-free bases (the neutral form of the alkaloid, such as the widely known cocaine freebase!). All of this was monitored using a pH meter, which measures the concentration of protons (H+) in solution using electrical conductivity.
Organic solvent extraction
The freebase harmine and harmaline solution was poured into a separatory funnel. Ethyl acetate, which is a less polar solvent than water, was added to get the relatively nonpolar alkaloids to migrate to this organic solvent. The two layers were mixed thoroughly and then left to separate. The aqueous and organic layers were separated and stored separately.
The moment of truth…
Like a kid impatient to open a present on a Christmas morning, I couldn’t wait to see if I really had extracted any alkaloids, and even more, I was anxious to see their fluorescence, which requires ultraviolet (UV) light of around 300nm.
I shone an old UV torch I found hidden in some old drawer on each of the solutions (organic and aqueous), but due to the brightness of the room and the visible light the torch was emitting, I couldn’t see any effect. Fortunately, months before, I had spent countless afternoons on a very special instrument working on my school chemical investigation: the mighty spectrophotometer. This instrument generates light of a discrete wavelength and passes it through a sample, measuring the amount of light absorbed. Water, which is (almost perfectly) transparent, for example, would absorb 0% of light at 500nm (greenish-blue).
I could programme it to pass ultraviolet light (I used 365nm) through my sample, and if I saw ANY LIGHT coming from it, then that’d imply fluorescence, as UV light is invisible to our human eyes. My molecule would be converting UV light to visible light!
I switched on the spectrophotometer. I had to wait 20 very long minutes for it to heat up (it uses a deuterium lamp to generate UV light). As an effort to make the laboratory as dark as possible, I took a poster of a scientific project I had presented at a conference with my friend (see the silver mirror post) and stuck it to a window that the blinds didn’t cover. I closed all the doors and switched off every single light, except the one of hope inside me.
Then, I placed my samples, one cuvette with the organic extract and another with the aqueous extract, inside the spectrophotometer, set the wavelength to 365nm, covered myself and the samples with a lab coat, and…
IT WORKED!
This minuscule speck of light my sample was re-emitting exhilarated me. This confirmed the presence of harmaline and/or harmine in the solution.
In that moment of ecstasy, I threw away the aqueous solution (which supposedly had no alkaloids) and bottled the organic one to keep it as a souvenir of my experiment. However, after this, I realised that only the aqueous solution fluoresced (probably due to an inadequate pH adjustment and a poor freebase conversion). I had thrown away all the alkaloids I had just spent hours extracting… luckily, I had no intention of using them.
Scientific Explanation
A compound, depending on its chemical structure and therefore its molecular orbitals, may absorb photons of a specific, discrete energy, which is related to its frequency by Planck’s equation:
E=hv
Where E is the energy, h is Planck’s constant, and v is the frequency of the light.
When a molecule absorbs a photon, it becomes excited, and the conformation of the molecular orbitals changes (some become antibonding, for example). When fluorescence occurs, the excited electron does not change its spin when it is promoted to higher levels and stays in a singlet state, denoted S, which means that all electrons are paired and hence that the net spin angular momentum is 0. The ground singlet state is designated as S0, and the excited states are labelled as S1, S2, S3, and so on.
Non-fluorescent molecules, when excited, return to their ground state via nonradiative transition, whereby the energy is released not as light, but usually as heat. On the other hand, fluorescent molecules, despite generally undergoing nonradiative transitions from higher excited states (S2, S3…) to the lowest excited state (S1), do release a photon when returning from S1 to their ground state (S0), as can be seen in the subsequent Jablonski diagram. As a side note, phosphorescence, a related but distinct phenomenon, is excluded from this discussion to keep it simple.
As our fluorescent molecules (harmine and harmaline) lose some of the energy they absorbed via nonradiative transition, the photon emitted during fluorescence is of lower energy and frequency (and hence of longer wavelength) than the one absorbed. This phenomenon is known as the Stokes shift, which explains why I could see the light my sample was emitting, which falls within the visible range (380nm to 750nm), rather than in the UV range (100nm to 400nm), like the photons that excited my sample in the spectrophotometer.
Conclusion
The minute speck of light that my sample emitted was a testament to something often overlooked in our quotidian lives: the truly incredible predictive power of Science. Owing to the invisible effort of a myriad of scientists and philosophers who laid (and lay) the foundation for our understanding of the world, we can know a fact so seemingly simple yet truly complex at heart (quantum mechanics, molecular biology, neuroscience, chemistry): that a plant’s chemical is psychoactive and will fluoresce under UV light.
Nature, like the peak of a towering mountain hidden by clouds, is perpetually enshrouded by an essence of change. In its never-ending chase, Science unveils fragments of its rugged terrain and shows us that even though the admirer and the admired do not share a synallagmatic contract, sometimes Nature does delight us with a glimmer of hope that we might know something of the intimidating cosmos we inhabit.
—Own
