Science: Sanguinarine

 

Mexican Poppy (Argemone mexicana, Papaveraceae)

Sanguinarine is the active toxin of the Mexican poppy plant (Argemone mexicana), and it is responsible for the deadly human disease, epidemic dropsy. Sanguinarine is a benzophenanthridine alkaloid. Examine the chemical structure of sanguinarine carefully in Figure 1, I’ve highlighted its benzophenanthridine skeleton in blue. A benzophenanthridine is a polycyclic (many rings) heteroaromatic compound (containing  O, N, and/or S atom in a ring) comprising of four hexagonal rings merged back to back, which I’ve labelled as A, B, C and D. Because all four rings of benzophenanthridine are connected to one another, electrons can delocalise and zip through the whole structure, a phenomenon chemists call extended conjugation. Highly conjugated molecules like benzophenanthridine absorb visible light, and they appear brightly coloured. This is the reason why sanguinarine is red in colour (sanguis is Latin for blood). Besides, because benzophenanthridine is aromatic, the sanguinarine molecule is mostly planar in 3D space.

Figure 1: Chemical structure of sanguinarine and dihydrosanguinarine.

Take note that ring A of sanguinarine contains a nitrogen atom, and it’s what makes sanguinarine an alkaloid. Remember that alkaloids must contain at least one nitrogen atom, and it is the nitrogen that confers most alkaloids alkaline property. However, sanguinarine’s nitrogen atom bears a positive charge (red symbol), and that renders sanguinarine more or less neutral in pH. Besides, the positively charged nitrogen is double bonded to a carbon [HC=N+], and this moiety is called an iminium cation (Figure 3). The iminium ion is key to sanguinarine’s chemical reactivity and toxicity, which we will see in a bit.  Finally, there are two methylenedioxy groups (coloured in purple) located at rings C and D, respectively. These two are also heterocyclic rings, but they are non-aromatic and do not contribute to conjugation. Methylenedioxy group is quite common, it can be found in natural products like safrole, and also synthetic drugs like ecstasy (MDMA, MD for methylenedioxy). The congener toxin of sanguinarine, dihydrosanguinarine has its iminium ion reduced by addition of a single hydrogen atom (HC=N+ becomes H2C-N–; hence dihydro). The nitrogen atom of dihydrosanguinarine bears an electron lone-pair, making it alkaline. 

Figure 2: Simplified biosynthesis pathway of sanguinarine.

Sanguinarine was first isolated, albeit accidentally by an American chemist James Freeman Dana in 1824 from blood-root (Sanguinaria canadensis, Papaveraceae). In fact, James was trying to experiment with blood-root to obtain novel dyes for artists, he wasn’t even trying to isolate an alkaloid! Regardless, James obtained a reddish extract he named ‘sanguinarine’, which turned pale green upon addition of alkali. Ten years later, German chemist Johannes Gadamer would examine this extract, isolated sanguinarine as a pure compound, and elucidated its chemical structure. Sanguinarine was later shown to be a prominent alkaloid of the poppy family (Papaveraceae), occurring in various genera like Argemone, Chelidonum, Macleaya, and Sanguinaria. The biosynthesis pathway of opium-related alkaloids including sanguinarine was extensively studied in the last century. Plants make sanguinarine from the amino acid tyrosine, which gives two most up-stream precursors called dopamine and 4-hydroxyphenylacetaldehyde (Figure 2). The two condense via Pictet-Spengler reaction to produce a simple benzylisoquinoline alkaloid called reticuline, which would furnish the benzophenanthridine skeleton of sanguinarine. We have encountered the same biosynthesis step in a previous post on aristolochic acid (aporphine alkaloid). Reticuline has three rings, and it would undergo ring closure give a tetracyclic (four rings) alkaloid called scoulerine. Scoulerine is then transformed into protopine, which is a minor alkaloid of Argemone mexicana. Then, a crucial enzyme inserts a hydroxy group next to the nitrogen atom of protopine, making an unstable intermediate alkaloid called 6-hydroxyprotopine. The intermediate spontaneously undergoes a series of rearrangement reactions (iminium ion formation followed by elimination reaction to favour aromaticity) to produce dihydrosanguinarine, essentially, ring B is constructed. Lastly, dihydrosanguinarine is oxidised by removing just one hydrogen atom to produce sanguinarine. I’ve attached the simplified biosynthesis pathway of sanguinarine in Figure 2, you may have fun deciphering it. If anything, dopamine fashions ring C and A, while 4-hydroxyphenylacetaldehyde fashions ring D, ring B is made near the end. 

 

Figure 3: Sanguinarine and the electrophilicity of its iminium group. 

The iminium cation of sanguinarine is strongly electrophilic, which means that it loves to grab hold of electrons. Once that happens, the positively charged nitrogen atom can be neutralised (electrons are intrinsically negative), contributing to chemical stability. In fact, sanguinarine is so reactive, it can even grab electrons from the oxygen atom of water (H2O), and is itself transformed into an alkanolamine (pseudobase). An alkanolamine has an alcohol group (–OH) next to an amine (alkaloid), you can study this reaction in Figure 3. When the iminium ion is quenched by water to form an alkanolamine, the extended conjugation is lost, and sanguinarine changes from red to white (colourless)! Interestingly, the reaction between sanguinarine and water depends on the ambient pH. When the surrounding is acidic, iminium ion predominates; when the surrounding is alkaline, alkanolamine prevails.  In the human body with a pH of ~7.2, both forms can exist, but it is the iminium that contributes to great toxicity. This is because many of the proteins and DNA base pairs of our body are rich in electrons (from S or N atoms), and they can react with sanguinarine to form stable adducts. In fact, we have previously encountered plant toxins of similar mechanism, i.e., aristolochic acid and pyrrolizidine alkaloids. 

Apart from its chemical reactivity, sanguinarine also affects other components of the human cell. It is a toxin of multiple actions, and we still don’t completely understand most of them. It is widely accepted that sanguinarine can inhibit a crucial ion-pump found in animal cells called the NaKATPase, which is the similar target for cardiac glycosides. Inhibition of NaKATPase causes a cell to be unable to perform signalling and transport nutrients. This in turn results in a cascade of bad events, leading to the disruption of heart rhythm, dysregulation of cellular energy production, and accumulation of toxic metabolic wastes or free radicals. The first to be poisoned is the liver because the liver tries to detoxify sanguinarine, but once the liver enzymes and antioxidants of the body are overwhelmed, epidemic dropsy ensues. Cell membranes begin to break down (lipoxidation), and tissues start to lose their integrity and function, particularly cells that line the blood vessels (endothelium). The blood vessels become leaky to water and protein, and that causes swelling (oedema, water build up) of surrounding tissues. The ‘leaking’ also causes blood to lose a lot of volume (hypovolemia), and the blood pressure plummets. The body then tries a last ditch attempt to save itself by instructing the kidneys to retain salt and water. However, this only makes worse, and soon every organs start to ‘flood’, a systemic swelling condition called anasarca. Sooner or later, even the lungs start to fill up with fluid. The blood pressure within the lungs gets higher and higher (pulmonary hypotension), and the heart has to work extra hard to compensate for gas exchange. Ultimately, the cells of the heart are stressed so much, they wear off and die (right cardiac failure). That’s how we think epidemic dropsy kills. Bear in mind that all these while, the liver is poisoned more and more, and the patient continues to consume sanguinarine from Argemone oil. In patients who develop glaucoma, fluids build in their eye so much, the retinal cells ‘pop’, and that results in permanent blindness.

There is no specific antidote for epidemic dropsy (sanguinarine poisoning). However, the general consensus on treatment is to remove the patient from sanguinarine exposure immediately, and start giving them high-dosage anti-oxidant supplements, in addition to a nutritious high protein diet for months. The body may or may not recover depending on the stages of toxicity. Even today, the mortality rate of epidemic dropsy remains around 2 – 40%. Prevention is certainly better than cure, with more law enforcement and public awareness in place, epidemic dropsy has become more and more sporadic in recent years. Today, researchers are still trying to uncover the toxicomechanics and pathophysiology of epidemic dropsy, in the hopes to find better cure and early detection modalities. Sanguinarine itself, despite being a toxin known to us for almost 100 years, has shown promise in treating certain diseases like cancer. However, more needs to be done before we tame this deadly genie in the bottle. 

A note to those who may wonder about sanguinarine’s LD50 value, after all that’s the gold standard measure for toxicity. Sanguinarine scores a whopping oral LD50 of more than 1000 mg/Kg on mice! If we extrapolate sanguinarine’s acute toxicity based on LD50 alone, it’s basically non-toxic, at least when ingested?! Early researchers cautioned that sanguinarine and Argemone oil appeared to affect rodents minimally, they were not a representative model to study epidemic dropsy. We are not rodents, so this is a lesson for us to NOT trust LD50 values entirely! Take LD50 as a crude guide, but treat it only with a grain of salt.