Profiling_for_bioactive_compounds_in_bramble

Profiling for Bioactive Compounds in Bramble (Rubus fruticosus)
Gregory Hammond
110107420
Submitted in part candidature for the degree of Biochemistry B.Sc., Institute of Biological and Rural
Sciences, Aberystwyth University.
April 9th
, 2014

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I certify that, except where indicated, all material in this thesis is the result of my own
work, presented in my own words, that any direct quotations are contained within
quotation marks with appropriate citation and that all sources of information used
(textual, graphic, etc) have been correctly cited in both the text and reference list in
accordance with the IBERS referencing style. The work has not previously been
submitted as part of any other assessed module, or submitted for any other degree
or diploma. I have read and abided by the University and Departmental statements
on plagiarism. I confirm that I have read, understood and abided by this declaration.
Abstract
Rubus fruticosus is an invasive species of blackberry that poses potential problems to other flora
growing around it. Berries, leaves and stems were harvested from a bird reserve in mid-Wales and
profiled for phenolics, sugars, proteins and antioxidant activity in the laboratory. It was found that
the main phenolic found in berries and stems was Sanguiin H-6 an ellagitannins with known
antioxidant activity. Further analysis of the smaller peaks found in the mass spectrometry need to be
studied to identify more phenolic compounds. The phenolic profiling of the leaves showed the
presence of casuarictin, the monomer of Sanguiin H-6 and other ellagitannins, but the presence of
these cannot be confirmed. Analysis using Dionex revealed the presence of glucose, fructose, xylose
and galactose as the main sugars within the samples, with fructose being the most abundant. Berries
were identified as the sample with the highest concentration of sugars. The Lowry assay showed the
presence of proteins within the samples, it was shown that the stems contained the highest
concentration of proteins compared with the other tissue types. FRAP and ABTS assays were used to
calculate the Trolox equivalent antioxidant capacity value (TEAC) for each of the samples. The FRAP
assay indicated that the berry samples had the highest TEAC, but the ABTS indicated that the leaf
samples had the highest TEAC. Further research is required into finding out which sample has the
highest overall antioxidant activity.

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Contents
Abstract...................................................................................................................................................1
Introduction ............................................................................................................................................3
Materials and Methods.........................................................................................................................10
Analysis and identification of sugars ................................................................................................10
Phenolic extraction with methanol in preparation for mass spectrometry .....................................11
Method for use of a Sep-pak ............................................................................................................11
Antioxidant assays ............................................................................................................................12
FRAP Assay....................................................................................................................................12
ABTS Assay ....................................................................................................................................12
Protein extraction and Lowry assay..................................................................................................13
Lowry Assay...................................................................................................................................13
Results...................................................................................................................................................14
Analysis of phenol composition of Rubus fruticosus ........................................................................14
Berry..............................................................................................................................................14
Leaves............................................................................................................................................21
Stem..............................................................................................................................................24
Sugar analysis....................................................................................................................................26
Protein analysis.................................................................................................................................37
Antioxidant testing............................................................................................................................38
FRAP assay ....................................................................................................................................38
ABTS assay.....................................................................................................................................40
Discussion..............................................................................................................................................42
Identifying compounds from mass spectrometry.............................................................................42
Analysis of Dionex results for sugars present in various tissues.......................................................44
Proteins.............................................................................................................................................45
Antioxidants......................................................................................................................................45
Conclusion.............................................................................................................................................46

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Future Research and Improvements ....................................................................................................47
Acknowledgments.................................................................................................................................48
Bibliography ..........................................................................................................................................49
Introduction
Rubus fruticosus is an invasive species of blackberry that can be found all over the world and is
native to Europe, it is commonly called bramble, due to its brambles on the stems of the plant. It is
very typical for bramble to be found in woodlands, hedgerows, meadows, and waste ground etc. It is
a deciduous shrub that can cover an area of up to nine metres squared in its first year (Clapham, et
al., 1987). In 1999 the blackberry plant was declared a weed of national significance in Australia and
is recognised as one of the worst weeds around (NSW Department of Primary Industries, 2010).
At an RSPB bird reserve in mid-Wales (Ynys-hir) the invasive properties of Rubus fruticosus are
becoming a problem and soon mass clearing of the plant will be required. Currently Rubus fruticosus
occupies around 20% of the flora at Ynys-hir. When the clearing of Rubus fruticosus happens the
plant material will mainly be thrown away at no benefit to the RSPB, however there is potential to
recover valuable commodities from the plant material. There are bioactive and nutritional
compounds within blackberries that could be exploited for a profit such as polyphenolics, sugars and
proteins.
Figure 1.1: An image of Rubus fruticosus (Species of the UK, 2013).

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The genus Rubus contains over 250 species (Sauer, 1993), not all species contain edible fruits, the
ones that do are known to be rich in vitamins A, B and C. It is also claimed that the roots and leaves
could have medicinal properties, it has been found that the leaves can also be made into herbal tea
when dried (Brown, 2002). “In recent years the antioxidative capacities of a number of Rubus species
have been investigated in vitro” (Byamukama, et al., 2005).
Figure 1.2: a map to show a small section of the diversity of the rubus genus (The Fruit Nut, 2012)
Blackberries have been a useful food source since the end of the last ice age; however they have also
been used for medicinal purposes. Uses of various species from the Rubus genus as a medicine has
been “documented in the writings of the ancient Greeks, Romans, Asian medicinal traditions,
traditional Chinese medicine and the Ayurvedic tradition of India” (Hummer, 2010). Branches were
made into a concentrated liquor by boiling them. This liquor was used to treat diarrhoea, vaginal
discharge and as an anti-venom for snake bites. This liquor was also used as a hair dye. (Hummer,
2010). The leaves of the Salmonberry plant (Rubus spectabilis) can be chewed and spat onto burns
to clean the wound and reduce pain. (Stevens & Darris, 2000). Rubus fruticosus is also used in tribal
medicine as an anthelmintic (an anthelmintic is a drug used to destroy parasitic worms) and as an
antispasmodic (used to relieve spasm of involuntary muscle) drug (Ali, et al., 2013).

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It is well known that in vivo reactive oxygen species are formed, for example superoxide anion,
hydroxyl radical and hydrogen peroxide, are potentially harmful to living tissue as well as being
highly reactive (Tacnakittirungrod, et al., 2007). There has been links between the presence of
reactive oxygen species and some degenerative disorders such as: Alzheimer’s disease, cancer,
atherosclerosis, diabetes mellitus, hypertension, AIDS and aging (Halliwell & Gutteridge, 1998).
One group of compounds that are found in Rubus fruticosus are a class of flavonoids called
anthocyanins; these are naturally occurring phenolic compounds. Phenolic compounds are
categorised as compounds containing one or more aromatic rings bearing one or more hydroxyl
groups, they can be simple molecules such as phenol (C6H5OH) or can be extremely complex such as
Lambertianin C (C123H80O78). They are responsible for the red, purple, and blue colours of many
plants and in particular in berries (Wang & Xu, 2007).
Figure 1.3: Functional group and basic structure of an anthocyanin (Tsuruda, et al., 2013)
Sellappan, et al., (2002) showed that blackberries and blueberries are major sources of
anthocyanins. The colouring effects and stability of anthocyanins can vary depending on many
factors such as pH, light, oxygen, enzymes, sugars, etc. For example certain anthocyanins in the
presence of acid will reflect red light but alter the pH to alkali and the same anthocyanin will reflect
blue light. An article published in 2010 claimed there are “more than 635 anthocyanins identified in
nature, featuring six common aglycones and various type of glycosylations and acylations” (He &
Giusti, 2010). The benefits of anthocyanins to the plant is that it acts as a “sunscreen” as it absorbs
blue-green and UV light, this is advantagous to the plant as it helps to inhibit oxidative stress
(Mulabagal, et al., 2009).

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Different types of anthocyanins are associated with producing different colours, a study conducted
in 1979 by Jennings and Carmichael, revealed which anthocyanins contribute to what colours
through out the Rubus species. The study was also very focused on the genetics, specifically as to
why each anthocyanin was produced from various phenotypes. Their results show that anthocyanins
that produce a black colour phenotype could be produced from two genotypes where as the other
mentioned colour phenotypes such as; deep purple, red, orange, and yellow; could only be signalled
by one genotype. (Jennings & Carmichael, 1980).
Table 1.1: The original table from Jennings and Carmichael’s study, showing the quantitative
variation in anthocyanin concentration associated with the major genes T, Y and B1 within the Rubus
genus (Jennings & Carmichael, 1980)
The abbreviations used in the table are: Cy – Cyanindin Gl – Glucose, Sop – Sophorose, Sam –
Sambubiose, RU – Rutinose, Xy – Xylosyl.
The data in table 1.1 shows that each one of these anthocyanins contribute towards the colours
relevant within this study, such as black and red. However this does not mean that all of these
anthocyanins will be present within Rubus fruticosus, but it is expected that there will be some of
these present. There are two major anthocyanins in blackberries that have long been established as
the major and minor anthocyanins in blackberries, these are cyanidin-3-glucoside and cyanidin-3-
rutinoside (Stintzing, et al., 2002; Mazza & Miniati, 1993). As well as the regular anthocyanins that
have been found, a novel zwitterionic anothocyanin was isolated in evergreen blackberries and has
been structurally characterized as cyanindin-3-dioxalylglucoside (Stintzing, et al., 2002).
There have been links made between the consumption of red wine and a decrease in coronary heart
disease (CHD), and the most accepted theory to explain this is that the antioxidising compounds
from the grapes that make the wine red in colour contribute to a decrease in the likelihood of
developing CHD (Wrolstad, 2001). In a study by Benvenuti, et al., (2004) it was shown that Rubus
fruticosus contains a high source of antioxidants, such as anthocyanins, (Conde, et al., 2007) so there
could be a similar link made to suggest that eating blackberries will lower the chance of developing
CHD. Despite these theories there is no definitive link to suggest that the consumption of anti-

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oxidising compounds will lower the risk of CHD, however a study conducted in 1993 by Hertog et al.,
achieved significant results when testing the flavonoid intake against the development of CHD and
fatal/non-fatal myocardial infarctions. It was shown by this study there is an inverse correlation
between the relative risk of CHD mortality and the intake of flavanoids (Hertog, et al., 1993).
Blackberries are known for their sweet taste, and use in many sugar rich products such as jam.
Cartier, et al., (1988) conducted a study with a purification experiment of Rubus fruticosus using
Barium hydroxide as the purification agent. The results showed that “Barium hydroxide yielded a
homogenous extracellular polymer which comprised of galactose, glucose and mannose” (Cartier, et
al., 1988). Sugars can have other uses, for example in the up and coming bioplastic industry.
Bioplastics are “biobased polyers derived from the biomass or issued from monomers derived from
the biomass and which, at some stage in its processing into finished products, can be shaped by
flow” (Vert, et al., 2012). The sugars that are expected to be found within Rubus fruticosus are:
glucose, fructose (Kafkas, et al., 2006), sucrose (Buysse & Merckx, 1993) and xylose (Koch, 1886).
Glucose is the main product of photosynthesis, because of this it is expected to be one of the most
abundant sugars within bramble. It has been found that yeast species such as Saccharomyces
cerevisiae can readily convert glucose into ethanol (Galazzo & Bailey, 1990). The exploitation of
photosynthesis and the conversion of glucose to ethanol by organisms such as yeast could
potentially be very useful to the biofuel industry.
The biofuel industry is one that is constantly growing in interest, especially with the supply of fossil
fuels running out very quickly (Shafiee & Topal, 2009). Biofuels are fuels that come directly from
living biomass; they can be gaseous, liquid or solid and are carbon based. Some examples of biofuels
are biodiesel, ethanol, methanol, methane and charcoal (Escobar, et al., 2009). Ethanol is the most
common biofuel as it can be produced from any organic matter of biological origin that contains a
large quantity of sugars or other materials that can be converted into sugars such as starch or
cellulose (Escobar, et al., 2009).
Ethanol is seen as an attractive alternative fuel as it has many benefits over more commonly used
fuels developed from fossil fuels such as petrol and natural gas. Some of the benefits of using
ethanol as a fuel include; being a renewable bio-based resource and being oxygenated. The
advantage of ethanol being oxygenated is that it could potentially reduce particulate emissions in
compression-ignition engines (Hansen, et al., 2005).
Fructose is most commonly used in its crystalline form as a nutritive sweetener in foods and
beverages (American Dietetic Association, 2006). Crystalline fructose has been seen as a nutritionally

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advantage sweetener because of how it is metabolised in the body, therefore it was a popular choice
of sweetener used by companies promoting “diet food”. It was not used by mainstream food
companies because it was considered an expensive source of sweetener (Hanover & White, 1993). In
addition to these fructose is also of use to the fuel industry, as it can be readily converted into furans
such as HMF(Hydroxymethylfurfural), which can then be converted into fuels, as well as solvents and
plastics (Ståhlberg, et al., 2011).
Figure 1.4: “Isomerization of glucose to fructose and its further derivatization to biopetrochemicals”
(Ståhlberg, et al., 2011)
Figure 1.4 shows how glucose, fructose and sucrose can be converted into useful compounds by the
dehydration of fructose into HMF a molecule that can be readily converted into plastics, solvents
and fuels. Glucose and fructose are both hexoses and are isomers of each other, whereas xylose is a
pentose.

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Figure 1.5: showing the ring and chain structure of glucose and fructose (Josephson & House, 2013)
Xylose is a main component of xylan; a hemicellulose. “Xylans constitute 25-35% of the dry biomass
of woody tissues” (Ebringerova & Heinze, 2000). “Hemicelluloses in plant cell walls that have beta-
(14)-linked backbones with an equatorial configuration” (Scheller & Ulvskov, 2010).
Xylose can also be converted into furfural by a dehydration reaction (O'Neill, et al., 2009). Furfural is
common furan; the functional group of a furan is a five membered aromatic ring consisting of four
carbon atoms and one oxygen atom (Brown, 2005). Furfural can be used for biological as well as
chemical purposes. Some of the biological uses for furfural include: “preservatives, fungicides,
herbicides, disinfectants and therapeutic agents” (Peters, 1939). The chemical uses of furfural have a
larger potential because of its chemical properties. For example, “its aldehydic nature suggested its
use in phenolic resin” (Peters, 1939). Phenol-furfural resins are particularly good at resisting
breaking down in the presence of an acid or an alkali; they also have a large mechanical strength and
useful electrical properties in comparison to other resins (Peters, 1939).
Figure 1.6: The chemical structure of furfural (Food-Info, 1999)

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Xylose can have other uses on a less industrial scale, for example it can be used in medicine to test
for malabsorption. It is administered in solution to a patient after a period of fasting, after a few
hours blood and urine tests reveal whether there is xylose in the blood or urine, which shows
absorption of xylose by the small intestine (MedlinePlus, 2008).
It is predicted that by 2050 the Earth’s population will reach 9.6 billion people, an increase of 2.4
billion from 2014 (UN News Service, 2013), this means there will be a huge increase on the demand
in the food industry. There is potential for plants previously considered to be waste material such as
the non-fruit tissues in Rubus fruticosus to be used as supplements for livestock. In particular the
amino acids from the “waste” tissue could be transformed into a potentially useful protein
supplement. “The food industry can contribute to a sustainable future by development of novel
plant protein products and continual innovations in food preservation” (Aiking, 2011).
The main aims and objectives of this study were to find a method of making a profit from plant
material would otherwise go to waste. There are various methods of analysis to discover the
compounds and molecules with Rubus fruticosus, the main groups of compounds to look for will be
sugars, proteins and phenolics. The methods used to identify the presence of these compounds will
be; Dionex analytical instruments, the Lowry assay, and Mass spectrometry. With using these
techniques it will also be possible to complete a molecular profiling of Rubus fruticosus.
Materials and Methods
Plant material was harvested on two occasions throughout early October 2013
After collection of plant material from Ynys-Hir mid-wales, it was frozen at -20°C and freeze dried to
dehydrate the material. After freeze drying, the plant material was separated into berries, stems and
leaves. The separated material was then ground into a fine powder using a spice grinder and stored
at lab temperature ready to be used.
Analysis and identification of sugars
Samples were analysed using Dionex HPAEC-PAD on an ICS-5000 system fitted with Eluent Generator
and CarboPac SA10 (4 x 250mm) column and appropriate guard column (Thermo Scientific). The
column was equilibrated with 1mM KOH and 10 µL samples run on an eight minute 1mM KOH
isocratic program.

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Phenolic extraction with methanol in preparation for mass spectrometry
Samples were analysed using the mass spectrometry software package Excalibur.
100mg of ground tissue was weighed out into tannin boiling tubes, add 3ml of 70% methanol, and
mix well with a vortex for around 10 minutes. Subject the boiling tubes to centrifugation at 3000rpm
for 10 minutes. Decant the supernatant; 1ml of 70% methanol was added to the pellet and which
was re-suspended and, subjected to further centrifugation for 10 minutes. Combine both
supernatants, and put into a vacuum centrifuge overnight to remove the methanol, repeat until
samples are dry. The samples are now ready to use with a Sep-pak.
Method for use of a Sep-pak
Sep-pak 500 mg C18 column was activated by drawing through 5ml of 100% methanol (activates C18
and removes bound compounds), column was washed with 3-5ml of water. If necessary, the column
was washed with further 2ml water or 5% HAc and added to sample. The sample was allowed to drip
through slowly to allow phenolic compounds to bind. The column was washed with 3-5ml water, to
remove non-phenolic compounds from the Sep-pak. Vial was inserted, eluted with 4ml methanol,
collected liquid in vial and removed and stored at 4°C. Wash column with 5ml 100% methanol. (Do
not let column run dry or overload the column. Ensure that the flow rate is reduced during loading
of the sample).
The samples were then analysed by liquid chromatography electro-spray mass spectrometry (LC-ESI-
MS) using a Thermo Finnigan LC-MS system (Finnigan Surveyor LC pump plus, PDA plus detector,
Finnigan LTQ linear ion trap) (Thermo Scientific, Massachusetts, USA) and a Waters Nova-Pak C18 4.0
µm, 3.9x100 mm column. Injection volume was 10 µL and the flow rate 1 mL/min. The mobile phase
consisted of purified water- 0.1% formic acid (solvent A) and MeOH-0.1% formic acid (solvent B) with
a linear gradient from 5 to 65%, B in A, over 30 min. Phenols were detected by UV absorption and
after the PDA detector the mobile was passed through a splitter with 100µL/min going the mass
spectrometer and 900µL/min diverted to waste. Phenolics were characterised by UV absorption
spectra, MS fragmentation patterns in negative ion mode and comparison with standards and
previously reported data in the literature.

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Antioxidant assays
In each Assay a standard curve was created using 6-hydroxy-2,5,7,8-tetramethylchroman-2-
carboxylic acid (Trolox), which is known for its antioxidising abilities. (Valyova, et al., 2009). The
assays were analysed using Swift II Fraction analysis software, connected to a Spectrophotometer.
FRAP Assay
The ferric reducing antioxidant power (FRAP) assay measures the antioxidant potential of
antioxidants to reduce the Fe3+
/ 2,4,6-tripyridyl-s-triazine (TPTZ) complex present in stoichiometric
excess to the blue ferrous form of TPTZ (Fe2+
) (Szôllôs & Varga, 2002). The FRAP reagent was freshly
prepared by mixing together 10mM TPTZ and 20mM ferric chloride in 0.25M acetate buffer, this
gives the FRAP reagent a pH of 3.6. The FRAP reagent is prepared by adding acetate buffer, TPTZ and
Iron chloride in the ratio of 10:1:1 respectively. Samples from the various tissues of Rubus fruticosus
were made up to a range of concentrations with water. For the assay, X volume of sample was
added to Y volume of the FRAP reagent. The mixture was let to stand at room temperature for 4
minutes. Presence of antioxidant activity is shown as the solution will change from being yellow in
colour to being blue/ purple in colour. Absorbance was measured at 593nm. Absorbance at 593nm is
directly related to the antioxidant activity of the sample.
ABTS Assay
The ABTS radical scavenging assay is based on the positive radical ion, ABTS•+
“The pre-formed
radical monocation of 2,2’-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS•+
) was
generated by oxidation of ABTS with potassium persulphate and was reduced in the presence of
hydrogen-donating antioxidants” (Re, et al., 1999). The radical form was prepared by adding
potassium persulphate to a 7mM stock solution of ABTS to give a final concentration of 2.45mM. The
mixture was then left to stand in the dark at room temperature for 16 hours. ABTS radical must be
kept in the dark while stored as there would be a reaction with the radical and visible light that
makes the assay ineffective, we can assume that it is the visible light doing this as UV cannot
penetrate the glass. At the time of the assay the ABTS•+
solution is diluted with water to give an
absorbance of 0.700 (±0.2) at 743 nm. 25µl of sample are added to 1500µl of reagent and
absorbance is measured after 6 minutes. Varying concentrations of Trolox were added to 1500µl of
reagent to give a standard curve. The higher the antioxidising activity of the sample, the greater the

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reduction in absorbance at 743nm, as the solution changes from being green in colour to becoming
colourless in the presence of antioxidants.
Protein extraction and Lowry assay
The Lowry assay was measured with a µQuant Microplate Spectrophotometer with KC4 software.
Samples were extracted in 1.8ml pH 7 McIlavaine buffer, then ground with a pestle and mortar.
200µl 20% lithium dodecyl sulphatewas added to the sample, grinding continued until the sample
was a fine paste, it was then transfered to a test tube. Extracts were subjected to centrifugation and
the supernatant was transferred to a clean tube. Protein samples were prepared by a precipitation
from McIlavaine buffer/Lithium dodecyl sulphate with an equal volume of 20% trichloroacetic acid
(TCA), 0.4% phosphotunistic acid (PTA), normally 125µl of each. The TCA/PTA was stored at 4°C and
was brought to laboratory temperature before use with samples containing Lithium dodecyl
sulphate. The sample/TCA mixture was then incubated at laboratory temperature for 30 minutes
and the precipitated protein was then subjected to centrifugation again at 15 000g for 10 minutes.
The supernatant was discarded – The pellet was re-suspended with 2 x 250µl TCA/PTA, the sample
was mixed well with a vortex, and was subjected to further centrifugation and the supernatant
discarded once more. This was repeated and the sides of the tubes were dried using paper towels.
The pellet was re-suspended in 750µ l of 0.1M sodium hydroxide and was mixed well.
Lowry Assay
The Lowry Assay method is particularly sensitive at reading concentrations ranging between 0.05 –
2mg of protein per ml (Dunn, 1992) (Price, 1996).
Bovine serum albumin (BSA) standard was made up in 0.1 M sodium hydroxide at a concentration of
0.5mg/ml. Increasing volumes of BSA and decreasing volumes of 0.1M sodium hydroxide were used
to create a standard curve to compare to the samples from the various tissues of Rubus fruticosus.
200µl of assay mixture was added to each tube. The mixture consisted of three reagents, the first
reagent was 4.8% sodium carbonate and 0.56% sodium hydroxide, the second reagent was 2.4%
copper sulphate, and the third was 4.8% sodium potassium tartaric acid. The three reagents were
added in a ratio of 10:0.1:0.1 respectively.
The solution was left for 10 minutes to allow the dissolved copper to bind to proteins in the solution.
After this 200µl of diluted Folin-Ciocalteu’s phenol reagent (one unit of concentrated Folin’s solution
with seven units of purified water) was added to each tube. The tubes were left for over an hour to

14
RT: 0.00 - 30.02
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
Time (min)
0
2000000
4000000
6000000
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10000000
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20000000
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30000000
Intensity
22.42
22.34
11.18
10.99 11.51
9.87 12.07
9.73
16.64
16.04
13.15 14.28
18.02
8.54
7.96
6.750.88 20.796.48
24.42 28.88
19.056.31
24.896.13
26.69
5.78
5.37
5.14
1.23 4.45
NL: 3.16E7
m/z= 200.00-1500.00
F: ITMS - c ESI Full
ms [ 95.00-1200.00]
MS
Berry1_13110514173
7
allow the reaction to take place and to produce the full colourisation. After this triplicate sub
samples (150µl per well) are transferred to a micro-titre plate (via pipette). Once loaded analyse the
micro-titre plate using a micro-titre plate reader. Absorbance was measured at 750nm.
Results
Analysis of phenol composition of Rubus fruticosus
The Rubus fruticosus plant material were analysed by CC-ESI-MS in triplicate. Molecular masses of
the phenolic compounds and their various fragmentation patterns were determined from the data.
Berry
Figure 3.1: A UV Chromatogram showing separation of berry phenolic compounds by C18 liquid-
chromatography
Figure 3.1 shows the UV absorption profile of the berry extract following separation by C18 liquid
chromatography. The largest peak shown by figure 3.1 is at a retention time of 9.75 minutes the
peak gives an absorbance 340 000uAU, the second largest peak occurs at a retention time of 11.17

15
minutes with an absorbance of 160 000uAU. These peaks were tentatively identified as phenolic
compounds and will be further investigatedto determine their identity.
Figure 3.2: shows the MS chromatogram of the berry extract following separation by C18 liquid
chromatography.
RT: 0.00 - 30.00
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
Time (min)
0
20000
40000
60000
80000
100000
120000
140000
160000
180000
200000
220000
240000
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300000
320000
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uAU
9.75
9.54
11.17
17.35
16.70
17.7414.91
12.528.476.72 24.036.09 19.93 20.44 24.840.97 27.384.854.13
NL:
3.43E5
Total Scan
PDA
Berry1_131
105141737

16
Berry1_131105141737 #2862 RT: 9.54 AV: 1 NL: 1.98E6 microAU
250 300 350 400 450 500 550 600
wavelength (nm)
0
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
1100000
1200000
1300000
1400000
1500000
1600000
1700000
1800000
1900000
uAU
379.00 517.00 531.00503.00392.00 551.00480.00439.00 580.00
Figure 3.3: UV spectrum of the compounds with a retention time of 9.54 minutes.
Figure 3.3 shows the UV absorbance of molecules with a retention time of 9.54 minutes. It shows
that there is a broad peak from 240 – around 330nm.
Berry1_131105141737 #632 RT: 9.56 AV: 1 NL: 6.10E5
F: ITMS - c ESI d Full ms2 933.64@35.00 [ 245.00-1880.00]
400 600 800 1000 1200 1400 1600 1800
m/z
0
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
550000
600000
Intensity
1566.51
300.69
632.71
1249.85
914.63
782.84
896.65
1098.88
616.98450.77 934.69857.55 1866.63764.74314.65 1698.641414.521160.00

17
Figure 3.4: Mass spectrum for the compounds with a retention time of 9.56.
Sanguiin H-6, C82H54O52 Antioxidant agent in raspberries and found in other rubus species such as
boysenberry, it was identified as one of the major ellagitannins (Kool, et al., 2010).
Figure 3.5: MS chromatogram for berry extract between the ranges of 200-1500 m/z
Very similar to figure 3.2 except for in figure 3.5 there is no peak at around 22 minutes, this is
because of the parameters of each ion trap, figure 3.2 is between 95-1500 ms and figure 3.5 is
between 200-1500 ms. From this we can conclude that the peak at 22 minutes is caused by a
molecule with an ms value between 95 and 200.
RT: 4.58 - 20.62
6 8 10 12 14 16 18 20
Time (min)
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32000000
34000000
36000000
38000000
40000000
42000000
44000000
46000000
48000000
50000000
52000000
54000000
Intensity
11.17
11.35
10.93
11.59
11.92
16.71
12.09 15.8110.65
13.18 17.11
17.837.95 15.716.73 14.84
14.046.42 7.08 20.47
8.44 8.52 19.9719.57
8.616.20
5.81
NL: 4.56E7
m/z= 200.00-1500.00
F: ITMS + c ESI Full
ms [ 200.00-2000.00]
MS
Berry1_13110514173
7

18
Figure 3.6: UV spectrum for compounds with a retention time of 11.14 minutes.
Figure 3.6 is an example of a Trademark UV spectrum of an anthocyanin.
Berry1_131105141737 #3342 RT: 11.14 AV: 1 NL: 3.94E5 microAU
250 300 350 400 450 500 550 600
wavelength (nm)
0
20000
40000
60000
80000
100000
120000
140000
160000
180000
200000
220000
240000
260000
280000
300000
320000
340000
360000
380000
uAU
279.00
515.00
Berry1_131105141737 #744 RT: 11.21 AV: 1 NL: 2.24E7
F: ITMS + c ESI Full ms [ 200.00-2000.00]
200 400 600 800 1000 1200 1400 1600 1800 2000
m/z
0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
8000000
9000000
10000000
11000000
12000000
13000000
14000000
15000000
16000000
17000000
18000000
19000000
20000000
21000000
22000000
Intensity
449.27
287.28
896.73
590.29 1344.74411.42 1793.89914.66 1037.94 1578.49697.45 1934.21878.18 1220.14

19
Figure 3.7: mass spectrometer results for compounds with a retention time of 11.21 minutes, size of
compounds range from 200-2000.
The obvious peak from the mass spectrum in figure 3.7 occurs at 449.27, this peak indicates the
presence of cyanidin-3-glucoside.
Figure 3.8: A zoomed in UV absorption for compounds from the berry extracts, ranges of retention
times are between 12.23 and 21.18.
RT: 12.23 - 21.18
13 14 15 16 17 18 19 20 21
Time (min)
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
11000
12000
13000
14000
15000
uAU
17.85
17.04
15.50
16.19
13.39 18.24
13.66
14.86
14.55
18.74 19.65
19.77
20.57
21.04
NL:
5.64E4
Total Scan
PDA
berryMSn

20
Figure 3.9: MS chromatogram for compounds with a mass/charge ratio between 136.85-900m/z
figure 3.10: UV spectrum for compounds with a retention time of 13.48 minutes. Another example
of a classic anthocyanin finger print spectra.
berryMSn #4044 RT: 13.48 AV: 1 NL: 3.47E4 microAU
250 300 350 400 450 500 550 600
wavelength (nm)
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
22000
24000
26000
28000
30000
32000
34000
uAU
279.00
516.00
454.00379.00 435.00402.00
581.00
RT: 0.00 - 30.01
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
Time (min)
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
55000
60000
65000
70000
75000
80000
Intensity
13.19
13.33
13.4913.04
13.61
12.79
12.66
12.46
12.35
12.15
12.0510.74
9.32
NL: 8.26E4
m/z= 136.85-900.00
F: ITMS + c ESI Full
ms2 595.00@35.00 [
160.00-1200.00] MS
berryMSn

21
Figure 3.11: Mass spectrum displaying fragments with a retention time of 13.38 minutes. 595
corresponds to Cyanidin 3 rutinoside, 449 corresponds to cyanidin glucoside, and 287 corresponds
to cyanidin aglycone.
Leaves
berryMSn #937 RT: 13.38 AV: 1 NL: 4.04E4
F: ITMS + c ESI Full ms2 595.00@35.00 [ 160.00-1200.00]
200 300 400 500 600 700 800 900 1000 1100 1200
m/z
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
22000
24000
26000
28000
30000
32000
34000
36000
38000
40000
Intensity
595.26
287.27
449.26
433.19213.54 375.52 854.88515.69 667.82 900.61

22
Figure 3.12: UV chromatogram showing the separation of leaf phenolic compounds by C18 liquid-
chromatography
Figure 3.13: UV spectrum for fragments with a retention time of 9.74 minutes.
Leaf1 #2924 RT: 9.74 AV: 1 NL: 2.28E6 microAU
250 300 350 400 450 500 550 600
wavelength (nm)
0
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
1100000
1200000
1300000
1400000
1500000
1600000
1700000
1800000
1900000
2000000
2100000
2200000
uAU
248.00
379.00 398.00 423.00 589.00444.00 560.00468.00 488.00
RT: 0.00 - 30.00
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
Time (min)
0
20000
40000
60000
80000
100000
120000
140000
160000
180000
200000
220000
240000
260000
280000
300000
320000
340000
360000
380000
400000
420000
uAU
9.71
9.47
5.94
11.68
16.62
18.88
8.33
7.34
18.27
16.37
12.09 13.10
19.50
20.08 21.32 23.70 24.195.320.98 25.64 27.213.95
NL:
4.31E5
Total Scan
PDA Leaf1

23
Figure 3.14: Zoomed in UV chromatogram of the two largest peaks from figure 3.12, separating the
two peaks.
Figure 3.15: Mass spectrum of the compounds with a 9.72 minutes retention time peak from figure
3.14. The large 934 peak is most likely to be the MS of Sanguiin H-6 but not in its dimer form.
RT: 9.19 - 10.23
9.2 9.3 9.4 9.5 9.6 9.7 9.8 9.9 10.0 10.1 10.2
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
RelativeAbsorbance
9.71
9.47
NL:
4.31E5
Total Scan
PDA Leaf1
Leaf1 #658 RT: 9.72 AV: 1 NL: 5.10E6
T: ITMS - c ESI Full ms [ 95.00-1200.00]
100 200 300 400 500 600 700 800 900 1000 1100 1200
m/z
0
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
2400000
2600000
2800000
3000000
3200000
3400000
3600000
3800000
4000000
4200000
4400000
4600000
4800000
5000000
Intensity
934.41
136.32
1017.89238.63 1137.09306.77 466.87 634.81578.79 674.77352.66178.52 782.93 888.81

24
Stem
Figure 3.16: UV chromatogram for stem phenolic compounds i.
Figure 3.17: UV spectrum for fragments with retention times of 9.19-9.74 minutes.
RT: 0.00 - 30.00
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
Time (min)
0
20000
40000
60000
80000
100000
120000
140000
160000
180000
200000
220000
240000
260000
280000
300000
320000
340000
uAU
9.72
9.51
16.6511.668.32 17.345.71
12.097.21 16.39
19.19 20.87 23.68 24.010.98 26.085.02 28.273.61
NL:
3.50E5
Total Scan
PDA Stem1
Stem1 #2757-2924 RT: 9.19-9.74 AV: 168 NL: 1.82E6 microAU
250 300 350 400 450 500 550 600
wavelength (nm)
0
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
1100000
1200000
1300000
1400000
1500000
1600000
1700000
1800000
uAU
379.00 589.00398.00 416.00 576.00440.00 549.00467.00 483.00 505.00

25
Figure 3.18: shows the MS chromatogram of the stem extract following seperation by C18 liquid
chromatography.
Figure 3.19: Mass spectrum for the highest peaks from figure 3.18, with retention times between
9.14-9.23 minutes.
Stem1 #592-618 RT: 9.14-9.23 AV: 2 NL: 3.31E5
F: ITMS - c ESI d Full ms2 933.58@35.00 [ 245.00-1880.00]
400 600 800 1000 1200 1400 1600 1800
m/z
0
20000
40000
60000
80000
100000
120000
140000
160000
180000
200000
220000
240000
260000
280000
300000
320000
Intensity
1249.89
1566.50
300.56
632.69
914.60
1098.88
782.76
896.58616.84
1866.521017.82314.59 468.58 764.61 832.86602.87 1396.67 1716.591208.73
RT: 0.01 - 30.03
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
RelativeAbundance
9.88
9.79
9.40 16.68
11.67
8.34
22.3814.845.72
12.780.79 8.16
17.71
6.37 19.98
14.64
18.897.85 20.89
23.88
26.7024.40
28.89
24.78
5.28 28.18
5.10
4.87
1.39
3.63
2.40
NL:
1.82E7
TIC F: ITMS -
c ESI Full ms
[
95.00-
1200.00] MS
Stem1

26
Sugar analysis
Figure 3.20: Chromatogram produced by DIONEX, showing sugars present within the Berry 1
sample.nC2
Table 3.1: Numerical representation of figure 3.20

27
Figure 3.21: Chromatogram produced by DIONEX, showing sugars present within the Berry 2 sample
Figure 3.22: Chromatogram produced by DIONEX, showing sugars present within the Berry 3 sample

28
Figure 3.23: Chromatogram produced by DIONEX, showing sugars present within the Leaf 1 sample

29
Figure 3.24: Chromatogram produced by DIONEX, showing sugars present within the Leaf 2 sample.

30
Figure 3.25: Chromatogram produced by DIONEX, showing sugars present within the Leaf 3 sample

31
Figure 3.26: Chromatogram produced by DIONEX, showing sugars present within the Stem 1 sample

32

33
Table 3.10: The total heights of the graphs from Dionex, dilution factors were: Berries, 500; Leaves,
20; Stems, 10.
1 2 3 Average
Adjusted Value for dilution
factor
Berries 135.631 97.203 106.164 112.9993 22599.86667
Leaves 216.959 238.278 233.185 229.474 4589.48
Stems 267.427 230.341 238.151 245.3063 2453.063333

34
Table 3.11: Values for the quantity of glucose in each tissue sample
1 2 3
Average
(µg/ml)
factor (µg/ml)
Berries 28.3627 20.0627 21.9815 23.46897 4693.793333
Leaves 45.5005 49.909 48.6461 48.01853 960.3706667
Stems 58.0292 49.4268 51.0364 52.8308 528.308
Table 3.12: Values for the quantity of fructose in each tissue sample
Table 3.13: Values for the quantity of xylose in each tissue sample
1 2 3
Average
(µg/ml)
(µg/ml)
Berries 0.2042 0.1335 0.1458 0.161167 32.23333333
Leaves 2.1721 2.5351 2.3731 2.3601 47.202
Stems 4.9268 3.5913 3.7995 4.105867 41.05866667
Table 3.14: Showing the calculated values of the response factor of each sugar found by Dionex
Sample
Area (nC^2
min)
Amount
ug/ml
Response
Factor
Galactose L1 0.313 0.8506 0.367975547
L2 0.437 1.1882 0.367783201
L3 0.592 1.6067 0.368457086
1 2 3
Average
(µg/ml)
(µg/ml)
Berries 34.4016 23.7384 26.0164 28.05213 5610.426667
Leaves 50.8169 57.5402 55.8109 54.72267 1094.453333
Stems 65.412 52.2666 54.756 57.4782 574.782

35
S1 0.609 1.6546 0.368064789
S2 0.437 1.1882 0.367783201
S3 0.445 1.2097 0.3678598
Average 0.472166667 1.283 0.368017667
Glucose B1 10.842 28.3627 0.38226262
B2 7.669 20.0627 0.382251641
B3 8.402 21.9815 0.382230512
L1 17.393 45.5005 0.382259536
L2 19.078 49.909 0.382255705
L3 18.595 48.6461 0.382250581
S1 22.182 58.0292 0.38225583
S2 18.893 49.4268 0.382242023
S3 19.509 51.0364 0.382256585
Average 15.84033333 41.43943333 0.382252653
Xylose B1 0.075 0.2042 0.367286974
B2 0.049 0.1335 0.367041199
B3 0.054 0.1458 0.37037037
L1 0.799 2.1721 0.367846784

36
L2 0.932 2.5351 0.367638357
L3 0.872 2.3731 0.367451856
S1 1.811 4.9268 0.367581392
S2 1.32 3.5913 0.367554924
S3 1.397 3.7995 0.367679958
Average 0.812111111 2.209044444 0.367630046
Fructose B1 7.769 34.4016 0.225832519
B2 5.361 23.7384 0.225836619
B3 5.875 26.0164 0.225819099
L1 11.476 50.8169 0.225830383
L2 12.994 57.5402 0.225824728
L3 12.603 55.8109 0.225816104
S1 14.771 65.412 0.225814835
S2 11.803 52.2666 0.225822992
S3 12.365 54.756 0.225820001
Average 10.55744444 46.751 0.225822858

37
Protein analysis
Figure 3.29: A graph showing the absorbance of varying concentrations of Bovine serum albumin to
create a standard curve
Figure 3.30: A graph showing the absorbance of the three types of tissues used in this study; Berry
(B), Leaf (L), and Stem (S).
y = 0.0101x
0
0.2
0.4
0.6
0.8
1
1.2
0 10 20 30 40 50 60 70 80 90 100
Absobrance(AU)
Percentage concentration of BSA (5mg/ml)
Absorbance of varied concentrations of BSA at
750nm
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
B1 B2 B3 B AVE L1 L2 L3 L AVE S1 S2 S3 S AVE
Absorbance(AU)
Rubus fruticosus tissue type
Absorbance of various tissues from Rubus
fruticosus at 750nm

38
Table 3.15: showing the calculation of the BSA equivalent concentration of the Rubus fruticosus
tissue samples and the BSA standard
Average
absorbance (AU)
Percentage BSA
equivalent
BSA equivalent
(mg/ml)
Berry 0.371333 36.76567657 2.65173E-05
Leaf 0.342444 33.90539054 2.44543E-05
Stem 0.638778 63.24532453 4.56157E-05
BSA 0.86 100 7.21251E-05
Antioxidant testing
During the preparation of the L2 sample was lost and was therefore not included in any result.
FRAP assay
Table 3.10: Absorbance of Trolox at varying concentrations
Trolox
Concentration (mM)
Absorbance (AU)
Run 1 Run 2 Average
0.41 0.344 0.355 0.3495
0.37 0.329 0.339 0.334
0.33 0.222 0.244 0.233
0.29 0.185 0.202 0.1935
0.25 0.135 0.157 0.146
0.21 0.117 0.137 0.127
0.16 - 0.096 0.096

39
Figure 3.31: Absorbance of varying concentrations of TROLOX, assayed with FRAP
Table 3.11: Absorbance of Rubus fruticosus tissues when assayed with FRAP
FRAP Assay results
Trial run run 1 run 2 Average
Trolox Equivalent
Antioxidant Capacity (mM)
B1 2.1 0.376 0.362 0.369 0.4861
B2 1.8 0.274 0.278 0.276 0.36359
B3 1.44 0.286 0.229 0.2575 0.3626
B AVE 0.300833 0.396302
L1 2.24 0.319 0.254 0.2865 0.37742
L2 N/A N/A N/A N/A
L3 1.89 0.237 0.29 0.2635 0.34712
L AVE 0.275 0.36227
S1 0.906 0.167 0.159 0.163 0.21473
S2 0.991 0.05 0.096 0.073 0.096167
S3 1.03 0.221 0.227 0.224 0.29509
S AVE 0.153333 0.20199
y = 0.7591x
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45
Absorbance(AU)
Concentration of TROLOX (mM)
FRAP assay TROLOX results

40
Figure 3.32: Absorbance of Berries, Leaves and Stems from Rubus fruticosus when assayed with
FRAP
ABTS assay
Table 3.12: Absorbance of varying concentrations of Trolox when assayed with ABTS
Trolox
Concentration (mM)
Absorbance (AU)
Run 1 Run 2 Average
0.616 0.619 0.622 0.6205
0.4928 0.654 0.656 0.655
0.3696 0.732 0.735 0.7335
0.2464 0.886 0.887 0.8865
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
B1 B2 B3 B AVE L1 L2 L3 L AVE S1 S2 S3 S AVE
Absobance(AU)
FRAP assay sample results

41
Figure 3.33: Absorbance of varying concentrations of TROLOX assayed with ABTS
Table 3.13: Absorbance of Rubus fruticosus tissues when assayed with ABTS
ABTS Assay results
Run 1 Run 2 Average
Trolox equivalent Antioxidant
Capacity (mM)
Control 0.859 0.852
B1 0.109 0.18 0.1445 1.2457
B2 0.225 0.24 0.2325 1.122
B3 0.135 0.306 0.2205 1.1389
B AVE 0.199167 1.2813
L1 0.156 0.273 0.2145 1.473
L2 N/A N/A N/A N/A
L3 0.08 0.293 0.1865 1.4488
L AVE 0.2005 1.4609
S1 0.468 0.483 0.4755 0.78043
S2 0 0.59 0.295 1.0342
S3 0.858 0.537 0.6975 0.46837
S AVE 0.489333 0.76103
y = -0.7114x + 1.0307
0
0.2
0.4
0.6
0.8
1
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
Absorbance(AU)
TROLOX conentration (mM)
ABTS assay from varying concentrations of
TROLOX

42
Figure 3.34: Absorbance of Berries, Leaves and Stems from Rubus fruticosus, when assayed with
ABTS
Discussion
Identifying compounds from mass spectrometry
The main aim of using mass spectrometry was to identify some of the various phenolic compounds
within Rubus fruticosus. Each of the tissue samples had varied UV chromatogram’s, but when further
analysed similarities were found.
The analysis of the berry tissue was the most in-depth as there is more literature published about
the profiling of the berries than any other tissue from the Rubus genus. Figures 3.1-3.4 indicate
towards the presence of Sanguiin H-6 in its dimer form. The standard MS peak for Sanguiin H-6 is at
934 m/z and this peak is not present within the spectra of figure 3.4, however Sanguiin H-6 has
multiple MS2
ions which are present within the spectra the largest of them being the peak at
1566.51 m/z. The other MS2
fragments that appeared in the spectra are 300.69, 632.71, 1249.85,
914.63, 782.84, 896.65 m/z (in order of intensity on the spectra). There is an assumption made that
the 782.84 m/z peak is doubly charged as it is half the molecular weight. The peaks at 300.69 and
632.71 are singly charged and are attributed to the sequential loss of ellagic units such as galloyl-bis-
HHDP glucoside (936 Da) which is a characteristic monomer of Rubus ellagitannins (Gasperotti, et al.,
2010). These fragments are all typical of Sanguiin H-6.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
B1 B2 B3 B AVE L1 L2 L3 B AVE S1 S2 S3 S AVE
Absorbance(AU)
ABTS assay sample results

43
Sanguiin H-6 is an isomer of agrimoniin and has the chemical formula of C82H54O52, it is also a dimer
of casuaricitin. Casuarictin is made up of two hexahydroxydiphenic acid units and one gallic acid unit
linked by a glucose molecule, it freely forms other oligomers as well as sanguiin H-6, such as
Lambertianin A-D (Tanaka, et al., 1993). The polymerization of casuarictin is caused by the “oxidative
linkage C-O between galloyl groups and HHDP with galloyl groups” (Aguilera-Carbo, et al., 2008;
Quideau & Feldman, 1996; Zhang, et al., 2001).
The next peak further analysed was the retention time of 11.17 from figure 3.1. When put through a
UV detector the result gave a trademark fingerprint of an anthocyanin the double peak. In figure 3.7
the two main peaks from the mass spectra are at 449.27 and 287.28 m/z. The 449.27 m/z peak is
representing cyanidin-3-glucoside (cyanidin-3-O-hexoside) (Yoshimura, et al., 2012). The peak at
287.28 m/z is 162 m/z lower than the peak representing cyaniding-3-glucoside so indicates that it
might be the same molecule with a hexose fragmented off of it.
Figure 3.9 shows the full ms2 of a peak found at 595 m/z, this can be identified as cyanidin-3-
rutinoside because of the presence of peaks at 449, and 287 m/z in figure 3.11 (retention time of
13.38 minutes) which are common fragments of this molecule, if there was no peak at 449 m/z then
this compound could easily be mistaken for cyanidin-3-O-6-coumaroylglucoside (Slimestead &
Solheim, 2002), the parameters of the ion trap were between 136.85-900 m/z. Figure 3.10 is the UV
spectrum of the molecules with the retention time of 13.48 minutes, and confirms that it is in fact an
anthocyanin. The fragment that was associated with the peak at 287 m/z could be cyanidin-3-
algycone, another anthocyanin (Byamukama, et al., 2005).
The UV chromatogram for the leaf tissue (figure 3.12) showed two main peaks with retention times
of 9.71 and 9.47 minutes. The minor peaks for the UV chromatogram of the leaf tissues had
retention times of 5.94, 11.68, 16.62 and 18.88. Figure 3.14 is a more specific spectrum showing the
close detail of the two largest peaks from the leaf spectra; 9.71 and 9.47. From this it was possible to
choose a retention time to be further analysed. The UV spectrum of this retention time does not
reveal much as there is just one peak at 248µAU (figure 3.13). Figure 3.15 shows the mass spectrum
for the retention time peak at 9.72 minutes, and gives a clear peak at 934.41 m/z. This peak alone is
undefinable as it could be the monomer casuarictin, which could mean the presence of any of its
polymers such as sanguiin H-6 or lambertianin C. Further experiments need to be conducted to fully
break down this peak and then to analyse the phenolic compounds in greater detail within the leaf
tissue. The other possibility with this peak is a slight error in the accuracy of the mass
spectrophotometer meaning that the peak could be rounded to 935 m/z which would correspond to
galloyl-bis-HHDP-glucose (Kajdžanoska, et al., 2010)

44
The UV chromatogram for the stem tissue (figure 3.16) is similar to that of the leaf tissue in regard to
the largest peak is in roughly the same place around 9.5 minutes, there are not any other peaks that
are thought to be worthy of further investigation. Another similarity the stem tissue has with the
leaf tissue is the UV spectrum produced from the largest peaks, but this is to be expected as they are
from the same retention time. However the ion trap mass spectrometry reveals more peaks that
could be worth investigation. The ms2 graph shown in figure 3.19 produced from the retention time
of 9.2 minutes shows an almost identical spectrum to the berry tissue for the presence of sanguiin H-
6, all of the trademark peaks are there to suggest its presence (Krauze-Baranowska, et al., 2010).
Analysis of Dionex results for sugars present in various tissues
The results from the Dionex are incredibly easy to analyse as the machine itself identifies the
compounds that have been extracted from the sample and also gives numerical values in the form of
a table for each of the samples. Figures 3.20 – 3.22 show the different sugars present within the
berries of Rubus fruticosus.
The overall trend of the prevalence of sugars within the various tissue types of Rubus fruticosus is
that glucose is the highest peak on each of the graphs with fructose being the second highest,
however in the leaves there is an unknown peak at a retention time of just under two minutes, this
is believed to be mannitol, xylitol or similar sugar alcohols (Mäkinen & Söderling, 1980).
However the peak heights are not proportional to the quantity of sugar present within the sample,
as each different compound has a different response factor to the system. Each particular sugar has
its own magnitude of response, so the assumption that the peak area will be equal to the abundance
cannot be made. Ignoring the variation in response factor, table 3.10 shows that the berry tissue
contained the largest total quantity of sugars (this was to be expected); followed by the leaves, and
then stems have the lowest value.
From the tables 4.2 and 4.3 it is obvious that although the peaks corresponding for glucose on the
Dionex results are larger than the ones corresponding to fructose, fructose is the more abundant
sugar in each of the three tested tissues. This suggests that glucose has a high response factor to the
system than fructose.
The sugar with the third largest abundance that was present in all of the tissues is xylose, the leaves
of the blackberry plant are the highest source of xylose within the plant; oddly the berries actually
have the lowest quantity of xylose, even lower than the stems. Fructose is the most abundant sugar

45
overall throughout the different tissues. It is interesting to see that the results showed no sign of
sucrose present in the tissues, this could be because it has been broken down into its monomers of
glucose and fructose.
From table 3.14 we can see that fructose has the lowest response factor meaning it has the lowest
response to the system, glucose has the highest response to the system. This is why at first glance of
the graphs from the Dionex results glucose appears to be the most abundant because of having the
largest peaks.
The results show that fructose is the most abundant sugar present within bramble, and is therefore
the most efficient sugar to be harvested from the plant, this means the use of Rubus fruticosus
berries could be used to synthesise furans for the biofuel industry or to create solvents or bioplastic.
Alternatively the fructose from the berries could be used as a sweetener for “diet foods” such as,
cereal bars or meal replacement shakes.
Proteins
From table 3.15 it is possible to see that the stem of the Rubus fruticosus plant is the richest in
proteins showing almost double the absorbance of the other two tissues.
The results of the Lowry assay show that the stem of the blackberry plant gives a 63% BSA
equivalent for the presence of proteins, this is the most viable tissue for protein extraction within
the plant. More research would be needed in this area to determine whether the stems of Rubus
fruticosus could be a potentially useful source of proteins to supplement livestock. There has been
very little research into the less popular protein plants, however there is evidence in this study to
suggest that there is potential in Rubus fruticosus for protein extraction.
It has been found that the average protein composition of Rubus spp. berries is between 20-40%
(Bushman, et al., 2004), the results agree with this statistic with the protein composition of Rubus
fruticosus being around 36%, this is near the top end of the suggested quota for the Rubus genus
suggesting that Rubus fruticosus berries are fairly rich in proteins in comparison to berries from
other Rubus species, further increasing the reasoning behind more research into protein extraction
from Rubus fruticosus.
Antioxidants
The results of the antioxidant assays give a clear indication that the main antioxidant activity in
Rubus fruticosus comes from the leaves and from the berries. The stems showed very little

46
antioxidant activity in comparison to the others. However knowing which of the leaves or berries has
the highest overall antioxidant activity is not clear, as each assay gave a different result. The FRAP
assay revealed that the berries had a higher antioxidant activity than the leaves, but the ABTS assay
showed the opposite. This is because the two assays have a very different mechanisms, ABTS
measures the ability to reduce a radical and the FRAP assay measures the ability to reduce the ferric
ion (Thaipong, et al., 2006). The results of the mass spectrophotometry showed that there are
similar molecules within each tissue that are known for antioxidant activity such as Sanguiin H-6.
Known anthocyanins were also found within the berries in addition to the ellagitannins, this could be
evidence towards a theory as to why berries give a higher antioxidant activity in the FRAP assay, as
anthocyanins could have a larger influence in the reduction of the ferric ion than the ellagitannins. It
is also possible to see a difference between the assays the standard curves created by the known
concentration of Trolox. However the individual mass spectra’s for the leaves and berries were very
different despite showing some similarities, further research into the mass spectrometry of
phenolics within the berries and leaves would need to be conducted to find other compounds with
antioxidant activity.
Conclusion
In summary the main phenolic compounds identified in berries were; sanguiin-H6, cyanidin-3-
glucoside, cyanidin-3-runtinoside: in leaves were; casuarictin (the monomer of sanguiin-H6 and
lambertanin C): and in stems; sanguiin-H6. There were many more peaks in the UV chromatograms
for each tissue that were not broken down and analysed this is one area that will require extra
research. The further research that should be conducted would involve the analysis of the smaller
peaks from the UV chromatogram of each tissue in particular the leaves and berries, as these were
the highest source of antioxidant activity.
In the sugar analysis it was found that fructose is the most abundant sugar throughout the plant, the
highest quantity was found the berries with a concentration of 5.6mg/ml. This is of interest as
fructose can be broken down to HMF and other furans to then be processed into plastics, solvents or
fuels. Glucose was the second most abundant compound with its highest concentration of 4.7mg/ml
being present within the berries. The other sugar of interest found within Rubus fruticosus was
xylose, the highest concentration of xylose was 47µg/ml and this was found within the leaf samples.
The use of xylose for industrial purposes such as the production of furfural will most likely be too

47
inefficient to be worthwhile. However blackberries could be a source of xylose for medical testing
(malabsorption).
The Lowry assay for proteins revealed that the stems of the blackberry plant were the richest source
of protein, with a BSA equivalent of 0.046µg/ml; this value initially seems too small to be of any use,
however in percentage terms it was 63% BSA equivalent, therefore 63% protein. From this evidence
the stems of Rubus fruticosus could potentially be a very viable source of protein to be used for
supplementation, in particular for livestock.
The two assays FRAP and ABTS showed that the berries and leaves have the highest antioxidant
activity respectively. The berries gave an average Trolox equivalent antioxidant capacity value of
0.40mM in the FRAP assay, in the ABTS assay the leaves gave an average Trolox equivalent
antioxidant capacity value of 1.46mM. Further analysis into antioxidant activity is required to
determine if bramble is a viable source of antioxidants.
Future Research and Improvements
If this study were to be conducted again it would be wise to consider harvesting the plant material
earlier in year as some of the leaves and berries were beginning to wilt as it was late in the season
when they were harvested.
To identify more phenolic compounds with in the samples it would require further analysis of the UV
chromatograms of the different samples. This means investigating further into the more minor peaks
present on the chromatograms and looking into the mass spectrometry and mass spectrometry two
results. By doing this it would be possible to identify many more phenolics and therefore have a
greater knowledge into the mechanisms behind the antioxidant activities they contribute to.
A method to improve the research into the phenolic compounds and their antioxidant activity,
would be to perform high performance liquid chromatography (HPLC) on the samples, this would
help to separate and isolate the various phenolics with the samples. Using HPLC allows the
extraction of compounds with particular retention times. With the use of HPLC a much more
detailed profile of phenolic compounds within Rubus Fruticosus could be found. The individual
extractions could then be analysed for antioxidant activity to show what compounds are responsible
for which mechanism within the assays. The separation technique of HPLC would also allow the use
of nuclear magnetic resonance (NMR) spectroscopy; this is used to investigate properties of organic
compounds, and can be used to identify the structure.

48
To increase the understanding of the content of proteins with in bramble, further assays could be
performed such as the Bradford protein assay (also called the Bradford Coomassie brilliant blue
assay) (Zor & Selinger, 1996). This assay is another method for determining unknown protein
samples. To increase the accuracy and reliability of the results another standard could be used as
well as BSA, such as α-chymotrypsin. The results would then give two equivalent concentrations.
NMR spectroscopy could also be used here to actually identify the proteins that are present in the
sample. The two antioxidant assays used different concentrations of the standard Trolox, this made
them harder to draw a comparison between them, if this was to be repeated the same
concentrations of Trolox. Another way the antioxidant assays could be improved would be to include
a second standard to measure the samples against as well as Trolox, such as; ascorbic acid (Khalaf, et
al., 2008).
An overall improvement for further research could be the inclusion of profiling the roots of Rubus
fruticosus for sugars, proteins, phenolics, etc. Roots of other species in the Rubus genus have been
found to be a large source of esters (Jung, et al., 2001).
Acknowledgments
A massive thank you to Dr Ana Winters for all of her help, support and overall supervision
throughout this project. Also thank you to Dr Barbara Hauck for her assistance in the laboratory, and
finally thank you to Jennifer King for assistance throughout the project.
Word count 6551

49
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