FIELD OF THE INVENTION
0001 The invention relates to cardioprotective agents. In particular, the
present invention relates to cardio-protective extracts and fractions thereof
prepared from kiwi fruit.
BACKGROUND OF THE INVENTION
2 It is known that high consumption of fruits and vegetables is an essential
preventive measure by which the risk of cardiovascular diseases and certain
nutritionally linked cancers, including stomach, colon, breast, and prostate
cancer, can be decreased[1, 2]. One factor involved in the initiation and
development of both cardiovascular diseases and cancers is abnormal oxidative
stress processes leading to the generation of hydroxy and peroxy free radicals
or compounds [3]. In part, the beneficial effect of eating fruits and vegetables
is explained by the antioxidants known to account for the inhibition include
vitamin C, vitamin E, and carotenoids such as alpha and beta carotenoids,
lycopene lutein, etc. [4]. However, many emerging data also indicate a role for
non-antioxidant properties of some compounds in fruits in different diseases [5,
6].
3 Considerable effort has been expended in identifying bioactive
compounds derived from fruits and vegetables that may have a role in
preventing some diseases. Fruits and vegetables have been thought to be
beneficial in cardiovascular disease. Antioxidants and non-antioxidant
components contained therein may explain the beneficial effects of fruits and
vegetables. These bioactives may function individually or in concert to protect
lipoproteins and vascular cells from oxidation or by other mechanisms (non-
antioxidant pathways) such as reducing plasma lipid levels (LDL cholesterol,
triglycerides) and platelet aggregation response [6, 7].

0004 Additional preparations from fruits and vegetables that provide cardio-
protective and other beneficial properties are needed. It may be noted that the
Kiwi Fruit Extract may be abbreviated as KFE.
References
5 [1] M.F. Chong, R. Macdonald, J.A. Lovegrove, Fruit polyphenols and
CVD risk: a review of human intervention studies, Br J Nutr 104 Suppl 3 (2010)
S28-39.
6 [2] A.K. Dutta-Roy, L. Crosbie, M.J. Gordon, Effects of tomato extract
on human platelet aggregation in vitro, Platelets 12(4) (2001) 218-227.
7 [3] I. Peluso, G. Morabito, L. Urban, F. Ioannone, M. Serafini,
Oxidative stress in atherosclerosis development: the central role of LDL and
oxidative burst, Endocr Metab Immune Disord Drug Targets 12(4) (2012) 351-
60.
8 [4] E.M. Alissa, G.A. Ferns, Dietary fruits and vegetables and
cardiovascular diseases risk, Crit Rev Food Sci Nutr 57(9) (2017) 1950-1962.
9 [5] A.K. Duttaroy, Cardioprotective properties of kiwifruit, Advances
in food and nutrition research 68 (2013) 273-82.
10 [6] A.K. Dutta-Roy, Dietary components and human platelet activity,
Platelets 13(2) (2002) 67-75.
11 [7] N. O'Kennedy, L. Crosbie, H.J. Song, X. Zhang, G. Horgan, A.K.
Duttaroy, A randomised controlled trial comparing a dietary antiplatelet, the
water-soluble tomato extract Fruitflow, with 75 mg aspirin in healthy subjects,
Eur J Clin Nutr (2016).
12 [8] D.P. Richardson, J. Ansell, L.N. Drummond, The nutritional and
health attributes of kiwifruit: a review, Eur J Nutr (2018).
13 [9] A.R. Ferguson, Kiwifruit: the wild and the cultivated plants,
Advances in food and nutrition research 68 (2013) 15-32.
14 [10] G.R. Buchanan, V. Martin, P.H. Levine, K. Scoon, R.I. Handin,
The effects of "anti-platelet" drugs on bleeding time and platelet aggregation in
normal human subjects, Am J Clin Pathol 68(3) (1977) 355-9.
15 [11] S.P. Jackson, The growing complexity of platelet aggregation,
Blood 109(12) (2007) 5087-95.

16 [12] A.K. Duttaroy, A. Jorgensen, Effects of kiwi fruit consumption on
platelet aggregation and plasma lipids in healthy human volunteers, Platelets
15(5) (2004) 287-292.
17 [13] L.L. Dizdarevic, D. Biswas, M.D. Uddin, A. Jorgenesen, E. Falch,
N.E. Bastani, A.K. Duttaroy, Inhibitory effects of kiwifruit extract on human
platelet aggregation and plasma angiotensin-converting enzyme activity,
Platelets 25(8) (2014) 567-75.
OBJECTIVES OF THE INVENTION
18 It is therefore, an object of the present invention is to find a solution
against cardiovascular diseases in particular against disease and/ or disorder in
the cardiovascular system
19 Another object of the present invention is to find the solution in
naturally obtained herbs, fruits, vegetables and the likes.
20 Yet another object of the present invention is to explore the
cardioprotective efficacy of de-sugared cardio-protective extracts and fractions
thereof prepared from Actinidia, more particularly Actinidia deliciosa.
21 Further object of the present invention is to design dosages for
individuals, healthy or diseased.
22 Still further object of the present invention is to verify and estimate the
efficacy of the naturally sourced, laboratory prepared extract.
SUMMARY OF THE INVENTION
0023 The invention relates to cardioprotective agents. In particular, the
present invention relates to cardio-protective extracts and fractions thereof
prepared from kiwi fruit.

24 In some embodiments, the fruit is a fruit of the family Actinidia. In some
embodiments, the present invention provides methods of using the extract
fractions to prevent or treat a disease state in a subject initiated or characterized
by platelet activation and/ or aggregation, for example, by inhibiting platelet
aggregation, anti-thrombotic activity, and/or blood thinning.
25 In one embodiment, the present invention discloses a process of
preparation of extract fraction of the family Actinidia, preferably kiwi fruit, said
process comprising the steps of homogenizing the fruits; centrifuging the
homogenized fruits at 9000xg for 15 minutes; boiling of the supernatant
obtained at 90 °C for 20 minutes; centrifuging again the boiled supernatant at
12000xg for 30 minutes at 4 °C; ultrafiltration through the filter with molecular
weight cut off of less than 1000; removing the soluble sugars from the
ultrafiltrate obtained through column chromatography; and freeze-drying.
26 In another embodiment, the present invention discloses a delipidated
fruit extract fraction of the family Actinidia, preferably kiwi fruit, as obtained
through the process, wherein said fraction comprises biological molecules with
a molecular weight of less than 1000 daltons.
27 In still another embodiment, the present invention discloses a method
of preventing or treating a disease state initiated or characterised by platelet
activation and/ or aggregation, comprising administering a delipidated fruit
extract fraction belonging to Actinidia family, wherein said fraction comprises
biological molecules with a molecular weight of less than 1000 dalton to a
subject in need thereof under conditions such that said disease state is prevented
or treated, wherein said administering inhibits platelet aggregation, resulting in
anti-thrombotic activity and blood thinning.

BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS/ FIGURES
28 Figure 1 shows platelet aggregation inhibition induced by ADP activity
by the extract.
29 Figure 2 shows the UV spectra of a delipidated, ultrafiltrated and sugar-
free kiwifruit extract (KFE). KFE was dissolved in PBS buffer, and the UV
scanning was done of the solution.
30 Figure 3 shows the freeze-dried sugar-free KFE obtained from
Kiwifruit.
DETAILED DESCRIPTION OF THE INVENTION
31 The invention relates to cardioprotective agents. In particular, the
present invention relates to cardio-protective extracts and fractions thereof
prepared from kiwi fruit. The principal biological material i.e. the kiwi fruits
were procured from New Zealand, China and India
32 Kiwifruit is the most well-known crop in the genus Actinidia [8].
Although Actinidia fruit sales in the international market are dominated by a
single kiwifruit cultivar Actinidia deliciosa "Hayward," there are many
cultivars and selections in the genus that have widely diverse shape, size, and
hairiness. They also offer a wide variation in sensory attributes such as flesh
color, flavor, taste, and nutritional facts such as the vitamin C level,
polyphenols, and carotenoid content[8, 9]. Few types of processed kiwifruit
food products are commercially available to consumers. Kiwifruits are mainly
eaten as whole fruits. The few examples where kiwifruit has been processed
into products include frozen desserts and blended juices and, more recently, a
few natural kiwifruit drinks such as Kiwi CrushTM (Vital Food Processors Ltd,
Manukau City, Auckland, New Zealand). Kiwifruit extracts containing the

fruit’s nutritional components and desirable bioactive, including polyphenols,
ascorbic acid, and water-soluble polysaccharides (pectic polysaccharides),
which may be advantageous for functional food applications, increasing the
range of kiwifruit products available to consumers. There are increased
consumer demands for acceptable nutritional foods with multiple consumer
benefits with growing health awareness, including defined health benefits,
increased convenience, and reduced additives.
0033 Platelets are involved in atherosclerotic disease development, and the
reduction of platelet activity by medications reduces the incidence and severity
of disease [10, 11]. Experiments conducted during development of
embodiments of the present invention evaluated whether consuming kiwi fruit
modulated platelet activity and plasma lipids in healthy human volunteers in a
randomized crossover study. It was reported that consuming two or three kiwi
fruits per day for 28 days reduced platelet aggregation response to collagen and
ADP by 18% compared with the controls[12]. Besides, consumption of kiwi
fruits lowered blood triglycerides levels by 15% compared with control,
whereas no such effects were observed in the case of plasma cholesterol levels.
All these data indicate that consuming kiwi fruit is beneficial in cardiovascular
disease. Incubation of KFE (expressed as weight of pulp used to prepare KFE)
inhibited platelet aggregation but was not an optimal preparation as it requires
a good amount of flesh, plus the activity can be lost in storage due to unwanted
reactions in the juice. Besides, it is thought that tannins and oil in the seeds
(and to a lesser extent the hair) can react with the highly acidic pulp to give rise
to unwanted smell and color. Many kiwifruit species have fine hair, which is
difficult to remove from a juice. Soft-pulping methods are preferred as it is
considered desirable to avoid both excessive cell disintegration and
fragmenting components of the fruit, such as the seed. Seeds may contain toxic
substances (e.g., apricot kernel) or contribute to off or undesirable flavours in a
juice.

34 Many fruits are acidic, and those with a pH of 6 or less are generally
most likely to be affected. Widely used and relatively inexpensive, sucrose is
alkaline and appears to induce or take part in further undesirable reactions when
added to an acidic pulp. The research leading to the present invention's
embodiments indicated that substances entering a juice from seed
fragmentation or excessive cell damage contribute to factors that adversely
affected the production of successful kiwifruit juice, such as browning and
catch factor. The kiwifruit is more acidic than most and has a pH of
approximately 3. This may also avoid any possible side reactions contributing
to catch, discoloration, etc. Glucose and fructose are commonly found in many
fruits. Typically, this is by masking some of the undesirable properties of the
fruit such as bitterness or excess acidity, partly due to the average human's
affinity for sweetness. In some embodiments, juice products are not pasteurized
as characteristic of most other juices and processes. Hence any preservative
effects which are contributed by the sweetening agent will help prolong the
shelf life of the product. It is possible that the alteration in pH resulting from
the combination may cause unwanted side reactions. Furthermore, it is noted
that under storage, the chemistry of most juices will vary. For kiwifruit, the acid
content of the juice will drop. The addition of a suitable buffering or pH
adjusting agent help to preserve the pH of the product over a longer period. This
also defers any undesirable long term reactions resulting in browning or
discolouration of the juice.
35 In some embodiments, the active fractions with the highest efficacy and
free from inert materials, with much longer shelf life, are utilized and are added
to any matrix for human consumption has been developed. As a further
modification to the process, a juice or pulp fraction was centrifuged, filtered,
and delipidated that produced highly enriched platelet inhibitors (more than 30
fold than the juice) with longer life, and stability to heating. It is an object of
embodiments of the present invention to address the preceding problems.

According to another embodiment of the present invention, a reconstituted
product from a fruit product is provided as described above. The present
invention has been developed for members of the genus Actinidia. Fruit
products, other than juice, are also within the scope of embodiments of the
invention. These fruits generally have a low pH (3.0-3.5), suffer from browning
upon exposure of a juice to the air, and have a chlorophyll content. It is
envisaged that while the process of the invention will be amenable to other fruit,
the most significant advantage is likely to be realized for fruit suffering
problems and characteristics in common with the kiwifruit, e.g., a pH of less
than 4.5, significant chloroplast levels, or catch (e.g., the fruit of Monstera
deliciosa). It should not be inferred that benefit from the invention is limited to
these types of fruit.
36 The invention has identified several problem areas, especially for
kiwifruit, and addresses their needs. A method of extracting individual
bioactive components of kiwifruit, the extract is retaining to a suitable extent
beneficial properties of kiwifruit in CVD.
37 Other studies on kiwifruit extracts have mostly focused on alcoholic
soluble polyphenols' roles as an antioxidant defence.
38 Experiments conducted during developments of embodiments of the
present invention demonstrated that the specially prepared extracts of kiwifruit
(KFE) exhibit an ability to inhibit platelet aggregation and reduce angiotensin-
converting enzyme (ACE) activity in vitro [13]. The results obtained to date
indicate that compositions containing extracts from kiwi fruits are used to
prevent cardiovascular diseases, such as myocardial infarctions, stroke, and
other thromboembolic events in patients who have suffered a myocardial
infarction, stroke, or unstable angina. Besides, such composition is of use in
preventing restenosis following angioplasty and bypass procedures. Moreover,
KFE is of service in the treatment of coronary disease resulting from thrombo-
embolic disorders such as MI in conjunction with thrombolytic therapy. Results

obtained to date indicate that compounds responsible for anti-platelet
aggregation activity are water-soluble compounds having a very different
structure to the lipid-soluble compounds. Many known anti-platelet
aggregating agents act at different stages of platelet production and action.
Aspirin (acetylsalicylic acid) is the most widely used and studied. Dipyridamole
and ticlopidine have also been used. Aspirin’s anti-platelet activity is due to
irreversible inhibition of platelet cyclooxygenase, thus preventing the synthesis
of thromboxane A2, a compound that causes platelet aggregation. Ibuprofen is
a reversible inhibitor of platelet cyclooxygenase. Some compounds are direct
inhibitors of thromboxane A2 synthetase, such as pirmagrel, or act as
antagonists at thromboxane receptors, such as sulotroban.
39 The present invention is not limited to a particular mechanism. Indeed,
an understanding of the mechanism is not necessary to practice the present
invention. Nonetheless, the results described herein indicate that the active
components in the fruit extract may affect one or more steps of the pathways
leading to the production of thromboxane A2 upstream from aspirin and other
anti-platelet drugs currently available. It is well known that the adverse effects
are common occurrences with therapeutic doses of aspirin, the principal results
being gastrointestinal disturbances such as nausea, dyspepsia, and vomiting.
Therefore, it is anticipated that the isolated platelet aggregation inhibition
compounds in fruit extract find use as a desirable alternative to aspirin and other
antiplatelet drugs in the prevention of thromboembolic events and coronary
disease.
40 Accordingly, in a first aspect, the invention provides a fruit extract,
active fraction thereof, or one or more active compounds isolatable therefrom,
for use in the prophylaxis or treatment of a disease state-initiated or
characterised by platelet aggregation.

41 In another aspect, the invention provides a fruit extract or active fraction
thereof or one or more compounds isolatable thereof for use as a platelet
aggregation inhibitor.
42 In a further aspect, the invention provides a fruit extract or active
fraction thereof or one or more compounds isolatable thereof for use as an anti-
thrombotic agent.
43 In a further aspect, the invention provides a fruit extract or active
fraction thereof or one or more compounds isolatable thereof as herein before
defined for the manufacture of a medicament for use in the prophylaxis or
treatment of a disease state-initiated or characterized by platelet aggregation; or
for use as a platelet aggregation inhibitor: or for using an anti-thrombotic agent.
As used herein the term ‘fraction’ refers to a partially purified extract or
compounds purified from an extract.
44 In another aspects the invention provides a process from the
manufacture of medication for use (i) in the prophylaxis or treatment of a
disease state-initiated, mediated or characterised by platelet aggregation, or (ii)
as a platelet aggregation inhibitor , or as (iii) an anti-thrombotic agent: which
process is characterised by the use, as an essential ingredient of the medicament,
of fruit, or an extract or active fraction thereof or one or more functional
components isolatable thereof as hereinbefore defined.
45 In a still further aspect, the invention provides a pharmaceutical
composition comprising an active component derived from a fruit or an extract
or active fraction or one or more active compounds isolatable thereof as a
hereinbefore defined and pharmaceutically acceptable carrier.
46 It is preferred that the fruit extract used following the invention are those
which are nontoxic to humans and typically the fruits which are usually
considered to be edible. Thus the fruits may or may not contain seeds or stones
but have an edible essentially non-oily flesh.

47 Kiwifruit is the most well-known crop in the genus Actinidia. The
extracts of embodiments of the invention can be prepared by homogenising the
flesh of a peeled kiwifruit and then removing solids therefrom, for example by
means of centrifugation. Thus the extract is a typically an aqueous extract,
which can consist or comprise the juice of the fruit, optionally with the addition
of extra water added during the homogenizing step. Such aqueous extracts can
be concentrated, enriched, or condensed by, for example, standard techniques,
e.g., evaporation under reduced pressure. Examples of concentrates are at least
2-fold concentrated, more usually, at least 4-fold, for instance at least 8-fold, or
at least 40-fold or at least 100-fold or at least 200fold or at least 1000 fold.
48 The extract can be fractionated to isolate one or more active fractions
therein by, for example, molecular weight filtration or chromatography on a
suitable support such as sepharose gel (for size exclusion chromatography), or
ion exchange column using HPLC on a suitably treated silica or alumina, for
example, ODS coated silica or solvent extraction.
49 Experiments carried out on kiwi fruit extract have revealed that the
extract pass’s active components pass through ultrafiltration having molecular
weight cut-off of 3000 is colourless, water soluble and does not lose activity
when boiled.
50 Accordingly, embodiments of the invention also provide for use an anti-
thrombotic agent, or use as a platelet aggregation inhibitor, or use in the
prophylaxis or treatment of a disease state-initiated or characterised by platelet
aggregation, an active fraction of a fruit extract (e.g., kiwifruit extract) the
active fraction containing a substantially heat-stable colorless or slightly straw-
coloured water-soluble compounds with a molecular weight less than 3000.
When incubated with the normal serum for 10 min, the extract also decreased
the ACE activity by 15% [13].
51 The active fraction has been found to be primarily associated with, or
extractable from, the juice, the flesh surroundings the pips and the pips of the

kiwifruit. Thus, the use of compositions prepared from an active fraction
consisting essentially of or comprising a homogenate or an extract thereof
derived from the flesh of a peeled kiwifruit or consisting essentially of or
comprising the juice and/or the flesh surrounding the pips, and or the pips,
represents a preferred embodiment of the invention.
0052 Embodiments of the present invention provide an active fraction of a
kiwifruit extract and are characterized in that it is
Substantially heat stable
Almost Colourless
Water-soluble compounds
Inhibits platelet aggregation
Inhibits angiotensin-converting enzyme
Pharmaceutical and nutraceutical preparations:
53 The extracts or active fractions thereof can be formulated in a variety of
ways. For example, they can have formulated for administrative orally,
sublingually, parenterally, transdermally, rectally, via inhalation or via buccal
administration. As such they can be formulated as solutions, suspensions,
syrups, tablets, capsules, lozenges, snack bars, inserts, and patches by way of
example. For example, the extracts or active fractions can be formed into syrups
or other solutions for administration orally. For example, in some embodiments,
extracts, fractions thereof, or active components isolated from the extracts can
be added to health drinks, yogurt, bread spreads, food supplements and the like.
54 The following examples are provided to demonstrate and further
illustrate certain preferred embodiments and aspects of the present invention
and are not to be construed as limiting the scope thereof.

Preparation of kiwifruit extract:
0055 An extract consisting of 100% kiwifruit juice was prepared. To prepare
100% fruit juice, the fruits were peeled and the flesh was homogenized. The
resulting homogenate was spun at 9000xg for 15 min at 4ºC on a centrifuge
after which the supernatant was removed and the pH of the juice was adjusted
to 7.4 with 1M sodium hydroxide solution. The anti-platelet activity of the kiwi
fruit extract (KFE) was determined initially.
Example 2:
Partial fractionation of Kiwi fruit extract:
0056 Kiwifruit extracts were then fractionated according to the general
scheme set out in the schematic diagram in the following paragraph. The
platelet aggregation inhibiting activity of the preparations was measured at
various stages. Thus, fresh kiwifruit juice, prepared from 100% fruit, was
centrifuged at 9000xg for 10 min. Following centrifugation, the supernatant
was freeze-dried and a portion of the dried material was dissolved in phosphate
buffer and pH was adjusted to 7.4. This was then subjected to ultrafiltration by
passing through a filter with molecular weight cut-off 1000 dalton. The
ultrafiltrate was collected, freeze-dried and reconstituted in water, and pH was
adjusted to 7.4. The platelet aggregation was measured using the extract at
different stages of fractionation. In a separate study, the extract was boiled for
10 min. and centrifuged, and the anti-platelet activity of the boiled sample was
determined.

Table-1 shows the yield of KFE
Steps Result
Weight whole fruit 325.19 gm
Weight of the peeled fruit 250.51 gm

Weight juice produced 235.92 gm
Weight of juice after centrifuged 141.87 gm
The pH of the supernatant 3,41
Total dry weight of KFE after lyophilization 17.654 1g m
0057 The schematic representation from a procedure for partial fractionation
of kiwi fruit extracts shows the kiwifruit extract preparation using different
fractionation procedures. The inhibitor(s) of platelet aggregation in kiwi
extracts were present in the water-soluble fraction and their size is smaller than
3000 daltons. Boiling of this fraction did not destroy the activity.


Example 3:
Platelet aggregation study, fractionation of kiwi fruit extracts and their
effects on ADP-induced platelet aggregation by ADP:
58 The effect of the fruit extracts on the aggregatory properties of human
platelets was investigated in healthy volunteers. Venous blood was collected
from volunteers who had not taken any medications for at least 14 days before
donation. Blood (20ml) was collected using a 19G butterfly needle and
coagulation was prevented by mixing the blood samples with acid citrate, (135
mM) in the ratio of 9 parts by volume of blood up to 1 part by volume of acid
citrate. Platelet-rich plasma (PRP) was prepared from the samples by
centrifuging the blood at 180xg for 15 min. Kiwi fruit juice (10-30 µl), the pH
was adjusted to 7.4 with 1M sodium hydroxide was mixed with the PRP to
make volume up to 500 µl, and incubated at 37ºC from 15 min. after which the
effect of the fruit extract on ADP induced platelet aggregation was monitored
with the addition of ADP to a final concentration 5 µM. Controls were run in
parallel using 10-30 µL phosphate buffer, pH 7.4 instead of the fruit extract.
Platelet aggregation in PRP was monitored using a Chrono-Log aggregation
(Chrono-Log, USA) at a constant stirring speed of 1000 rpm at 37C.
59 To determine the effect of KFE on platelet aggregation in vitro, PRP
(450µl) was incubated with different concentrations of KFE (in volume 50µl
for 15 min at 37°C prior to the addition of an aggregating agent. The IC50 for
different fractions of KFE was determined by incubating these platelets with
different concentrations of KFE for 15 min. Controls were run in parallel
replacing fruit extract with 50µl of phosphate. Inhibition of platelet aggregation
is expressed as the decrease in the area under the curve compared with the
control. Figure 2 shows the effect of fractionated kiwifruit extract on platelet

aggregation induced by ADP. The experimental conditions are described in
Table-1.
60 Figure 2 shows the effect of different volumes of kiwi fruit extract on
platelet aggregation by ADP in vitro. PRP (450 ml) was incubated with
different volumes (0, 10, 20 and 30 ul) of KFE for 15 min at 37C prior to the
addition of agonists, arachidonic acid (500 µg/ml), ADP (3 mM, and collagen
(1 µg/ml). KFE inhibited ADP-induced aggregation in a dose-dependent
manner (Table-1). ADP induced aggregation was inhibited by 45% with 10 µl
KFE, 65% with 20 µl KFE, and 95% with 30 µl KFE, compared with controls.
Similarly, KFE inhibited collagen-induced platelet aggregation; the inhibition
level was lower with 10 and 20 µl incubations. Inhibition of arachidonic acid–
induced platelet aggregation exhibited 38% inhibition at the highest KFE level
tested and very little inhibition at lower concentrations of KFE.
61 Table 2 shows the dose-response of kiwifruit extract on inhibition of
platelet aggregation by different agents. It demonstrated a dose-response effect
with ADP-induced aggregation: increasing the kiwifruit extract led to a greater
reduction in platelet aggregation. The fraction isolated from kiwifruit was
equally effective against all three platelet aggregating agents, collagen, ADP,
and arachidonic acid. Boiling of kiwifruit extract at 100° C for 10 min did not
affect the anti-platelet aggregation of the extract.
Table-2:
Kiwifruit extract Inhibition of platelet aggregation by three different
Volume (µl) agonists (mean %)
Arachidonic Collagen ADP
acid
10 12 18 45
20 25 45 65
30 38 90 95

Example 4:
Inhibition of angiotensin-converting enzyme (ACE) by KFE:
0062 Angiotensin I-converting enzyme (ACE, EC 3.4.15.1), an exopeptidase, is
a circulating enzyme that participates in the body's renin-angiotensin system,
which mediates extracellular volume (e.g., that of the blood plasma, lymph and
interstitial fluid), and arterial vasoconstriction. It is secreted by pulmonary and
renal endothelial cells and catalyzes the conversion of decapeptide angiotensin
I to octapeptide angiotensin II. ACE inhibitors block the conversion of
angiotensin I to angiotensin II. They therefore lower arteriolar resistance and
increase venous capacity; increase cardiac output and cardiac index, stroke
work and volume, lower renovascular resistance, and lead to increased
natriuresis (excretion of sodium in the urine). With ACE inhibitor use, the
effects of angiotensin II are prevented, leading to decreased blood pressure.
0063 To determine the effects of KFE on the ACE activity of human serum,
incubation of serum is done with 20 µl of KFE for 15 min inhibited more than
15% activity compared with control. Figure-3 shows UV scanning of the
delipidated, ultrafiltrated purified active fractions of the Kiwifruit extract.
0064 Table-3 shows the effect of KFE on the serum Angiotensin Converting
Enzyme. Typically, the serum was incubated with different amounts or
volumes of fruit extract or captopril for 15 min at 37C in 50 uL.
Table-3: Effect of fruit extract on the ACE activity
KFE (Sugar-free) ACE Activity (%)
0 mg 96
0.75 mg 33
1 mg 19
2 mg 10

0065 Various modifications and variations of the described method and
system of the invention will be apparent to those skilled in the art without
departing from the scope and spirit of the invention. Although the invention has
been described in connection with specific preferred embodiments, it should be
understood that the invention as claimed should not be unduly limited to such
specific embodiments. Indeed, various modifications of the described modes
for carrying out the invention that is obvious to those skilled in the field of this
invention are intended to be within the scope of the following claims.

We Claim:
1. A process of preparation of extract fraction of the family Actinidia,
preferably kiwifruit, said process comprising the steps of:
Homogenizing the fruits;
Centrifuging the homogenized fruits;
Boiling of the supernatant obtained;
Centrifuging again the boiled supernatant;
Ultrafiltration through the filter with molecular weight cut off of less than
1000;
Removing the soluble sugars from the ultrafiltrate obtained through column
chromatography; and
Freeze-drying.
2. The process as claimed in claim 1, wherein the centrifugation of the
homogenized fruits is performed at 9000xg for 15 minutes.
3. The process as claimed in claim 1, wherein the boiling of supernatant is
performed at 90 °C for 20 minutes.
4. The process as claimed in claim 1, wherein the centrifugation of the boiled
supernatant is performed at 12000xg for 30 minutes at 4 °C.
5. A fruit extract fraction of the family Actinidia, preferably kiwi fruit, as
obtained through the process as claimed in claim 1, wherein said fraction
comprises biological molecules with a molecular weight of less than 1000
daltons.
6. The fruit extract fraction as claimed in claim 5, wherein said extract is
delipidated.

7. A method against platelet activation and/ or aggregation, comprising
administering a delipidated fruit extract fraction belonging to Actinidia
family as claimed in claim 5, wherein said fraction comprises biological
molecules with a molecular weight of less than 3000 dalton to a subject in
need thereof under conditions such that said disease state is prevented or
treated.
8. The method as claimed in claim 7, wherein said administering inhibits
platelet aggregation, resulting in anti-thrombotic activity and blood
thinning.