WO2019193614A2

WO2019193614A2 - The process of achieving the highest degree of drug's purity of c-phycocyanin from spirulina alga

Info

Publication number: WO2019193614A2
Application number: PCT/IR2019/050010
Authority: WO
Inventors: Fahime HOSEINI; Saeide HOSEINI
Original assignee: Hoseini Fahime; Hoseini Saeide
Priority date: 2018-04-01
Filing date: 2019-03-31
Publication date: 2019-10-10
Also published as: WO2019193614A3

Abstract

Spirulina platnaseus, is a multi-striated cyanobacterium prokaryotic and photosynthetic. The extraction and purification of c-phycocyanin from spirulina due to a wide range of drug activity, which is mainly attributed to C-phycocyanin, has definitive therapeutic properties. It provides 20 times more antioxidant activity than ascorbic acid (Karkos.2011). Among these, Spirulina has been reported to exhibit anti-inflammatory (Remirez.2002), anti- hyperlipidemic (Torres-Duran.2007), hypoglycemic(Lima.1999), antihypertensive (Torres- Duran.2007), anti-neoplastic (Mittal.1999), antiviral(Lee.2001), antianemic (Simsek.2009). All of these benefits are attributed to substances in Spirulina known as phycobiliproteins (C- phycocyanin, allophycocyanin, phycoerythrin and phycoerythrocyanin), which called phycobilisome (Hoseini.2013). C-phycocyanin is the major phycobiliprotein in Spirulina. This blue colored red fluorescing biliprotein of algae was first reported in 1928 by Lemberg. The isolation and testing of these phyco billy proteins has shown that the beneficial effects of all microalgae depend on them.Hence, they are considered as a real and effective biological agent (Eriksen.2008;Hoseini.2013). Therefore, maintaining valuable properties and improving the benefits of active biological substances such as C-phycocyanin is essential. Because the nature of its protein structure, is sensitive to temperature, light, pH, etc makes it difficult to achieve purity, especially the purity of the drug. To divine grace, We have devised a simple, fast and innovative way for the separation and purification of this bioactive material from its natural resources. The benefits and merits of the new invention is that we were able to achieve the highest purity of the drug in the world, which has a significant difference with the previous achievements in the world (The new achievement is more than 7 times the purity of the drug in the world). It is important that we achieved this degree of purity and maximum yield by minimizing the number of extraction and purification steps.

Description

The process of achieving the highest degree of drug's purity of C-phycocyanin from spirulina alga

2) Technical field of the invention:

The extraction and purification of c-phycocyanin from spirulina (aerospira) due to a wide range of drug activity, which is generally attributed to the phycobiliprotein content of its, and is mainly attributed to C-phycocyanin, has definitive therapeutic properties, such as antioxidants. Therefore, maintaining valuable properties and improving the benefits of active biological substances such as C-phycocyanin is essential. Because the nature of its protein structure, is sensitive to temperature, light, pH, etc., makes it difficult to achieve purity, especially the purity of the drug. So far, several methods have been developed for the separation and purification of C-PC, but many are only able to produce phycocyanin of food grade and cosmetics, and only a few limited methods have been reported that can be of purity Phycocyanin medicine. Since the C-phycocyanin purity base in the world is 4 and above 4, it is obvious that any method that can increase purity from this level is preferable to other methods. Because increased purity of the drug means increasing the effectiveness of the drug on the one hand and reducing its dosage on the other, which are important indicator in the design of drugs. Therefor, for this purpose, to achieve the highest degree of purity of C- phycocyanin (more than 7 times the base drug purity) was done by us for the first time in the world. It is necessary to achieve the C-phycocyanin, two principal mechanisms, such as extracting the substance from the algae and purifying it, should be applied in the best method. To this end, we preferred to reduce the number of steps in these two mechanisms, Because the participation of more stages of extraction and purification is a major weakness for protein purity so that to touch of the protein increases its denaturation and reduces its efficiency. Therefore, in this method, we have reduced the number of purification steps to at least one stage. By the study of scientific literature in the last fifty years in the world, with the subject of extraction and purification of C-phycocyanin from spirulina algae for medicinal use, we found that the highest purity of C-phycocyanin in the world is 6.69, which is by which is by Mr. Patel et al Contributors have been present and registered. To divine grace, our in a unique method, we were able to achieve a degree of purity near to 7 equals the purity of the drug and 4 equals the highest purity of the drug obtained in the world, namely 28.8, That achievement to this degree of drug purity is unmatched.

3) Technical problems and express purpose:

Spirulina is considered a functional food because of its broad spectrum of biologic effects, which have been demonstrated in vitro and in vivo. On the other hand, the extraction and purification of c-phycocyanin from spirulina (aerospira) due to a wide range of drug activity, which is mainly attributed to C-phycocyanin, has definitive therapeutic properties. Therefore, maintaining valuable properties and improving the benefits of active biological substances such as C-phycocyanin is essential. Because the nature of its protein structure, is sensitive to temperature, light, pH, etc., makes it difficult to achieve purity, especially the purity of the drug. Although several methods have been developed for the separation and purification of these phycobiliproteins (Boussiba and Richmond. 1979; Herrera et al. 1989; Bermejo et al.l997; Zhang and Chen. 1999; Anamika et al. 2005; Minkova et al. 2007; NIU et al. 2007; Soni et al. 2008), they are either more tedious or time consuming. The major drawback of almost all these methods is the large number of steps involved, and it is known that higher the number of steps higher is the loss of product yield (Kula et al., 1982). Furthermore, the scale- up of these methods is very difficult and also expensive. On the other hand, attention should be paid to the point approved by the competent authorities (valid scientific articles), And that is, the amount of purity obtained can indicate the type of use of this protein. Purity 1 can be used in the food and cosmetic industry, while purity of 3.9 as a reactivity grade and greater than 4 as analytical grade can be used in pharmaceutical industry and research. So we can conclude that, first, the substance we are a protein, and because of the rapid denaturation of the protein, any method for purifying and extracting it can not be used. On the other hand, any degree of purity of C- phacocianine does not have the required efficacy for pharmaceutical use, but the resulting product should be maximally purified so that its medicinal effects can be effected in an effective and desirable manner. 4) Description of previous knowledge and previous history;

Spirulina is a blue-green microalgae strand, with a spring-like, photosynthetic, and very small 2-8 micron filament that belongs to the family of cyanobacteria and is commercially produced around the world (Belay.2002&2004). After the spirolina algae was successfully used by the space agency as a food supplement for astronauts in space travel, Known and has gained significant popularity in the food industry as a supplement to vitamin and protein (Karkos.2008). They collect this micro-algae with a special centrifuge from the water. Spirulina contains a lot of protein, essential fatty acids, essential amino acids (Belay. 1993). Spirulina as a food supplement is due to its high protein content (about 70% of its dry weight) and high biological value (containing essential amino acids like phenylalanine and methionine). Spirulina also contains vitamins: B12 (cyanocobalamin), B6 (pyridoxine), Bl (thiamine) and B2 (riboflavin), as well as beta carotenes (precursors of vitamin A); minerals (e.g., iron, zinc, selenium, calcium and magnesium), phytochemicals (phenolic acids and tocopherols), and essential fatty acids (such as gamma linoleic acid) (Belay. 2002; Dillon et al. l995; Habib et al. 2008).

Spirulina is considered a functional food because of its broad spectrum of biologic effects, which have been demonstrated in vitro and in vivo. Among these, Spirulina has been reported to exhibit anti-inflammatory (Remirez, Ledon, & Gonzalez. 2002), anti-hyperlipidemic (Torres- Duran et al. 2007), hypoglycemic (Lima, Facchinetti, & Santos. 1999), antihypertensive (Torres- Duran et al. 2007), antineoplastic (Mittal, Suresh Kumar, Banerjee, Rao, & Kumar. 1999), antiviral (Lee et al. 2001), anti anemic (Simsek et al. 2009), and antioxidant activity (Karkos et al. 2011). All of these benefits are attributed to substances in Spirulina known as phycobiliproteins (C-phycocyanin, allophycocyanin, phycoerythrin and phycoerythrocyanin), which called phycobilisome (Gantt et al. 1979; Hoseini, Khosravi- Darani, & Mozafari. 2013). Isolation and testing of these phycobiliproteins has shown that they possess same beneficial effects as the whole microalgae. Hence, they are considered as the actual bioactive agents (Eriksen. 2008; Hoseini et al. 2013; Khan et al. 2006).

Obtention of C-phycocyanin: brief historical perspective

Different methods of extraction, isolation and purification (summarized in Figure 1) have been assayed in order to obtain these phycobiliproteins (especially C-phycocyanin) from Spirulina (Glazer et al. 1983; Khan et al. 2006). The first attempts involved simple chromatography by using precipitations previously obtained with ammonium sulphate. Subsequently, phycobiliproteins were isolated by crystallization (Ocarra. 1965). However, these methods lacked specificity as they extracted the whole phycobilisome without separating each pigment.

Years later, a thermal shock-based separation technique allowed pigment separation via density- gradient centrifugation with sucrose (Bekasova et al. 1984). Although these processes represented an advance, the overall purity and yield of the process was still low.

With the use of reversed phase high-performance liquid chromatography (RP-HPLC), it was possible to achieve an isolation of up to 85% of C-phycocyanin and allophycocyanin. (Swanson & Glazer. 1990) Nevertheless, other compounds apart from the phycobilisome were present in the final products due to the sample pre-treatments needed to perform HPLC. In order to optimize phycobiliprotein isolation, the chromatographic method was modified by adapting resins used in the solid phase, varying the polarity and pH of the eluent solution (Moreno et al. 1997), and using magnesium chloride precipitation with further diffusion in polyethylene glycol gel. By enhancing overall specificity, the isolation of pure C-phycocyanin and allophycocyanin was obtained, but the yielding mass was significantly decreased.

Towards the end of the 20th century, electrophoresis-based techniques were tested with the addition of laser-induced fluorescence (LIF) detectors. This novel achieved separation with a fairly good yield (~90 - 100%). Nevertheless, such extractions corresponded to a mixture of both C-phycocyanin and allophycocyanin (Viskari & Colyer. 2002). Yet another type of electrophoresis was used with polyacrylamide/dodecyl sulphate gel, pre-treating the samples by precipitation with ammonium sulphate followed by further separation in chromatographic columns by Sephadex (Minkova et al., 2003). This technique achieved an isolation of pure C- phycocyanin with a yield of ~ 45%.

Afterwards, different combinations of methods were tested in order to increase the ease of separation and isolation as well as the grade of purity and final yield. One such method, HPLC coupled with a flame ionization detector (Zolla & Bianchetti. 2001), was able to separate C-phycocyanin from allophycocyanin, yet it destroyed the original sample. In order to resolve this problem, an integral procedure was devised that extracted the phycobiliproteins with sodium phosphate neutral buffer, and then further purified them via dialysis and gel filtration chromatography (Bhaskar, Gopalaswamy, & Raghu. 2005). This procedure yielded C- phycocyanin with a purity of 4.98. Another method, pre treated the sample in the same manner, but the purification process consisted of ion-exchange chromatography (Patel, Mishra, Pawar, & Ghosh. 2005), yielding C-phycocyanin with a purity of 4.42.

Another widely studied method for isolating C-phycocyanin from Spirulina platensis combined chromatography with expanded bed adsorption, anion interchange, and hydroxyapatite columns (Niu, Wang, Lin, & Zhou. 2007). These techniques yielded 4.45 mg of C-phycocyanin per gram of dried S. platensis with a purity of 3.2. This method offers several advantages, such as the possibility of using different resins with special charge characteristics (i.e., anionic or cationic). For instance, by using Q-Sepharose (Silveira, de Menezes Quines, Burkert, & Kalil, 2008) it was possible to isolate C-phycocyanin from Spirulina platensis with -75% yield and a purity of 3.4. The problem with these kinds of techniques is that they are often strongly dependent on the pH and temperature of the eluent solutions.

On the other hand, hydrophobic interaction chromatography with ammonium sulphate and liquid nitrogen precipitation pretreatments (Soni, Trivedi, & Madamwar. 2008) are capable of isolating C-phycocyanin with a purity of 4.5 but with poor yields. This may be due to the original cyanobacterium (Phormidium fragile) from which the phycobilin was isolated. To improve the yield, C-phycocyanin was extracted from Spirulina platensis with high-speed counter current chromatography (HSCCC) (Yin et al. 2011), obtaining 78.7 mg per 200mg of crude extract with a purity of 4.25.

By the study of scientific literature in the last fifty years in the world, with the subject of extraction and purification of C-phycocyanin from spirulina algae for medicinal use, we found that the highest purity of C-phycocyanin in the world is 6.69, which is by which is by Mr. Patel et al Contributors have been present and registered, which involves pre treating vegetable samples of Spirulina platensis with two aqueous phases(Patil, Chethana, Sridevi, & Raghavarao. 2006), leading to a purity of 6.69.

By the grace of God, we devised an innovative, simple, fast and efficient method for the separation and purification of this bioactive material from its natural resources our in a unique method, we were able to achieve a degree of purity near to 7 equals the purity of the drug (Namely 4) and 4 equals the highest purity of the drug obtained in the world, namely 28.8, That achievement to this degree of drug purity is unmatched. Advantages and benefits of the new invention as compared to previous inventions. Firstly, this is a purity of drugs for the first time, which is carried out in Dear and Islamic Iran, and secondarily, In the world, it is the highest purity of the drug for this substance, which has a significant difference with the previous achievements in the world. This is achieved by minimizing the number of extraction and purification steps so that the maximum purity and yield of the product can be achieved.

5) Provide a solution along with a detailed description of the invention;

C-Phycocyanin (C-PC), the major light harvesting biliprotein from Spirulina platensis is of greater importance because of its various biological and pharmacological properties. Since the C-phycocyanin purity base in the world is 4 and above 4, it is obvious that any method that can increase purity from this level is preferable to other methods. So it was necessary to achieve the C-phycocyanin, two principal mechanisms, such as A- extracting the substance from the algae and B- purifying it, should be applied in the best method.

A. Extraction and preparation of raw extract of phycocyanin;

For this purpose, among the methods for extracting and breaking the cell wall, such as extraction with organic solutions and buffers, enzyme digestion with lysozym, ultrasonication,homogenizer, osmotic shock, and ... all of them were done by the test and error method. The best method that minimize damage to the protein was selected, which was the use of the freeze-thaw and homogenizer mechanism for 5 minutes.

freshly harvested Spirulina biomass was washed with distilled water for about 2-3 times to remove the culture media components. After that, in order to break the cell wall, samples were placed in a freezer at -28 °C. then They were three times freeze-thaw. At first, the specimens were placed in a freezer to allow for complete freezing. Then, at laboratory's temperature, the freezing was removed. This action was repeated three times. After this The biomass was homogenized for about 5 min to break the cells (Homogenizer with mark MiCCRA) and centrifuged at 11,000 rpm for about 20 min and temperature 4°C to separate the phycocyanin from the cell debris. The raw extract obtaineds of C-PC was stored at 4-5 °C for further use. B. Phycocyanin purification;

At this stage, we considering the minimum damage to phacocianine and its medicinal properties, after extensive research and performing several methods and methods including ; aqueous two- phase systems (ATPS), organic solutions and buffers, enzyme digestion with lysozom, activated carbon and chitosan That we did But each of them for some reason Including; 1 - ammonium sulfate precipitation generally takes 1-2 days, and easily denatures the proteins so that loss of the target protein is increased. 2- aqueous two-phase extraction facilitates separation of the phycocyanin, but the poly(ethylene glycol) used can stably bind to phycocyanin, so their complete separation is not easy. 3- In addition, some operations may cause structural and functional changes of proteins. For example, after ultrasonication (ultrasonic waves) the surface charge increased, the amount of free sulfhydryl groups decreased, the particle size increased up to 3.4 times, and the surface hydrophobicity increased. Therefore, at this stage, after obtaining the results mentioned, We decided that for purification of phycocyanin, to use of the chitosan with chemical formula (C6H11N04) Which is obtained from the deacetylation of chitin. Because it is used in clinical products because of the compatibility of biology with other materials, easy digestibility, non toxicity, high absorption capacity and availability as a carrier. We then Chitosan solution (2%, w/w) was added to the crude extract of phycocyanin and the sample was stirred for 5 min.In the next step, supernatant was collected by centrifugation.

Ion-exchange chromatography;

Ion-exchange chromatography was prepared using Anionic Resin (amberlite) in a column of 2.5 cm diameter and 50 cm length. The C-PC purified by chitosan, 5 ml was loaded on to the anionic resin column and elution with NaCl solution of linearly increasing ionic concentration from 0- 0.35M at 10 uiM potassium phosphate buffer at a flow rate of 1 mL min ¹. Samples based on NaCl concentration between 0.20 and 0.30M was collected.

Spectroscopic measurements;

The absorption spectra of phycocyanin were measured on a UV-vis Spectrophotometer (UV - 2550,SHIMADZU). By studying numerous papers, how to calculate the degree of purity is that we measured optical absorption of samples, or their OD in the wavelength range (200-800 nm). wherein A280 is the absorbance of total proteins and A620 is the maximum absorbance of C-PC. Then, after calculating the A620 / A280 ratio, you can see the degree of purity of the sample.

By measuring the optical absorption after each step, in addition to determining the purity of C- phycocyanin, the obtained numbers also confirm the accuracy of the process steps of the mechanism of action. Which are listed below.

Calculation of the purity of the first stage (crude extract); degree of purity = OD 620/ OD280= 3.072 / 0.703 = 4.37

Calculation of the purity of the second stage (purification); degree of purity = OD 620/ OD280= 4.119 / 0.316 = 13

Calculation of the purity of the third stage (chromatography); degree of purity = OD 620/ OD280= 2.595 / 0.09 = 28.8

On the other hand, attention should be paid to the point approved by the competent authorities (valid scientific articles), And that is, the amount of purity obtained can indicate the type of use of this protein. Purity 1 can be used in the food and cosmetic industry, while purity of 3.9 as a reactivity grade and greater than 4 as analytical grade can be used in pharmaceutical industry and research. It is clear that, the greater the purity, the greater its effectiveness on the one hand, and on the other hand, reducing its drug dose. Now in this method, as described above, we were able to achieve a degree of purity with two simple and very efficient steps. The highest purity of 6.69 which has been achieved in recent 50-year studies in the world is far superior and is unique in its kind. It is noteworthy that only by performing the first two steps we were able to achieve purity of 13 (more than three times the purity of the drug). We do not need to do the third step, ion exchange chromatography. But brilliantly, by doing the third step, which is the same phase of ion exchange, we were able to achieve purity of 28/8 (more than 7 times the purity of the drug), which is very unique in its kind. Because this method in large- scale can be economically and drug is unique and affordable.

It is from the grace of my god

Claims

Claims; 1 We claim that for the first time, we achieved the process of the highest degree of purity of C-phacocianine from Spirulina algae, namely 28.8, more than7times the purity of the drug(The global drug base is 4).

2-We also claim that this achievement, this is done with mechanical technique and free of chemicals and toxic materials and Maximum drug effect of the product with its lowest dose, and in this technique, important items such as: a)the minimum number of steps; b)the maximum efficiency of purification; c)the minimum amount of algae consumed, were considered.

3-We claim that in this process, we were able to achieve this unique purity(Namely 28.8)with a minimum amount of fresh algae, about5/7 grams, which is equivalent to 1 gram of powder dry.The minimum amount of algae consumed, that its cost-effectiveness, is an important factor in the pharmaceutical industry and commercialization in large volume and scale.

4-We claim that in this technique with reducing the time factor, this maximum purity was achieved within a period of less than one day.

5-We claim that we used two main mechanisms to achieve the C-phycocyanin,that is the phrase; A)Extraction technique of the substance from algae tissue. B purification technique.

6-According to claim5,we claim that in the Extraction technique of the substance from algae tissue, of the two mechanisms freeze-thaw and homogenizer used.

7-According to claim6,we claim that this extraction process is completely innovative and has no internal or external similar.

8-According to claim6,to break the cell wall, we first frozen the samples in a freezer at -28 c· then They were three times freeze-thaw. After that the biomass was homogenized for about 5min to break the cells and centrifuged at H000rp_m for about 20_mm and temperature 4^°c to separate the phycocyanin from the cell debris. The raw extract obtaineds of C-PC was stored at 4-5^°c for further use.By calculating the degree of purity of the extract obtained at this stage, we reached 4.37.

9- According to claim8,we claim that we only with done the stage extraction and even without entering the purification step, reached a purity higher than the purity grade of the drug. 10-According to claim5,we for Phycocyanin purification, Chitosan solution(2% w/w) was added to the crude extract of phycocyanin and the sample was stirred for 5_mjn.Then supernatant was collected by centrifugation. At this stage, by calculating the purity of the supernatant, we reached 13.

11-According to claimlO,we claim that in the first stage of purification, we reached a much higher purity than the global drug base.

12-According to claimlO,we claim that we have reduced the number of purification steps to at least one step.

13-According to claim8 and 10, we claim that the extract obtained due to the mechanical mechanism of action, does not contain any toxic and chemical waste So that the production of C-phycocynin is unique in this world of health and naturality, which is an important factor in the food-medicine industry.

14-According to claim5 and 10, we continued the purification process using ion exchange chromatography.

15-According to claiml4, chromatography column using anion resin with a diameter of 2.5_cm and length 50_cm was made.The C-PC purified by chitosan, 5_mi was loaded on to the anionic resin column and elution with Nacl solution of linearly increasing ionic concentration from 0- 0.35_M at lO_mM potassium phosphate buffer at a flow rate of 1 .Samples based on Nacl

concentration between 0.20 and 0.30_M was collected. By measuring optical absorption at this stage, we reached the highest purity in the world 28.8.

16-According to claim8, 10 and 15, we claim that by measuring the optical absorption after each step, in addition to determining the purity of C-phacocianine, the obtained numbers also confirm the accuracy of the process steps of the mechanism of action, so that this technique can be used as a new and unique method commercially, with high efficiency.

It is from the grace of my god