FORM 2
THE PATENT ACT 1970
(39 of 1970)
&
The Patents Rules, 2003
PROVISIONAL SPECIFICATION
(See section 10 and rulel3)
1. TITLE OF THE INVENTION:
"NOVEL COMPOSITIONS FOR PREVENTION AND TREATMENT OF
MASTITIS AND METRITIS"
2. APPLICANT (S)
(a) NAME: ADVANCED ENZYME TECHNOLOGIES LIMITED.
(b) NATIONALITY: an Indian Company incorporated under the
Indian Companies ACT, 1956
(c) ADDRESS: Above Navneet Motors, Gokul Nagar, P. O. Box 182,
Thane (W) -400 601. Maharashtra, India
3. PREAMBLE TO THE DESCRIPTION
The following specification particularly describes the invention.

Technical Field of the Invention:
The present invention relates to novel stable compositions used for the prevention and/or treatment of mastitis and metritis in mammals comprising combination of therapeutically effective amount of Serratiopeptidase, Lysozyme, Oscimum sanctum and Azadirecta indica having synergistic effect. The present invention further relates to a method of treatment and/or prevention of an infective condition in a fluid containing organ having a natural exterior orifice, such as the udder of a milk producing animal.
Background and prior art of the Invention:
Mastitis is an inflammation of the mammary glands of milk-producing animals, for example dairy cows, most often caused by bacterial infection Caused by Streptococcus agalactiae, Staphylococcus aureus, Streptococcus dysgalactiae, Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter aerogenes, Streptococcus uberis, Streptococcus bovis, Streptococcus disgalactiae, Enterococcus faecium, Enterococcus faecalis.
Bacteria enter through the teat canal of the animal and can cause acute, clinical, or subclinical mastitis. Over 135 organisms have been documented as causative pathogens for bovine mastitis. Three of the major groups of pathogens are gram-positive cocci, gram-negative bacilli and gram-positive bacilli. Hygiene, environmental factors and metabolic disturbances deriving from high milk yield combine to create conditions favorable to the onset of mastitis. An increased somatic cell count, associated with mastitis, is positively correlated with infection and negatively correlated with milk production.
Symptoms of mastitis includes inflammation of the mammary glands along with other symptoms like,
- Mild signs flakes or clots in the milk may have slight swelling of infected quarter.
- Severe signs secretion abnormal, hot, swollen quarter or udder; cow may have a fever, rapid pulse, loss of appetite, dehydration and depression; death may occur.
- Somatic cell count (SCC) of the milk will be elevated.
- Bacteriological culturing of milk will detect bacteria in the milk.
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- Lowered milk production.
Effects of mastitis on milk Production, milk composition and quality of milk:
Mastitis reduces milk yield and alters milk composition. The magnitude of these changes in individual cows varies with the severity and duration of the infection and the causative microorganisms. Mastitis is almost always caused by bacteria. These microorganisms produce toxins that can directly damage milk-producing tissue of the mammary gland, and the presence of bacteria initiates inflammation within the mammary tissue in an attempt to eliminate the invading microorganisms. The inflammation contributes to decreased milk production and is primarily responsible for the compositional changes observed in milk from infected quarters and cows. In general, compositional changes involve an increase in blood components present in milk and a decrease in normal milk constituents.
Metritis is inflammation of the endometrium (the lining of the uterus), the underlying glandular tissues and the muscular layers. Metritis also causes inflammation of the ovaries.
It is commonly observed that cattle like cows and buffalos suffer their entire life with mastitis and metritis, which ultimately results in their bad health and tremendously decreased milk production. Mastitis is the most hindering disease affecting the economy of the dairy industry, with losses estimated at lacs of rupees annually in the asian countries alone. As for every clinical case of mastitis, there will be 15 to 40 sub-clinical cases. The majority of these losses are due to reduced milk production.
There are several allopathic as well as synthetic drugs such as Benzyl penicillin, Phenoxymethyl penicillin, Cloxacillin, Nafcillin, Methicillin, Oxacillin, Amoxycillin, Temocillin, Ticarcillin, Indol and Indene acetic acid derivatives, Acetylsalicylic acid derivatives, Fenamates, heteroaryl acetic acid derivatives, propionic acid derivatives, enolic acids para-aminophenol derivatives alkanones nimesulide proquazone known and available to treat the disease.
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US6414036 discloses a pharmaceutical composition for treating mastitis wherein oil extract from plants from the Labiatae family are used. The compositions are formulated by combining extracts of essential oils from plants of the Labiatae family with an organic acid or a Group I salt.
CN1390554 discloses an anti-inflammatory paste for treating epidemic parotitis, acute mastitis prepared from fresh cactus, rhubarb natural indigo and sodium sulphate powder.
W09913892 discloses antimastitic pharmaceutical composition of natural origin comprised of plant extracts for veterinary medical applications in order to treat mastitis in bovine, ovine and caprine animals, and process for obtaining such composition. The composition comprises juice or gel of liliacious plants (Aloe vera), aqueous extract of maguey (Agave atrovirens), essential lemon oil (Citrus limon), essential oil of tea tree (Melaleuca alternifolia), comfrey extract (Symphytum officinale). The composition comprises additionally zinc sulphate, sodium salt of ethylene-diamino tetra-acetic acid, citric acid, ascorbic acid and sodium benzoate.
US3636194 discloses compositions for treating mastitis by intramammary infusion, comprising an antibiotic, a vegetable oil, an alcohol-soluble fraction of natural lecithin phospholipid material for promoting dispersion of the oil in milk, the phospholipid being selected from the group consisting of phosphatidyl choline and phosphatidyl ethanolamine and mixtures thereof and present in an amount of at least 0.25% in said oil.
EP1656159 discloses a method of treatment and/or prevention of an infective condition in a fluid-containing organ having a natural exterior orifice, such as the udder of a milk-producing animal or an ear of a subject. This invention also provides a dispersible pharmaceutical composition suitable for infusion into the organ and a process for preparing such a composition. Compositions contain one component from antimicrobial class (antibiotics) in combination with anti-inflammatory analgesic compound like Non Steroidal Anti Inflammatory Drugs (NSAIDs).
GB1181527 discloses a composition for treating mastitis comprising an active substance and a pharmaceutically acceptable oil base. The compositions contain phospholipid
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material consisting substantially entirely of alcohol-soluble material for promoting dispersion of the composition in milk. Specified active agents include penicillin, streptomycin, dihydrostreptomycin, neomycin, polymyxin, tetracyclines, nitrofurazone, cortisone, hydrocortisone, prednisolone, sulpha methazine, sulphamerazine and sulphathiazole.
There are several disadvantages associated with these types of compositions for the treatment of mastitis. All such products are well known to affect negatively on general immunity of subject. It is very commonly observed that the subject looses its immunity after administration of the product. It is also observed that the subject becomes extremely lethargic after administration of the said allopathic or synthetic products. Moreover it is also observed that administration of such product negatively affects milk yield and quality. The cost of these commonly known allopathic products is very high.
Very few antibacterial agents possess anti-inflammatory, anesthetic, antipyretic or analgesic properties in addition to their antibacterial activity. Therefore, treating an infective condition with an antibacterial agent alone typically does not alleviate the inflammation, pain, swelling, fever and other complications that often accompany such an infective condition. These problems are usually not totally resolved until the causal organism of the infective condition has been eliminated or reduced to a sub pathogenic population by the antibacterial agent. The commonly known compositions for treatment of mastitis lack stability and does not provide an extended chemical and/or physical stability. The formulations comprises of pharmaceutically active agent and/or excipient that is prone to oxidative degradation.
Therefore, in view of the aforementioned drawbacks associated with prior art compositions for the treatment of mastitis and metritis, it is apparent that there exists a need for compositions which are effective and devoid of side effects as well as rejuvenate general health and immunity of the dairy animals.
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Object of the Invention:
The main object of the present invention is to provide novel stable compositions used for the treatment of mastitis and metritis comprising combination of therapeutically effective amount of Serratiopeptidase, Lysozyme, Oscimum sanctum and Azadirecta indica having synergistic effect.
As per another object, the novel compositions of the present invention are effective at lower doses of the active agent, provides targeted delivery of the active agent to sites of infection with minimal/no irritation upon administration and minimal/no side effects in comparison to the synthetic and allopathic drugs used for the treatment of mastitis and metritis.
As per yet another object of the invention, the novel compositions rejuvenates the general health and immunity of the subject naturally on administration and are effective against a wide variety of infectious organisms and inflammatory and infectious components like pain, inflammation, fever, edema.
Further object of the present invention is to provide novel compositions having economic significance in comparison to the commonly available allopathic and synthetic drugs.
Yet further object of the invention is to provide a method of treatment and/or prevention of an infective condition in a fluid containing organ having a natural exterior orifice, such as the udder of a milk producing animal.
Summary of the Invention:
The present invention discloses novel stable compositions used for the treatment of mastitis and metritis in mammals comprising combination of therapeutically effective amount of Serratiopeptidase, Lysozyme, Oscimum sanctum and Azadirecta indica having synergistic effect. The present invention further discloses a novel method of treatment and/or prevention of an infective condition in a fluid containing organ having a natural exterior orifice, such as the udder of a milk producing animal. The present invention still
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further discloses evaluation of the antibacterial properties of the novel compositions on a wide variety of causative organisms.
Description of the Invention:
The present invention describes novel stable compositions for the treatment of mastitis and metritis in mammals.
The novel composition as per the present invention comprises of combination of absolutely Natural products such as Serratiopeptidase, Lysozyme (Muramidase), Oscimum sanctum and Azadirecta indica.
The novel compositions of the present invention have minimal or no side effects and also have economic significance in comparison to the commonly known compositions.
The present invention further relates to a method of treatment and/or prevention of an infective condition in a fluid containing organ having a natural exterior orifice, such as the udder of a milk producing animal. The novel method of treatment for mastitis and metritis comprises of administering a natural combination of Serratiopeptidase, Lysozyme(Muramidase), Oscimum sanctum and Azadirecta indica, as an antibacterial and anti-inflammatory agent through oral and/or topical route.
Described below are the ingredients and qualities of the natural products used in the composition for the treatment of mastitis and metritis.
1] Serratiopeptidase
Serratiopeptidase also known as Serrapeptase is a proteolytic enzyme that stimulates immunity, reduces edema, and fights inflammation. Serratiopeptidase is isolated from the non-pathogenic Enterobacteria Serratia El5. The enzyme is found naturally in the intestine of the silkworm, which is used by the silkworm to dissolve the cocoon and emerge as a moth. When consumed as uncoated tablets or capsules, the enzyme is
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destroyed by the acid in the stomach. However, when enteric coated the enzyme passes through the stomach unaffected, and can be absorbed in the intestine.
Serrapeptase when given in combination with antimicrobial agents delivers increased concentrations of the antimicrobial agent to the site of the infection. The mechanism of antibacterial action of serratiopeptidase can be explained, as an inhibitor of biofilm formation of bacterial cell wall. Bacteria often endure a process called biofilm formation, which results in resistance to antimicrobial agents.

Chemical characterization of active component Microbial fermentation enzyme preparation from bacterial culture.
Source/ Origin Serratia spp.
Systematic Name (IUB) Serratiopeptidase
IUB/CAS number CAS 37312-62-2
Hazardous ingredients None
Classification Enzyme protease
Pharmacopoeial Status Martindale Pg no 1662
2] Lysozyme
Lysozyme is also known as Muramidase and is isolated from extract of purified chicken egg white along with naturally occurring biologically active proteins. Lysozyme acts as a "natural" antibacterial. The therapeutic effectiveness of lysozyme is actually based on its ability to control the growth of susceptible bacteria and to modulate host immunity against infections. The ability to control the growth of the susceptible bacteria is due to the biological activity of the enzyme. Antibiotic activity and immune stimulating effects of lysozyme impart therapeutic benefits.
Lysozyme hydrolyzes preferentially the P-l, 4 glucosidic linkages between N-acetylmuramic acid and N-acetylglucosamine which occur in the mucopeptide cell wall structure of certain microorganisms, such as Micrococcus lysodeikticus. A somewhat more limited activity is exhibited towards chitin oligomers. Lysozyme is of widespread
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distribution in animals and plants. Lysozyme is also found in mammalian secretions and tissues, saliva, tears, milk, cervical mucus, leucocytes, kidneys, etc

Chemical characterization of active component Enzyme preparation from animal origin.
Source/ Origin Extracts of purified chicken egg white
Systematic Name (IUB) Peptidoglycan N-acetylmuramoylhydrolase
IUB/CAS number 5 CAS 3.2.1.17
Hazardous ingredients None
Classification Own Antibiotic
Pharmacopoeial Status Martindale Pg no 1638
3] Oscimum sanctum
Oscimum sanctum acts as a COX-2 inhibitor and provides the benefits of an analgesic owing to active constituent Eugenol (l-hydroxy-2-methoxy-4-allylbenzene). Studies have shown Oscimum sanctum to be effective for the treatment of diabetes, as it reduces the blood glucose levels and this benefit is due its antioxidant properties. The same study showed that there is a significant reduction in total cholesterol levels with Oscimum sanctum.
Oscimum sanctum extracts are used for common colds, headaches, stomach disorders, inflammation, heart disease, various forms of poisoning, and malaria. Oscimum sanctum can be consumed in various forms like herbal tea, dried powder, fresh leaf, or mixed with ghee. Essential oil extracted from Karpoora Oscimum sanctum is mostly used for medicinal purposes and in herbal toiletry. The dried leaves of Oscimum sanctum being an excellent insect repellant are mixed with stored grains to repel insects.
4] Azadirecta indica
Azadirecta indica plant has numerous medicinal properties hence used for various conditions like digestive disorders, diabetes, high cholesterol, cancer, etc.
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Azadirecta indica has anti malarial properties hence used for malaria. Oil of Azadirecta indica is used extensively by the cosmetic industry for the preparation of cosmetics like soap, shampoo, balms and creams. All parts of the tree (seeds, leaves, flowers and bark) are used for preparing many different medicinal preparations. Azadirecta indica twigs are used for brushing teeth in India-perhaps one of the earliest and most effective forms of dental care. In some parts of Sub-Saharan Africa the bark is used as both toothbrush and toothpaste. Azadirecta indica tree is of great importance for its anti-desertification properties and possibly as a good carbon dioxide sink.
The primary interest of research scientists is the insecticidal activity of Azadirecta indica. The secondary metabolites of various trees have biological activity, but azadirachtin of Azadirecta indica has utmost ecological importance. Studies have shown wide spectrum of insecticidal activity for Azadirecta indica and the numerous species affected. Azadirecta indica acts by breaking the insect's lifecycle. Research has increased in the past few years as the desire for safer pest control methods increases and it becomes apparent that this tree will be able to play a role in integrated pest management systems.
Azadirecta indica is deemed very effective in the treatment of scabies although only preliminary scientific proof exists which still has to be corroborated, and is recommended for those who are sensitive to permethrin, a known insecticide which might be irritant. Also, the scabies mite has yet to become resistant to Azadirecta indica, so in persistent cases Azadirecta indica has been shown to be very effective. There is also anecdotal evidence of its effectiveness in treating infestations of head lice in humans.
In view of the above qualities of the individual substances, the present inventors have developed novel compositions from natural product for the treatment of mastitis and metritis in mammals. The novel stable compositions containing the combination of therapeutically effective amount of Serratiopeptidase, Lysozyme, Oscimum sanctum and Azadirecta indica provides synergistic activity.
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The present invention is more specifically explained by following examples. However, it should be understood that that the scope of the present invention is not limited by the examples in any manner. It will be appreciated by any person skilled in this art that the present invention includes the following examples and further can be modified and altered within the technical concept of the present invention.
EXAMPLES
Example 1.
CI = Serratiopeptidase 1-5 %
C2= Lysozyme 10%
C3 = Oscimum sanctum 10 %
C4 = Azadirecta indica 10%
C5 = Citric acid 10 %
C6 = Malt dextrin Q.S.

Sr. C1% C2% C3% C4% C5% C6% Stabilityfor 1month Stabilityfor 6 months
1 1 10 10 10 10 Q.S. Stable Stable
2 2 10 10 10 10 Q.S. Stable Stable
3 3 10 10 10 10 Q.S. Stable Stable
4 4 10 10 10 10 Q.S. Stable Stable
5 5 10 10 10 10 Q.S. Stable Stable
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Example 2.
CI = Serratiopeptidase 2 %
C2= Lysozyme 10-30%
C3 = Oscimum sanctum 10 %
C4 = Azadirecta indica 10 %
C5 = Citric acid 10 %
C6= Malt dextrin Q.S.

Sr. C1% C2% C3% C4% C5% C6% Stabilityfor 1month Stabilityfor 6 months
1 2 10 10 10 10 Q.S. Stable Stable
2 2 15 10 10 10 Q.S. Stable Stable
3 2 20 10 10 10 Q.S. Stable Stable
4 2 25 10 10 10 Q.S. Stable Stable
5 2 30 10 10 10 Q.S. Stable Stable
Example 3.
CI = Serratiopeptidase 2 %
C2= Lysozyme 20%
C3 = Oscimum sanctum 10-30 %
C4 = Azadirecta indica 10 %
C5 = Citric acid 10 %
C6 = Malt dextrin Q.S.

Sr. C1% C2% C3% C4% C5% C6% Stabilityfor 1month Stabilityfor 6 months
1 2 20 10 10 10 Q.S. Stable Stable
2 2 20 15 10 10 Q.S. Stable Stable
3 2 20 20 10 10 Q.S. Stable Stable
4 2 20 25 10 10 Q.S. Stable Stable
5 2 20 30 10 10 Q.S. Stable Stable
•
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Example 4.
CI = Serratiopeptidase 2 %
C2= Lysozyme 20%
C3 = Oscimum sanctum 20 %
C4 = Azadirecta indica 10-30 %
C5 = Citric acid 10 %
C6= Malt dextrin Q.S.

Sr. C1% C2% C3% C4% C5% C6% Stabilityfor 1month Stabilityfor 6 months
1 2 20 20 10 10 Q.S. Stable Stable
2 2 20 20 15 10 Q.S. Stable Stable
3 2 20 20 20 10 Q.S. Stable Stable
4 2 20 20 25 10 Q.S. Stable Stable
5 2 20 20 30 10 Q.S. Stable Stable
Example 5.
CI = Serratiopeptidase 2 %
C2= Lysozyme 20%
C3 = Oscimum sanctum 20 %
C4 = Azadirecta indica 20 %
C5 = Citric acid 10 %
C6= Zeolite Q.S.

Sr. C1% C2 % C3% C4% C5% C6% Stabilityfor 1month Stabilityfor 6 months
1 2 20 20 10 10 Q.S. Unstable Unstable
2 2 20 20 15 10 Q.S. Unstable Unstable
3 2 20 20 20 10 Q.S. Unstable Unstable
4 2 20 20 25 10 Q.S. Unstable Unstable
5 2 20 20 30 10 Q.S. Unstable Unstable
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Example 6.
CI = Serratiopeptidase 2 %
C2= Lysozyme 20%
C3 = Oscimum sanctum 20 %
C4 = Azadirecta indica 20 %
C5 = Citric acid 10%
C6 = DCP Q.S.

Sr. C1% C2% C3% C4% C5% C6% Stabilityfor 1month Stabilityfor 6 months
1 2 20 20 20 10 Q.S. Stable Stable
2 2 20 20 20 10 Q.S. Stable Stable
3 2 20 20 20 10 Q.S. Stable Stable
4 2 20 20 20 10 Q.S. Stable Stable
5 2 20 20 20 10 Q.S. Stable Stable
Example 7.
CI = Serratiopeptidase 2 %
C2 = Lysozyme 20%
C3 = Oscimum sanctum 20 %
C4 = Azadirecta indica 20 %
C5 = Citric acid 10%
C6 = Malt dextrin Q.S.
C7 = Saccharomyces boulardii. 5 %

Sr. C1% C2% C3% C4% C5% C6% C7% Stabilityfor 1month Stabilityfor 6 months
1 2 20 20 20 10 Q.S. 5 Stable Stable
2 2 20 20 20 10 Q.S. 5 Stable Stable
3 2 20 20 20 10 Q.S. 5 Stable Stable
4 2 20 20 20 10 Q.S. 5 Stable Stable
5 2 20 20 20 10 Q.S. 5 Stable Stable
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The novel compositions of the present invention used for the treatment of mastitis and metritis has the following advantages.
1) Efficacy against a wide variety of infectious organisms.
2) Effective treatment for inflammatory as well as infectious components of mastitis and metritis like pain, inflammation, fever, edema.
3) Novel compositions containing the combination of therapeutically effective amount of Serratiopeptidase, Lysozyme, Oscimum sanctum and Azadirecta indica provides synergistic activity.
4) Potential to administer a lower dose of an active agent while still providing efficacy.
5) Targeted delivery of the active agent to sites of infection.
6) Minimal to no irritation after administration of the composition.
7) Removal / or reduction in side effects in comparison to the synthetic and allopathic drugs used for the treatment of mastitis and metritis.
8) Short milk out times following mastitis treatment for lactating cows.
9) Rejuvenates the general health and immunity of the animal.
10) Economic in comparison to the commonly available allopathic drugs used for the
treatment of mastitis and metritis.
Evaluation of the antibacterial properties
1] Testing with a Gram positive organism
Formulations: Polyenzyme formulations.
Organisms used for the test: Streptococcus spp., Staphylococcus spp. Klebsiella spp.
Enterococcus spp. E. coli.
Method: drop inoculation (20mg/ml) on lawn culture.
Procedure: The test organism was inoculated into nutrient broth and incubated overnight.
This inoculum was matched with 0.5 Macfarland standards and then inoculated onto
respective Agar plates.
A penicillin disk (potency 10 units; HiMedia Laboratories, Mumbai) was placed onto the
plate as a control. Drops (20^1) of various concentrations of polyenzyme formulations,
and components were applied separately onto the inoculated plates. The plates were
incubated at 37° C for 24 hours.
Results: Polyenzyme formulations produced a zone of clearance on the lawn culture plate
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2] Lab Trial Results

Example Trial Causative organism Growth 2.5 mg/ml Growth 5 mg/ml Growth 10 mg/ml Growth 15 mg/ml
E2T1 E. coliS. aureusK. pneumoniaeStreptococcus spp. +++ +++ ± ± + ± —
E2T2 E. coliS. aureusK. pneumoniaeStreptococcus spp. ± ± + + ± ± ± ± —
E2T3 E. coliS. aureusK. pneumoniaeStreptococcus spp. -H-+ ± ± ± ± ± ± —
E2T4 E. coliS. aureusK. pneumoniaeStreptococcus spp. +++ — — —
E3T3 E. coliS. aureusK. pneumoniaeStreptococcus spp. ± ± ± ± ± ± —
E4T3 E. coliS. aureusK. pneumoniaeStreptococcus spp. ± ± ± ± ± ± ~
E6T3 E. coliS. aureusK. pneumoniaeStreptococcus spp. +++ ±± ± ± ± —

*
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative examples and that the present invention may be embodied in other specific forms without departing from the essential attributes thereof, and it is therefore desired that the present embodiments and examples be considered in all respects as illustrative and not restrictive, reference being made to the appended claims, rather than to the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Dated this 10th day of July 2006


LUU+'
Dr. Gopakumar G. Nair Agent for the Applicant

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