Plant Phenolic Compounds, Part II: Phenolic Acids
Chaga
Introduction: Commoditized Love Doctors
Is there some sustenance which they give, which our bodies crave? Is it something fundamentally human? Why do I love yerba mate, coffee, and tea such as I do? The answer: ritual and caffeine. Humans crave ritual. What better than a psychoactive ritual which brings us together and enhances our performance; in the physical, mental, and spiritual. There are so many such rituals, yet few, if not any, which perform all three of the aforementioned, without any punitive consequences: either social, economic or biological (within reason).
The amont of effort, energy, and resources behind every sip of coffee, yerba mate, or tea that we take is nothing short of phenomenal - from farm to cup. So why not take some time and try and learn some more about the intricacies of these "commoditized love doctors" which, if for better or for worse, are such an important part of so many of our lives.
Our last post introduced some of the most desirable chemical compounds to be found in yerba mate, tea, and coffee - in most of our plant-based diets, actually - polyphenols. This post is going to look more in depth at phenolic acids, the first of the four major groups of these dietary polyphenols: where to find them, their biochemistry, and some fun examples of their significance.
Phenolic Acids
Phenolic acids (PA's) account for nearly one third of the polyphenols in our diet and are found in all plant materials (1,2); with particular abundance in acid-tasting fruits (3). The primary sources of dietary phenolic acids are: chaga, blueberry, cranberry, pear, cherry, apple, orange, grapefruit, lemon, peach, potato, lettuce, spinach, coffee, yerba mate and tea. PA's are sub-divided into two classes: derivatives of benzoic acid and derivatives of cinnamic acid.
(Hydroxy)Benzoic Acid Derivatives
Dietary Sources
The hydroxybenzoic acid (HBA) content of edible plants is generally low (Table 1), with the exception of certain fruits of the Rosaceae family, herbs and spices belonging to the Apiaceae family (anise, fennel, coriander), green and black tea, black radish and onions (3).
Table 1
Major Sources of HBAs in Rosaceae Fruits (4)
HBA Biochemistry
Common dietary HBAs are characterized by the presence of a single carbon side chain on the phenolic C6 carbon position, and they include, most notably: 4-hydroxybenzoic acid, gallic acid, ellagic acid, protocatechuic acid, vanillic acid and syringic acid (those of you who read our posts on whisky production - An Interview: How whisk(e)y gets its flavour and The Secret Science of Vanilla: Connecting Whisky and Vanilla Flavoured Foods - will understand the significance of the aromas associated with these former compounds) (4) (Fig 1).
Figure 1
(Left) Hydroxybenzoic Acid Base with Derivative Branch Points; (Right) Hydroxybenzoic Acid Derivative – Vanillin
From the sum of data, it is evident that several pathways for benzoic acid biosynthesis may co-exist in a single plant and alternations from one pathway to another may be controlled by hormones or abiotic stressors (4). Two of the widely accepted biosynthetic pathways, starting from trans-cinnamate (see previous post, Plant Phenolic Compounds, Part I: Their Relevance, Diversity and Biosynthesis, for information on the shikimate and phenylpropanoid pathways), involve a β-oxidative pathway (5,6) and a non-oxidative pathway (7).
Variations to the basic HBA structure include hydroxylations and methoxylations of the aromatic ring (4). Despite their detection in certain fruits as free acids (e.g. gallic acid in parsimmons), unless having undergone some sort processing, HBAs are typically found naturally in plants in conjugated form. For example, gallic acid (GA) may be conjugated in its dimer form (attached to another one of itself) to give ellagic acid (EA) (Table 2). Futhermore, both EA and GA can be esterified to tannins (e.g. most of the EA present in raspberries is present as elagitannins) (8). Many HBAs become available, or are derived, through the degradation of certain lignified parts of the plant, as a result of processing (4).
Table 2
Gallic Acid Contents in Teas (8)
HBA in your Alcoholic Beverages:
Four common HBAs - 4-hydroxybenzoic acid, vanillic acid, syringic acid and protocatechuic acid - are constituents of lignin, and plants lacking lignin are also lacking these four acids; even following any sort of hydrolysis or degradation technique.
This is an interesting overlap with our previous discussion on whisky maturation. If you recall, wood is composed of two relevant fractions: the sapwood and the heartwood. The heartwood is the section which coopers (ones who build wooden casks) desire due to its additional hardness and flavor contributors.
The heartwood is comprised of three main components: cellulose, hemicellulose and lignin. These are all polymeric structures that make up the cell walls, with lignin accounting for most of the intercellular space. Combined, these three components make up roughly 90% of the heartwood, with the remaining being “extractives” (e.g. volatile oils and acids, sugars, tannins, pigments); mostly in the form of ellagitannins and other volatile compounds which are free to diffuse into the maturing spirit, wine, or beer, over time (Fig 2). All of these components will play a role in the flavor development of the maturing liquid contained within the wooden cask.
Figure 2
Oak Wood Components
For the purpose of our discussion, the most important group here of which to take notice are the lignin degradation products and the tannins. Lignin degradation products (e.g. vanillin) enter the maturing spirit primarily through heat treatment and to a lesser extend through oxidation, hydrolysis, and ethanolysis. These compounds typically provide “vanilla”, “floral”, “spicy”, and “smooth” attributes to the flavour or the maturing contents.
As HBAs occur in intact plant tissues mainly as conjugates (conjugates of which may then be hydrolysed during processing) any subsequent hydrolysis can significantly impact the bioavailability of the HBAs. This is a fact which is not exclusive to HBAs; a fact of which one should remain cognisant when deciding on how to consume one's food. For example, the GA content in tea leaves have been shown to increase drastically following fermentation of the leaves; as we can see from experiments involving pu'er tea. Several studies have demonstrated a characteristic considerable microbial contribution to the leaf processing, in this regards (9,10,11,12). The impact of out gut bacteria on these compounds is not less important to consider.
While on the topic of processing, and for the sake of a more technical throw-back to our discussion on potatoes (see You say bad potato, I say good potato), preparing our food in the kitchen is another form of food processing; preparations such as the peeling of potato tubers. It has been shown that the skin of fresh potatoes contain 100-400 mg/kg of protocatechuic acid, 20-200 mg/kg vanillic acid, and 30 mg/kg GA and syringic acid; while the flesh contains only 50-200 mg/kg protocatechuic acid and a small amount of vanillic acid (4). Of course, there are other factors to consider when deciding to peel your fruits and vegetables but these numbers do not represent an isolated case.
(Hydroxy)Cinnamic Acid Derivatives
Dietary Sources
Hydroxycinnamic acids (HCAs) are present in all parts of fruits and vegetables, although the highest concentrations are found in the outer part of ripe fruits. While overall amounts of HCAs increase as the size of the fruit increases, concentrations in the outer portions are associated with a decrease during ripening. Among the richest sources of HCAs are: kiwis, blueberries, plums, cherries, apples, pears, chicory, artichokes, carrots, lettuce, eggplant, wheat and coffee (1,2,4,5).
The dietary intake of HCAs has been associated with the prevention of the development of certain chronic diseases (e.g. cardiovascular disease, cancer, type-II diabetes) (13,14,15,16,17). An association due not only to their antioxidant activity (dependant on the hydroxylation pattern of the aromatic ring), but also other important mechanisms (e.g. reduction in intestinal absorption of glucose, the modulation of secretion of gut hormones) which will be discussed in future posts.
HCA Biochemistry
The basic structure of HCAs are characterized by the presence of a three carbon side chain on the aromatic ring; a structure that is referred to as C6-C3 (in contrast to the HBAs which were characterized by a single carbon side chain, and referred to as C6-C1 (2). The main dietary HCAs include, most notably: caffeic acid (3,4-dihydroxycinnamic acid), ferulic acid (4-hydroxy-3-methoxycinnamic acid), sinapic acid (found most abundantly in citrus peel) and p-coumaric acid (4-hydroxycinnamic acid; most abundant in eggplant) (1,2,3,4) (Fig 3). Caffeic acid is the most abundant HCA (being particularly abundant in coffee, yerba mate, carrots, lettuce, potatoes, and berries), accounting for roughly 75-100% of the HCAs found in fruits and vegetables.
Figure 3
Cinnamic Acid and Common Hydroxycinnamic Acid Derivatives
Similarly to the HBAs, HCAs are rarely found in fruits and vegetables in their free acid forms; they are typically associated with other molecules as conjugates, such glycosylated derivatives and esters (e.g. with tartaric, quincic or shikimate acid). Free form HCAs are typically only found in foods that have undergone some sort of processing (e.g. fermentation, freezing, pasteurization).
For example, an overly long storage of blood orange fruits leads to a significant hydrolysis of hydroxycinnamic derivatives, releasing the free form acids along with the associated conjugate. As these HCAs are highly reactive, this typically leads to their bonding with other coumpounds, or being metabolized by microorganisms present. Both of which can lead to the formation of malodorous compounds, such as vinyl phenols; which at concentrations greater than the olfactory threshold, can impart to products such as wine and beer aromas described as barnyard and plastic.
Relevance for Brewers & Distillers:
An example of this mechanism, pertinent to brewers, is the overly abundant formation of the phenolic compounds 4-vinylphenol, 4-vinylguaiacol and ethyl phenols, by the yeast genus Brettanomyces, with p-coumaric acid as the pre-cursor (Fig 4).
Figure 4
Biotransformation of P-Coumaric Acid by Brettanomyces (18)
When produced in high quantities - especially 4-vinylphenol - this is typically seen as a sign of contamination in most beer styles and leads to "off-flavours" described as "medicinal", "band-aid", and "mousy". In fact, p-coumaric acid is sometimes added to microbiological media, enabling the positive identification of Brettanomyces by smell.
In contrast, 4-vinylguaiacol, aside from being the compound responsible for the natural aroma of buckwheat, is actually produced by many common brewing strains of Saccharomyces cerevisiae, from the HCA precursor, ferulic acid.
When produced in the right context, and at the right levels, this can help to enhance the "cloviness" of German and Belgian wheat beers; as 4-vinylguaiacol is know to exhibit properties similar to those of eugenol, the phenylpropanoid responsible for the characteristic aroma in clove buds. At varying levels, 4-vinylguaiacol has been described as: apple, spicy, plastic, peanut, clove, and curry-like.
Balancing the clove and banana characteristics of a good wheat beer is a perfect example of the marriage of art, science, and engineering. A balance most often not easily achieved.
Ferulic acid is the most abundant HCA in cereals and represents roughly 90% of the total polyphenol content in wheat - 98% of which is found in the aleurone layer and pericarp, which makes whole wheat a more attractive choice for those seeking this polyphenol. The molecule is present primarily esterified with arabinoxylans and hemicelluloses, with the soluble free form representing only roughly 10% of the total amount in the grain.
So how do brewers get this molecule into their fermentation, to get those desired "clove-like" aromas? The answer lies freeing up the molecule via a calculated control of temperatures, pH, time, and yeast selection.
Ferulic Acid Rest During Mashing
The biosynthesis of HCAs consists of a series of enzymatic reactions, subsequent to the production of phenylalanine from the shikimate pathway, and as part of the phenylpropanoid metabolism. A good starting point would therefore be the deamination of phenylalanine to yield trans-cinnamic acid; a reaction catalysed by the phenylalanine ammonia lyase (PAL) enzyme.
From this point, things get very interesting and nearly all other dietary polyphenols can trace their origins to HCAs (Fig 5).
Figure 5
Central Role of HCAs in Plant Polyphenol Biosynthesis (19)
The first step, following the production of trans-cinnamic acid, involves the hydroxylation at the 4 position to form p-coumaric acid (catalysed by cinnamic acid 4-hydroxylase). Subsequently, the addition of a second hydroxyl group at the 3 position (catalysed by phenolase) leads to caffeic acid. Further methylation of this hydroxyl group at the 3 position (catalysed by catechol-O-methyltransferase) yields ferulic acid (Fig 6).
Figure 6
Ferulic Acid (Right) Biosynthesis From 4-Hydroxycinnamic Acid (p-coumaric acid) (Left) and Caffeic Acid (Middle)
Finally, a similar hydroxylation followed by methylation at the 5 position will yield sinapic acid (Fig 7).
Figure 7
HCA (Sinapic Acid) Biosynthesis
It is nice to know how the various HCAs come to be, however, as I mentioned earlier, HCAs are not found in high quantities in free form as they are rapidly converted to glucose esters or coenzyme A (CoA) esters. This is where we see the linkage between HCAs and other types of polyphenols. As we can see from the diagram below, these activated intermediate esters are essentially branch point sites, being able to participate in a vast number of reactions (e.g. condensation with malonyl-CoA to form flavonoids, reduction to form lignans - the precursors to lignins). Figure 9 represents the general influence of HCAs on other forms of plant derived polyphenols; try and follow the main HCA pathway.
Figure 8
General Biosynthesis Map of HCA Derivatives in Plants (19)
Conclusion
This post was intended to help conceptualize the vastness and complexity of plant based polyphenols as a whole, by introducing their most basic group - phenolic acids. Phenolic acids can be primarily found in coffee, tea, yerba mate, berries and citrus fruits (among others) however are rarely present, or consumed, in their free acid state. For this reason, the "take home" message from this post should be that one needs to consider food preparation when trying to gain dietary advantage from the foods which they are consuming. With so many different compounds, each susceptible to different processing, with differing results and function in the human body, some research is required by the consumer to try and best optimize this consumption for their own self benefit.
Literature has shown that some degree of processing is beneficial when trying to consume the bioactive free-forms of these polyphenols, with beverages boasting the highest numbers. Among them, yerba mate infusions boast the highest levels of free phenolic acids at 1520 mg/l (by contrast green and black tea are closer to 300 mg/l (21). The large majority of PAs in beverages are liberated from hydrolysis, implying that nearly all PAs are conjugated or bound in their original forms.
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