Student copy

Determination of the polyphenols and caffeine contents recovered from spent

coffee grounds and their antioxidant capacities.

Gareth Fenn BSc

Department of Analytical Chemistry, School of Pharmacy and Life Sciences, Robert Gordon University, The Sir Ian

Wood Building, Garthdee Road, Aberdeen, AB10 7GJ, Scotland

Abstract

The feasibility of utilizing spent coffee grounds (SCGs) as a natural antioxidant resource was

determined. A solvent extraction was performed to extract the polyphenols and antioxidants

present in Costa® Robusta and Nandos Arabica SCG. The antioxidant capacities of the SCGs

were determined using the FRAP and DPPH assays. The Robusta mix displayed higher

scavenging and electron transfer abilities (EC50 = 20.70 +/- 1.49 µg/ml and a trolox equivalent

(TE) of 495.11 TE/100µg) compared to the Arabica sample (EC50 = 24.36 +/- 1.24µg/ml and

326.06 TE/100µg). The FC assay was used to determine the total polyphenol count (expressed

as Gallic acid equivalent GAE/100mg) for the Robusta and Arabica samples obtaining values of

7.40+/-1.68 GAE/100mg and 6.25+/-1.22 GAE/100mg. Finally, the caffeine and

neochlorogenic acid levels were determined by HPLC-DAD using an Agilent C18 5µm column

at 233nm for caffeine, (obtaining values of 70.01mg/ml and 67.18mg/ml respectively), and a

Phenomenex C18 3µm column at 325nm for Neochlorogenic acid obtaining values of 9.02mg/ml

and 8.91mg/ml. These values provide promising results for the feasibility of utilizing SCGs as a

source of natural antioxidants.

Keywords: Spent coffee grounds, Chlorogenic acid, polyphenols, Caffeine, DPPH, FRAP, Folin–

Ciocalteau.

† To whom correspondence should be addressed.E-mail: 1201058@rgu.ac.uk

2

Introduction

Thought to have originated in Ethiopia over

1000 years ago, coffee is now the world’s 2nd

most traded commodity1 with Coffea Arabica

and Coffea Canephora varieties (commonly

known as Robusta coffee) holding the most

economic value2.

Spent Coffee Grounds (SCG).

The International coffee organisation’s (ICO)

world consumption report 20153 estimated

that in 2014 over 8.9 billion Kg of green

coffee bean (GCB) were consumed,

generating the equivalent waste in the form

of spent coffee grounds (SCG). This

generated waste is equivalent to 1.25Kg of

coffee per person in the world or 86

espressos per person per annum.

Due to the health benefits associated with

coffee, many studies have focused on the

extraction and identification of these

beneficial compounds. However, in recent

years the SCGs produced have become of

great interest due to the presence of these

beneficial compounds and the mass of SCG

produced.

Health benefits associated with coffee

consumption.

In the past decade there has been an increase

in reports focusing on the harmful effects of

coffee consumption. However, a thorough

review of these reports4 refutes most of the

studies based on uncontrolled variables and

irreproducible results.

In contrast to these challenged reports, there

have been numerous studies investigating the

health benefits related to the consumption of

coffee, more specifically the polyphenols

within the coffee. The antioxidants and

polyphenols in coffee are of great interest due

3

to the well documented health benefits of

other plant polyphenols. Most of these

benefits relate to the scavenging capabilities

and metal chelating abilities of these

compounds5. These abilities have been linked

to the prevention of oxidative related stress

diseases and degenerative brain diseases such

as Alzheimer’s and Parkinson’s.

This paper will focus on two of the major

components of coffee and the benefits

associated with them, caffeine (1,3,7-

Trimethylpurine-2,6-dione) and a group of

polyphenols named Chlorogenic acids

(CGAs) more specifically Neochlorogenic

acid6 (5-CQA).

Health benefits associated with caffeine

The stimulant effects of caffeine commonly

known and thought to be linked to the

popularity of coffee as a beverage, this is

however only one of the positive benefits of

caffeine. In a review4 conducted by Higdon

and Frei, the authors concluded that in

moderate doses caffeine has been shown to

reduce the risk of cardiovascular diseases and

helped prevent type 2 diabetes as well as

Parkinson’s. Caffeine’s role as an adenosine

antagonist is thought to be the pathway

through which it helps prevent neurological

degeneration and diseases such as

Alzheimer’s, Huntington’s, Parkinson’s and

schizophrenia7.

The metal chelating abilities of caffeine

become clearer upon reviewing the

relationship between caffeine and

osteoporosis. In high doses, caffeine was

found to reduce the calcium concentration

within the test subjects increasing the

subjects risk of developing osteoporosis.

Nevertheless, a review published in 20128

suggested that caffeine was not solely

responsible for the prevention of type 2

4

diabetes as decaffeinated coffee also reduced

the risk suggesting polyphenolic

involvement.

Health benefits associated with CGAs

CGAs have been of great scientific interest

due to their antioxidant and biological

properties. According to Ky et al.9 the three

main sub groups of CGAs are caffeoylquinic

acids (CQAs), feruloylquinic acids (FQA)

and dicaffeoylquinic acids (diCQAs) (Fig

1.1). with 5-CQA being the most abundant. It

was also noted that Robusta coffee contained

a higher percentage of these components on a

dry mass basis (%dmb) compared to Arabica.

Table 1.1- Comparison of CGA components

between species (%dmb)

Component Arabica

(%dmb)

Robusta

(%dmb)

CQA 3.26 7.66

diCQA 0.19 1.43

FQA 0.60 2.31

* figures adapted from Ky et al.9

Fig. 1.1 Chemical structures of: CQAs1, FQAs2 and

diCQAs3 in coffee10

Research suggests that the antioxidant and

the radical scavenging properties of the

CGAs found in coffee play a role in

preventing degenerate brain diseases11.These

scavenging properties are split into the

following sub groups: metal chelating

scavengers, nitrous oxide (NO) scavengers

and reactive oxygen scavengers (ROX), each

5

group responsible for its unique preventative

abilities. Although essential for bodily

functions, reactive oxygen species (ROS)

such as peroxides can, if allowed to

accumulate, cause cell damage and disease

due to free radical attack. If allowed to

accumulate, these species can cause cancers

and degenerate neurological diseases12.

Moreover, a review conducted by

Nichenametla et al.13 suggested that although

CGAs possess anti-carcinogenic properties,

these properties are highly dose dependant,

with too high a dose stimulating certain types

of tumour cells. Nevertheless, further studies

have shown caffeic acid, a minor CGA

component, to be a highly effective

chemosensitizer. Caffeic acid has been

shown to dramatically increase the efficiency

of certain breast cancer treatments reducing

the IC50 of the treatment drug from 10.8µM

to just 0.83 µM13. This reduction not only

reduces the adverse effects of the treatment

for the patient, it also reduces the overall cost

of the treatment.

It is clear from the above studies that the

polyphenols and antioxidants in coffee, are

beneficial to the health of the consumer.

Hence, if we consider that in procedure

associated with making coffee, as only water

is passed through the ground beans, it is

logical to assume, that the resulting SCGs

would contain some of these compounds.

This assumption suggesting that SCGs are a

possible source for antioxidant recovery.

Many studies14-18 have focused on optimising

extraction procedures in order to recover

these compounds from SCG. Experimental

work carried out by Nayak et al.19 showed

that ultrasonic assisted extraction (UAE)

increases the extraction efficiency of CGAs.

Hence, UAE was factored into the

experimental work of this paper.

6

Determination of the antioxidant and

polyphenolic activity of the SCG

To determine if SCGs are indeed a viable

source for antioxidant recovery, and to assess

the effect of the extraction method on the

antioxidants, the activity of the sample

extract must be investigated. As previously

mentioned, the antioxidants, caffeine and

polyphenols in coffee have specific beneficial

qualities, whether that be metal ion chelation

or NO scavenging. These abilities are

exploited in order to assess the health

benefits of the extract. The following assays

are known to be the most common methods

of assessing antioxidant and polyphenolic

activity in samples. This is not only due to

the simplicity of the colorimetric

determination, but as each of these assays

react via a different mechanism20-23 it allows a

more accurate determination of the overall

health benefits. The total polyphenolic count

(TPC) is determined by the Folin-Ciocalteu

(FC) assay while the antioxidant activity is

determined by the DPPH and FRAP assays.

DPPH assay (2,2-Diphenyl-1-picrylhydrazyl)

The DPPH assay measures the antioxidants

radical quenching ability. The purple DPPH*

radical reacts with the antioxidant (Ao)

converting it to the yellow DPPH-H22. (Eq.

1). The results are commonly expressed as

the EC50. This is the concentration required to

reduce the absorbance and hence the

concentration of DPPH* molecule by 50 %.

FRAP assay (Ferric Reducing Antioxidant

Power)

The FRAP assay determines the antioxidants

electron transfer ability by assessing its

ability to reduce the Fe(III) complex (ferric-

tripyridyltriazine) to the Fe(II) complex

(ferrous-tripyridyltriazine). This reduction

(Eq. 2) prompts the colour change from pale

7

yellow to blue. FRAP results are expressed as

an antioxidant equivalent per 100µg with

Trolox being the favoured standard.

FC assay (Folin-Ciocalteau)

The FC assay, like the FRAP assay, relies on

the antioxidants electron transfer abilities.

However, unlike the FRAP assay, the FC

mechanism requires the oxidation of the

yellow molybdenum (VI) complex to form

the blue molybdenum (V) complex (Eq. 3).

The FC assay is however non-specific,

allowing the Mo(VI) to react with non-

polyphenolic compounds23, which in turn

would lead to a higher TPC. Again, similar to

the frap assay the FC results are expressed as

an antioxidant equivalent per 100mg with

Gallic acid (GA) being the favoured standard.

DPPH*(purple)+AH→DPPHH(yellow)+Ao (1) Fe(III)(yellow)+Ao→Fe(II)(blue)+Ao (-) (2) Mo(VI)(yellow)+Ao→Mo(V)(blue)+Ao(+) (3)

It was the purpose of this paper to determine

the antioxidant capacities of two different

species of coffee in order to assess the

feasibility of utilising SCG extract as a

natural source of antioxidants.

Experimental

Reagents and chemicals

The chemicals and reagents used were

obtained from three suppliers.

Acetonitrile (HPLC grade), Acetic Acid

(Analytical grade), Citric Acid monohydrate

(Analytical grade), Ethanol (HPLC grade),

HCl (Analytical grade), Methanol (HPLC

grade), Sodium acetate trihydrate (Analytical

grade) were all purchased from Fisher

scientific (Loughborough, UK). Caffeine

(99%) was purchased from Alfa Aesar

(Heysham, Lancaster). With DPPH(2,2-

Diphenyl-1-picrylhydrazyl), FC-reagent,

8

Gallic acid (97%), Iron(III)chloride

hexahydrate (97%), Neochlorogenic acid

(>98% HPLC), sodium carbonate (97%),

TPTZ [2,4,6-Tris(2-pyridyl)-s-triazine

(98%)] Trolox [(±)-6-Hydroxy-2,5,7,8-

tetramethylchromane-2-carboxylic acid

(97%)] all purchased from Sigma Aldrich

(Dorset, UK).

Preparation of SCG extracts

SCG samples were donated by Costa®

(Robusta mocha mix) and Nandos (Arabica).

These samples were reduced to a dry mass by

freezing the samples (-30oC) then the ice was

removed by dry freezing (Edwards

Cryogenics, UK). The extraction process for

the SCGs was based on an optimised

method18 with the following modifications.

Extraction temp. 47°C, extraction time- 150

min, solvent/sample ratio- 48mL/g and a

58% aqueous ethanol solvent. For each

method samples were prepared in triplicate.

This first set of extractions were aided by

agitation from magnetic stirring with the

second set aided by UAE.

A third set of samples were prepared in a

closed container conditions. This time, the

first set was agitated by magnetic stirring

(150 min) the second set by UAE (15 min).

After the extraction, the samples were

transferred into centrifuge tubes (50ml) and

centrifuged at 3000rpm for 5 min (IEC

Centra-4x centrifuge, Bedfordshire, UK). The

supernatants were transferred into pre-

weighed round bottom flasks then the solvent

removed by rotary evaporation (Buchi,

Switzerland). The samples were then frozen

and freeze dried to obtain a dry extract.

It was from this resulting dry mass which

1mg/ml standards were created by dissolving

the samples in methanol. These were

prepared fresh daily.

Determination of caffeine and 5-CQA

9

HPLC data was obtained with a Shimadzu

LC-20AD Prominence Liquid

Chromatograph fitted with a Shimadzu

DGU-20A5 Prominence Degasser, Shimadzu

SIL-20A Prominence Autosampler and a

Shimadzu SPD-M20A Prominence DAD

(USA). The buffers were 10mM Citric acid

(Solvent A) and 50:50 Methanol:Acetonitrile

(Solvent B).

For the determination of caffeine, the

samples were dissolved in methanol and the

conditions were set as follows. The flow rate

was set at 1.5mL/min and solvent ratio of

70:30 (A:B). The column for the

determination of caffeine was an Agilent C18

5µm column (4.6x150mm) at 233nm.

For the determination of 5-CQA, the samples

were dissolved in the mobile phase in a ratio

of 85:15 and the conditions were set as

follows, flow rate of 0.5mL/min and solvent

ratio of 85:15 (A:B). The column for the

determination of 5-CQA was a Phenomenex

C18 3µm column (4.6x150mm) at 325nm.

Determination of the Total Polyphenol

content (TPC)

To determine the TPC the FC assay was

performed on a 96 well plate. GA standards

along with the samples were prepared in

methanol with serial dilutions of standard

prepared to create a calibration curve. This

allowed the TPC of each sample to be

expressed as the GAE. Samples and

standards were repeated in triplicate with

samples also being used at 1mg/ml and

0.5mg/ml

Briefly, the FC assay was performed as

follows, 270µL of dH2O was pipetted into the

outer wells while 25µL of sample of interest

along with 200µL of dH2O were pipetted into

the inner wells. To the inner wells 20µL of

FC reagent was added and the plate incubated

10

at room temperature (3 min). Finally, 25µL

of 20% sodium carbonate solution was added

(total volume for each well was 270µL) and

the plate incubated at 37°C (30 min) before

the absorbance was determined using a BIO-

RAD iMark plate reader (Japan). Methanol

was used as a control.

Antiradical activity of SCG extracts

The antiradical scavenging ability of the SCG

samples (EC50) were determined by the

DPPH assay. As with the FC assay, the

DPPH assay was performed on a 96 well

plate and values determined by a BIO-RAD

iMark plate reader (Japan). Serial dilutions of

each sample along with a serial dilution of

GA were carried out. A 50 µL sample of each

dilution was pipetted into the desired well

along with 100 µL of 0.1mM DPPH solution.

The outer wells contained 150 µL methanol

with the negative controls consisting of 50

µL methanol and 100 µL of 0.1mM DPPH

solution (total well volume 150 µL). The

plate was incubated in the dark at room

temperature (30 min) before the absorbance

was read at 490nm. The linear range of the

calibration was extracted and the IC50

calculated. Samples and standards were

repeated in triplicate.

Reducing power of SCG

The FRAP values, expressed as trolox

equivalent (TE), were used to assess the

reducing power of the samples. The Frap

reagent was prepared by mixing 25 mL of

300mM acetate buffer at pH3.6 with 2.5 mL

of 10mM TPTZ dissolved in 40mM HCL and

2.5 mL of 20mM FeCl3.3H2O. Serial

dilutions of the trolox standard were prepared

and the wells of the 96 well plate set up as

follows. The outer wells contained 10µL of

dH2O and 190 µL FRAP reagent with the

inner wells containing 10 µL of the sample or

11

standard and 190 µL FRAP reagent (total

volume of well 200 µL).

Statistical analysis of results

F, T and Q tests were carried out on the raw

data to determine the validity and remove any

outliers. The data was analysed using Excel

Analysis ToolPak.

Results and Discussion

Effect of UAE on extraction efficiency of SCG

The effect of UAE on the antioxidant

capacities, as well as the caffeine and 5-CQA

concentrations, were investigated. It is clear

from the results obtained as seen in Table 1.2

(for samples 1-4) that prolonged exposure

(150 min) to UAE had a negative impact on

the antioxidants scavenging abilities as well

as the extraction of caffeine and 5-CQA.

However, in agreement with Nayak et al.19 if

we compare samples 5 and 3, we can see that

short term exposure (15 min) to ultrasonic

agitation increases the caffeine and 5-CQA

yields by around 50% (Refer to table 1.2).

If we compare samples 4 and 6, it is clear that

the open container extraction method had

very little impact on the extraction yields.

Taking into account that the closed container

differences are minimal, if we then compare

samples 3 (UAE 150 min) and 5 (UAE 15

min), the decrease in antioxidant capacity,

given that the TPCs are similar, can be

related to the length of time exposed to

ultrasonic waves. It is not clear how UAE

interferes with the antioxidant capacities nor

how it effects the extraction yield of caffeine

and CGA. However, the similar TPCs could

be explained by the fact that the oxidation of

the Mo(VI) complex is not very specific23, it

could be suggested that UAE causes the

analytes to collide slightly altering their

chemical structure, reducing their scavenging

12

and electron transfer abilities but still

allowing the oxidation of the Mo(VI). This

could also explain the low caffeine and 5-

CQA yield.

FC assay results

The FC assay was used to determine the TPC

of each of the samples. A gallic acid standard

calibration curve was created (500, 250, 100,

50 and 25µgml-1 respectively) (Fig. 3.1). The

resulting equation of the line was used to

determine the sample count expressed as the

GAE. The GAEs were then converted to the

GAE/100mg to allow for comparison (Eq. 4,

5). Due to the nature of this work the

calibration was determined to be valid based

on it adhering to the equation of a straight

line with acceptable y-intercept and R2 value.

(Refer to table 1.2).

Sample GAE=Sample|−c|

1.8308 (4)

GAE100 mg

= X (GAE)[Conc . of sample ]

(5)

It can be clearly seen from the results that

both Nandos Arabica and Costa® Robusta

SCGs contain a small quantity of

polyphenolic compounds. These results are

lower than those cited in the literature9,15,18 24.

However, as the extraction methods between

these papers are different, as well as the

species of SCGs investigated, no true

statistical comparison can be made. Again,

by referring to the samples aided by UAE

comparisons if we compare the sonicated

samples to the stirred samples little

difference is seen suggesting that the UAE

has no positive effect on the TPC.

13

Figure 3.1 TPC determination of SCG samples by

UV absorption using iMark plate reader at 750nm

(Gallic acid calibration).

Table 1.2 Summary of results from SCG extract

* All errors expressed as SD

a-Extraction aided by magnetic stirring

(150min).

b - Extraction by UAE (150min).

c-Closed container extraction by UAE

(15min).

d-Closed container extraction aided by

magnetic stirring (150min)

Sample Species TPC GAE/100mg EC50 µg /ml TE/100µg caffeine (µg /ml) 5-CQA (µg /ml)

1 Arabica a 6.25+/-1.22 24.36 +/-1.24 362.06 67.18 8.91

2 Arabica b 6.49+/-1.22 32.68 +/-1.92 304.7 56.17 5.98

3 Robusta b 5.39+/-1.00 33.81 +/-0.93 251.34 38.75 4.88

4 Robusta a 7.4+/-1.68 20.7 +/-1.49 495.11 70.01 9.02

5 Robusta c 5.09+/-0.78 20.56 +/-0.42 350.07 78.52 9.64

6 Robusta d 7.3+/-1.95 20.7 +/-0.45 388.86 64.61 8.89

14

DPPH scavenging abilities of SCG sample

extracts

The DPPH radical scavenging capabilities of

each of the six samples along with the GA

standard are depicted in Figure 3.2. The EC50

of 5-CQA was also determined (72.7 +/- 1.23

µg/ml) however this is not shown as, due to

its weak antioxidant capacity a further

standard point at (100µg/ml) was introduced.

The EC50 of each sample was determined by

extrapolation using the equation of the line

relating the linear range of the sample in

question (Eq. 6). GA was used as a standard

for the samples and 5-CQA

Figure 3.2 DPPH* scavenging capacities of SCG

samples and GA standard by UV absorption using

iMark plate reader at 490nm

However, it is clear that due to the gradual

slope decrease of the SCG samples, they

possess only weak scavengers hence a

weaker standard could be used to reduce any

error and increase the accuracy of the assay.

It should also be pointed out that the ƛmax for

DPPH* is 520 nm. However, the Bio-Rad

iMark plate reader used was unable to scan at

this range hence the closest wavelength

490nm was used instead. For this reason, the

results could not be compared directly to

literature values.

15

Figure 3.3 FRAP determination of SCG samples

by UV absorption using iMark plate reader at

595nm (Trolox calibration).

.

EC50=50−c

M(6 )

Upon evaluation of results, it is clear that

both samples 2 and 3 have a higher than

expected value yielding a 34% and 63%

increase when compared to samples 1 and 4

respectively. This again could potentially be

explained by the prolong exposure to the

ultrasonic waves although due to insufficient

data this cannot be confirmed. Nevertheless,

we can see from sample 5 that UAE used for

15 min has no apparent negative effects

suggesting that the negative effects are time

dependent.

FRAP capacities of SCG sample extract

To determine the TE values a Trolox

calibration curve was prepared (fig. 3.3).

Based on the nature of this work, as the

calibration followed the equation of a straight

line, with an acceptable y-intercept and R2

value the calibration was deemed to be valid.

To allow for comparison the TE values were

converted to TE/100µg. This was performed

in a similar fashion to Eq. 4-5. Sample 4

(Table 1.2) appeared to have a value of

almost twice that of its sonicated counterpart

(495.11 and 251.34 TE/100µg, respectively).

However, when compared to the closed

container sample 6 (388.86 TE/100µg) as we

already established the open container had a

16

limited effect, the large TE value suggests an

error.

It has been reported24 that a correlation

between the TPC and the trolox equivalents

exists, suggesting the electron transfer

abilities are directly proportionate to the

concentration of the active compounds. The

findings of this report are in agreement with

the literature. Similar to the DPPH results, it

is clear that the 150 min sonication had a

negative impact on the results whilst the

negative effect was limited in the 15 min

sample. This again provides further evidence

that the benefits of UAE are highly time

dependent.

HPLC capacities of SCG sample extract

The HPLC parameters were chosen from a

comparison of literature25-26. A citric acid

organic phase was chosen with the 50:50

ACN:MeOH solvent B.

A caffeine calibration graph was created and

the resulting equation of the line used to

calculate the sample concentrations.

However, to determine and overcome

interference caused by the coffee browns, the

method of standard addition was applied.

(Fig. 3.4).

Figure 3.4 Determination of Caffeine content of

SCG samples by HPLC-DAD at 233nm (1)

method of standard addition (2) method of

determination using caffeine calibration curve.

It is clear from the comparison (results

unpublished) of the two methods that the

coffee browns did create some interference.

These browns lead to the concentration

obtained using the calibration curve to be on

17

average 20ug/ml lower than the alternate

method.

Due to the nature of the CGAs and the high

degree of similarity between structures10 it

was decided that a 3µm column would help

resolve the CGAs more efficiently. As with

caffeine, standards were used to aid

optimization. To help reduce interference, the

samples were dissolved in 15% A: 75% B.

(fig. 3.5). Due to the low levels of 5-CQA

present and the high level of noise on the

baseline, manual integration was used to

create a base line for the 5-CQA.

Figure 3.5 Absorbance chromatogram of the

CGAs present in SCG by HPLC-DAD at

325nm.5-CQA (1) Rt=4.1 min

Contrary to literature, which states that 5-

CQA should be the most abundant analyte9,

figure 3.5 suggests otherwise. However, upon

analysing the absorbance signature, it was

suggested that the peak could be a member of

the di-CQA or, more likely, the co-elution of

two different sub-group CGAs. However,

given the limited data obtained, and as no

standards were available for all subgroups the

larger peak could not be identified.

Again, in referring to the literature, the

results obtained are consistent with those of

Ky et al.9 showing that the Robusta SCGs

contain a higher concentration of caffeine

and 5-CQA than the Arabica SCG. Although

the caffeine and 5-CQA concentrations are

substantially lower than those found in a

fresh coffee beverage, this is to be expected.

1

18

The lower yields can be related to the fact

that the grounds have already been through a

filtration stage during the preparation of the

beverage.

When the results (table 1.2) are compared it

is clear that the UAE (150 min) dramatically

reduces the extraction yield. That being said,

the highest values for caffeine and 5-CQA

were obtained from UAE (15 min) yet this

was the sample with the lowest TPC.

Conclusions

It is clear from the results that agitation by

magnetic stirring provided a higher TPC as

well as a better quality yield displaying

higher antioxidant activity. However,

although a lower TPC, and TE value was

obtained by UAE (15 min), there was a

notable increase in the levels of caffeine and

5-CQA extracted. Although these extraction

yields are relatively low for both species, (6-

8% for caffeine and 0.8-0.9% for 5-CQA) if

applied to the annual SCG waste, 0.63 billion

kg of caffeine and 71.92 million kg of 5-

CQA are wasted each year.

These figures along with the antioxidant

scavenging and electron transfer abilities

provide substantial evidence that SCGs are

indeed a feasible source of natural

antioxidants as well as caffeine.

Future work

As mentioned, the results of this paper

suggest that the benefits of UAE are highly

time dependent, given the slight variances

between the stirring for (150 min) and the

UAE (15 min). An optimized UAE method

would not only increase the yield of the

products it would reduce the extraction time

increasing efficiency. Hence future work

should be focused on developing and

optimizing an UAE method for the use on

SCG. Further work should also compare the

activities and concentrations of brewed

beverage against the associated SCG. This

19

would allow for a true comparison between

the coffee and the SCG.

Acknowledgments

This work was funded by the Robert Gordon

University, Aberdeen. Another vote of thanks

is extended to Costa® and Nandos Chicken

Land for the donation of the SCGs.

References

1. P. S. Murthy and M. Madhava

Naidu, Conservation and Recycling,

2012, 66, 45-58

2. L. R. Cagliani, G. Pellegrino, G.

Giugno and R. Consonni, Talanta,

2013, 106, 169-173.

3. International coffee organisation,

“World coffee consumption report”,

Last accessed 20/04/16, from

http://www.ico.org/prices/new-

consumption -table.pdf.

4. J. V. Higdon and B. Frei, Critical

Reviews in Food Science and

Nutrition, 2006, 46(2), 101-123.

5. P. Parras, M. Martinez-tome, A.

Jimenez and M. Murcia, Food

Chem., 2007, 102(3), 582-592.

6. D. Komes and A. Bušić, Elsevier,

2014, 25-32.

7. M. Sebastião and J. A. Ribeiro,

Journal of Alzheimer’s Disease,

2010, 20(s1), s3-15.

8. Muley, P. Muley and M. Shah,

Current Diabetes Review, 2012, 8(3),

162-168.

9. Ky, J. Louarn, S. Dussert, B. Guyot,

S. Hamon and M. Noirot, Food

Chem., 2001, 75(2), 223-230.

10. Stalmach, H. Steiling, G. Williamson

and A. Crozier, Archives of Biochem.

and Biophysics, 2010, 501(1), 98-

105.

20

11. R. Cabezas, M. Fidel Avila, D.

Torrente, J. Gonzalez, R. Santos El-

Bacha, R. Guedes and G. E. Barreto,

“Diet and Nutrition in Dementia and

Cognitive Decline”, ed. C. R. Martin

and V. R. Preedy, 2015, Elsevier BV,

827–836.

12. L. E. Cassagnes, P. Perio, G. Ferry,

N. Moulharat, M. Antoine, R. Gayon,

J. A. Boutin, F. Nepveu and K.

Reybier, Free Radical Biology and

Med., 2015, 89, 126-134.

13. S. N. Nichenametla, T. G. Taruscio,

D. L. Barney and J. H. Exon, Critical

Reviews in Food Science and

Nutrition, 2006, 46(2), 161-183.

14. J. Bravo, C. Monente, I. Juaniz, M.

P. Be Pena and C. Cid, Food

Research International, 2013, 50(2),

610-616.

15. S. I. Mussatto, L. F. Ballesteros, S.

Martins and J. A. Teixeira,

Separation and Purification Tech.,

2011, 83, 173-179.

16. M. Pinelo, A. G. Tress, M. Pedersen,

A. Arnous and A. S. Meyer,

American Journal of Food and Tech.,

2007, 2(7), 641-651.

17. M. Ranic, M. Nikolic, M. Pavlovic,

A. Buntic, S. Siler-Marinkovic and S.

Dimitrijevic-Brankovic, Journal of

Cleaner Production, 2014, 80, 69-79.

18. Zuorro, Separation and Purification

Tech., 2015, 152, 64-69.

19. Nayak, F. Dahmoune, K. Moussi, H.

Remini, S. Dairi, O. Aoun and M.

Khodir, Food Chem, 2015, 187, 507-

516.

20. K. Thaipong, U. Boonprakob, K.

Crosby, L. Cisneros-Zevallos and D.

Hawkins Byrne, Journal of Food

Composition and Analysis, 2006,

19(6-7), 669-675.

21

21. L. Y. Chen, C. W. Cheng and J. Y.

Liang, Food Chem., 2015, 170, 10-

15.

22. Floegel, D. O. Kim, S. J. Chung, S. I.

Koo and O. K. Chun, Journal of

Food Composition and Analysis,

2011, 24(7), 1043-1048.

23. M. R. Rover and R. C. Brown,

Journal of Analytical and Applied

Pyrolysis, 2013, 104, 366-371.

24. Sanchez-Gonzalez, A. Jimenez-

Escrig and F. Saura-Calixto, Food

Chem., 2005, 90(1-2), 133-139.

25. Fujioka and T. Shibamoto, Food

Chem., 2008, 106, 217-221.

26. N. P. Rodrigues and N. Bragagnolo,

Journal of Food Composition and

Analysis, 2013, 32(2), 105