The Sources and Mechanisms of Bioactive Ingredients in Coffee

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Page 1 of 48 Food & Function
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The Sources and Mechanisms of Bioactive Ingredients in Coffee DOI: 10.1039/C9FO00288J
G. L. Hu,1,2 X. Wang,1,2 L. Zhang,1 and M. H. Qiu1,2*
1 State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of
Botany, Chinese Academy of Sciences, Kunming 650201, Yunnan, China
Food & Function Accepted Manuscript

Published on 17 May 2019. Downloaded by Syddansk Universitetsbibliotek on 5/17/2019 9:22:44 PM.
2 University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China
Corresponding author: Tel: +86-0871-65223327. Fax: +86-0871-65223325.
E-mail: mhchiu@mail.kib.ac.cn (M. H. Qiu).
1
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Abstract: Coffee bioactive components include caffeine, chlorogenic acid (CGAs), trigonelline,
tryptophan alkaloids, diterpenes and other secondary metabolites. During roasting, coffee
metabolites undergo complex Maillard reactions, producing melanoidins and other degradation
products, the most controversial among which is acrylamide, an ingredient widely found in baked

food and listed as second class carcinogen. Green and roasted coffee ingredients have good
biological activities in the prevention of cardiovascular disease, antibacterial, anti-diabetic,
neuroprotection, anti-cancer. To better understand the relationship between coffee ingredients and
human health, and to effectively use the active ingredients, it is essential to understand the sources
of coffee active ingredients and their mechanisms of action in the organism. This paper
systematizes the available information and provides a critical overview about the sources of coffee
active ingredients and the mechanisms of action in vivo or in vitro, and their combined effects on
common human diseases.
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1. Introduction
Coffee is the most popular beverage around the world. Coffee drinking is closely related to
human health. Long-term researches show the bioactivity of coffee is closely related to CGAs,
caffeine, trigonelline, and coffee melanoidins. In addition, diterpenoids in coffee, especially cafestol

and kahweol (C&K) and their derivatives, significantly affect the biological activity of coffee. In
recent years, with the deepening of coffee research, more and more coffee diterpenoids, which have
anti-cancer, anti-oxidation or other functions, have been discovered. Our team discovered a variety
of diterpenoids from coffee Arabica which was planted in Yunnan Province, China, and found that
most of them have significant biological activity. Additionally, we firstly discovered the presence of
triterpenoids from green beans and concluded they were derived from seed coats, unfortunately, we
did not find their biological activity.1
All along, although in different articles there are biosynthetic pathways involved in caffeine,
CGA. There is no article detailing the sources of various bioactive compounds in coffee. In
particular, the source of trace components newly discovered from coffee in recent years, such as
diterpenes, are rarely summarized.
At present, according to research scales, research methods for the physiological functions of
coffee are mainly divided into observational studies of large samples, crude studies based on coffee
extracts, and detailed studies focusing on individual ingredient. In the past, when summarizing
coffee ingredients and diseases, researches tend to focus more on observational studies and meta
analyses. With the deep understanding of the mechanisms of all ingredients, it is necessary to
3
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respectively summarize the bioactive mechanisms of coffee ingredients. In addition, it is worth
noting that the prevention of various diseases by coffee is usually the joint actions of multiple
components, and sometimes the synergistic effect of various types of compounds is better than the
activity alone,2 Therefore, it is necessary to summarize the mechanisms of coffee active ingredients

from the perspective of diseases.
In this paper, we systematically summarized the biosynthetic pathways of coffee active
ingredients and, for the first time, classified the sources of coffee diterpenes and speculated their
biosynthetic pathways. At the same time, we separately detailed the mechanisms of the main active
ingredients in coffee based on existing research data, and summarized their joint roles in the
prevention of common human diseases.
2. Bioactive ingredients in coffee
Green coffee beans are mainly composed of carbohydrates (59-61%), lipids (11-17%), proteins
(10-16%), phenols (6-10%), minerals (4%), fatty acids (2%), caffeine (1-2%), trigonelline (1%) and
free amino acids (<1%). During the roasting process, carbohydrates (38-42%), proteins (8-14%),
phenols (3-4%) and free amino acids are reduced, while the change of lipids (11-17%), minerals
(5%), fatty acids (3%), caffeine (1-2%) and trigonelline (1%) was small (Fig. 1A, 1B).3 The process
of coffee roasting is accompanied by the Maillard reaction, resulting in complex and diverse coffee
melanoidins, which account for 29% of the total weight of roasted coffee (Fig. 1B). The lipid part
of coffee consists mainly of triacylglycerols and esters of diterpene alcohols, which account for
75.2% and 18.5% of the total lipid content, respectively, and contains small amount of esters of
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steroids (3.2%), diterpene alcohols (~0.4%), sterols (2.2%), tocopherols (~0.05%), phosphatides
(~0.4%), and tryptamine derivatives (~0.8%) (Fig. 1C). So far, most of the diterpenes found in
coffee have been present in the form of fatty acid esters, with the highest content of cafestol,
kahweol, and 16-methoxycafestol (16-OMC) in all coffee diterpenes. The content of these three

diterpenes varied among different coffee species. The content of kahweol in Arabica was
significantly higher than that of Robusta. 16-OMC is only present in Arabica coffee or in
microdistribution in Arabica coffee (Figure 1D).4, 5
2.1 Caffeine
Caffeine is a trimethylxanthine compound whose xanthine skeleton is derived from purine
nucleosides. The purine nucleoside is converted to xanthine nucleoside, which is the first key
intermediate in the caffeine biosynthetic pathway. There may be multiple biosynthetic pathways in
caffeine (Fig. 2), all possible pathways are caused by xanthosine or xanthine undergoing three
methylations and eventually forming caffeine. The main biosynthetic pathway found in coffee so far
is: Xanthosine→7-methylxanthine→theobromine→caffeine, which is catalyzed by three
methyltransferases from the SABATH (salicylic acid, benzoic acid, theobromine methyltransferase)
family.6
Almost all consumed caffeine can be distributed throughout the body through the absorption of
the stomach and intestine. Once absorbed, caffeine will play multiple roles in the body, especially in
the brain by inhibiting the adenosine receptors which enable to regulate multiple physiological
activities through endogenous adenosine.7 Adenosine receptor include subtypes A1, A2A, A2B and
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A3, caffeine A1 subtypes are mainly distributed in the central nervous system DOI:
and10.1039/C9FO00288J
peripheral
nervous system, especially in the hippocampus, cerebellum, hypothalamus and cortex, and they are
also distributed in the kidney, lung, bladder and heart. Long-term studies have shown that inhibition
of the A1 subtype can alleviate hypertension, enhance cognitive function, relieve Alzheimer's

disease, ease anxiety, and can also treat congestive heart failure and polar renal dysfunction. The
A2A receptor is mainly distributed in the dopamine-rich region of brain tissue while A2B receptor is
mainly distributed in the digestive system, and the A3 receptor is widely distributed in the spleen,
lung, heart, kidney and other organs and the surface of inflammatory cells. A large number of
experiments have shown that, as an adenosine receptor inhibitor, caffeine administered at different
doses and different phases can alter the expression of different subtypes of adenosine receptors,
enhance or attenuate their biological effects, and it plays the most prominent role in the A1 and A2A
subtypes. Both the A1 and A2A subtypes are associated with the mental excitatory effects of
caffeine, but the A1 subtype is more critical. Animal model experiments show that caffeine can
effectively Inhibit A1 and A2A subtypes receptors of microglial to reduces microglia-mediated
inflammatory environment and finally prevent neurodegenerative diseases (Fig. 2).8 The core
structure of caffeine is also widely used by researchers as the basic skeleton of novel adenosine
receptor antagonists. In addition to affecting adenosine receptors, acute and chronic caffeine
intervention can also cause different changes in the expression of a series of receptors such as
5-hydroxytryptamine (5-HT) receptor, cholinergic receptor, opioid receptor, and GABA receptor.
2.2 CGAs
The synthesis starting material of CGAs is D-glucose, and D-glucose produces

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phosphoglycerate acid and erythrose 4-phosphate through the glycolytic pathway (EMP) and
pentose phosphate pathway (PPP) cycle, respectively, and they can react to synthesize
5-dehydroquinic acid. 5-dehydroquinic acid is catalytically converted to quinic acid and shikimic
acid. Quinic acid is used as a raw material for the synthesis of CGAs, and shikimic acid is converted

to phenylalanine by the shikimic acid pathway. Phenylalanine can further form hydroxycinnamic
acid and caffeic acid through the cinnamic acid pathway. Finally, hydroxycinnamic acid or caffeic
acid is esterified with quinic acid to form CGAs (Fig. 3).
So far, the biological activities discovered by CGAs in human and animal researches can be
summarized as the following categories:
1) Prevent diseases caused by free radical damage, such as free radical induced cardiovascular
damage, nervous system damage, liver damage and tumors.
2) Control the inflammatory response in the body by inhibiting the release of certain
inflammatory mediators such as TNF-α, IL-6 and IL-1β,9, 10 thereby controlling various diseases
caused by inflammation,11-13 for example, acute liver injury, gastrointestinal disease, and
endothelial dysfunction.9, 14
3) Promote glucose metabolism by activating or inhibiting enzyme activity in the body,
reducing insulin tolerance, and increasing insulin secretion,15 thereby lowering blood glucose
concentration and treating diabetes. One of the most important metabolic pathways is thought to
inhibit the glucose-6-phosphate system and subsequently delay glucose uptake in the intestine.16, 17
In addition, a rat model test found CGA can down-regulate blood glucose levels by directly
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inhibiting G6Pase activity and related effects of hepatic gluconeogenesis.18
4) Prevent obesity and cardiovascular disease by promoting lipid metabolism. For instance,
oxidative modification of low-density lipoprotein (LDL) is an important risk factor in the
pathogenesis of arteriosclerosis,19 while an in vivo study in 11 healthy male students found that

CGAs can prevent cardiovascular-related diseases by lowering the level of LDL-cholesterol in the
body.20
5) Additionally, CGAs can also lower blood pressure by promoting the release of NO and
inhibiting the activity of angiotensin-converting enzyme.21
2.3 Maillard products
Maillard reaction, also known as non-enzymatic browning reaction, is widely happened in the
roasting process of daily foods such as coffee, tobacco, bread, etc. When the temperature of beans
rises to about 154 °C, Maillard reaction begins (Fig. 4). It is a complex reaction between amino
compounds and reducing sugars, which can take place between aldehydes, ketones, peptides,
proteins, and even ammonia. To date, people only know the chemical process of producing low
molecules and medium molecules in this reaction, and the research on the polymers produced by
this reaction still remains unknown. The Maillard reaction can be divided into three stages: early
stage, middle stage and final stage.
In 1950s, Hodge proposed the Maillard reaction route, which proposed the initial steps of the
Maillard reaction and was widely used to explain the production process of low molecular weight
(LMW) Maillard reaction products. In the early stage, the carbonyl group of the reducing sugar is
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added to the amino group, dehydrated to form a schiff base, and the schiff base is cyclized to form
N-glucosamine, which is converted into 1-Amino-1-deoxy-2-ketosaccharide through Amadori
rearrangement. In the middle stage, Amadori compounds are degraded to produce reducing ketones
or furfural compounds, and the numerous reactive intermediates formed by the reaction can

continue to react with the amino compounds. In addition, the condensate of the dicarbonyl
compound and the amino acid produced by the degradation of the Amadori compound can further
undergo strecker degradation (Fig. S. 1).
The final stage of Maillard reaction in coffee may produce a range of compounds that are
beneficial to human health, especially the HMWM which habitually called melanoidins. Exploring
the formations of such compounds and their mechanism of action is a valuable but challenging area
of coffee research. The origin and detailed structural features of the HMWM remain uncertain, and
three hypotheses for melanogenesis have been proposed, all based on the Maillard reaction. One
theory is that HMWM is produced by a condensation reaction of LMW, and the condensation
reaction occurs at final stage of the Maillard reaction (Fig. S. 1B).22 Another theory is that HMWM
is obtained by crosslinking reaction of LMW with protein side chains.23 The third theory holds that
the skeleton of HMWM is produced in the early stage of the Maillard reaction by sugar degradation
products.24 Apart from LMW, proteins and polysaccharides, phenols, which exist in the form of
glycosidic linkages, are also major players in the formation of melanoidins, accounting for nearly
10% of the weight LimaI, 2003.25 A recent study showed that transglycosylation reactions (TGRs)
are the main mechanism for the addition of phenolic substances to HMWM.26 Galactomannan and
type II arabinogalactan are the most abundant coffee bean polysaccharides, which react with LMW,
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proteins and phenolic compounds to form melanoidins during baking.27
Animal model experiments show that melanoidins can exert antioxidant effects in the body.
The mechanism may be due to its free radical scavenging and metal chelation ability. It is also
found that its antioxidant activity is largely due to the copolymerization of phenolic substances such

as CGAs. Low molecular weight compounds released from melanoidin after gastrointestinal
digestion exerted the highest antioxidant activity, even higher than compounds bound ionically to
melanoidins.28 Melanoidins have a good preventive effect on free radical-induced diseases such as
liver injury, atherosclerosi, colon cancer, this activity may related to the bonded-CGAs.29, 30
Melanoidins can also inhibit diseases caused by inflammation in the body by inhibiting the release
of inflammatory factors. In addition, melanoidins may also increase the antioxidative capacity
inside the cell by inducing Nrf2-regulated signaling pathways not only in different cell types, but
also in intact gut tissue.31
Both soluble and insoluble melanoidins can act as functional dietary fiber to quench radical
species in the gas-trointestinal tract due to their antioxidant activity.32 Melanoidins escape digestion
and pass through the upper gastrointestinal tract where they can interact with the different microbial
species present in the hindgut,33 and part of the coffee melanoidins are fermented by
microorganisms in the gut and exhibited the properties of soluble dietary fiber.34,35
Melanoidins can produce antimicrobial activity through chelating with metal irons, and three
different mechanisms were found: 1) Melanoidins mediate antibacterial activity by chelating with
iron in the medium at low concentrations. 2) In a bacterial strain capable of producing a
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3+
siderophore, melanoidins reduces the activity of the strain by chelating with the siderophore-Fe
complex. 3) higher concentrations of coffee melanoidin can remove Mg2+ from the outer
membrane, promote cell membrane destruction and release intracellular molecules.36
Melanoidins have also been found to lower blood pressure by modulating the in vitro activity

of angiotensin-I converting enzyme (ACE) and prevent colon cancer by inhibiting the in vitro
activity of matrix metalloproteases (MMPs), a family of endo-peptidases that play a central role in
tumor growth and metastasis.28 In general, coffee melanoidins have diverse biological activities in
living organisms, however, the complex the structures largely limit the study of their biological
activities, and it is worthwhile to conduct some more systematic researches in the future.
Acrylamide is mainly produced by Maillard reaction of asparagine and reducing sugars, and is
considered to be a substance having neurotoxicity and potential carcinogenic activity. Once
ingested, it can be rapidly distributed to whole body tissues and metabolized by cytochrome P450
2E1 to glycidamide,37, 38 glycidamide can also produce genotoxic character via forming adducts
with proteins and DNA in vivo.39 To reduce the produce of acrylamide during the roasting
processes of coffee is a challenging work and has become a hot research area. Xu et al. found
content of acrylamide in the whole programme increased at first but decreased near by the first burst
until finished (Fig. 4).40 Properly shortening the roasting time before 1st Crack stage and prolonging
the time after 1st Crack stage may help reduce the production of acrylamide.
Furan and furan derivatives are also produced during the roasting of coffee beans via the
degradation or oxidation of carbohydrates, amino acids, ascorbic acid, and polyunsaturated fatty
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acids.41-43 Furan in coffee has recently received significant attention due to it being classified as
being “possibly carcinogenic to humans” (Group 2B), based on animal trials, by the International
Agency for Research on Cancer (IARC, 1995). Their concentration presented in beverage is closely
related to the variety, pretreatment method, roasting degree and brewing method.44, 45 Due to its

strong volatility, furans and furan derivatives compounds will decrease significantly during
processing.45, 46
2.4 Diterpenoid
In 1930s, researchers found C&K in coffee and incorrectly identified it as steroid. In the
1940s-1950s, several researchers spent nearly 20 years to determine the chemical structure of coffee
alcohol. Subsequently, in 1989, 16-OMC was found in the coffee oil of Robusta coffee, and its
structure was determined by synthesis. The 16-OMC, which is an ideal biomarker for distinguishing
Robusta from Arabica, has been considered to exist only in Robusta coffee in the past. However,
recently, it was also found in Arabica coffee. C&K and 16-OMC are mainly esterified with various
fatty acids, rarely exist as free forms.
In the 21st century, more and more researchers, including our team, found that there are
various forms of diterpenes in coffee, most of them are derivatives of C&K and 16-OMC. So far,
nearly 100 diterpenes with different structures have been identified in coffee. Through structural
classification, we divided them into oxidized diterpenoids, rearranged diterpenes,
tetrahydrofuran-type diterpenes, pyrolyzed diterpenes, γ-lactone-type diterpenes, Δ4,18-type
diterpenes, degraded-type diterpenes and Villanova-type diterpenes (Fig. 5), and speculated their
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biosynthetic pathways (Fig. S. 2). Until now, most of the diterpenes found in coffee are derived
from the kauran skeleton via oxidation, condensation, rearrangement and other catalytic reaction in
coffee plant, a few of them may also be produced during the roasting processes.
Because of the rich content in coffee, the bioactivity of C&K has been extensively studied.

C&K can increase serum cholesterol levels of gerbils and rats by increasing the activity of
cholesterol transfer proteins and accumulating extracellular cholesterol.47 In addition, C&K were
shown to have anticarcinogenic, antioxidant, anti-inflammatory and hypercholesterolemic
activities.48, 49 C&K palmitates are potent inducers of glutathione S-transferase activity and induces
xenobiotic detoxification in the gut and mucosa of mice. It is worth mentioning that the biological
activity seems to be closely related to the furan ring of cafestol and kahweol, the compound loses its
activity after the furan ring is destroyed. C&K were also found to reduce the metabolic toxic
substances by inhibiting N-acetyltransferase in vitro and model rats, thereby playing a role in
glycosidic protection.50 The good bioactivity of C&K and their rich content in coffee means that
they have the potential to be the starting material for drugs.
Studies have found that other types of diterpenes also have good biological activity.
Interestingly, the degree of oxidation at 19 site has a large effect on the intensity of the activity, for
instance, 6a,17-dihydroxy-ent-kauran-19-al showed strong in vitro inhibitory activity against human
ovarian cancer cell line A2780 (IC50 0.38 μM) and human lung cancer cell 95-D (IC50 19.38 μM),
while when the aldehyde group located at 19 site is oxidized to a carboxyl group, it loses activity to
human ovarian cancer cell A2780 and has a reduced activity against 95-D (IC50 39.83 μM).51
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2.5 Trigonelline
The amount of trigonelline synthesized in the pericarp is much higher than that in the seeds,
but some of the trigonelline synthesized in the pericarp can be transported to the seeds, leading to
the amount of trigonelline in seeds is higher than that in pericarp. Quinolinic acid, an intermediate

synthesized from the pyrimidine nucleotide, enters the pyridine nucleotide cycle,
nicotinamide—nicotinic acid—nicotinic acid mononucleotide (NaMN)—nicotinic acid—adenine
dinucleotide (NaAD)—NAD—nicotinamide mononucleotide (NMN)—nicotinamide, and
trigonelline is finally synthesized from the nicotinic acid produced by this cycle (Fig. 6).52
Studies on the bioactivity of trigonelline are focused on the prevention of cardiovascular
disease, neuroprotection and anticarcinogen. Trigonelline regulates key enzymes of glucose and
lipid metabolism, such as glucokinase, glucose-6-phosphatase, fatty acid synthase, and carnitine
palmitoyl transferase in diabetic rats, which leads to its activity in lowering blood sugar, cholesterol
and blood lipid.53, 54 There are two cardinal mechanisms for trigonelline to exert anticancer activity
which do not directly exert cytotoxicity to kill cancer cells. One is to prevent cell invasion, an
important means of cancer cell proliferation.55 The other is to regulate the expression of
transcription factor Nrf2. The transcription factor Nrf2 is activated to protect cells from harm when
cells are invaded. However, Nrf2 factor is also present in cancer cells, so there is often inhibition
during chemotherapy. An experiment using pancreatic carcinoma cell lines and H6c7 pancreatic
duct cells as models found that trigonelline can inhibit the expression of Nrf2 in cancer cells and
improve the effect of chemotherapy.56 Experiments have shown that trigonelline enters the brain
through the blood-brain barrier, which can improve the memory of rats and exert neuroprotective
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activity, but does not clarify its mechanism of action.
3. Coffee active ingredients and diseases
3.1 Cardiovascular disease

The relationship between coffee drinking and cardiovascular disease has been controversial.
Several studies have shown that drinking coffee may increase the incidence of cardiovascular
disease. A study surveyed 24,710 Finns without record of drug treatment for hypertension and
assessed their daily coffee consumption through a questionnaire. During the survey, 2,505
participants started antihypertensive drug treatment. The result showed that drinking coffee seems
to increase the risk of antihypertensive drug treatment, and this risk was higher among subjects with
low to medium coffee intake.57 However, most follow-up studies did not have direct evidence that
coffee can cause cardiovascular disease. Subsequent researches showed that moderate coffee
consumption could reduce blood pressure,58-60 prevent stroke and heart diseases,61-63 reduce the risk
of type 2 diabetes.64 The main components of anti-cardiovascular disease in coffee are considered to
be CGAs, trigonelline, caffeine, and melanoidins (See Table S. 1 for more information about their
Anti-cardiovascular disease mechanisms).
Blood pressure of spontaneously hypertensive rats injected with coffee extract or chlorogenic
acid can be significantly improved. The mechanism may be that ferulic acid, a metabolite of CGAs,
acts on NO from the inner wall of blood vessels, and regulates the tension of blood vessels by
improving the bioavailability of NO.65, 66 In addition, the antihypertensive effect of CGAs has been
observed in both animal models and hypertensive patients to be affected by hydroxyhydroquinone,
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which produced during coffee roasting.66-68
According to a study conducted by Shamil et al., CGA (5-CQA) inhibited platelet activity at
50 nM, suggesting it can be used as a good antithrombotic agent.69 Pharmacokinetics studies have
shown that the consumption of 2-3 cups of coffee can reach the 5-CQA active concentration, but its

metabolism in human body is fast, and it is necessary to consume regularly to maintain its plasma
concentration.70 It was found that the pyridinium compounds 1,3- and 1,4-dimethylpyridine formed
during the heat treatment of trigonelline or the roasting of coffee also showed slight antithrombotic
activity in rat model.71
Some coffee melanoidins showed significant ACE inhibitory activity, and its activity was
positively correlated with the degree of heating. The ACE inhibitory activity is associated with the
complex structure of melanoidins, but is also attributable in part to low molecular weight
compounds such as natural phenolic compounds that are not chemically bonded to melanoidins,
therefore, melanoidins can also act as a carrier protectant.72
Although it has been thought in the past consumption of a large amount of coffee containing
C&K may cause cardiovascular disease because C&K consumption can increase the amount of
cholesterol in human body.47 However, the recommendation for daily intake of C&K not exceeding
300 mg in the 1960s lacked sufficient scientific evidence. In the latest version of the US Dietary
Guidelines (2015-2020), the cholesterol "300 mg daily intake limit" was removed, and cholesterol
is no longer considered to be "a worried nutrient", meaning that moderate drinking of coffee
containing C&K will not contribute to cardiovascular disease.
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3.2 Antibacterial activity
A secondary analysis of the 2003-2004 U.S. health and nutrition survey data and found that
compared with those who do not drink coffee, coffee drinker’s MRSA nasal cavity risk is reduced
by about a half,73 suggesting that the coffee components can be used to resist the invasion of human

pathogens. CGAs, melanoidins, dicarbonyl compounds, polysaccharides and some other trace
ingredients play great roles in the antibacterial activity of coffee (See Table S. 2 for more
information about their antibacterial activities).
Suarez-Quiroz, et al. screened the in vitro antimicrobial activity of CGAs and dodecyl chloride
(DCGA) against different bacterial strains.74 CGAs have inhibitory activity against both
gram-positive and gram-negative bacteria, among which, it exhibits bactericidal activity against
Pseudomonas fluorescens and Staphylococcus aureus; DCGA is active against Gram-positive
bacteria and inactive against Gram-negative bacteria. Both CGA and DCGA exhibit
anti-Aspergillus activity and have higher inhibitory activity against ochratoxin A than aflatoxin B1.
This work reported for the first time that CGA and DCGA were used as antibacterial agents,
antifungal agents and antimycotic agents, suggesting that CGA compounds are promising as
preservatives in foods or non-food products. In addition to the antibacterial activity alone by
chelating with metal ions as mentioned above, when coffee is roasted, CGA can also be part of
HMWM by Maillard reaction, in combination with HMWM to exert antibacterial activity.36
3',4'-dihydroxyacetophenone is an antimicrobial substance in coffee grounds, and the
antimicrobial activity of the 3',4'-dihydroxyacetophenone isomers depends on the binding site of the
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hydroxyl group.75 Glyoxal, methylglyoxal and diacetyl and other α-dicarbonyl compounds formed
during the roasting of coffee are the main active substances in brewing coffee against Sa. aureus
and St. mutans, adding caffeine with weak intrinsic antibacterial activity to a mixture of
alpha-dicarbonyl compounds may synergistically enhance the antibacterial activity of

alpha-dicarbonyl compounds. 2
3.3 Anti-diabetic
Extensive epidemiological studies have shown that coffee intake reduces the risk of diabetes in
humans. Among all the active ingredients, caffeine, CGAs and trigonelline play the most prominent
roles to prevent diabetes.76-79 (See Table S. 3 for more information about their anti-diabetic
mechanisms).
It was found in the study of streptozotocin-induced niacinamide-induced type 2 diabetes in
mice that metabolites of caffeine in the gastrointestinal tract protected the pancreas from type 2
diabetes.80 Besides, Cheng, et al. 2011 proposed that the intake of caffeine and caffeic acid could
inhibit the misfolding of human amylin polypeptide (hIAPP) and reduce the incidence of type 2
diabetes.81
CGAs have a similar protective effect on the pancreas as caffeine.82 CGAs can increase the
body's insulin response and may be the component that normalizes the acute glucose response.
However, randomized crossover studies have shown that there is no significant difference in the
effect of different CGAs levels on glucose and insulin in humans.83 Henry-Vitrac, et al. found that a
coffee extract, Svetol, inhibited glucose-6-phosphatase hydrolysis in human liver microsomes,
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which could reduce the production of hepatic glucose through this inhibitionDOI:and
10.1039/C9FO00288J
achieve
antidiabetic effects, and CGAs (caffeoylquinic acid and di-caffeoylquinic acid) were the main
ingredients that promoted this process.84 Acetylcholinesterase (AChE) activity and lipid
peroxidation will increase in the cerebral cortex in diabetic rats, while Stefanello, et al. found that

CGA and CA inhibited AChE activity in diabetic rats, prevented diabetes-induced lipid
peroxidation, improved memory and reduced anxiety, suggesting that it can be used to prevent
diabetes-induced brain disorders.85
Trigonelline can improve symptoms of diabetes in rats by regulating glucose and lipid
metabolism key enzymes (such as glucokinase, glucose-6-phosphatase, fatty acid synthase, etc.).86
Treated with trigonelline for 4 weeks, the blood glucose, cholesterol, and triglyceride levels in the
diabetic rats were significantly reduced.87 Hong, et al. found that trigonelline in coffee can improve
auditory threshold changes caused by diabetic neuropathy and delay the latency of auditory evoked
potentials in mice, which may help improve diabetes-induced hearing impairment.88 However,
another study found that trigonelline (50 mg/kg daily for four weeks) increased bone mineral
density and cancellous bone strength in nicotinamide/streptozotocin-treated rats, which means the
improper intake of trigonelline may increase diabetes-induced skeletal system diseases.89
3.4 Neuroprotection
A large number of epidemiological studies have shown that coffee has the efficacy of
preventing neurodegenerative diseases such as Parkinson's disease (PD), Alzheimer's disease and
depression.90 However, most of the research results are from observational experiments, and there
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are few studies on the mechanisms of specific secondary metabolites in coffee (SeeDOI: 10.1039/C9FO00288J
Table S. 4 for
more information). The association between coffee and PD risk is related to gender, and it is usually
more related to men than women. This gender difference may be related to the secretion of
estrogen. Women who use coffee during the use of postmenopausal hormones will increase the risk

of PD. Although the cause of this adverse effect of estrogen is currently unclear, the more common
current guess is that estrogen replacement therapy may inhibit cytochrome P-450
(CYP1A)-mediated metabolism.
Among all the secondary metabolites of coffee, caffeine has the most significant effect on the
nervous system, and it may be the main active substance of the coffee components to prevent PD. It
has been demonstrated that caffeine is resistant to dopaminergic neurotoxicity in animal models,
and its mechanism of action may be to block A2 adenosine receptors to stimulate dopamine release,
thereby improving the function of the dopaminergic system.91 Recently, a study showed that
eicosanoyl-5-hydroxytryptamide, which purified from coffee as an agent that leads to enhanced
enzymatic activity of the specific phosphatase PP2A that dephosphorylates the pathogenic protein
α-synuclein, works in synergy with caffeine in protecting against mouse models of PD and
Dementia with Lewy bodies. The mechanism of this synergy is also through enhancing PP2A,
which is dysregulated in the brains of individuals with these α-synucleinopathies.92
Extensive studies have shown that caffeine and other biologically active molecules in coffee
can reduce the risk of depression.93-97 For example, Hall, et al. studied the effects of caffeine on the
behavior of depressed mice. Behavioral studies showed that caffeine-injected mice did not undergo
tail suspension or forced swimming tests.98 Although many studies have provided evidence that
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caffeine has antidepressant activity, the mechanism of action has not yet been fullyDOI: 10.1039/C9FO00288J
elucidated. As
the understanding of the pathogenesis of depression continues to deepen, it is expected to have a
deeper understanding of the antidepressant mechanism of caffeine.
3.5 Anticancer activity

Although laboratory data indicate that certain components of coffee may cause DNA mutations
and inhibit tumor suppression mechanisms,99-101 subsequent researches have not confirmed this
point. Recent studies based on human and animals have shown that coffee may play a role in
preventing colon cancer, rectal cancer, breast cancer, hepatocellular carcinoma, and prostate
cancer.102-104 Caffeine, coffee melanoidins, CGAs, C&K, and trigonelline are the main substances
that produce anticancer activity (See Table S. 5 for more information about their anticancer
mechanisms).
Hirose, et al. examined the relationship between coffee intake and hormone-related cancer risk
in Japanese women and used a multivariate logistic regression model to determine whether there
was a correlation between caffeine intake and endometrial cancer, and found that coffee
consumption reduces the risk of endometrial cancer in subjects.105 Interestingly, in another
epidemiological survey, opposite results were obtained. A survey of 5,847 postmenopausal women
with effective cancer status by Yuan, et al. found that the incidence of endometrial cancer in women
consumed vary degrees of caffeine increased significantly compared with women without caffeine
intaking.106
Zinc-containing matrix metalloproteinases MMP-1, MMP-2, and MMP-9 play a pivotal role in
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DOI: 10.1039/C9FO00288J
the pathogenesis of colorectal cancer. De Marco, et al. found that melanoidins isolated from coffee
beans can effectively inhibit the activities of MMP-1, MMP-2 and MMP-9 with IC (50) values
ranging between 0.2 and 11 mg/mL in vitro, indicated that coffee melanoidins have important
values in the study of colorectal cancer.107 CGAs can induce selective killing of lung cancer cells

(A549) while normal lung fibroblasts (MRCS) are unaffected.101 Another study in vitro found that
CGAs can inhibit CT-26 colon cancer cell-induced lung metastasis by blocking the phosphorylation
of extracellular regulatory protein kinase (ERK).108 In addition, CGAs can also exert anti-cancer
effects in vitro through other mechanisms, such as reducing DNA methylation by inhibiting DNA
methyltransferases, the IC50 values of CGAs was 0.75 µM.109
The transcription factor Nrf2 can be involved in regulating the drug resistance of cancer cells,
and trigonelline can increase the sensitivity of pancreatic cancer and colon cancer cell lines to
anticancer drugs by inhibiting Nrf2 activity in tumor-bearing mice.56 In another study, rat ascites
liver cancer cell line AH109A was used to infect rats, trigonelline was able to resist the invasion of
liver cancer cells at a concentration of 2.5–4.0 μM, but it did not inhibit the differentiation of liver
cancer cells.110 However, studies have also shown that trigonelline has a proliferative effect on
breast cancer cells. Trigonelline produces phytoestrogens, which activates the estrogen receptor
(ER) at a concentration of 100 pM and significantly induces the proliferation of MCF-7 breast
cancer cells.111
C&K is a relatively important part of coffee anti-cancer research in recent years, but the
mechanism of apoptosis they mediate is not fully studied, most of anti-cancer researches about
C&K or other diterpenoids were limited to in vitro trails. Park et al. found that kahweol inhibited
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DOI: 10.1039/C9FO00288J
markedly the proliferation of human colorectal cancer cell lines such as HCT116, SW480 via
inhibiting cyclin D1 protein level in HCT116 and SW480 cells. In the following study, they found
kahweol can also induces apoptosis through activating transcription factor 3-mediated pathway in
human colorectal cancer cells.112 In some other studies, C&K were also found to exert anticancer

activity by increasing overall glutathione transferase, inhibiting CYP450s, inducing phase II
detoxifying and antioxidant enzymes.113-115
4. Conclusions
In addition to the HMWM, the sources of other types of coffee main bioactive substances have
been clarified. The complexity of the reaction process causes the possibility that the structure of
HMWM formed under the same reaction conditions may be different, which leads to different
mechanisms.
From the existing epidemiological literature reports, despite a small amount of negative
reports, coffee consumption tends to benefit human health. For single coffee component, some
specific trace constituents in roasted coffee like acrylamide and furan, although no convincing
evidence to prove, may have negative impact on human health, while caffeine, CGAs, melanoidins,
diterpeniod and trigonelline showed anti-cardiovascular disease, anti-diabetic, neuroprotection and
anticancer activity through multiple mechanisms of action. It is worth being mentioned that, as with
most of drug researches, the mechanisms of action in model animals or in vitro are often hard to
repeat in humans. In vivo studies, the effects of coffee active ingredients are greatly affected by the
location, gender, and individual differences of the subjects. The results obtained are less
23
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reproducible, which is a factor that needs attention when conducting mechanism DOI: 10.1039/C9FO00288J
research in the
future.
Conflict of interest statement
The authors are not aware of any affiliations, memberships, funding, or financial holdings that

might be perceived as affecting the objectivity of this review.
Acknowlegement
This study was supported financially by the National Natural Science Foundation of China,
China (No. 31670364), Foundational Project of Key Laboratory of Tobacco Chemistry of Yunnan
Province, R&D Center of China Tabacco Yunnan Industrial Co., Ltd.( KCFZ-2017-1096), Project
of Key New Productions of Yunnan Province, China (No. 2015BB002), the STS Programme of
Chinese Academy of Sciences, China (KFJ-SW-STS-143-8), Special Fund Project of Pu’er
municipal government, China (2018), as well as Foundation of State Key Laboratory of
Phytochemistry and Plant Resources in West China, China (P2015-ZZ09). Compliance with Ethical
Standa.
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Figure captions
Fig. 1 A) Main ingredients of green coffee, B) Main ingredients of roasted coffee. The ingredients
marked with green indicates a decrease in content; C) Main ingredients of coffee lipids; D) The
content of major diterpenes in Arabica and Robusta coffee.
Fig. 2 Twelve possible synthetic pathways and neurodegenerative diseases prevention mechanisms
for caffeine. The main biosynthetic pathway is marked in colors.6, 8
Fig. 3 Biosynthesis pathway of CGAs. Quinic acid is an intermediate of the shikimic acid pathway,
and hydroxycinnamic acid or caffeic acid is produced by the cinnamic acid pathway. They serve as
starting materials for the synthesis of CGAs.
Fig. 4 The curve of coffee baking process. Melanoidins and Acrylamide are mainly produced in
maillard stage, and the amount of acrylamide is decreased near by the first burst until finished.
Fig. 5 Eight types of diterpenes from coffee. All the coffee diterpenes are derived from the kauran
skeleton via oxidation, condensation, rearrangement and other catalytic reactions.
Fig. 6 The biosynthetic pathways of trigonelline in coffee. NaMN, nicotinic acid mononucleotide;
40
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NaAD, adenine dinucleotide; NMN, nicotinamide mononucleotide; NAD, nucleosidase; PRPP,
5-phosphoribosyl-1-pyrophosphate.52

41
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DOI: 10.1039/C9FO00288J

Fig. 1 A) Main ingredients of green coffee, B) Main ingredients of roasted coffee. The ingredients marked
with green indicates a decrease in content; C) Main ingredients of coffee lipids; D) The content of major
diterpenes in Arabica and Robusta coffee.
121x110mm (300 x 300 DPI)

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DOI: 10.1039/C9FO00288J

Fig. 2 Twelve possible synthetic pathways and neurodegenerative diseases prevention mechanisms for
caffeine. The main biosynthetic pathway is marked in colors.6, 8
161x92mm (96 x 96 DPI)

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DOI: 10.1039/C9FO00288J

Fig. 3 Biosynthesis pathway of CGAs. Quinic acid is an intermediate of the shikimic acid pathway, and
hydroxycinnamic acid or caffeic acid is produced by the cinnamic acid pathway. They serve as starting
materials for the synthesis of CGAs.
187x228mm (300 x 300 DPI)

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DOI: 10.1039/C9FO00288J

Fig. 4 The curve of coffee baking process. Melanoidins and Acrylamide are mainly produced in maillard
stage, and the amount of acrylamide is decreased near by the first burst until finished.
178x132mm (96 x 96 DPI)

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DOI: 10.1039/C9FO00288J

Fig. 5 Eight types of diterpenes from coffee. All the coffee diterpenes are derived from the kauran skeleton
via oxidation, condensation, rearrangement and other catalytic reactions.
142x142mm (96 x 96 DPI)

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The biosynthetic pathways of trigonelline in coffee. NaMN, nicotinic acid mononucleotide; NaAD, adenine
dinucleotide; NMN, nicotinamide mononucleotide; NAD, nucleosidase; PRPP, 5-phosphoribosyl-1-
pyrophosphate.52
169x87mm (300 x 300 DPI)

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DOI: 10.1039/C9FO00288J
Graphical Abstract

Detailed the sources and mechanism of action of coffee active ingredients and summarized their joint roles in
the prevention of common human diseases.

The Sources and Mechanisms of Bioactive Ingredients in Coffee

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