1. Introduction
Coffee remains the most valuable primary product globally, employing over 26 million people along the chain from cultivation mainly in developing countries to consumption in developed countries [
1]. By 2017, 160 million bags of coffee were exported, of which arabica (
Coffea arabica L.) accounted for 63.3% of the total and the rest being mainly robusta (
C. canephora Pierre ex A. Froehner). It has been estimated that the coffee industry value is over 100 billion USD worldwide with up to 20 billion USD involved in export alone. Coffee is mainly traded as raw or processed beans. Therefore, it is not surprising to give considerable attention to the fluctuations in the phytochemical profile that occur during fruit growth and maturation [
2,
3,
4,
5,
6], as well as changes induced during processing such as fermentation and roasting of the coffee beans after harvest [
7].
In mature coffee beans, accumulation of sucrose, caffeine, chlorogenic acids and trigonelline has been well investigated due to the involvement of these compounds in flavour formation and the characteristic bitter taste of the coffee beverage [
7,
8]. Sucrose and its derivatives fructose and glucose provide the carbonyl group that combines with the hydrazyl group from amino acids to form a number of odorants during caramelisation in Strecker and Maillard reactions [
8]. The thermal degradation of trigonelline into pyrroles and pyridine derivatives has also been reported to contribute to coffee aroma [
9,
10]. On the other hand, products of chlorogenic acids thermal degradation as well as caffeine, which is barely affected by high temperatures during roasting, are responsible for the characteristic bitter taste of the beverage [
11]. Moreover, the ratio of chlorogenic acids to caffeine has also been described as an important parameter that might have an effect on taste, antioxidant potential and preference of a given type of beverage [
12].
On the other hand, previous reports have suggested that coffee leaves may contain all the major metabolites present in the seeds [
6]. In addition, they contain mangiferin, a xanthonoid with great antioxidant potential as well as therapeutic and pharmacological properties, and although present in the fleshy fruit parts, this compound is absent in the endosperm [
13,
14,
15]. As a result, the use of coffee leaves as a dietary source of antioxidants in form of “coffee leaf tea” might become more common. Moreover, several processing methods like those used in tea processing have since been suggested [
16]. Coffee leaves also contain carotenoids and chlorophyll pigments whose antioxidant value for dietary intake can be harnessed by using appropriate processing methods such as in green tea processing [
16,
17,
18,
19]. It has been demonstrated that food processing generally affects the content of the phytochemicals and the corresponding antioxidant capacities in foods [
20]. Until today, coffee beans and recently leaves are the main sources of antioxidants from coffee plants. These undergo either roasting or some form of drying before utilisation. The two processes may result in significant changes in the biochemical composition and/or antioxidant capacities of the resultant beverage [
7,
16]. For example, Strecker and Maillard reaction may increase oxidant scavenging abilities due to the formation of new compounds [
21].
Although it is likely that other organs of the coffee plant such as roots or stems may accumulate similar phytochemicals, their utilisation and/or investigations have not received similar attention. In fact, only a few reports have elucidated accumulation of some alkaloids and phenolic compounds in other organs such roots of seedlings or juvenile coffee plants [
22,
23]. Moreover, reports on the accumulation of the major phytochemicals in all coffee plant organs in comparison with the content in green and roasted coffee beans are still lacking. In addition, studies comparing the relationship between this phytochemical composition and the antioxidant capacities in the organs to the typical dietary antioxidant sources (beans and leaves) remain uninvestigated whatsoever. Similarly, phytochemical changes with corresponding effects on the antioxidants capacities, resulting from bean or leaf processing, still remain unclear.
This study therefore aims at assessing the phytochemical composition and antioxidant capacities of coffee plant organs such as coffee seeds, leaves, stems and roots. In addition, the changes induced by roasting and leaf senescence on the phytochemical composition and antioxidant capacities of beans and leaves, respectively, are investigated. Coffee is mainly consumed as a beverage processed from coffee beans; therefore, we also investigated whether other organs, especially leaves, might have high antioxidant activities and/or phytochemical content in relation to roasted coffee beans. Finally, relationships amongst plant organs in terms of phytochemical composition and antioxidant capacities are evaluated.
4. Discussion
Coffea species contain several phytochemicals such as caffeine, trigonelline, chlorogenic acids, mangiferin, sucrose (
Figure 1), which render their wide exploitation for pharmacological and health promoting benefits [
6]. The utilization of coffee plants has mainly focused on a single organ, the coffee seeds, as the source of the phytochemicals with related health benefits [
7]. However, utilization of other organs, including those evaluated in the current study, is also increasingly becoming common because of their possible therapeutic values [
16]. Moreover, organs such as leaves have always been used traditionally in many coffee-growing regions for mitigation of a number of illnesses including cardiovascular, gastrointestinal, cancer, diabetes, dermatological and obesity amongst others [
6]. In addition to containing all the known phytochemicals in the coffee seeds, the leaves contain exclusive compounds such as mangiferin (1,3,6,7-tetrahydroxyxanthone-C2-β-D-glucoside) and pigments like chlorophylls and carotenoids, whose antioxidant potency is also widely reported [
13,
14,
15,
16,
17,
18,
19].
The current study revealed that raw beans contained the highest amounts of the phytochemicals, especially 5–CQA, sucrose, caffeine and trigonelline. Coffee seeds remain the most important organ in coffee trade because of their extensive use in the coffee beverage processing [
7]. Phytochemicals in seeds accumulate as a result of metabolism within the fleshy parts of the fruits during maturation but also due to deposition, having been processed from leaves and young buds of the coffee plant [
2,
3,
4,
5]. Coffee seeds contain mainly carbohydrates; sucrose being the main constituent, whose role is to provide nourishment for the embryo in case the seeds germinate [
29]. Also, similar to the findings in the current study (
Table 2), high amounts of phenolic compounds have been reported in the coffee fruits and seeds. Chlorogenic acids are the main phenolic compounds that accumulate in the beans during the maturation of coffee fruits and seeds [
2,
5]. Of these, 5–CQA forms the main constituent of these hydroxycinnamic acid esters [
14]. On the other hand, although mangiferin, another phenolic compound, was reported in the fruits, its accumulation in the seeds has been disputed [
14], which explains its absence in all the beans in our study (
Table 1). Phenolic compounds in the seeds and fleshy parts of the young coffee fruits are associated with their role in defence against oxidative stress and for future use in the synthesis of cell wall–bound phenolic polymers after seed germination and during seedling development [
22]. However, as the fruits mature, a decline in the total chlorogenic acids content from between 5–7 mg g
−1 DW by up to 7% or more occurs as a result of development of an elaborate enzymatic antioxidant system and reduced activities of polyphenol peroxidase and oxidase activities, hence warranting a lesser role of the phytochemicals in defence against reactive oxygen species [
30,
31]. In the current study, the content of 5–CQA in the mature GB was 3 mg g
−1 DW (
Table 2), which is consistent with the above findings. Before utilization as a beverage, coffee beans normally undergo processing that includes roasting under high temperatures [
7]. During such processes, a number of reactions such as Maillard, Strecker’s and caramelisation occur at temperatures over 200 °C during roasting, which result in the characteristic aroma and bitter taste of the coffee beverage [
8,
32]. In the current study, sucrose content reduced by 97% when the green beans were roasted, due to its participation in the above reactions (
Table 2). During roasting, free amino acids in the coffee beans react with fructose and glucose produced from sucrose digestion beforehand during fermentation to form a number of odorants such as 2–furfurylthiol, 2, 3 butanedione amongst others, whose identity is rather determined by the type of amino acid involved in the reaction [
8,
9]. Additionally, coffee flavour is also determined by pyrroles and pyridines from trigonelline degradation [
9,
10]. However, only a small fraction of trigonelline is involved in this reaction, hence minor reductions in the content between raw and roasted beans occurs, similar to what was observed in the current study (
Table 2). On the other hand, coffee taste is determined by caffeine, which is thermally stable, and phenolic derivatives from chlorogenic acid degradation into melanoidins [
11]. The content of 5–CQA dropped sharply when the beans were roasted, supporting their degradation into melanoidins and other related compounding at high temperatures (
Table 2). However, the slight increase in caffeine in the roasted samples could have resulted from the differences in the moisture content when the GB were roasted.
Leaves are associated with high rates of metabolism due to their role in photosynthesis. For this, they contain chlorophylls to facilitate photosynthetic activity. The concentration of total chlorophylls in the leaves or proportions of their respective types (
a and
b) varies with leaf age or position. Chlorophyll
a is normally highest in the youngest leaves, whereas chlorophyll
b is highest in mature leaves. The latter is normally found in the reaction centres of photosystem I, II and in the pigment antenna system, whereas the former is found only in the pigment antenna system [
27]. In the current study, there was a general increase in the concentration of the chlorophyll
b in the older leaves (
Table 3). It is suggested that this is meant to maximize light capture because of the quaternary arrangement of leaves on the orthotropic stem, which dictates older leaves receive less incident light than their younger counterparts [
33]. Chlorophylls are normally unable to utilize all the photosynthetically active radiation (PAR) and, therefore, plants have evolved mechanisms to avoid or detoxify ROS that result from excess excitation energy. In addition to energy evasion, by accumulating less amounts of chlorophylls [
34], leaves contain carotenoids that serve to protect the chlorophylls against oxidative stresses [
19]. These pigments have also been reported to contribute to health benefits such as decreasing disease risk due to their high antioxidant activities when consumed [
19,
35]. The presence of high amounts of other phytochemicals such as alkaloids, phenolic compounds and sugars has also been reported in the coffee leaves [
13,
14,
16]. Our results also showed similar findings, especially in the youngest leaves. Like carotenoids, these compounds protect the leaves against ROS that are by-products of aerobic metabolism, more so in the young leaves [
36,
37,
38]. These compounds normally complement the enzymatic defence system in detoxifying the ROS [
39]. It has recently been shown that unlike the older counterparts, young coffee leaves have a poorly developed enzymatic antioxidant defence system and hence the reliance on oxidant scavenger compounds is inevitable [
40,
41]. In addition to defence, some phytochemicals in the current study have other functions in coffee plants. Sucrose, a highly soluble disaccharide, is synthesised in the leaf cytosol, and hence its accumulation is directly related to photosynthesis [
42]. By virtue of their position, the youngest leaves accumulated the highest content of sucrose, which reduced with leaf maturity (
Table 3). It is also a storage reservoir molecule and a transportation solute, which is readily broken down to provide energy for growth and other cellular functions [
43].
In the current study, relatively higher amounts of sucrose accumulated in the HS compared to roots, while WS had the least content (
Table 2). This could be due to the presence of active meristems in both the HS and the roots that require the energy for growth [
43]. Accumulation of phytochemicals such as caffeine, 5–CQA and mangiferin in other organs of the coffee plants like the stem (especially WS) and roots is less reported. Nevertheless, this study confirmed the presence of high amounts of sucrose and 5–CQA in the roots (
Table 2). Although evidence of chlorogenic acid metabolism in the roots remains uninvestigated, their accumulation has been suggested to be because of their regulatory role in root hair formation [
44]. Mangiferin and caffeine were essentially absent in the WS and the roots. This observation is in agreement with similar findings that have suggested that mangiferin, a bioactive xanthonoid compound, accumulates in the photosynthetic tissues so as to protect the organs against ultraviolet stress [
14]. On the other hand, caffeine is known to protect against herbivory and therefore accumulates only in the forage tissues, especially leaves and beans and hence less in lignified tissues such as WS and roots [
21]. Though in lesser amounts, trigonelline was present in the HS, roots and WS, in that order. This pyridine alkaloid accumulates in coffee organs as a reservoir for nicotinamide adenine dinucleotide (NAD) biosynthesis, which plays a key role in sub–cellular energy metabolism [
4].
Biosynthesis of these phytochemicals is normally limited to specific organs. Phenolic compounds accumulation mainly occurs via the phenylpropanoid biosynthetic pathway [
45]. However, just like in Campa et al. [
14], this study found no correlation between chlorogenic acids (5–CQA) and mangiferin accumulation in the plant organs (
Table S1). This is owed to the absence of metabolite competition for the two phenolic compounds and the silencing of the gene that encodes 3–ketoacyl–CoA thiolase (PhKAT1) protein, which catalyses the committed step for benzoic acid production in the benzenoid biosynthetic pathway [
46] from which mangiferin biosynthesis proceeds. Moreover, unlike chlorogenic acids that are distributed in all organs of the coffee plant [
22], recent reports have reported the presence of mangiferin only in the photosynthetic tissues of the coffee leaves and the receptacle of the young fruits of arabica coffee, which is in agreement with our findings [
14]. The two phenolic compounds are however degraded during senescence, which could explain the decrease in the content of mangiferin and 5–CQA in BL (
Table 2). On the other hand, alkaloids are metabolised in young leaves and the growing tips of the coffee plants and therefore accumulation of caffeine in older leaves is as a result of deposition rather than active biosynthesis where they are protective against herbivory [
23]. Although it was earlier suggested that trigonelline also acts as a chemical defence against herbivory [
47], recent reports have suggested that trigonelline biosynthesis results from detoxification of excess nicotinic acid and therefore is reconverted to the required substrate whenever the need for NAD biosynthesis arises [
48]. Moreover, trigonelline accumulation was almost equally distributed in all the plant organs, especially those with active meristems. Our results agree with Ashihara and Watanabe [
48] who have also reported presence of trigonelline in all coffee plant organs, with higher amounts especially in the upper stem and relatively lower amounts in the roots. Metabolism of the two alkaloid compounds occurs through two pathways, the
de novo pathway and the salvage pathway. These two pathways for the alkaloids have been reported to occur simultaneously in the youngest buds and expanding leaves, hence resulting into high accumulation of alkaloids in such organs. On the other hand, the mature leaves contain only the salvage pathway, which is further constrained by reduced endogenous supply of the necessary substrates during biosynthesis [
23]. Caffeine and trigonelline are degraded by demethylation into xanthine and nicotinic acid in mature plant organs. Our results suggest that caffeine degradation could be occurring at higher rates compared to that of trigonelline, hence a higher degradation percentage in BL due to loss in biological value in dried leaves [
23,
49]. The pattern of biosynthesis and accumulation of the two main alkaloid compounds, caffeine and trigonelline, in coffee seeds, especially the pericarp, follows a similar trend [
4]. It has been reported that, largely, the two alkaloids are biosynthesized elsewhere and transported to the fruits and the seeds during maturation [
4]. Therefore, the difference in caffeine and trigonelline content in the seeds corresponded with the difference in the youngest leaves, which are the main sites of alkaloid biosynthesis.
Coffee plants are an important source of dietary antioxidants. Antioxidant capacity of several foods including coffee is reported to be as a result of polyphenol accumulation [
20], which include mangiferin and 5–CQA. The current study revealed that coffee leaves contained the highest of total phenolic content compared to other organs (
Table 4). This could due to exposure of the leaves to oxidative stresses resulting from ultraviolet radiation and/or pathogens which the polyphenols protect against [
50]. In coffee beans, roasting significantly increased the total phenolic content which could be due to thermal degradation of complex phenolic compounds such as chlorogenic acids into simpler ones like melanoidins with several hydroxyl components and glycosylic linkages [
11]. As a consequence, the ROS scavenging capacity determined by DPPH, FRAP and ABTS was highest in the leaves, followed by beans, HS, roots, and least in WS. It is presumed that this order is dependent on the risk of ROS accumulation and hence an increase in total phenolic content. Moreover, Alvarez-Jubete et al. [
20] also reported a strong positive correlation between TPC and oxidant scavenging capacity, while these parameters strongly negatively correlate DPPH IC
50, as indicated in
Figure 2. Phenolic compounds contain hydroxyl components and glycosylic linkages that scavenge ROS [
40]. Antioxidant capacity and related benefits on human health are however dependent on bioavailability of the phytochemicals after consumption, which in turn is dependent on the soluble parts of the sample also known as extraction yield [
5,
16]. The results in the current study suggest that in addition to coffee beans, other coffee organs, especially the leaves, are also a major source of phytochemicals and bio–available antioxidant compounds.