Sample Paper
Flavonoids in Camillia sinensis
PBIO405/Spring '98
Magdalena M. Malak
Next to water, tea is the second most consumed beverage in the world. Being so popular it is hard to believe that so little is known about the pathways that lead to the aroma and taste that draws us to it. In this paper I propose to use the literature available on tea, scientific name Camillia sinensis, and studies performed on different plants to elucidate and describe the properties that are tea.
Tea Manufacture
Camillia sinensis is a white-flowering evergreen that thrives in tropical and semitropical climates, as China, India, Argentina, and Java (1966, Segal.) When picked it could be manufactured in three different ways to produce: green, oolong, and black teas. The most popular of the three, black tea, a product of withering and fermentation, is consumed by the majority in the western hemisphere. Black tea is dried immediately after picking on withering racks to remove excess moisture. The leaves are then rolled in machines. Here an enzyme called polyphenol oxidase catalyzes the oxidation (fermentation) of catechins, polyphenols characteristic to green tea, to theaflavins and thearubigens.
Less popular, green tea, is prepared by steaming the leaves immediately after picking, to inactivate enzymes that catalyze the oxidation of catechins. Oolong is a combination of the two manufacturing methods, with a characteristic greenish-brown color, resultant of a short fermentation time and a combination of the three main types of polyphenols.
Flavonoids as Antioxidants
Flavonoids, polyphenols, are externally derived "nonnutrient" antioxidants (1996, Dreosti) common to Camillia sinensis. Flavonoid function in humans is still speculative, but in plants flavonoids have defined functions. Primarily, flavonoids account for the accumulation of pigment in flower petals, seeds and leafs. Flowers are pollinators and must be able to attract pollen carriers. Secondly, they protect plants from UV damage, by absorbing UV at the epidermal layer. Lastly, flavonoids serve to protect against insects and pathogens.
Flavonoids are characterized by their C15 skeleton. They are modified by enzymes to yield glycosides, conjugated polyphenols, which are aroma precursors (1997, Winterhalter). Upon picking tea leaves, the conjugated groups are hydrolyzed off and the phenols and their groups exist in free form. They have medicinal properties that delay the natural process of oxidation in human tissue, that may lead to acute as well as chronic ailments as cancer and ageing. Another study proposes that phenolics in free-form are potential antimicrobial agents and toxins (1979, Harborne). Plants exhibit toxicity, phenolics in free-form, on leaf and root in order to effect nearby germination. Harborne also noted, that phenol has been previously used in the first antiseptic surgery as an antimicrobial agent.
Flavonoids, because of their antioxidant properties, have the ability of chelating free radicals and trace metals found in blood, to render them inactive and unable to do oxidative damage to the body. Many people equate antioxidative with antimutagenic properties. The antimutagenic potential of tea is undetermined and studies are conflicting. One study published in Mutagenesis compared antimutagenic activity in both black and green tea and found no correlation between blood antioxidant activity and tea variety (1996.) Thus, since green tea has a higher flavonol content, their conclusion is that flavonol may not be the antioxidative agent. This conflicts with studies, like the one published in the American Journal of Clinical Nutrition, claiming that the oxidized derivative of the flavonol, theaflavin, has very high antioxidant properties (1997.) Yet, if there is no correlation between anticancer activity and flavonol, how do we know these are the compounds to seek?
Flavonoids as Flavor Components
Earlier studies suggest that free-form amino acids, not the phenols, lead to tea aroma (1966, Roberts, G.R.). Roberts reported an increase in free amino acids during the withering period and a decrease during and at the end of fermentation. These free amino acids were being converted to other substances, perhaps volatile compounds that are important constituents of tea aroma. Ammonia and methyl butyraldehyde, the oxidized forms of amino acids' leucine and isoleucine, respectively, contribute to the bitter quality of the drink made from the hot water effusion of tea. Likewise, amino acid oxidation is involved in a non-enzymatic browning reaction (1966, Roberts), common to black tea color.
The same study purported that more mature leaves have a low concentration of free amino acids. A more recent study by Ogawa, et.al. reported an abundance of aroma precursors, conjugated phenols (not flavonoids yet), in young leaves (1995.) Both these studies indicate an indirect relationship between leaf ageing and aroma precursor content. But are the aroma precursors the free polyphenols or their free conjugate groups, as sugars and amino acids? And, is the color the result of amino acid or flavonoid oxidation?
Phenylpropanoid Pathway
Flavonoid biosynthesis is the best understood and characterized plant secondary metabolism pathway (1992, Gerats). The key enzyme of this pathway is Phenylalanine-Ammonia Lyase (PAL). Phenylalanine, final result of the Shikimic Pathway, serves as a primary substrate flavonoid biosynthesis. PAL converts phenylalanine into cinnamic acid. It also, controls the total flow of carbons into phenolics, it has been hypothesized as being the limiting step in this pathway (1974, Creasy). More recent research in apples supports this hypothesis (1996, Lister). PAL had the lowest enzymatic activity and its activity was found to be proportional to total flavonoid concentration. A poor correlation with PAL in the early stages of apple development suggest that PAL is a key enzyme in more than one biosynthetic pathway. In latter stages of apple development PAL correlates with flavonoid content, confirming its role in flavonoid biosynthesis.
The key enzyme of the flavonoid pathway is the Chalcone Synthase (CHS). It condenses three molecules of malonyl-CoA with one molecule p-courmaroyl-CoA to form an C15 intermediate, naringenin chalcone, with a R stereochemistry at carbon number two. CHI, Chalcone Isomerase, transforms the intermediate into the first flavonoid of the pathway, 2S-flavonone (naringenin). This reaction is part of all major flavonoid biosynthesis pathways. CHS and CHI appear as a complex, to ensure the right stereochemistry (1996, Lyster).
Wound response is another biosynthetic pathway where cinnamic acid is channeled. Lignin is a product in the stem and leaves of plants. Its biosynthesis increases upon wounding. Likewise, another pathway, discovered in legumes under the influence of Rhizoba, synthesizes a toxic substance as a defense response to this pathogen.
Study of the Pathways
Inhibitors
Studies have been performed on the lignification of plant cell wall and the functions of PAL (Takabe.) A specific inhibitor of PAL, 2-aminoidan-2-phosphonic acid (AIP), was used to treat the plants. The lignification was greatly reduced. Analogous studies could be performed on tea and the flavonoid production could be assayed from crude fractions using HPLC or the production of cinnamic acid itself could be assayed. Enzymes further in the flavonoid biosynthesis could likewise be inhibited and the resultant flavonoids studied. A similar study was performed in Cucumis sativus, but instead of using a PAL inhibitor a substrate analog was used to compete with phenylalanine for the active site on PAL (1983, Lamb).
These studies can be used to study Camillia sinensis, to assay whether the aroma in tea stems from flavonoid production or the amino acids and if this process is independent of browning. CHS, the key enzyme of flavonoid biosynthesis, can be inhibited so it will not synthesize flavonoids. Then, the tea components produced will be oxidized and the resultant aroma, could be noted with a hot water efflux. If the tea has lost the aroma, it can be concluded that the flavor component is due to flavonoids. Then, if we oxidize with polyphenol oxidase, and a change in color results, then we can be assured that flavonoids are also responsible for the color of tea.
Similarly anti-oxidant properties could be assayed. If PAL is inhibited and if the tea's metal chelating abilities were assayed, a correlation could likewise be made about antioxidant properties and flavonoids.
Light Induction
The phenylpropanoid pathway is also influenced by light illumination (1974, Grisbach). Two primary places for photo control are the two key enzymes PAL and CHS. The genes for the two enzymes were first isolated in parsley and were found to be transcriptionaly activated by light (1992, Douglas). Upon induction with light, PAL mRNA and CHS mRNA concentrations increased and correlated with their enzymatic activity (1997, Kalbin). PAL mRNA reached its peak before CHS mRNA supporting the findings that PAL came before CHS in the phenylpropanoid pathway (1983, Lamb, C.). In legumes DNA itself can act as a photoreceptor to stimulate flavonoid biosynthesis (1986, Beggs). Damage to DNA mediates the response to UV stress and the concentration of PAL, reported Beggs. Further research into regulation of flavonoid biosynthesis was performed in Arabidopsis. Two mutants were isolated, each for a photoreceptor in the flavonoid biosynthetic pathway. The results of this recent study confirmed that the light signal pathway controls the levels of CHS in plants (Fuglevand).
Tea leaves induced with light, can be measured by northern blotting, for PAL and CHS activity. A PAL mutant can be created, that does not respond to light, to see if sole CHS activity is responsible for flavonoid production. Likewise CHS can be mutated, and the response to light can be measured by southern, to see if any DNA of the light receptor/enzyme was made. Then by northern to see the amounts of mRNA produced in PAL and CHS could be measured. This could determine if CHS and PAL are the light receptors and likewise how their expression can effect tea quality. Conversely, the light source could be removed and then the enzyme activity studied.
Transcriptional Manipulation
Phenylpropanoid metabolism is primarily regulated at the level of transcription (1992, Douglas). In legumes transcription factors interact with promoters of phenylpropanoid genes. The transcription factors, as Douglas explained, are regulated by developmental and environmental signals. Similarly, studies performed on Petunia hybrida show how the pigmentation in petals is influenced by changes in the environment (1992, Gerats). CHS gene has been cloned for P.hybrida and eight genes have been isolated as part of the CHS enzyme, four of which were structural genes. Petunia's pattern, determined by the four structural genes, consists of colored and white sectors. Gerates, et.al. determined that there is no active CHS mRNA in the white sectors, where no flavonoid synthesis occurred and hence no pigmentation was made.
Further studies on CHS were performed using sense and anti-sense suppression. CHS was over expressed using a strong promoter, as cauliflower mosaic virus (CaMV) 35S promoter. The accumulation of the chs transcripts shut of the function of CHS enzyme and no flavonoids were produced (1997, Que). Likewise anti-sense suppression could be used to decrease the activity of CHS and similar findings would most likely result.
Structural genes are the last genes expressed in flavonoid biosynthesis, they are preceded by the expression of regulatory genes. The regulatory gene products act as transcription factors for the structural gene products. To further experiment on the flavonoid biosynthesis in C.sinensis, regulatory gene products can be constructed so as to inactivate a specific structural gene. Then you could see which sequence of PAL or CHS, is responsible for flavonoid content, if any part at all.
The activity of CHI (Chalcone Isomerase) was also assayed in P.hybrida. Studies were performed using Bovine Serum Albumin (BSA), as a repressor. BSA bound to an upstream sequence before the CHI operon, prevented the CHI gene product from being expressed. Naringenin chalcone, the substrate for CHI, accumulated and no flavonoids were made (1992, Gerats).
In tea this could be utilized to assay the activity of chalcone conversion to flavanone. If BSA has been proven to inhibit the activity of CHI, then the expression of CHI genes can be assayed and related to the activity of flavonoid production. Again, if flavor in tea remains, then it is another compound that is the tea pre-cursor or CHI is not one of the enzymes that controls the flavonoid concentration in tea.
CHI and the other enzymes, CHS and PAL, could also be assayed in tea by utilizing sense and anti-sense knock out strategies. This would be mainly to test whether the three enzymes play a major role in flavonoid biosynthesis in tea and if the aroma produced correlates to their individual activity. mRNA can be assayed by northern blotting to determine the amount of expression of each.
Conclusion
Plenty of research has been performed on the flavonoid biosynthesis and the phenylpropanoid pathway. Species used for the identification of the pathways were those that have easily detectible phenotypic markers, as Petunia hybrida, or a model organisms which are easy to work with, as Arabidopsis. Findings in these plants bear significant relevance onto plants of similar pathways as Camillia sinensis, because similar genes occur in different plants as was noted early in the history of molecular biology (1930, Vanilov).
Modern science has made great advances in the field of plant genetics, where with each new discovery, new research opportunities surface. Studies performed in transformations for example, already studied in model systems, could be extrapolated to tea. Thus, we could possibly express the aromas that are ideal for our personal tea drink.
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