WO2024025479A1

WO2024025479A1 - A process of extraction of porcine placenta protein and development of encapsulating and transporting system of porcine placenta protein extract to control an enteric release

Info

Publication number: WO2024025479A1
Application number: PCT/TH2022/000044
Authority: WO
Inventors: Wannapa CHINCHOOSAK; Nattika SAENGKRIT; Paweena DANA; Prattana TANYAPANYACHON; Nutthanit THUMRONGSIRI; Walailuk CHONNIYOM
Original assignee: Saraburi Farm Company Limited
Priority date: 2022-07-25
Filing date: 2022-11-15
Publication date: 2024-02-01

Abstract

A method of extraction of porcine placenta protein and development of encapsulating and transporting system of porcine placenta protein extract to control an enteric release is provide.The protein extract from porcine placenta is encapsulated to obtain the nanocarrier in the form of enteric microbeads having bioactivities, anti-oxidant and anti-inflammatory effects. The enteric microbeads are formed via ionic gelation technique and are used for transporting and maintaining the property of protein extract from the porcine placenta. The enteric microbeads can help control the enteric release of the protein extract, prevent the degradation in the stomach, and become releasable or degradable in the intestine.

Description

A PROCESS OF EXTRACTION OF PORCINE PLACENTA PROTEIN AND DEVELOPMENT OF ENCAPSULATING AND TRANSPORTING SYSTEM OF PORCINE PLACENTA PROTEIN EXTRACT TO CONTROL AN ENTERIC RELEASE

Field of the Invention

The present invention is related to the field of nanotechnology chemical, pharmaceutical science, and zoological, particularly to the process of extraction of porcine placenta protein and the development of encapsulating and transporting system of porcine placenta protein extract to control an enteric release.

Background of the Invention

Presently, Thailand's swine-raising industrial sector has tended to expand and grow yearly since Thai people prefer to consume swine, which is regarded as the main raw material for meals rich with protein and other plentiful nutrients. The parts of swine can generate income for the agriculturalists who are the swine raisers, such as meat, leather, liver, entrails, including bone and joints, etc., but some parts, which are seldom preferred to be utilized and are numerous agricultural leftovers of swine raising farms, are placenta.

The placenta is a special organ that is a body part of a female mammal, which connects the embryo with the uterus of a mother; and acts as a provider of oxygen and nutrients that are essential for growth from a mother to an offspring, that is a fetus, including the crucial factor synthesis for producing fetus hormone and immunization. Within the placenta, there is a source rich in various types of nutrients, such as protein, peptides, amino acids, vitamins, and minerals, including enzymes and growth factors, etc. In ancient times, the placenta was used as a medicine because of its many active substances that help maintain good health. In modern times it is used in health or beauty. In addition, the research on the placenta found biological effects, such as antioxidant, antiinflammation, vulnerary effect, skin nourishment and beautification, fatigue recovery, immunization, etc.

From searching for information from academic articles and research related to the placenta of mammals such as horses, sheep, cattle, and pigs in various fields as follows: the placenta has often been used in the extraction of vital substances for development to be capsules ready to nourish skin for women. Regarding cow and sheep placenta, there has still been the issue concerning the infection risk of Bovine Spongiform Encephalopathy (BSE), which is a cause of Dementia. Thus, in Japan, there has been a law prohibiting the manufacture of products made from cow and sheep placenta. Therefore, the products from porcine and horse placenta have been particularly remained to be permitted for manufacturing. The key role of the placenta in hormone accumulation affects embryo growth and development (Anthony et al. , 1995) . Such hormones consist of several types of Growth Factors, such as Hepatocyte growth factor (HGF), Epidermal growth factor (EGF), and Transforming growth factor-a; TGF-a,-0 (TGF-b)), where a receptor for these hormones has been found in human placenta (Tonello G et al., 1996; Pal P et al., 2002; Wolf HK et al., 1991) . In addition, in the placenta, other substances in the group of Autocrine and/or Paracrine, with therapeutic agent property that stimulates the tissue recovery process, are also available (Tonello G et al. , 1996; Shukla VK, et al. , 2004; Saito S et al. , 1995) . Thus, human placenta extract is used in treatment according to oriental medicine (Oriental therapeutic agent) . The human placenta extract has been used in wound healing, relief of Psoriasis or Rheumatoid arthritis, etc., (Tonello G et al., 1996; Kang SS, 2007; Yeom MJ, 2003). In addition, the placenta has also been utilized in health since the placenta is a temporary organ connected with a developing fetus to transport nutrients and antibodies and exchange gas via blood from a mother to an offspring. In Asia, placenta extract has been preferred to use in wound healing and anti-aging (Hong et al. , 2002; Datta and Bhattacharyya, 2004) . In addition, the placenta extract has also helped to boost immunity in mammals, where the placenta extract can induce the secretion of an intercellular messenger called Interleukin- 8 (IL- 8) in human monocytic cell line by stimulating through transcription factors and enzyme kinase. It was also found that macrophages could be stimulated in guinea pigs. (Chakraborty et al., 2006; Kang et al., 2007). When the placenta extract is used in raising postpartum piglets by mixing with swine feeds, it was found that a large quantity of the protein content in the immunity system of the body can resist pathogens and foreign bodies (Immunoglobulin G) in plasma and milk in piglets has been induced (Lee et al., 2006), etc.

From the search of patents relating to the animal placenta concerning extraction and utilization in various areas, the related information was found, for example:

Republic of Korea Patent Publication No. KR20100011850A stated about the manufacture of porcine placenta extract for reducing the complexity of the manufacturing process using calcium salt, phosphate salt, or mixed solution, where the procedures are porcine placenta conditioning and protein hydrolysis using Papain, Bromelain, or Alcalase, and enzyme inactivation at a temperature of 20-30°C, and then removal of fatty acid with calcium salt, phosphate salt or mixed solution, and adjust the pH concentration to 7, and filtration through a membrane filter, and autoclave using temperature to obtain the purified porcine placenta extract.

Chinese Patent Publication No. CN101309695 A stated the preparation method of porcine placenta extract and use as animal feed, where the porcine placenta extract in the present invention results in boosting the animal growth and is used as an animal drinking water additive or food additive. This porcine placenta is used as supplementary food that can produce quality meat. Thus, the present invention is very beneficial in the animal feed industry.

Chinese Patent Publication No. CN10699411 OB stated about the food processing from porcine placenta by applying freeze drying technology and producing freeze-dried powder of porcine placenta with antioxidant activity for producing skin care product to help diminishing wrinkles on skin with simple production method and capability to maintain a high level of nutritive value in the porcine placenta. The detected heavy mental matter content, such as lead, arsenic, and mercury, in freeze-dried powder of the prepared porcine placenta was in the range defined by the national standards. Calcium and phosphate contents improve almost ten times compared with the fresh weight of the porcine placenta. Moreover, the dried placenta powder also has an antioxidant effect, and the weight of finished goods is light. Dried placenta powder for porcine placenta is convenient for moving, transporting, and processing and is mainly beneficial to the economy.

Chinese Patent Publication No. CN 103565839B stated that the extraction of a porcine placenta comprising the procedures of cleaning to remove blood and contaminants and downsizing, then quickly freezing at -65 °C, dissolving, and downsizing again, washing with deionized water, blend to be homogenous at 10,000 revolutions per minute and a temperature not exceeding 40 °C, then centrifuging placenta with ultrasonic at a temperature of 40 - 60 °C, electric power of 200 - 350 watt for 4-10 minutes long, and freezing at 4 °C, and then centrifuging, and then collect the upper liquids, where the soluble protein content is 2 - 4 milligrams/milliliter, then passing membrane ultrafiltration to obtain a composition from porcine placenta with low molecular weight, and carrying out microwave vacuum freeze dry.

Chinese Patent Publication No. CN101837005B stated that the porcine placenta extract comprising the preparation procedures as follows: cleaning a placenta and pasteurizing with steam at 121 °C for 30 minutes long and then carrying out hydrolysis by adding 10 times water down in the treated placenta, and then adding sodium hydroxide or sodium carbonate to obtain pH of 7.0- 9.0, heat at 45-65°C, crushing by adding pancreatic enzyme and adding chloroform or chloroform mixed with 5%wt n-butanol, letting it digest for 2 hours long, cleaving protein, adding 1-10% of trichloroacetic acid, and centrifuging, purifying sediment portion by adding ethanol at a concentration of 70-85% for 2-3 times of quantity, and sterilized by steam sterilization at 121°C for 30 minutes long. It could be seen from the information mentioned above that the porcine placenta protein extracts have not been stated together with the development of encapsulating and transporting system of porcine placenta protein extract to control an enteric release like the present invention. Enteric encapsulation/coating is the alimentary tract's entrapment and release control process and is utilized in numerous pharmaceutical industries, which transport active compounds in the form of oral administration, in which the transported compounds are formed into pellets encapsulated with a material that can prevent degradation in the stomach (acidity) but can release or degrade in the intestine (alkalinity) to be used to protect the medicaments or active ingredients destroyable by acids in the stomach, and must be coated or stored in the transport system to release the medicaments at small intestine for improved efficacy of activity. The present invention has developed the transport system of medicaments or active compounds in the animal alimentary system, particularly porcine placenta protein extracts stored in the form of enteric microbead, by applying an Ionic gelation technique in controlling enteric release to maintain the property of porcine placenta protein extract through oral administration and controlling to make it released or degraded in the intestine.

Summary of the Invention

A process of extraction of porcine placenta protein and development of encapsulating and transporting system of porcine placenta protein extract to control an enteric release, the process comprises preparation of a porcine placenta sample, an extraction of protein from the porcine placenta and development of the system for encapsulating the porcine placenta protein extract to control the enteric release, wherein:

The preparation of a porcine placenta sample is performed by cleaning a porcine placenta to remove blood clots or waste attached therewith, then reducing the size of the porcine placenta, and dry it.

The extraction of protein from the porcine placenta can be performed by finely grinding the prepared placenta and then immersing in homogenizing buffer comprising DMEM high glucose, sucrose and EDTA, where the piece of placenta is suspended in the homogenizing buffer, and disrupting cells using sound waves, then centrifuging to separate cell pellet, collect only supernatant, pass-through pasteurization, and then filtrating it.

The development of the system for encapsulating the porcine placenta protein extract to control the enteric release is performed by increasing the concentration of the porcine placenta protein extract by freeze drying, then adding water into the dried protein extract, and homogeneously dissolving it, next, encapsulating the concentrated porcine placenta protein extract in the form of an enteric microbead by mixing the concentrated porcine placenta protein extract with the chemical used in encapsulating, then stirring until both solutions are homogeneous, and spraying into a container containing the solutions to form enteric microbead particles having the concentrated porcine placenta protein extract encapsulated therein.

The object of the invention aims at solving the problems of agriculturalists who have agricultural leftovers by adding higher values with innovation to these leftovers, resulting in a new product model that can access a target group in the type of animal feed industry for immunization, such as dog, cat, and fish, and piglet, etc. According to the present invention, the enteric microbead particles that entrap the porcine placenta protein extracts can be used to manufacture a product for boosting growth, immunizing, resisting free radicals, and inflammation for vertebrates, particularly domestic animals.

Brief Description of the Drawings

Figure 1 A porcine placenta extract undergoes an extraction process with (a) liquid nitrogen and (b) freeze drying

Figure 2 A microbead particle synthesized from the formulations under a Scanning Electron Microscope at lOOx magnification and having a scale bar size equaling 200 micrometers by Formulation Fl (a), Formulation F2 (b), and Formulation F3 (c)

Figure 3 A size of microbead particle analyzed by Mastersizer shows the result in the form of a graph showing an average particle size by Formulation Fl (a), Formulation F2 (b), and Formulation F3 (c)

Figure 4 Test result of anti -oxidation action with superoxide anion scavenging assay, which reacts to concentrations by comparing Quercetin (a) and microbead particles (b)

Figure 5 shows the result of cytokine inhibition relating to inflammation of microbead particles and porcine placenta extracts, where the statistical results were compared in the significant difference with control Raw cell incubated with lipopolysaccharide (C-LPS), A)), IL- 6 (***P-value<0.001), B), IL-8 (**P-value<0.002; and

* P-value<0.033) and C) TNF-a (***P-value<0.001 ), where lipopolysaccharide is the control unit of the system.

NC = non-treated cell, BLK = 0.1M sodium citrate, and Dexamethasone (Dexa) = positive control, by inhibition of cytokine IL-6 (a), inhibition of cytokine IL-8 (b), and inhibition of cytokine TNF-a (c). Figure 6 A result of protein release out of microbead particles compares the porcine placenta extraction in pH 2.0-6.8 simulated alimentary fluid within 0-24 hours of the test period.

Detailed Description of the Invention

A process of extraction of porcine placenta protein and development of encapsulating and transporting system of porcine placenta protein extract to control an enteric release, the process comprising: a. preparation of a porcine placenta sample clean a porcine placenta to remove blood clots or waste attached therewith, then reduce the size of the porcine placenta, and dry it.

In one aspect, the preparation of the porcine placenta sample, the cleaning using washing buffer is performed by the followings: washing with tap water, followed by deionized water and phosphate buffer saline (PBS) at pH 7.4, respectively. In one aspect of the preparation of the porcine placenta sample, the reducing of size is performed using a blender.

In one aspect, the preparation of porcine placenta sample, the method of drying is selected from drying by using liquid nitrogen or by freeze drying.

In another aspect, the freeze drying is performed by freeze dryer. b. extraction of protein from the porcine placenta finely grind the placenta prepared from step a, and then immerse in homogenizing buffer comprising DMEM high glucose, sucrose at a concentration of 0.1-1 Molar (M), and EDTA at a concentration of 1-2 millimolar ( mM) , where the piece of placenta is suspended in the homogenizing buffer for 20-40 minutes,

In one aspect, the extraction of protein from the porcine placenta, the placenta is suspended in a homogenizing buffer, which said homogenizing buffer is immersed in ice.

Disrupting cells using sound waves, then centrifuge to separate cell pellet, collect only supernatant, pass-through pasteurization, and then filtrate it.

In one aspect, the extraction of protein from the porcine placenta, the cell disruption is performed by using sound waves from Probe-type Ultrasonicator with an amplitude of 10-20% for 10-30 minutes.

In one aspect, the extraction of protein from the porcine placenta, ice bathing is performed during disrupting the cell by using sound waves to prevent excessive cumulative heat. In one aspect, the extraction of protein from the porcine placenta, the centrifugation of cell pellet is performed at a speed of 8 ,000-12,000 revolutions per minutes (rpm) at a temperature of 4°C for 20-30 minutes.

In one aspect, the extraction of protein from the porcine placenta, the pasteurization is performed with heat at a temperature of 50-60°C for 20-30 minutes.

In one aspect, the extraction of protein from the porcine placenta, the filtration is performed by using a thin white cloth and vacuum filtration through a filter with a porous size of 10-20 micrometers, respectively.

C. development of the system for encapsulating the porcine placenta protein extract to control the enteric release increasing the concentration of the porcine placenta protein extract derived from step b by freeze drying for 1-2 days, then add water into the dried protein extract, and homogeneously dissolve it.

In one aspect, the freeze drying is performed by freeze dryer. next, encapsulating the concentrated porcine placenta protein extract in the form of an enteric microbead by mixing the concentrated porcine placenta protein extract with the chemical used in encapsulating, the ratio of the porcine placenta protein extract to the chemical used in encapsulating is 10-20 to 70-80 %wt, then stir until both solutions are homogeneous, and spray into a container containing the solutions to form enteric microbead particles having the concentrated porcine placenta protein extract encapsulated therein.

In one aspect, the chemical used in encapsulating can be selected from any one of or more of Sodium Alginate, Bentonite, Calcium chloride or water soluble chitosan in combination thereof.

In another aspect, the chemical used in encapsulating is provided as a mixture of Sodium Alginate and Bentonite and a mixture of Calcium Chloride and water soluble chitosan, wherein the mixture of Calcium Chloride and water soluble chitosan having a ratio of Calcium Chloride to water soluble chitosan at 1-2 %wt to 0.5-2.0 %wt.

In one aspect of the development of the system for encapsulating the porcine placenta protein extract to control the enteric release, the spraying is performed into the container containing 0.5-3%wt/v of Calcium Chloride solution, wherein the container containing the Calcium Chloride solution is immersed in an ice bath and immersed in the Calcium Chloride solution for 30-40 minutes. Centrifuge the solutions having the enteric microbead particles, then wash with distilled water, collect the obtained enteric microbead particles to mix with 0.05-0.3%w/w talcum and dry it to obtain nanocarrier in the form of enteric microbead.

In one aspect, the development of the system for encapsulating the porcine placenta protein extract to control the enteric release, the centrifugation is performed at a speed of 5 ,000-7,000 rpm, the temperature of 4°C for 15-20 minutes.

In one aspect, the development of the system for encapsulating the porcine placenta protein extract to control the enteric release, the drying of nanocarrier in the form of enteric microbead is performed by the freeze drying method, wherein the drying of nanocarrier in the form of enteric microbead is performed by the freeze drying method for 1 -3 days, and the said freeze drying is performed by freeze dryer.

In addition, the development of the system for encapsulating the porcine placenta protein extract to control the enteric release further comprises an antioxidant, wherein the antioxidant is Vitamin C.

According to the present invention, enteric microbead particles encapsulating the porcine placenta protein extract from the process are used for producing a product to promote growth, increase immunization, anti-oxidant, and anti-inflammation for vertebrates, particularly domestic animals.

The followings are the examples relating to the process of extraction of porcine placenta protein and the development of encapsulating and transporting system of porcine placenta protein extract to control an enteric release, the evaluation of the biological quality of nanocarrier in the form of microbead, which store the porcine placenta protein extract. However, the present invention is not limited to these examples.

Example 1 Porcine Placenta Protein

Preparation Method of Porcine Placenta Sample

Clean a porcine placenta to remove blood clots or wastes partially attached to the placenta with washing buffer 3 times as follows: washing with tap water, following with deionized water and pH 7.4 phosphate buffer saline (PBS), respectively, and then cutting the porcine placenta into small pieces, drying by freeze drying by 2 methods with the following detail.

Method 1 Immerse the placenta in liquid nitrogen for sudden deep freezing. Method 2 Dry the placenta using Freeze Dryer to make water sublimation of the biopsy overnight until the placenta is dried.

Extraction Method of Porcine Placenta Protein

Finely grind the prepared placenta using a blender, and stop every 15 seconds during the blending process in order to prevent excessive accumulation of heat, then immerse the ground porcine placenta in homogenizing buffer (DMEM comprising high glucose, sucrose at a concentration of 0.1-1 Molar (M), and EDTA at a concentration of 1-2 millimolar (mM)), at a volume of 300 milliliters, where the piece of placenta is suspended in the homogenizing buffer for 20-40 minutes in an ice bath.

Disrupt cells using sound waves from Probe-type Ultrasonicator with an amplitude of 10- 20% for 10-30 minutes, then simultaneously perform an ice bathing to prevent excessive cumulative heat, and then centrifuge to separate cell pellet at a speed of 8,000-12,000 revolutions per minute (rpm) at a temperature of 4°C for 20-30 minutes, then collect the supernatant to perform pasteurization with heat at a temperature of 50-60°C for 20-30 minutes.

Filtrate using a thin white cloth and vacuum filtration through a filter with a porous size of 10-20 micrometers, respectively, to sterilize, then analyze the supernatant to determine the total protein with BCA™ protein assay kit.

From the experimental results of extracting the protein from the porcine placenta and comparing the two extraction methods consisting of Method 1 - put the ground porcine placenta through the process of immersing in liquid nitrogen, and Method 2 - freeze drying the placenta, then entering protein extraction process by disrupting cell wall with sonication, and then conducting the quantitative analysis of the obtained extract to determine protein content (with BCA™ protein assay Method) , it was found that the protein content from Method 1 (Liquid Nitrogen) (Figure 1 (a)) is greater than the Method 2 (freeze drying) (Figure 1 (b)) with the detail in Table 1. However, when the antioxidant effect test for qualitative comparison is conducted with the extract from both sources, it was found that the placenta protein extract from Method 2 had an antioxidant effect at 50.37% while the placenta protein extract from Method 1 did not detect the said effect. Therefore, the porcine placenta protein extract obtained from the freeze drying process is a suitable method for extracting the protein for further development as an encapsulating system of porcine placenta protein extract in the form of enteric microbead.

Table 1 Comparison of extracts and protein content obtained from Method 1 (Liquid Nitrogen) and Method 2 (Freeze Drying) Sample of Porcine Placenta Method 1 Method 2

Fresh Weight of Placenta (Gram) 399.30 447.87

Dry Weight (Post-freeze drying) (Gram) - 15.55

Post-Pasteurization Characteristic Dark Brown-Red Dark Red

Final Volume (Milliliter) 550 250 protein concentration (Milligram/Milliliter) 3.28 3.09

Protein Content in Percent 0.45 0.17

Antioxidant effect N/D* 50.37

*N/D; Oxidation inhibition value cannot be detected since the inhibition result was lower than the analyzable value.

Example 2 Development of Porcine Placenta Protein Extract Entrapment System by Making Enteric Microbead

The chemicals used for encapsulating are as follows: Sodium alginate, Bentonite, Calcium chloride, and Water soluble chitosan. The particle preparation system will use the principle of Ionic gelation and the principle of reaction of the force of different charges, cations and anions, which mainly use Sodium alginate and Calcium chloride to create nanocarrier particles in the form of microbead that encapsulate the porcine placenta protein extracts.

From forming the porcine placenta protein extract into carrier particles in the form of microbead or Alginate capsules, which is a transport technique of active compounds via medicine or food, helping to preserve flavor and value in the food, including controlling the release of active compounds by using Spraying Method for encapsulating the porcine placenta protein extract containing Sodium alginate as composition and spraying in Calcium chloride solution. The said principle is a mechanism of ion exchange between sodium in the alginate molecule and calcium ion dissolved in the solution, causing alginate bond formation and encapsulating of active compounds inside the beads. As the developed Formulations in Table 2, Bentonite was added to act as a binder to improve the protein encapsulating in the beads, including the use of Water soluble chitosan as a composition which also has the enteric polymer property. So as to allow particles to further have the anti-oxidant property, the bead formation formulation containing Vitamin C as a composition in combination with porcine placenta extracts is designed, then analyzed to compare the efficiency of all three particle formulations to determine the possibility of utilization in encapsulating of porcine placenta extracts and anti-oxidant. Preparation Method

Increasing the concentration of the porcine placenta protein extract by using the porcine placenta protein extracts of 100-milliliter volume, taking into Freeze Dryer for water sublimation for 1-2 days, adding water of 50-milliliter volume in the dried protein extract, then dissolving to be homogeneous, and conducting the quantitative analysis of total protein (by BCA™ protein assay kit method) by calculating from an equation obtained from standard solution graph between absorbance value at 562 nanometers, measured by Microplate Reader.

Encapsulating the concentrated porcine placenta protein extract in the form of enteric microbead by mixing the concentrated porcine placenta protein extract with sodium alginate according to the defined ratio in Table 2 using glass rod to stir the substance to be mixed until both types of solutions are homogeneous, then pouring the prepared solutions in Air Blow Gun with the nozzle size of 1.5 millimeters, then spraying into a container containing 0.5-3%wt by volume calcium chloride solution which immersed in an ice bath to form as microbead particles in which the concentrated porcine placenta protein extracts are encapsulated, and immersing in calcium chloride solution for 30-40 minutes for a complete bond formation.

Centrifuging the solutions having microbead particles by Centrifuge at a speed of 5,000 revolutions per minute, a temperature of 4°C for 15-20 minutes, then washing with distilled water 2 times, then collecting a portion of the obtained microbead particles to be mixed with 0.05- 0.3%w/w Talcum, then drying by Freeze Dryer for 1-3 days to obtain nanocarrier in the form of enteric microbead required.

Table 2 Formulation of microbead particles that encapsulate the Fl-3 porcine placenta protein extract

Formulation S

od um g nate o r c n e

Calcium Water soluble + P l a c e n t a chloride chitosan

B t it P t i

From the experimental result in preparing microbead particles that encapsulated the Formulation Fl, F2, and F3 porcine placenta protein extract after drying with freeze drying in Table 3, it was found that all three formulations had the light brown loose powder characteristic. Formulation Fl was a darker brown, and the obtained matter content was less than the formulation containing Water soluble chitosan as a component. When dissolving the obtained microbead particles to conduct the quantitative analysis of protein content encapsulated with 0.1 molars, pH 7.4 sodium citrate solution, it was found that Formulation Fl- F3 had the protein concentration of 2,156.3, 57.5, and 72.1 micrograms per milliliter, respectively, which can be estimated in total percentage of protein in microbead powder equaling to 6.38, 0.17, and 0.21 percent, respectively, and when estimating in percentage of protein encapsulating efficiency, the values equaling to 99.49, 99.72, and 99.60%, respectively, were obtained. As results, it was found that Formulation Fl had a maximum concentration of protein content.

Table 3 Result summary of microbead particles that encapsulate Formulation 1-3 porcine placenta protein extract

Formulation Fl F2 F3

Protein Concentration

2,156.3 57.5 72.1

(mi crogram/ mil lil iter)

%Protein in Microbead Powder 6.38 0.17 0.21

%Protein Encapsulating Efficiency* 99.49 99.72 99.60

(Initial Protein Extract Content- Protein Content in Fluid)

* Analy

^J sis ot %Entrapment efficiency J = ⁱ _: - - - - x 100% Initial Protein Extract Content

Example 3 Physical Property Analysis of Microbead particles that Entrap the Porcine Placenta Protein Extracts

3.1 Study of Physical Characteristics of Microbead particles that Entrap the porcine Placenta Protein Extract

In the present invention, the microbead nanoparticles of the Formulation Fl -3 were analyzed on the particle morphology and surface by Environment Scanning Electron Microscope (E-SEM) by adjusting to the lOOx magnification, and having a scale bar size of 200 micrometers. The samples were prepared by evenly diffusing dry powder of microbead nanoparticles on Carbon tape, then glazing the samples with gold (Au) to make the samples conductive at a high vacuum condition and reduce damage from the electron beam heat hitting the samples. From the morphology analysis of the synthesized microbead particles by E-SEM, it was found that the microbead particles of Formulation Fl -3 had similar characteristics for all three formulations. The shape had a roundish feature, and the particle size was in the range of 1 -60 micrometers (Figure 2).

3.2 Particle Size Evaluation

The size of the microbead particles of the Formulation Fl -3 is measured with a Mastersizer relying on the principle of laser diffraction from a laser source by preparing the dry powder samples.

From the analysis of the size of microbead particles measured after passing drying with Spray Dryer, it was found that the size of particles obtained from Formulation Fl, F2, and F3 had similar average sizes which are approximately 14.53±0.11, 14.65±0.08, and 13.32±0.08 micrometer, respectively (Figure 3).

Example 4 Evaluation of Biological Properties of the Nanocarrier in the Form of Microbead that Encapsulates the porcine Placenta Protein Extract

4.1 Anti-Oxidation Agent Effect Test

The antioxidant effect of porcine placenta protein extract is analyzed and tested by comparing both pre- and post-encapsulating in microbead particles with the superoxide anion scavenging assay method, where the test is conducted in comparison with the standard substance, which is Quercetin. The test is performed by the following methods: mixing Nitroblue Tetrazolium (NBT) solution at a concentration of 200-300 micromolar with Nicotinamide adenine dinucleotide (NADH) solution at a concentration of 600-650 micromolar in a ratio of 1 : 1 by volume, where both solutions will be prepared in Sodium Phosphate solution at a concentration of 100-150 micromolar, pH 7.4, and newly prepared every time before testing; and then adding liquid medicine obtained above to be combined with standard substance or sample substance at different concentrations in a ratio of 2: 1 by volume before mixing with Phenazine methosulfate (PMS) solution at a concentration of 50-100 micromolar in the same ratio as sample substance (preparing PMS solution in Sodium Sulfate at a concentration of 100-200 micromolar, pH 7.4); and then reading the obtained results within 3-5 minutes after adding PMS solution to cause a reaction to change into magenta pink, but in the case that the tested substance has anti-oxidant effect occurred in the reaction, its color will be lighter, checking absorbance value at 560 nanometer.

From the result of the action test with the superoxide anion scavenging method in porcine placenta extracts and microbead particles compared with Quercetin, a substance in the flavonoid group often found in vegetation and fruits, such as grape, ginkgo, or Berries, having a good antioxidation property, as shown in Figure 4 and Table 4, it was found that, in the equal concentration level, Formulation F3 had the best anti-oxidation action property followed by Formulation F2, porcine placenta extract, and Formulation Fl, respectively; while Formulation F3 and F2 showing the good anti -oxidation result might be caused by Water soluble chitosan, which is a composition in the particle formulation since there has been academic information stating that Water soluble chitosan can contribute to boosting the anti-oxidants (Ngo and Kim, 2014). When the concentration value that caused anti-oxidation inhibition at 50% (IC50) was statistically tested by comparing the values of the porcine placenta protein extracts and Formulation F2 and F3, it was found that both formulations had the action with a significant difference at ***P-value<0.001.

Due to the test required to first dissolve microbead particles, then the test was further conducted in the determined method. However, Formulation Fl contains the alginate ingredient in the highest amount; thus, when beads are dissolved, the solution is viscous. As the dissolved particle quantity is high resulting in a higher viscosity of the solution, it will cause an obstacle to increase the concentration in the test. The substance’s viscosity may also affect the free radical reaction that occurred in the determined method, causing an inability to determine the concentration that can cause the anti -oxidation inhibition at 50%. None of the said problems occurred while dissolving F2 and F3. However, the experimental result indicated the tendency of anti -oxidation action capability when increasing the concentration of tested substances in all formulations, indicating that the encapsulating of active compounds in microbead particles has maintained the anti-oxidation action property, not affecting the disappearance of those properties.

Table 4 shows the concentration value causing anti-oxidation inhibition at 50% obtained from the anti -oxidation action test, where the statistical results are compared in the difference of concentration values causing anti -oxidation inhibition at 50% of the porcine placenta extract (PP extract) and Formulation F2 and F3, respectively (***P-value<0.001). Concentration value causing anti-oxidation inhibition at 50% (ICso) (microgram/milliliter)

Quercetin 81.45

Porcine Placenta

424.8

Extract

Fl N/A

F2 202.7***

F3 175.5***

5.2 Anti-inflammatory Action Test

The anti-inflammatory agent effect test was analyzed by taking porcine placenta extracts of microbead particles for all three formulations that are Fl, F2 and F3 to conduct the antiinflammatory agent effect test, where the test started from culturing Macrophage of RAW mice 264.7 in 48- well plate for 100,000 cells in culture medium containing 10% of fetal bovine serum ingredient for 24 hours, then aspirating culture medium and adding new medium not containing fetal bovine serum with porcine placenta extract or particles containing porcine placenta of all three formulations that are F1-F3 in 250 micrograms per milliliter of protein content, including medicine used as positive control unit of inflammatory inhibition, which is 50 micromolar of Dexamethasone, further incubating cells for 2 hours in cell culture incubator, treating with Lipopolysaccharides (LPS) at a concentration of 2 micrograms per milliliter when completing 2 hours, and then incubating in Cell Culture Incubator for 24 hours, then collecting culture medium to be centrifuged with Centrifuge at a speed of 2,000 revolutions per minute, 4°C for 5 minutes, and then particularly collecting supernatant to conduct cytokine TNF-a, IL-6, and IL-8 secretion inhibition test to determine the capability of anti-inflammatory effects with ELISA (enzyme-linked immunosorbent assay) Technique according to the test method specified in the test kits, then analyzing the obtained absorbance values by comparing the cells cultured with LPS to be 100%.

From the test result, nitric oxide was a chemical released when cells or organs were injured or inflammatory. Therefore, it was used to test anti-inflammatory action. As the test result of the anti-inflammatory effect of microbead particles of all three formulations in comparison with porcine placenta extracts in the suitable concentration content of dissolved protein of beads equaling to 250 micrograms per milliliter as Table 5, it was found that Formulation F2 and F3 had similar nitric oxide inhibition values equaling to 13.5% and 13.1%, respectively, which is higher than porcine placenta extract (2. 1 %) and have a statistically significant difference. However, at the said concentration levels, it was observable that Formulation Fl was clotted during the reaction, which may be caused by alginate, which is a composition in the largest amount when compared with other formulations, resulting in obstacles to the test and the inability to analyze the results like the antioxidant effect test.

Table 5 shows the result of inhibiting nitric oxide of porcine placenta extract and microbead particles at protein concentration equaling to 250 micrograms per milliliter, where the statistical result was compared in the difference of placenta extract action with Formulation F2 and F3 , respectively (***P-value<0.001)

% Nitric oxide inhibition (at protein concentration equaling to 250 micrograms per milliliter)

Quercetin 60.0

Porcine Placenta Extract 2.1

Fl N/A

F2 13.5***

F3 13.1***

In addition, Fl, F2, and F3 microbead particles were also tested for anti-inflammatory effects at the cell level with the inhibition result analysis in the level of 3 types of cytokine, i.e., TNF-a, IL-6, and IL-8, where lipopolysaccharide is control unit of the system, NC is a non-treated cell, BLK is 0.1 of molar sodium citrate, and Dexamethasone is a positive control unit. The experimental results are shown in Figure 5, where the percentage of cytokine inhibition of all three types can be concluded when compared with the controller as follows.

The porcine placenta extract could inhibit the secretion of cytokine IL-6 at 47.3 ± 1.63%, while the particles containing Formulation Fl, F2, and F3 placenta extracts could inhibit IL-6 secretion at 55.1± 2.39, 44.3 ± 0.02 and 47.3 ± 0.49 %, respectively.

The porcine placenta extracts could inhibit the secretion of cytokine TNF-a at 42.7 ± 1 .8 %, while the particles containing Formulation Fl, F2, and F3 placenta extract could inhibit TNF- a secretion 70.6 ± 1.6, 37.6 ± 2.1 and 36.9 ± 3.2%, respectively. The porcine placenta extract could inhibit secretion of cytokine IL-8 for 35 .7 ± 2.0 % , while the particles containing Formulation Fl, F2, and F3 placenta extract could inhibit IL-8 secretion at 48.1 ± 2.4, 40.1± 12.1, and 33.2 ± 22.2%, respectively.

From the said test results, it could be concluded that microbead particles and porcine placenta extracts had an anti-inflammatory effect through the mechanism of nitric oxide inhibition and cytokine inhibition relating to inflammation for all of TNF-a, IL-6 , and IL-8 , where the encapsulating could maintain this property.

Example 5 Study of Protein Release Profile from Microbead Particles that Encapsulate the Porcine Placenta Protein Extracts

The study is conducted in the simulated alimentary tract in part of the stomach and intestine with simulated gastric fluid (SGF) and simulated intestinal fluid (SIF), respectively. The release rate of protein encapsulated in the particles with a changing acidity-alkalinity according to the alimentary system in the following defined period.

The 2^st-3^rd Hour in the simulated stomach

The 8^th Hour in the simulated small intestine

The 14^th Hour in the simulated cecum and colon

The condition for testing dissolution of these developed nanocarrier determines pH equaling to pH 2-5 and 6. 5-7 with the release control goal, that is, the extracts were not released or slowly released out of the particles in the first 2-3 hours in the stomach acidity; after that, they started releasing more extracts according to the higher pH condition of the intestine until they were entirely released within 24 hours. The test herein will be conducted with a dry sample substance in the amount of 20 micrograms, where the samples are added in a buffer that simulates the condition in the alimentary tract. The composition of the buffer are as follows.

Release Test in Stomach

- Simulated gastric fluid (SGF): 0.2-0.3% of sodium chloride (NaCl) + 0.01-0.1 molars of hydrochloric (HC1) (adjusting pH with hydrochloric to be in the range of 1.5-2.0).

Release Test in Small Intestine

- Simulated intestinal fluid (SIF): 0.5-1% potassium dihydrogen phosphate KH2PO4) + 0.1-0.2 normality sodium hydroxide (NaOH) (adjusting pH with NaOH to approximately 6.0).

Release Test in Cecum and Colon - Simulated colonic fluid : 0.5-1% potassium dihydrogen phosphate (KH2PO4) + + 0.1 -0.2 normality sodium hydroxide (NaOH) (adjusting pH with NaOH to approximately 6.8).

The samples are collected at 0, 0.25, 0.5, 1 , 2 , 4, and 20 hours, and then analyzed to determine protein content by centrifuging samples at 5,000-7,000 revolutions per minute for 5-10 minutes before conducting the quantitative analysis of the obtained supernatant. The aspirated supernatant samples are then added with buffer according to the condition defined in different pH, and further incubated for a specified period.

The release test results of the protein of microbead particles were compared with the placenta extracts in simulated gastric fluid pH 1.5 at 0-2 hours, simulated intestinal fluid (SIF) pH 6.0 at 2-8 hours, and simulated colonic fluid pH 6.8 from the 8- 24 hours. It was found that Formulation 1 microbead particles released protein less than 20% in the first 2 hours and started increasing when the pH of the buffer was changed in the following hour. While the release of Formulation F2 and F3, where 2 hours elapsed, are at 38.7 and 74.0, respectively, where those porcine placenta extracts were released until almost up to 100% within the first 15 minutes of the test (Figure 6).

When considering the consistency with the duration in the alimentary tract and the capability to protect active compounds of particle formulations, it was found that in the first 2 hours period where the particles were in simulated gastric fluid, Formulation Fl could best reduce the release of the encapsulated protein followed by Formulation F2 and F3, respectively. However, it might be due to the Formulation Fl containing the alginate ingredient which had the property of poor decomposition in acidity, but it would gradually decompose and release active compounds in the condition near neutral pH. Therefore, Formulation Fl with the most alginate in the matter could well slow down the release of active compounds, where the Formulation F2 and F3 increasingly contain the composition of water-soluble chitosan in the matter, respectively. Even though water- soluble chitosan has the enteric polymer property, but this property is not as good as alginate, therefore, resulting in a reduction of enteric property.

Example 6 Protein Stability Test after Encapsulating in Microbead particles

To verify the hypothesis that after the protein was encapsulated in the particles, the encapsulating system can help to protect protein stability better than the group of protein that is not encapsulated in the particles. The samples are preserved at three temperatures, i.e., 4°C when time is elapsed for 14, 30, and 60 days, and at room temperature (25°C) when time is elapsed for 7, 14, 30, and 60 days, including stability test of the particles at a temperature of 45°C when time is elapsed for 0, 3, 5, 15 and 30 minutes. The following results can be analyzed by comparing the total protein content of all three temperatures.

According to the protein stability test from the porcine placenta after encapsulating in nanocarrier in the form of enteric microbead by preserving at three temperatures, i.e., 4, 25, and 45°C and analyzing to determine protein content after preserving, it was found that, at temperatures when the time was elapsed for 60 days and at a temperature of 45°C when the time was elapsed for 30 minutes, the protein content encapsulated in particles in the form of microbead could be analyzed and determined. This shows that by applying the encapsulating technology to the active biological compounds, it could help to protect the protein to be more stabilized, not being destroyed in the environments, when compared with the group of protein that is not encapsulated in particles.

Best Mode of the Invention

As aforementioned in the detailed description of the invention.

Claims

Claims A process of extraction of porcine placenta protein and development of encapsulating and transporting system of porcine placenta protein extract to control an enteric release, the process comprising: a. preparation of a porcine placenta sample clean a porcine placenta to remove blood clots or waste attached therewith, then reduce the size of the porcine placenta, and dry it, b. extraction of protein from the porcine placenta finely grind the placenta prepared from step a, and then immerse in homogenizing buffer comprising DMEM high glucose, sucrose at a concentration of 0.1-1 Molar (M), and EDTA at a concentration of 1-2 millimolar (mM), where the piece of placenta is suspended in the homogenizing buffer for 20-40 minutes, disrupt cells using sound waves, then centrifuge to separate cell pellet, collect only supernatant, pass-through pasteurization, and then filtrate it, c. development of the system for encapsulating the porcine placenta protein extract to control the enteric release, increase the concentration of the porcine placenta protein extract derived from step b by freeze drying for 1-2 days, then add water into the dried protein extract, and homogeneously dissolve it, next, encapsulate the concentrated porcine placenta protein extract in the form of an enteric microbead by mixing the concentrated porcine placenta protein extract with the chemical used in encapsulating, the ratio of the porcine placenta protein extract to the chemical used in encapsulating is 10-20 to 70-80 %wt, then stir until both solutions are homogeneous, and spray into a container containing the solutions to form enteric microbead particles having the concentrated porcine placenta protein extract encapsulated therein, centrifuge the solutions having the enteric microbead particles, then wash with distilled water, collect the obtained enteric microbead particles to mix with 0.05-0.3%w/w of talcum and dry it to obtain nanocarrier in the form of enteric microbeads. The process of extraction of porcine placenta protein and development of encapsulating and transporting system of porcine placenta protein extract to control an enteric release according to claim 1 , wherein, in the preparation of porcine placenta sample, the cleaning is performed by using washing buffer comprising tap water, deionized water, and phosphate buffer saline (PBS) at pH 7.4. The process of extraction of porcine placenta protein and development of encapsulating and transporting system of porcine placenta protein extract to control an enteric release according to claim 2 , wherein, in the preparation of porcine placenta sample, the cleaning using washing buffer is performed by the following: washing with tap water, followed by deionized water and phosphate buffer saline (PBS) at pH 7.4, respectively. The process of extraction of porcine placenta protein and development of encapsulating and transporting system of porcine placenta protein extract to control an enteric release according to claim 1 , wherein, in the preparation of porcine placenta sample, the size reduction is performed using a blender. The process of extraction of porcine placenta protein and development of encapsulating and transporting system of porcine placenta protein extract to control an enteric release according to claim 1 , wherein, in the preparation of porcine placenta sample, the method of drying is selected from drying by using liquid nitrogen or by freeze drying. The process of extraction of porcine placenta protein and development of encapsulating and transporting system of porcine placenta protein extract to control an enteric release according to claim 5, wherein the freeze drying is performed by freeze dryer. The process of extraction of porcine placenta protein and development of encapsulating and transporting system of porcine placenta protein extract to control an enteric release according to claim 1 , wherein, in the extraction of protein from the porcine placenta, the placenta is suspended in a homogenizing buffer, which said homogenizing buffer is immersed in ice. The process of extraction of porcine placenta protein and development of encapsulating and transporting system of porcine placenta protein extract to control an enteric release according to claim 1 , wherein, in the extraction of protein from the porcine placenta, the cell disruption is performed by using sound waves from probe-type ultrasonicator with an amplitude of 10-20% for 10-30 minutes. The process of extraction of porcine placenta protein and development of encapsulating and transporting system of porcine placenta protein extract to control an enteric release according to claim 1 , wherein, in the extraction of protein from the porcine placenta, ice bathing is performed during disrupting the cell by using sound waves to prevent excessive cumulative heat. The process of extraction of porcine placenta protein and development of encapsulating and transporting system of porcine placenta protein extract to control an enteric release according to claim 1 , wherein, in the extraction of protein from the porcine placenta, the Centrifugation for separating cell pellet is performed at a speed of 8 ,000-12,000 revolutions per minutes (rpm) at a temperature of 4°C for 20-30 minutes. The process of extraction of porcine placenta protein and development of encapsulating and transporting system of porcine placenta protein extract to control an enteric release according to claim 1 , wherein, in the extraction of protein from the porcine placenta, the pasteurization is performed with heat at a temperature of 50-60°C for 20-30 minutes. The process of extraction of porcine placenta protein and development of encapsulating and transporting system of porcine placenta protein extract to control an enteric release according to claim 1 , wherein, in the extraction of protein from the porcine placenta, the filtration is performed by using a thin white cloth and vacuum filtration through a filter with a porous size of 10-20 micrometers, respectively. The process of extraction of porcine placenta protein and development of encapsulating and transporting system of porcine placenta protein extract to control an enteric release according to claim 1 , wherein, in the development of the system for encapsulating the porcine placenta protein extract to control the enteric release, the freeze drying is performed by freeze dryer. The process of extraction of porcine placenta protein and development of encapsulating and transporting system of porcine placenta protein extract to control an enteric release according to claim 1 , wherein, in the development of the system for encapsulating the porcine placenta protein extract to control the enteric release, the chemical used in encapsulating can be selected from any one of more of Sodium Alginate, Bentonite, Calcium chloride or water soluble chitosan in combination thereof. The process of extraction of porcine placenta protein and development of encapsulating and transporting system of porcine placenta protein extract to control an enteric release according to claim 14, wherein the chemical used in encapsulating is provided as a mixture of Sodium Alginate and Bentonite, and a mixture of Calcium Chloride and Water soluble chitosan. The process of extraction of porcine placenta protein and development of encapsulating and transporting system of porcine placenta protein extract to control an enteric release according to claim 15 , wherein the mixture of Calcium Chloride and water soluble chitosan having a ratio of Calcium Chloride to water soluble chitosan at 1 - 2 %wt to 0.5-2.0 %wt. The process of extraction of porcine placenta protein and development of encapsulating and transporting system of porcine placenta protein extract to control an enteric release according to claim 1 , wherein, in the development of the system for encapsulating the porcine placenta protein extract to control the enteric release, the spraying is performed into the container containing 0.5-3 %w/v of Calcium Chloride solution and immersed in the Calcium Chloride solution for 30-40 minutes. The process of extraction of porcine placenta protein and development of encapsulating and transporting system of porcine placenta protein extract to control an enteric release according to claim 1 , wherein, in the development of the system for encapsulating the porcine placenta protein extract to control the enteric release, the container containing the Calcium Chloride solution is immersed in an ice bath. The process of extraction of porcine placenta protein and development of encapsulating and transporting system of porcine placenta protein extract to control an enteric release according to claim 1 , wherein, in the development of the system for encapsulating the porcine placenta protein extract to control the enteric release, the centrifugation is performed at a speed of 5,000-7,000 rpm, the temperature of 4°C for 15-20 minutes. The process of extraction of porcine placenta protein and development of encapsulating and transporting system of porcine placenta protein extract to control an enteric release according to claim 1 , wherein, in the development of the system for encapsulating the porcine placenta protein extract to control the enteric release, the drying of nanocarrier in the form of enteric microbeads is performed by the freeze drying method. The process of extraction of porcine placenta protein and development of encapsulating and transporting system of porcine placenta protein extract to control an enteric release according to claim 20 , wherein the drying of nanocarrier in the form of enteric microbeads is performed by the freeze drying method for 1-3 days. The process of extraction of porcine placenta protein and development of encapsulating and transporting system of porcine placenta protein extract to control an enteric release according to claim 21, wherein the freeze drying is performed by freeze dryer. The process of extraction of porcine placenta protein and development of encapsulating and transporting system of porcine placenta protein extract to control an enteric release according to claim 1 , wherein, in the development of the system for encapsulating the porcine placenta protein extract to control the enteric release, further comprises an antioxidant. The process of extraction of porcine placenta protein and development of encapsulating and transporting system of porcine placenta protein extract to control an enteric release according to claim 16, wherein the antioxidant is Vitamin C. The enteric microbead particles encapsulating the porcine placenta protein extract derived from the process according to any one of claims 1-25. A use of enteric microbead particles encapsulating the porcine placenta protein extract according to claim 2 5 for manufacturing a product to boost growth, increase immunization, anti-oxidant and anti -inflammation for vertebrates.