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MAQUI NEW LIFE

MAQUISELECT/DELPHINOL
Scientific/Technical Report
CONFIDENTIAL

Scientific/Technical Report
CONFIDENTIAL

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MAQUI NEW LIFE


MAQUISELECT/DELPHINOL
Scientific/Technical Report
CONFIDENTIAL

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MAQUI NEW LIFE


MAQUISELECT/DELPHINOL
Scientific/Technical Report
CONFIDENTIAL

SUMMARY
The present report contains a brief exposure of scientific and technical
information about MAQUISELECT/DELPHINOL, a product from MAQUI NEW LIFE
S.A. a bio-science company from Chile.
Our product, MAQUISELECT/DELPHINOL, is a standardized extract
from Maqui berry, Aristotelia chilensis, a fruit with high antioxidant capacity.
The report includes generalities about Maqui berry, as well as the
composition of our product MAQUISELECT/DELPHINOL, a dry standardized
extract of freezed Maqui berry fruit.
General information on Maqui and in particular on MAQUISELECT/
DELPHINOL will be shown and discussed.
Also included in the report is scientific evidence of the effect of the
different antioxidant components present in MAQUISELECT/DELPHINOL, such as
anthocyanins and delphinidins, in four main aspects of human health: as an immune
system booster, as an inflammatory control, as anti-cancer treatment, and as a
glucose balance composition.
Further technical information includes Industrial Property Protection of our
product, MAQUISELECT/DELPHINOL, and a general brief of patent documents
and scientific publications about delphinidins and their uses and effects.

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Scientific/Technical Report
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Contents
Summary..................................................................................................................3
Product Description..................................................................................................7
MAQUISELECT/ DELPHINOL ...............................................................................7
Background Information............................................................................................9
Pharmacognosy.....................................................................................................9
Traditional use..................................................................................................9
Current situation.................................................................................................10
Use as food ingredient........................................................................................12
Scientific research...................................................................................................12
Antioxidant content of berries.............................................................................12
Delphinidins........................................................................................................13
Maqui berry, Aristotelia chilensis.........................................................................14
Pharmacodynamics and pharmacological effects of anthocyanins........................15
Application 1: Immune System Booster................................................................17
Our Research...................................................................................................17
Application 2: Anti-Inflammatory Effect...............................................................29
General Research.............................................................................................29
Our Research...................................................................................................29
Application 3: Cancer Treatment.........................................................................33
General Research.............................................................................................33
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Scientific/Technical Report
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Our Research...................................................................................................33
Application 4: Glucose Balance............................................................................38
General Research.............................................................................................38
Our Research...................................................................................................40
Clinical Study...................................................................................................41
Toxicology..............................................................................................................43
Safety in relation to traditional and well established use......................................43
Repeated Dose toxicity, Short term Toxicity testing.............................................43
Subchronic toxicological study in pigs of MAQUISELECT/ DELPHINOL...........43
Sampling and testing.......................................................................................44
Results................................................................................................................45
Body weight.....................................................................................................45
Food intake.....................................................................................................45
Behaviour and appearance...............................................................................45
Mortality..........................................................................................................45
Haematological tests.......................................................................................45
Blood chemistry...............................................................................................45
Histopathological examination........................................................................46
Chronic toxicity testing...................................................................................46
Patent Applications.............................................................................................47
References..............................................................................................................48

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MAQUISELECT/DELPHINOL
Scientific/Technical Report
CONFIDENTIAL

PRODUCT DESCRIPTION
MAQUISELECT/ DELPHINOL
MAQUISELECT/ DELPHINOL is a purified, concentrated, standardized
dried extract from Maqui berry freezed fruit, Aristotelia chilensis.
MAQUISELECT/ DELPHINOL contains fruit solids, is a free flowing deep
purple powder , 100% soluble in water, with good taste, typical of Maqui fresh fruit.
The total content of anthocyanins in MAQUISELECT/ DELPHINOL is NLT
35%, and the total content of delphinidins is NLT 25%. MAQUISELECT/ DELPHINOL
also shows less than 5% of humidity (Certificate of Analysis 10/0174/LRF).

GENERAL ISSUES OF MAQUISELECT/ DELPHINOL


Botanical name
Aristotelia chilensis
Plant part extracted
the fruit
Indian name
MAQUI
Fruit origin
Patagonia area in southern Chile, South America
Manufacturer
Indena Group
Extract ratio
25 40 : 1

PHYSICAL PROPERTIES OF MAQUISELECT/ DELPHINOL


Humidity ( w/w % )
5.0
Appearance
Free flowing deep purple powder
Taste
Good taste, typically maqui fruit fresh
Water solubility
Good solubility in water

BIOACTIVE COMPOUNDS OF MAQUISELECT/ DELPHINOL


Average value of ORAC FN (mol TE/g of extract)
25,000
Total ANTHOCYANINS %
35 NLT
Total DELPHINIDINS %
25 NLT

MICROBIOLOGICAL SPECIFICATIONS OF MAQUISELECT/ DELPHINOL


TAMC Total Aerobic Microbial Count
3,000 CFU/g
TYMC Total Combined Yeast/Moulds Count
500 CFU/g
Bile-tolerant gram negative Bacteria
100 CFU/g
Escherichia Coli
Absent /g
Salmonella
Absent /25g
Pseudomonas aeruginosa
Absent /g
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MICROBIOLOGICAL SPECIFICATIONS OF MAQUISELECT/ DELPHINOL
Staphylococcus Aureus
Absent /g

Another information about MAQUISELECT/ DELPHINOL


Pesticides determination (for food products)
complies

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MAQUISELECT/DELPHINOL
Scientific/Technical Report
CONFIDENTIAL

BACKGROUND INFORMATION
Pharmacognosy
In southern Chile, a wild fruit grows which boosts the immune system,
contains anti-inflammatory properties, is effective to control blood sugar and has
higher levels of antioxidants than any other berry fruit: the Maqui berry.
The Maqui berry is just 4 mm in diameter. It has a dry flavor and contains
four seeds. It grows on an evergreen bush that reaches a height of about 4 meters,
which is also known as Maqui (Aristotelia chilensis).
Traditional use
Maquis therapeutic qualities have been known for centuries to the
Mapuches, indigenous people who have traditionally lived in the southern part of
Chile. Besides eating the fruit, they also consumed fresh and fermented Maqui juice.
They used it to treat stomach ailments, sore throats or wounds, and also as an
analgesic and fever reducer, and as a natural colorant.
Mapuches also used dry leaves infusions or directly powdered dry leaves to
treat wounds. Fresh leaves infusions were used to alleviate feverish conditions,
diarrhea, dysentery, indigestion, to alleviate sore throat symptoms,
tonsil
inflammation, and mouth ulcers. The juice of fresh leaves was also drank or used as
ointment topically.
The research team at Pharmacology and Morphophysiology Institute, of the
Faculty of Veterinary Sciences of Universidad Austral de Chile, one of the partners of
Maqui New Life developing MAQUISELECT/DELPHINOL, is conducting ongoing
research that started three years ago. Their findings have proven the exceptional
properties of Maqui, revealing their chemical origin and identifying other properties
which were not known to the Mapuches.
MAQUISELECT/DELPHINOL extract is obtained from the wild Maqui fruit,
gathered in southern Chile. It has a standardized content of anthocyanins (35% NLT)
and an astonishing level of delphinidins (25% NLT), the highest among all food
ingredients which are currently available.

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Scientific/Technical Report
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Current situation
The oxidation processes in our cells produce free radicals, which are atoms
or molecules with an unpaired electron. These free radicals are highly reactive with
other substances and therefore damaging to the human organism.
Antioxidants are molecules which can neutralize free radicals, stopping the
stress caused by oxidants and slowing down the cell aging process.

Figure 1: ORAC capacity of different berry fruits.

The ORAC (Oxygen Radical Absorbance Capacity) index measures the


antioxidant activity and free radical neutralization properties in food. Also, a high
ORAC value for a food is often associated with protection against various chronic
inflammatory diseases or metabolic disorders.
Maqui fresh fruit has the highest ORAC score among berries known so far
(Figure 1). Ronald L. Prior and Guohua (Howard) Cao from the Jean Mayer USDA
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Scientific/Technical Report
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Human Nutrition Research Center on Aging at Tufts University in Boston suggest a
daily intake of 3,000 to 5,000 ORAC units. However, based on average fruit and
vegetable consumption and their ORAC content, most people consume only about
1,200 ORAC units.Recently, Chilean Government, through the Health Ministry
included Maqui in a list of Traditional Herbal Medicines, recognizing its antiinflammatory, antispasmodic, astringent and analgesic properties.
In the United States of America, the use of Maqui berries in food is
approved as safe by the FDA and the import directives established by the USDA, as
established in the list of Approved Fruits and Vegetables version 04/2010.
In Europe, Maquie berries have been imported before 1997, and thus
would not fall in the category of novel food, according to regulation 258/97/EC.

Use as food ingredient


The idea to commercialize Maqui was introduced by our team to the US
market. Given the high content of antioxidants in the Maqui fruit, its use as an
ingredient in different food products has increased in the last years.
Alone, or in combination with other plants or fruit extracts, Maqui is
present in drinks, capsules, makeup powders, dietary supplements, nutraceuticals,
with different marketing focus, and in different configurations, from lyophilized
powder, concentrated juice extracts, fruit pulp, etc.
More than 60 products containing Maqui are available, such as Monavie's
Pulse, Monavie's EMV, which are marketed as energizing and anti-oxidant beverages;
Lancome's Maqui Loose powder, commercialized as a makeup powder; Tahiti
Trader's Maqui 100 and Organic Maqui 100, sold as beverages with high antioxidant
content; Swanson's Full Spectrum Maqui Berry capsules; Live Superfoods' Maqui Berry
powder; Genuine Whole Food Now Maqui Super Antioxidant Juice; S4Labs'
MaquiMaxx; Health Spark Limited's Maqui Extreme; MritzMayers Laboratories' Super
Maqui-1200; Health Essentials' Maqui Berry Active; Biovea's Maqui Berry capsules;
Dr. Scurr's Zinopin capsules; Honest Tea's Kombucha beverage; sold as dietary
supplements, among many others. Nevertheless, none of the available products
contains a Maqui concentrated extract standardized in its delphinidin content as
MAQUISELECT/DELPHINOL does.

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Scientific/Technical Report
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SCIENTIFIC RESEARCH
Antioxidant content of berries
The intake of super berries has a positive impact on human health, well
being and the prevention of certain diseases, including cardiac, neurodegeneration ,
aging, obesity, and also certain types of cancer, such as esophagus and digestive
system. The biological properties are due to the content of polyphenols. Polyphenols
include flavonoids (anthocyanins, flavonoles, and flavonoids), condensed tannins
(proanthocyanidins) and hydrolized tannins (ellagitannins and galotannins),
estilbenoids and phenolic acids [61].
Among polyphenols, anthocyanin (pigments that give the attractive blue
violets, red colors), have demonstrated antioxidant, anti cancer, and antiinflammatory effects [62-65]. The concentrations of polyphenols, either condensed
proanthocyanidines or ellagitannins, vary considerably among berries [65]. For
example, blueberries contain principally proanthocyanidines, whereas blackberries,
black raspberries, raspberries and strawberries contain principally ellagitannins.
Therefore, the type and specific chemical structure of the compounds present in a
berry can contribute significantly to the biological properties. For example, the
antibacterial anti-adhesive properties of red cranberry are explained by the
oligomeric proanthocyanidins, that possess a type A structural link [66]. Similarly,
the biological effects of blueberries (rich in proanthocyanidine) compared to
strawberries (rich in elagitannins) on neuronal and cognitive deficit in adult rats can
be due to the effect of these compounds in different regions of the brain [67,68]. A
diet rich in pterostilben (present in berries and grapes) is able to revert the negative
effects of cognitive involution and behavior performance [69].
The phenolic compounds from berries are better known for their antioxidant capacities [70]. In fact, they regulate the activity of the enzymes that
metabolize and modulate the nuclear receptors, genetic expression, signalization
paths, and DNA oxidative damage repair [70, 71]. Animal and human studies have
shown that polyphenols are poorly absorbed due to the low levels found in blood.
However, the polyphenols from berries are also metabolized and converted by the
micro flora of the colon in other related compounds. These compounds can persist
in vivo, accumulating in the tissues and contributing to the biological effects.
Polyphenols from berries have a role in the prevention and treatment of different
chronic diseases [72].

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Scientific/Technical Report
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Delphinidins
Delphinidins are a class of anthocyanidines of which Maqui fruit, A.
chilensis has the highest content of all known fruits, and our product,
MAQUISELECT/DELPHINOL contains up to 28.6%.

Berry

mg of
anthocyanin
per 100mg
fruit

Blueberry

25-495

Cranberry

45-100

Raspberry

20-60

Blackberry

83-326

Maqui

1400

Main kind of anthocyanins


delphinidin-3-galactoside(13,5%),malvidin-3galactoside(12,9%),malvidin-3-glucoside(11,9%),malvidin-3arabinoside(11,9%),delphinidin-3-arabinoside
peonidin-3-galactoside, peonidin-3-arabinoside, cyanidin-3galactoside,cyanidin-3-arabinoside
cyanidin-3-sophoroside, cyanidin-3-glucoside, cyanidin-3rutinoside,cyanidin-3-glucosylrutinoside
cyanidin-3-glucoside(57%),cyanidin-3-rutinoside(30%)
delphinidin-3-sophoroside-5-glucoside(33,4%),delphinidin-3-5diglucoside(16,2%),cyanidin-3-5-diglucoside(5,8%),cyanidin-3sophoroside-5-glucoside(14,8%),delphinidin-3sophoroside(9%),delphinidin-3-glucoside(12,4%),cyanidin-3sophoroside(4%),cyanidin-3-glucoside(4,4%)

Maqui berry, Aristotelia chilensis


A. chilensis is an endemic plant of the Elaeocarpaceae family present
geographically in central and southern Chile, up to 2,500 m altitude, also in the
archipelago of Juan Fernndez and south-western of Argentina. It grows in soil rich
in organic matter. Frequently A. chilensis forms pure communities that receive the
name of macales. The fruit has more pulp than other berries and its flavor is
described as astringent but fresh''. The leaves and the fruits of A. chilensis, have
been used for sore throat, ulcer, fever, hemorrhoids, inflammation, diarrhea, lesions,
migraines and scars [74-76].
Maqui has become a top super berry in the food and beverage industry in
USA. This fruit contains one of the highest contents of anthocyanins, polyphenols
and has a great capacity to trap free radicals (ORAC), with values between 4 to 30
times higher than other berries (mangosten, acai, etc.) [77].
The anthocyanins present in the fruit of Maqui constitute a 0.2% and are
associated to a great anti-oxidant capacity. In the fruit 8 pigments are found:

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Scientific/Technical Report
CONFIDENTIAL
1. Delphinidin-3-O-samb-5-O-gluc;
2. Delphinidin-3,5-O-diglucoside;
3. Cyanidin-3-O-samb-5-O-gluc;
4. Cyanidin-3,5-O-diglucos;
5. Delphinidin-3-O-sambubioside;
6. Delphinidin-3-O-glucoside;
7. Cyanidin-3-O-sambubioside;
8. Cyanidine-3-O-glucoside
Where the principal anthocyanin is delphinidin-3-sambubioside,5glucoside, that constitute 34% of total anthocyanins. In the fresh fruit, the mean
total anthocyanins is 137.6 0.4mg/100g; and in dried fruit 211.9 0.6 mg/100g.
[78]. Moreover, the fruits contain a high content of minerals, 100gr of fruit, provide
27% of the daily recommended dose of calcium, 28% of potassium and 70% of iron,
demonstrating a potential as a dietary supplement and functional food [82]
The juice of Maqui can inhibit the oxidation of lipoproteins of low density
(LDL) and protect the endothelial cells against intracellular oxidative stress, as antiatherogenic [73]. Methanolic extract of the fruits in in vivo studies have
demonstrated a preventive protective effect in reperfusion/ ischemia in the heart of
rats, probably due to a reduction in the lipidic oxidation and oxidative stress[79].
MAQUISELECT/DELPHINOL

has a high concentration of delphinidins

(28.6%).
It is known that anthocyanins have a positive effect in many diseases.
Recently, anti-inflammatory [68], antiviral [69], and antitumoral [67] effects were
demonstrated for anthocyanins. It is known that anthocyanins, activate PPAR
modulating the inflammatory responses and inhibiting NfB, a nuclear transcription
factor that controls the expression of several proinflammatory proteins. When NFB
is inhibited, the pro inflammatory cytokines (Interferon gamma, Interleukin 2) and
pro inflammatory agents (COX-2, iNOS) are reduced.
MAQUISELECT/DELPHINOL has demonstrated the capacity to inhibit
COX2 and to reduce inflammation induced by carrageen using the rat paw model. It
is experimentally proven that MAQUISELECT/DELPHINOL activates PPAR and
inhibits NFB without toxicity and collateral effects. NFB is activated in the
inflammation of upper respiratory tract as asthma and allergies and in autoimmune
diseases
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Pharmacodynamics and pharmacological effects of anthocyanins


In the murine model Apcmin, which is highly susceptible to spontaneous
intestinal adenoma formation, the effect of anthocyanins obtained from red grape
pomace extract (oenocyanin) were tested for its cancer chemopreventive properties.
Mice received oenocyanin (0.3%) in their diet until week 16, when adenoma number
and burden were recorded. In mice which had consumed oenocyanin, overall
adenoma burden was halved and adenoma number was marginally reduced when
compared with mice on control diet. Oenocyanin anthocyanins and glucuronide
metabolites were found in the urine and intestine but not in plasma [132].
A further study [131] analysed the effect of mirtocyan, an anthocyanin
extracted from bilberries, in colorectal cancer human patients, considering the
pharmacodynamics. The study found that anthocyanin concentrations in plasma and
urine were roughly dose-dependent, reaching approximately 179 ng/gram in tumor
tissue at the highest dose. In tumor tissue from all patients on mirtocyan,
proliferation was decreased by 7% compared with preintervention values. The low
dose caused a small but nonsignificant reduction in circulating IGF-I concentrations.
In conclusion, repeated administration of bilberry anthocyanins exerts
pharmacodynamic effects and generates concentrations of anthocyanins in humans.
The pharmacological effect of delphinidins has been widely researched,
and scientific evidence has been found, either in vitro or in vivo, relating the
application of delphinidins with antioxidant effects [1, 2, 3, 5, 6, 7, 11, 12, 13, 14,
19, 20, 21, 24, 25, 26, 28, 29, 30, 31, 32, 35, 36, 37, 38, 39, 41, 43, 44, 47, 52, 54,
56, 57 and 59]; anti-inflammatory effects [5, 24, 34, 50, 51, 53 and 55], prevention
in tumor generation, cancer suppressor, and apoptosis induction in cancerous cells
[5, 8, 10, 15, 17, 18, 22, 24, 32, 33, 40, 42, 44, 45, 46, 49, 50, 51, 55, 58 and 60];
angiogenesis inhibition in cancer [9, 23, 25, 27 and 56]; inhibition of nitric oxide
production [4, 16 and 25]; and effect on metabolic syndrome [48].
Our team identified four main application fields for anthocyanins, more
particularly delphinidins, in human health. These fields are application of
delphinidins as an immune system booster, application of delphinidins with an antiinflammatory effect, application of delphinidins in cancer treatment, application of
delphinidins to maintain glucose balance in metabolic syndrome associated
conditions.
In the following sections, further detail will be given to the use of
delphinidins in the fields of application identified by our team, as well as results
from our own research, demonstrating the effects of our product, MAQUISELECT/
DELPHINOL, in these situations.

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Application 1: Immune System Booster


As previously discussed in this report, our product, MAQUISELECT/
DELPHINOL , comprises up to 28.6 % of delphinidins, thus, analysing the function
or effect of delphinidins is of major importance.
Current research on the effect of delphinidins in the immune system is
non-existent. Nevertheless, Maqui New Life is analysing the effect of delphinidins in
the immune system, firstly in an in vitro set of experiments.
Our Research
Our research group has performed several studies to determine the effect
of the main components of our product, MAQUISELECT/DELPHINOL, on the
immune system.
Calcium, apart from being important as a major bone constituent, is also
relevant as a second messenger in many cell types. In particular, lymphocytes.
In cell signaling, calcium is maintained at a low intracellular concentration,
and in the case of lymphocytes, the activation of antigen receptors induces an influx
of calcium from the extracellular space. One of the main routes is the StoreOperated Calcium (SOC) channels.
Therefore, the research of our team has been focused in studying the
effects of the main components of MAQUISELECT/DELPHINOL in calcium
mobilization in lymphocytes, more particularly, T cells. This includes analysing the
effect of delphinidin in Jurkat cells, which are an immortalized line of T lymphocyte
cells frequently used for in vitro studies of immune system, as well as similar studies
using human lymphocytes obtained from peripheral blood.
In a first approach, our research determined that delphinidin induces
calcium mobilization in a dose-dependent manner in Jurkat E6-1 cells. As can be
seen in Figure 2, with increasing concentration of delphinidin, from 10 micromolar to
100 micromolar, the calcium fluxes are also increased.

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3.20
3.0

Ratio 340/380

2.8
2.6
2.4
2.2

In t

2.0
1.8
1.6

100 M Delphinidin
50 M Delphinidin
10 M Delphinidin

1.4
1.2
1.0

0.90
50.0

80

100

120

140

160

180

200

220

240

260

280

300

320

340

360

380 400.0

Seconds
Figure 2: Calcium fluxes in Jurkat cells. A dose-dependent effect of delphinidin is shown.

As mentioned earlier, the mobilization of calcium in lymphocytes is


primarily through Store-Operated Calcium Entry (SOCE) system. In order to prove
that delphinidins induce calcium flux at SOCE level, Jurkat cells were incubated with
gadolinium, BTP-2, and 2-APB, known as SOCE inhibitors.
Figures 3 and 4 show the effect of known SOCE inhibitors, 2-APB and
gadolinium, in delphinidin-induced calcium fluxes in Jurkat cells.

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B
1.8

Delphinidin

1.7

Delphinidin

1.6

2.4

Ratio

Ratio

2.2
2.0
1.8
1.6

1.5
1.4
Int

340/380

340/380

Int

1 mM CaCl2

1.9

2.8
2.6

EGTA +
0 mM CaCl2

2.00

3.00

1.3
1.2
1.1
1.0
0.9

1.4

0.8
1.2
1.00
50.0

0.7

80

100

120

140

160

180

200

220

240

260

280

300

320

340

0.60
50.0

360 380.0

100

150

200

250

300

350

400

450

480.0

Seconds

Seconds

2-APB
0 M + 50 M Delphinidin
2-APB 10 M + 50 M Delphinidin
2-APB 50 M + 50 M Delphinidin
2-APB 100 M + 50 M Delphinidin
2-APB 0 M + 0 M Delphinidin

2-APB
0 M + 50 M Delphinidin
2-APB 10 M + 50 M Delphinidin
2-APB 50 M + 50 M Delphinidin
2-APB 100 M + 50 M Delphinidin
2-APB 0 M + 0 M Delphinidin

Figure 3: Jurkat cells incubated with delphinidin and 2-APB SOCE inhibitor. A) Calcium flux in
response to delphinidin in the presence of variable concentrations of 2-APB, B) Calcium flux
response in Jurkat cells to an increase in extracellular calcium.
3.60
3.4

3.2
3.0

Ratio 340/380

2.8
2.6

Delphinidin

2.4
Int

2.2
2.0
1.8
1.6
1.4
1.2
1.0

0.80
50.0 60

80

100

120

140

160

180

200

220

240

260

280

300.0

sec

Seconds
Gd3+
0 M + 50 M Delphinidin
Gd3+ 1 M + 50 M Delphinidin
Gd3+ 3+
5 M + 50 M Delphinidin
Gd 10 M + 50 M Delphinidin

Figure 4: A) Jurkat cells incubated with delphinidin and gadolinium, a SOCE inhibitor.
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Apart from SOCE, there are also other types of calcium mobilization which
are modulated by the application of delphinidins. Intracellular calcium as well as PLC
are essential in delphinidin-induced calcium mobilization. This is clear when Jurkat
cells are incubated with variable concentrations of U-73122, a known PLC inhibitor,
and delphinidins as shown in Figure 5.

2.50
2.4
2.3
2.2
2.1

Ratio 340/380

2.0

Int

Delphinidin

1.9
1.8
1.7
1.6
1.5
1.4
1.3
1.2
1.1
1.0
0.9
0.8
0.7

0.60
50.0

80

100

120

140

160

180

200

220 240

260

280

300

320

340

360

380 400.0

Seconds
U-73122 0 M + 50 M Delphinidin
U-73122 1 M + 50 M Delphinidin
U-73122 5 M + 50 M Delphinidin
U-73122 10 M + 50 M Delphinidin
U-73122 0 M + 0 M Delphinidin
Figure 5: Calcium release in response to delphinidin, challenged with PLC inhibitor U-73122.

Further research was focused in following the effects of calcium signaling


through SOCE system in the production of cytokines involved in immune response,
IL-2 and Interferon .
Intracellular calcium in resting cells is approximately 0.1 micromolar, while
in an activated cell the concentration of intracellular calcium increases to 1
micromolar. This increase in turn induces the expression of different cytokines. As
can be seen in the plot below, IL-2 production is significantly increased with 50
micromolar delphinidin (Figure 6).

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900

800

IL-2 (pg/ml)

700
600
500
400
300
200
100
f
D
el

f
M

D
10
0

M
50

M
30

el

f
D
el

f
el
D
M

10

C
on

tr
ol

Figure 6: Delphinidin-induced IL-2 production.

It is postulated that delphinidin-induced IL-2 production is through


calcium mobilization through SOCE, since addition of known SOCE inhibitor BTP-2
(1, 5, 10 M), greatly affects IL-2 production, as can be seen in Figure 7.

IL-2 (pg/ml)

All previous results are experiments using Jurkat cells. Nevertheless, a


similar effect is observed using T-cells. We observed an increase in IL-2 and INFgamma induced by delphinidin in human lymphocytes obtained from peripheral
blood, as shown in Figure 8.
300
250
200
150
100
50
20
15
10
5

**
**

**

D
C
el
on
B
fin
TP
tr
ol
i
-2
di
na
1

50
B
M
TP
+
M
-2
D
e
5
lf.
M
B
5
TP
0
+
M
-2
D
10
el
f
.5
M
0
+
M
D
el
f.
50
M
B
TP
-2
M

Figure 7: Delphinidin induced IL-2 is inhibited with BTP-2.


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900

800
700

2000

600

INF- (pg/ml)

500
400
300
200

1500
1000
500

f
M

el

f
10
0

M
50

M
30

D
el

f
D
el

el
f
D

0
M

D
el
f

el
f
10
0

50

D
el
f

30

D
el
f
M
10

C
on

tr
o

10

100

C
on
tr
ol

IL-2 (pg/ml)

**

2500

Figure 8: Delphinidin-induced IL-2 and INF- production in human peripheral blood


lymphocytes.

Figure 9: Delphinidin induced IL-2 and INF-gamma production is mediated by SOCE.

In order to prove that human peripheral blood lymphocyte IL-2 and IFN-
production induced by delphinidins is also mediated by SOCE system, human
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peripheral blood lymphocytes were incubated with a SOCE inhibitor, BTP-2. The
results, shown in Figure 9 prove this hypothesis.
Further to our research on the effects of delphinidin on the immune
system, our group found that delphinidin-induced production of cytokines (IL-2 and
INF-) is through the NFALT pathway, as was measured by dephosphorylation of
NFAT and the activation of pNFAT/pRL.
Our research has demonstrated that delphinidins elevate the release of
intracellular calcium in Jurkat cells, which may activate the production of cytokines
such as IL-2 and IFN- in this cell line and in human T lymphocytes. Since cytokine
production in T lymphocytes is activated through the NFAT transcription factor, and
production of IL-2, induced by the delphinidins, is significantly reduced by the
cyclosporin A (CsA) calcineurin inhibitor, it is evident that delphinidins have the
ability to activate NFAT. All of these effects result in strengthening the cells of
the immune system.

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Application 2: Anti-Inflammatory Effect


General Research
Inflammation processes are biological responses of tissues to harmful
stimuli. These processes are mediated by many different enzymes, the most
common pathways in inflammation including cycloxygenases 1 and 2 (COX-1, COX2) and 5-lipoxygenase (5-LOX). Thus, the inhibition of these enzymes using different
drugs has been proved to alleviate the symptoms of inflammation.
There are several reports describing specific effects of delphinidins in
inhibiting the above mentioned enzymes COX and LOX, thus, an anti-inflammatory
effect is expected.
A research reports the inhibition of up to 12% of COX-1 and COX-2 using
concentrations of delphinidins of 40 micromolar, compared to common use antiinflammatory drugs, such as ibuprofen (10 micromolar) with an inhibition of nearly
40% [5]. A similar research, this time using 100 micromolar delphinidins reported an
inhibition of COX-1 and COX-2 of up to 49% [24].
Other study reports that delphinidins inhibit TNF-alpha induced COX-2
expression [51], while other study shows that delphinidins block the activation of
NF--B by UVB, and thus blocked the NF--B induced COX-2 expression.
Also, delphinidins have been found to act as lipoxygenase inhibitors [53]
suggesting that delphinidins could be used for anti-inflammatory therapy.

Our Research
We have conducted preliminary tests to find the
MAQUISELECT/DELPHINOL in an in vivo model of inflammation.

effect

of

The anti-inflammatory effect of A. chilensis using the acute subplantar


model of inflammation in rats was studied. Carrageenan 1% was injected
subplantarly. The degree of inflammation was evaluated with the measurements of
the width of the mice leg using a digital caliper, from time 0 (before injection) till 24
hrs. The maximum inflammatory effect was observed at 3 hours post injection. A.
chilensis, was injected i.p. at 100 mg/kg. A. chilensis had a significant antiinflammatory effect at 4 hours after injection. This effect was similar to the one
observed with diclophenac between 4 and 24 hours (Figure 10).

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Edema (mm)

10.0

7.5

* *

**
**

****
*

5.0

2.5

0.0

0.0

1.0

2.0

3.0

4.0

5.0

6.0

24.0

Hora
SS
A. chilensis 100
mg /kg

Diclofenaco2mg/kg
A. chilensis
0,025 mg/kg

Figure 10: Evolution of edema size in time for SS saline solution, A. chilensis 100mg/kg, A.
chilensis 0.025 mg/kg and diclophenac.

Figure 11: Histological comparison of rat paw tissue between experimental groups at 6 hours
after treatment with carrageenan. (a) control subject; (b) treated with A. chilensis 100mg/kg at
time 0; (c) treated with A. chilensis 0.025mg/kg at time 0; (d) treated with diclofenac 2mg/kg.
All pictures with 10X magnification.

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Application 3: Cancer Treatment


General Research
Inflammation has been identified as a critical component in tumor
progression, where tumor microenvironments are frequently invaded by
inflammatory cells. Also, quantitative aspects of wound repair or inflammatory gene
expression often correlate negatively with cancer stage and prognosis. Furthermore,
NF--B signaling pathway has been described in inflammatory processes as well as
cancer.
Delphinidins, apart from research focused in inflammatory processes, have
been widely studied to determine their effect in cancer. There are reports suggesting
that delphinidins have a tumor suppressor function, can induce apoptosis, or can
have a potential role inhibiting angiogenesis in cancer.
For example, a study with 3-O-beta-galactopyranoside, a delphinidine,
shows inhibition of COX-1 and COX-2, suggesting a protecting role in tumor
formation and a role as anti-inflammatory compound [5].
Other reports show the effect of delphinidines present in berries inducing
apoptosis in leukemia cells HL60 [8, 10, 22], humane uterine carcinoma, colon
adenocarcinoma [15], hepatoma [18] and colon cancer cells HCT116 [8, 51].
A very important discovery, by Dr. Beliveau team, showed that delphinidins
inhibit VEGF receptor 2 in neovascularization processes of tumor genesis [130].
Further studies show the effect of delphinidins inhibiting the proliferation
of mammary cancer cells [17], human umbilical cord endothelial cells [25], colon
cancer, lung, stomach, and mammary [24], adenocarcinoma and HT-29 cells [45]
and murine hepatoma cells [33].
Thus, the effect of delphinidins in cancer is at many different levels, acting
as a tumor suppressor, inhibiting proliferation of cells, inducing apoptosis, and
inhibiting angiogenesis among others.

Our Research
In the study, conducted by our research group, we evaluated the effects of
a juice berry A. chilensis on COX-2 expression, intracellular signalling pathways, and
cell viability in colon cancer cells. Caco-2 cells were incubated with 5 g/ml A.
chilensis and the COX-2 expression was analyzed by immunoblot and real time PCR.
The effect of A. chilensis on NF-B pathway activation was studied by immunoblot of
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IB, p65 NF-B localization by immunocytochemistry and NF-B activity by
luciferase reporter plasmid. Also, the effect of A. chilensis on ERK1/2 and Akt
phosphorylation, and c-fos expression was analyzed by immunoblot. The cell
viability and apoptosis in Caco-2 and HT-29 cells was assessed by MTT assay, flow
cytometry of annexin V-propidium iodide and immunoblot of apoptotic proteins. It
was observed that A. chilensis reduced expression of protein and mRNA COX-2 and
inhibited the TNF--induced activation of NF-B. A. chilensis transiently reduced the
levels of IB in cytoplasm at 2 and 4 h of incubation and mildly increased the
nuclear localization of p65 NF-B at 4 h. The treatment with A. chilensis transiently
increased the ERK1/2 and Akt phosphorylation, at 4 h and 30 min, respectively, and
an increase in the c-fos expression after 4 h of treatment was observed. The MTT
and annexin V-PI analysis showed that A. chilensis did not affect Caco-2 cell
viability, at concentrations that reduced COX-2 expression. On the contrary, high
concentrations of A. chilensis induced an increase in apoptosis and necrosis of
Caco-2 and HT-29 cells. In conclusion, we demonstrated that A. chilensis reduces
COX-2 expression and TNF--induced activation NF-B in Caco-2 cells suggesting a
putative anti-carcinogenic and anti-inflammatory effect of A. chilensis.

Figure 12. Effect of A. chilensis on COX-2 expression. Caco-2 cells were incubated with 5 g/ml
A. chilensis (A.ch.) for 0, 24, 48 or 72 h and total proteins or RNA was isolated. The COX-2
protein level (A) was analyzed by immunoblotting using an antibody against COX-2, and the
blot was stripped and re-probed with anti--actin antibody. (B) The mRNA expression of COX2 at 24 h of treatment with A. chilensis was assessed by real time PCR. The graphs show the
mean S.E. from three independent experiments. * p < 0.05 compared to the control.

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Figure 13. Effect of A. chilensis on NF-B activation. Caco-2 cells were transiently transfected
with the pGL3-NF-kB and pRL-TK plasmids for 24 h, treated with A. chilensis or vehicle for 30
min and TNF- or solvent was added for 16 h. Luciferase activity was measured in a
luminometer. Each bar represents mean S.E., n=3, * p<0.05 compared with TNF-.

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Figure 14. Effect of A. chilensis on cell viability. Caco-2 cells were incubated with 5 g/ml A.
chilensis for 24 h and then stained with AnnexinV-FITC and PI, or analyzed by MTT assay. The
AnnexinV-FITC and PI signal was detected by flow cytometry (A-C) and the formazan crystal
produced from MTT was registered in a microplate lector (D). Each bar represents the mean
S.E., n=8. ** p<0.01 vs control.

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Application 4: Glucose Balance


General Research
Hyperglycemia is the central characteristic of all metabolic imbalances of
sugar, lipids and ketones and amino acids [83]. This pathologic condition is the most
common and known as diabetes type 2 (about 90% of all types of diabetes), it is
considered and epidemic disease, especially in occidental countries with an incidence
of 2 to 6% [84].
Diabetes type 2 is preceded during years by an abnormal condition known
as glucose intolerance , that is characterized by higher levels of glucose than normal,
with a concentration (between 140 mg / dL and 199 mg / dL) 2 hours after a
standard charge of glucose, but not as high as in diabetes (> 200 mg / dL) [25].
There are characteristics that are associated to a mayor risk in the progression of
glucose intolerance to diabetes type 2. Among these, alteration in the secretion of
insulin, insulin resistance, obesity and aging occur [86-88 y 127]. Physical exercise
and some diets can be of help in the control of pre-pathologic hyperglycemia [89].
Many diets and natural product can be of help in the control and prevention of
hyperglycemia [90]. In particular, there are many medicinal plants with hypoglycemic
effects [91-92]. In this context, our product MAQUISELECT/DELPHINOL could be
significant for the control of hyperglycemia [93-94].
Diabetes mellitus is a chronic disease caused by an inherited deficiency
and/or acquired in the production of insulin, or a lack of response to insulin. This is
caused by an increase in the concentration of glucose in the blood that affects
various tissues and organs. This is a global epidemic affecting approximately 143
million people [89,95,96]. In Chile the prevalence of diabetes estimated by the OMS
is 6-8%, similar to countries as USA, Canada, Argentina and Uruguay [97].
The effect of A. chilensis in diabetes or renal pathologies is unknown.
However, it is known that the fruit and vegetable intake rich in polyphenols
diminishes the incidence of diabetes type 2. It is possible that the anthocyanins exert
an antioxidant/anti-inflammatory effect at the kidney level since the concentrations
of these substances is 3 times higher than the ones reported in the plasma [98]. It is
known that antioxidants from diet, including anthocyanins, protect the pancreatic
beta cells from oxidative stress induced by the increase in glucose. Studies have
described that anthocyanins are able to induce the release of insulin and therefore
reduce hyperglycemia. Polyphenols from the skin of grapes or whole grapes are able
to increase the secretion of insulin and inhibit COX-2. Anthocyanins can reduce the
pathologies associated to elevated glycemia such as cataracts. In this sense,
combination of semi synthetic anthocyanins and natural can treat cataracts. In
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diabetes there is microcirculation problems (thickening of capillary layers) that are
involved in the pathogenesis of micro vascular diseases associated to diabetes.
Delphinidin induces an increase in the microvascular permeability and a reduction in
the leukocyte adhesion in the venules in diabetic hamsters [99]. In diabetes and
other diseases, vasodilatation of the endothelium induced by agonists is reduced
[40], due to a decrease in the release of nitric oxide [100]. Wine extract, or products
from grapes and plants with high polyphenolic content (among them anthocyanins),
increase the vasorelaxation probably due to an increase in nitric oxide via liberation
of NO or increase of its biological effects [101-104]. Anthocyanins from V. myrtillus
has vasodilatating [105], and ophthalmological [106] activities. Furthermore, 10
mg/L delphinidin increase the expression of eNOS in adherent BAEC, augmenting the
nitric oxide production, exerting vasorelaxation and preventing cardiovascular
diseases[129].
Antioxidant molecules diminishes the oxidative stress and hypertension
associated to the resistance to insulin in rats fed with fructose [102]. Oranges
complex supplements (anthocyanins, flavanones and hidroxinamic acid) improved
significantly the activity of glutathione in the serum reducing the levels of free
radicals [103]. Pomegranate juice consumption in diabetic patients showed
antioxidant effects in the serum, and in the oxidative state of monocytes and
macrophages [104], probably due to the presence of anthocyanins [105].
The sugar fraction of pomegranate juice, in difference to the juice of White
grapes, diminishes the oxidative state in macrophages in normal and diabetic
conditions suggesting an anti tetarogenic effects. Anthocyanins and procyanidins,
lower triglycerides and increases the HDl cholesterol in rats. In acute studies in
animals, the glycosides of anthocyanins (derived from leucopelargonine) from F .
bengalensis showed hypocholesterolemic and antioxidant properties [116-118].
Adipocytes are the primary place for energy storage and due to the excess of
nutritional triglycerides. In recent years, the adipocyte dysfunction plays a key role in
the development of obesity and insulin resistance. Adipocites sensitize and secret
biologically active molecules known as adipokines [119]. Adiponectin is one of the
most important and highly expressed in adipocitokines in adipocytes. The plasmatic
concentration of adiponectin and the level of expression of ARNm are inferior in
obese and insulin resistant individuals [120, 121].
To the best of our knowledge, we have found only one study [48] directly
linking the effects of delphinidin, among other compounds, in the prevention of
metabolic syndrome. The study considered administration of 2.1mg/kg of
delphinidin to a rat model of metabolic syndrome, where said administration
prevented insulin resistance.

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Our Research
Our research group conducted a study where a diabetic rat model was
used. After 4 months of treatment with 20 mg/kg, a decrease from 380 mg/dl to 90
mg/dl in glycemia was found (Figure 16), a 76% reduction. Triglycerids also showed
a decrease of a 52.5% (Figure 15).

Figure 15: Triglycerids measured as mg/dl in a rat diabetic model after 4 months of treatment
with 20 mg/kg of MAQUISELECT/DELPHINOL.

Figure 16: Glycemia measured as mg/dl in rat diabetic model after 4 months of treatment with
MAQUISELECT/DELPHINOL.

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Clinical Study
The results obtained in the laboratory using a rat diabetic model provided
sufficient evidence to initiate a human clinical trial.
Our research group, in association with a local University, Universidad
Austral, started a clinical trial for a dried standardized extract of 300mg of
MAQUISELECT/DELPHINOL, for oral administration previous dissolution in fresh
water.
All studies and clinical trials are performed in accordance with the approval
of the Scientific Ethical Committee of the Occidental Metropolitan Health Service,
Ministry of Health.
Phase I Trial is designed to evaluate the efficacy of the oral administration
of MAQUISELECT/DELPHINOL on the post-prandial levels of glucose and insulin
and tolerability in individuals with glucose intolerance.
The design of the study is a double blind randomized crossover, and the
event under study is the change in the post-prandial plasmatic blood levels of
glycemia and insulin. The principal parameter to evaluate is the measurement of
post-prandial plasmatic blood levels of glucose and insulin, and the second
parameter is the determination of adverse effects and/or events.
Individuals selected for the study comprise individuals with high levels of
glycemia between 110 y 125 mg/dL, age 18 and 55 years, with a total of 10 patients.
Preliminary results for glycemia and insulinemia are shown in Figure 17.
Preliminary results show a first effect of MAQUISELECT/DELPHINOL
increasing AMPK activity. In fact, MAQUISELECT/DELPHINOL increases
phosporilation of AMPK which acts like obesity control (Figure 18). AMPK is a key
target in the effect of well known drugs such as metformin and thiazolidinediones.

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pAMPK/actina
(veces del control)

pAMPK/actin
(times the
control)

de
lp
hin
id
in

co
nt
ro
l

Figure 17: Preliminary results of insulinemia and glycemia of patients receiving 300 mg of
MAQUISELECT/DELPHINOL.

1.75
1.50
1.25
1.00
0.75
0.50

Figure 18: Phosphorylation of AMPK is shown as a consequence of treatment with insulin and
delphinidin. The control shows no phosphorylation of AMPK.

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TOXICOLOGY
Safety in relation to traditional and well established use
Several preparations with Maqui are used in Chile. Furthermore, chilean
government, through the Health Ministry, recognized the use of Maqui as a
traditional herbal medicine, promoting its use for treatment of diarrhea, dysentery,
indigestion, to alleviate sore throat symptoms, tonsil inflammation, and mouth
ulcers. The way of administration is internal or topical.
For internal preparations, the recommended dosage is adding 1 tea spoon
of dried or fresh ground leaves, or 1 spoon of Maqui berries prepared to 1 liter of
boiling water, and drink 3 to 4 cups per day.
For External preparations, the same infusion prepared for drinking is used
to wash wounds, or for gargling. Crushed fresh leaves are also used as cataplasm for
back pain.

Repeated Dose toxicity, Short term Toxicity testing


Repeated-dose administration of Maqui to albino rats or rabbits at 1g/kg
PO once daily for 7 days did s not significantly change the body weight, blood
chemistry, hepatic and renal functions and histology of important organs. Similar
results were obtained with the injection administered to rats at 84 mg/kg i.p. for 10
days also produced no toxic effects.
Subchronic toxicological study in pigs of MAQUISELECT/ DELPHINOL
The aim of the study was to evaluate the sub-chronic peroral toxicity of
MAQUISELECT/ DELPHINOL. This study was carried out in Landrace piglets for 2
months using MAQUISELECT/ DELPHINOL. The product was stored in a dark
room, in sealed plastic containers, under controlled conditions of humidity (18%) and
temperature (215C).
Test samples of MAQUISELECT/ DELPHINOL were prepared mixing the
product with the food in 3 final different doses, 0,171 g/kg, 0.857 g/kg and 1,379
g/kg using powdered industrial food as vehicle. Group size was 6 piglets. Group 1
control, group 2; 0171g/kg group 3: 0857 g/kg and group 4: 1.371 g/kg. The
dosage scheme was based on the group size for all calculations of dosages. Each
week MAQUISELECT/ DELPHINOL was mixed in a known quantity of food and
given during the whole week. The animal used in the test were landrace weaned
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piglets, three per sex, per dose, with an initial body weight of 15 3 kg, supplied by
Lorenzini Piglet Farm, Molina, Chile.
All animals were individually identified by ear tattoo. Upon arrival all
animals were caged in groups according to the dose of MAQUISELECT/
DELPHINOL and control. A quarantine period of 7 days of acclimatisation was used
before the test started. The animals received a prefabricated food mix of high
quality. Water was given ad libitum. Environmental conditions were as follows:
temperature (ambient), humidity (relative) 40% RH and natural light.
Ventilation: The level of ammonia and other volatile products from
excrements were kept at a low level due to the design of the building and strict
cleansing activities twice a day.
Sampling and testing
Sampling of blood from each animal was performed at day 0, 15, 30 and
60. The blood was drawn from the vena cava cranialis with the animal resting on its
back.
Haematological tests:
EDTA was utilized for the hemogram in solutions of 1%, 1:9 and the blood
samples were analysed by standard laboratory procedures.
Red Blood Cells, White Blood cells, Vol. Packed RBC. Haemoglobin,
differential count of leukocytes were controlled in heamatological tests.
Blood chemistry:
The samples were centrifuged in order to obtain serum for analysis of the
following parameters.
Urea-N, Glucose, Protein, Alkaline phosphatises AP, Aspartate
aminotransferase (ASAT), Alanine aminotrasnferase (ALAT) were controlled in blood
chemistry tests.
All haematological and biochemical data were evaluated to the following
statistical methods: basic statistics including mean (M), standard error of the mean
(SEM), standard deviation (SD), number (N), etc. Where appropriate, Bartlett test for
homogeneity or variance, ANOVA, one way and multiple comparison test of Tukey
were used. A level of p<0.05 was used for significance. A two way ANOVA (factorial
2x4) was utilised if the variables (effects of treatment) were significant a Scheffes
test was also used. The non-parametric test, Kruskal-Wallis was utilised for all
variables. Sequentially a box-design graphical statistical was employed. For the
evaluation of body weight the same descriptive statistics as above were used.

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Results
Body weight
In all three doses of MAQUISELECT/ DELPHINOL, no significant
changes in the weight of the piglets were observed. The treatment lasted 42 days.
Food intake
Initially due to the bitterness of the herb extract, a decrease in the intake
of food was observed, but this was recovered after two to three days. The intake of
food was restricted for the animals in all groups.
Behaviour and appearance
All piglets looked healthy, playful with no changes in behaviour in all
groups during treatment.
Mortality
Not observed.
Haematological tests
Apart from some minor changes observed in white blood cells (dose:
0171g/kg, day 15; and 0.857g/kg, day 30) and differential leukocyte counting, these
are not due to the effect of MAQUISELECT/ DELPHINOL, as they are within the
normal ranges for the piglet species.
Blood chemistry
With all doses of MAQUISELECT/ DELPHINOL there was a significant
decrease in proteins at day 0 which recovered afterwards. At day 30, a mild decrease
with the dose of 0171g/kg was observed.
At day 15 all doses of MAQUISELECT/ DELPHINOL induced lower levels
of ALT enzyme as compared to the control, a fact already described in the literature.
Alkaline phosphatase showed a mild decrease at a dose of 1.370g/kg at day 15.
Histopathological examination
No changes were observed in the histopathological examination

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Chronic toxicity testing
In chronic toxicity test carried out in dogs, MAQUISELECT/ DELPHINOL
at doses 10-15 times those used clinically exhibited no toxicity. The LD 50 of a water
extract (1:1) samples varied from 5.4 to 6.8 g kg -1 (Wagner).

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Patent Applications
An international patent application, PCT/IB2010/002698 has been filed on
October, 21st, 2010. The matter protected by this application comprises a
composition of a plurality of anthocyanins and/or anthocyanidins, wherein (a) at
least or about 35% of the composition, by weight, is an anthocyanin or
anthocyanidin, (b) at least or about 15% of the anthocyanins and/or anthocyanidins,
by weight, are sugar-free or sugar-containing delphinidins, and (c) the composition
is non-toxic and non-naturally occurring.
The patent application also claims protection over an oral formulation,
either tablet, capsule, or food product, method of treatment or use as an immune
system booster, cancer or acquired immunodeficiency conditions, improving
inflammation in a subject, and for improvement on the condition of a patient
suffering of metabolic syndrome.
A further composition also covered in the patent application, is the initial
composition plus andrographolides, and another plus Vacciunum myrthilus

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REFERENCES
1. Noda, Y Univ Calif Berkeley RESEARCH COMMUNICATIONS IN MOLECULAR
PATHOLOGY PHARMACOLOGY 102 (2): 175-187 NOV 1998 Antioxidant activity
of nasunin, an anthocyanin in eggplant
2.

Noda, Y Univ Calif Berkeley TOXICOLOGY 148 (2-3): 119-123 AUG 7 2000
Antioxidant activity of nasunin, an anthocyanin in eggplant peels

3.

Noda, Y Univ Calif Berkeley JOURNAL OF AGRICULTURAL AND FOOD


CHEMISTRY 50 (1): 166-171 JAN 2 2002 Antioxidant activities of pomegranate
fruit extract and its anthocyanidins: Delphinidin, cyanidin, and pelargonidin

4.

Wang, J Agr & Agri Food Canada JOURNAL OF AGRICULTURAL AND FOOD
CHEMISTRY 50 (4): 850-857 FEB 13 2002 Inhibitory effects of anthocyanins
and other phenolic compounds on nitric oxide production in LPS/IFN-gammaactivated RAW 264.7 macrophages

5.

Seeram, NP Michigan State Univ JOURNAL OF AGRICULTURAL AND FOOD


CHEMISTRY 50 (9): 2519-2523 APR 24 2002 Characterization, quantification,
and bioactivities of anthocyanins in Cornus species

6. Matsumoto, H Meiji Seika Kaisha Ltd JOURNAL OF AGRICULTURAL AND FOOD


CHEMISTRY 50 (18): 5034-5037 AUG 28 2002 Antioxidant activity of black
currant anthocyanin aglycons and their glycosides measured by
chemiluminescence in a neutral pH region and in human plasma
7.

Kahkonen, MP Univ Helsinki JOURNAL OF AGRICULTURAL AND FOOD


CHEMISTRY 51 (3): 628-633 JAN 29 2003 Antioxidant activity of anthocyanins
and their aglycons

8.

Kobori, M Natl Food Res InstJ ARQ-JAPAN AGRICULTURAL RESEARCH


QUARTERLY 37 (3): 159-165 JUL 2003 In vitro-screening for cancersuppressive effect of food components

9. Favot, L Univ Louis Pasteur Strasbourg 1 CARDIOVASCULAR RESEARCH 59 (2):


479-487 AUG 1 2003 Involvement of cyclin-dependent pathway in the
inhibitory effect of delphinidin on angiogenesis
10.
Hou, DX Kagoshima Univ INTERNATIONAL JOURNAL OF ONCOLOGY 23
(3): 705-712 SEP 2003 Anthocyanidins induce apoptosis in human
promyelocytic leukemia cells: Structure-activity relationship and mechanisms
involved
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