Photosynthetically controlled spirulina extract for treatment of cytokine storm syndrome

Abstract

The invention relates to a photosynthetically controlled spirulina extract for use in the treatment of cytokine storm syndrome. Spirulina extracts and / or fractionated compounds thereof, sublingual spray formulations thereof, spray devices for using the formulations, and methods of making a Spirulina extract and methods of treating TNF-alpha associated inflammation using a Spirulina extract are provided. Spirulina extracts are prepared by aqueous extraction of Arthrospira spp. Biomass accompanied by strong UV illumination and strong continuous mixed culture under controlled, ultra high density conditions, and are characterized by high levels of c-phycocyanin, sorbitol and adenosine derivatives-which was found to have a strong low dose effect of reducing TNF-alpha secretion. The spirulina extract can be correspondingly used for preventing or relieving cytokine storm related to various infections or autoimmune diseases.

Description

[0001] This application is a divisional application of an application filed on May 19, 2021, with application number 202180036384.0, and invention name “Photosynthetically controlled Spirulina extract for the treatment of cytokine storm syndrome”. Background of the Invention 1. Technical Field The present invention relates to the field of photosynthetically controlled Spirulina extracts, and more particularly to the use of Spirulina extracts in the treatment of cytokine storm syndrome.

[0003] 2. Discussion of related technologies Inflammatory diseases are common due to external infections or autoimmune diseases. Specifically, cytokine storm (CS) or hypercytokinemia triggered by macrophage activation syndrome (MAS) involves high levels of macrophage- and monocyte-induced tumor necrosis factor (TNF)-α and can lead to life-threatening conditions. SUMMARY OF THE INVENTION

[0004] The following is a brief summary to provide an initial understanding of the present invention. This summary does not necessarily identify key elements nor limit the scope of the present invention, but merely serves as an introduction to the following description.

[0005] One aspect of the present invention provides a Spirulina extract comprising an aqueous extract of Arthrospira spp. cultured under photosynthetically controlled conditions to produce upregulated bioactive compounds including c-phycocyanin, sorbitol, and adenosine derivatives, wherein the Spirulina extract has a concentration of less than 10 μg / ml and is effective as an anti-inflammatory agent.

[0006] One aspect of the present invention provides a sublingual spray formulation comprising a spirulina extract and / or fractionated compounds thereof at a concentration of less than 10 μg / ml, and a spray device configured to apply the sublingual spray formulation to a sublingual mucosa.

[0007] One aspect of the present invention provides a method for preparing a Spirulina extract, the method comprising: culturing Arthrospira species cyanobacteria, wherein the Arthrospira species cyanobacteria are cultured under photosynthetic control conditions to produce up-regulated bioactive compounds including c-phycocyanin, sorbitol, and adenosine derivatives, and preparing a water-based extract of the cultured Arthrospira species cyanobacteria to produce a Spirulina extract having a concentration of less than 10 μg / ml and being effective in treating TNF-α cytokine storm.

[0008] These, additional and / or other aspects and / or advantages of the invention are set forth in the detailed description which follows; can be inferred from the detailed description; and / or can be learned by practice of the invention.

[0009] This application provides the following: Item 1. A Spirulina extract comprising an aqueous extract of Arthrospira spp. cultured under photosynthetically controlled conditions to produce upregulated bioactive compounds including c-phycocyanin, sorbitol, and adenosine derivatives, wherein the Spirulina extract has a concentration of less than 10 μg / ml and is effective as an anti-inflammatory agent.

[0010] Item 2. The Spirulina extract according to Item 1, wherein the photosynthetic control conditions include 31±2°C, 10.8±0.2 pH and 700 μmol / (m 2 s) and 1500 μmol / (m 2 s).

[0011] Item 3. The Spirulina extract according to Item 1 or 2, wherein the photosynthetic control conditions include ultra-high density culture with a density between 3 g / l and 10 g / l and at 70 μmol / m 2 s - 150 μmol / m 2 Arthrospira species were cultured under UV radiation intensities between s.

[0012] Item 4. The Spirulina extract according to any one of items 1-3, wherein the water-based extract is produced by subjecting cultured Arthrospira species to water extraction and freeze-thaw cycles.

[0013] Item 5. A pharmaceutical composition comprising the fractionated compounds of the Spirulina extract according to any one of Items 1 to 4.

[0014] Item 6. Use of the Spirulina extract or a fractionated compound thereof according to any one of Items 1 to 4 for reducing the level of tumor necrosis factor (TNF)-α induced by macrophages and monocytes.

[0015] Item 7. The use according to Item 6, for treating virus-induced TNF-α cytokine storm.

[0016] Item 8. The use according to Item 6, for treating at least one of the following: acute respiratory distress syndrome (ARDS), heart failure, Crohn's disease, thrombophilia and gingivitis.

[0017] Item 9. A sublingual spray preparation comprising the Spirulina extract or the fractionated compounds thereof according to any one of Items 1 to 4 at a concentration of less than 10 μg / ml.

[0018] Item 10. The sublingual spray preparation according to Item 9, further comprising a pharmaceutically acceptable carrier.

[0019] Item 11. The sublingual spray preparation according to Item 10, wherein the sublingual spray preparation has a spirulina extract concentration of 0.4%-4.0% by volume.

[0020] Item 12. A method for treating TNF-α-related inflammation, the method comprising applying the sublingual spray formulation according to any one of Items 9 to 11 to the sublingual mucosa of a patient suffering from inflammation.

[0021] Item 13. The method according to Item 12, further comprising applying a spray in an amount that produces a blood concentration of 0.1 μg / ml-1 μg / ml in the patient.

[0022] Item 14. A spray device configured to administer the sublingual spray formulation according to any one of Items 9 to 11 to the sublingual mucosa of a patient suffering from inflammation.

[0023] Item 15. According to the spray device of Item 14, the spray device is also configured to be able to apply a certain amount of spray in one or a specified number of spray actions, and the amount produces a blood concentration of 0.1 μg / ml - 1 μg / ml in the patient.

[0024] Item 16. The spray device according to Item 15, wherein the sublingual spray formulation has a concentration of spirulina extract of 0.4%-4.0% by volume, and the spray device is also configured to deliver between 0.05 ml and 0.5 ml per spray action.

[0025] Item 17. A method for preparing a Spirulina extract, the method comprising: culturing an Arthrospira species cyanobacterium, the Arthrospira species cyanobacterium being cultured under photosynthetically controlled conditions to produce upregulated bioactive compounds including c-phycocyanin, sorbitol, and adenosine derivatives; A water-based extract of cultured Arthrospira sp. cyanobacteria was prepared to produce a Spirulina extract having a concentration of less than 10 μg / ml and effective in treating TNF-α cytokine storm.

[0026] Item 18. The method according to Item 17, wherein the Arthrospira species is cultured at 31±2°C, at a pH of 10.8±0.2 and at 700 μmol / (m 2 s) and 1500 μmol / (m 2 s).

[0027] Item 19. The method according to item 17 or 18, wherein the Arthrospira species is cultured in an ultra-high density culture having a density between 3 g / l and 10 g / l and at 70 μmol / m 2 s - 150 μmol / m 2 s under ultraviolet radiation intensity between s.

[0028] Item 20. The method according to any one of items 17-19, wherein the water-based extract is prepared by subjecting the cultured Arthrospira species to water extraction and freeze-thaw cycles.

[0029] Item 21. The method according to any one of items 17-20, further comprising using the Spirulina extract as an anti-inflammatory agent to reduce the level of TNF-α induced by macrophages and monocytes.

[0030] Item 22. The method according to Item 21, further comprising treating at least one of the following: acute respiratory distress syndrome (ARDS), heart failure, Crohn's disease, thrombophilia and gingivitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] For a better understanding of embodiments of the invention and to show how embodiments of the invention may be effected, reference will now be made, by way of example only, to the accompanying drawings in which like numerals refer to corresponding elements or parts throughout.

[0032] In the attached picture: Figure 1 is a high-level schematic diagram of cytokine storm (CS) and its treatment by the disclosed Spirulina extract according to some embodiments of the present invention.

[0033] Figure 2 Comparative metabolomic profiles are provided indicating compounds that are down-regulated and up-regulated in the disclosed Spirulina extracts according to some embodiments of the present invention compared to a "daylight" Spirulina extract grown under sunlight illumination.

[0034] Figure 3A-3C Data demonstrating the anti-inflammatory activity of Spirulina extracts as measured by TNF-α and IL-6 secretion from a murine macrophage cell line (RAW 264.7) are presented.

[0035] Figure 4A and Figure 4B Data are presented demonstrating the anti-inflammatory activity of Spirulina extracts as measured by TNF-α secretion from a human monocytic cell line (THP-1).

[0036] Figure 5A , Fig. 6A and Figure 6B is a high-level schematic diagram of a culture system according to some embodiments of the present invention.

[0037] Figure 5B is a high-level schematic flow chart illustrating methods of culture, extraction, and treatment according to some embodiments of the present invention. Detailed Description of the Invention

[0038] In the following description, various aspects of the present invention are described. For the purpose of explanation, specific configurations and details are set forth to provide a thorough understanding of the present invention. However, it will also be apparent to those skilled in the art that the present invention can be practiced without the specific details shown herein.

[0039] In addition, well-known features may be omitted or simplified so as not to obscure the present invention. Where specific reference is made to the drawings, it is emphasized that the details shown are by way of example and for the purpose of illustrative discussion of the present invention only, and are presented in order to provide what is considered to be the most useful and easily understood description of the principles and concepts of the present invention. In this regard, no attempt is made to show the structural details of the present invention in more detail, except for attempting to show what is necessary for a basic understanding of the present invention, and it is obvious to those skilled in the art how several forms of the present invention can be implemented in practice in conjunction with the description of the drawings.

[0040] Before explaining at least one embodiment of the present invention in detail, it should be understood that the present invention is not limited in its application to the details of the arrangement of construction and parts set forth in the following description or illustrated in the accompanying drawings. The present invention is applicable to other embodiments and combinations of disclosed embodiments that can be practiced or executed in various ways. In addition, it will be understood that the words and terms used herein are for descriptive purposes and should not be considered limiting.

[0041] Provided are a spirulina extract, a sublingual spray formulation thereof, a spray device for using the formulation, and a method for preparing the spirulina extract and a method for using the spirulina extract to treat TNF-α-related inflammation. The spirulina extract is prepared by water extraction of Arthrospira species biomass under controlled, ultra-high density conditions accompanied by strong UV illumination and strong continuous mixed culture, and is characterized by high levels of c-phycocyanin, sorbitol, and adenosine derivatives, which are found to have a strong low-dose effect of reducing TNF-α secretion. The spirulina extract can be used accordingly to prevent or alleviate cytokine storms associated with various infections or autoimmune diseases.

[0042] The disclosed Spirulina extracts and / or fractionated compounds thereof, for example in the form of sublingual spray formulations, can treat a range of inflammatory conditions, including cytokine storm (CS) or hypercytokinemia triggered by macrophage activation syndrome (MAS) caused by the novel coronavirus (SARS-CoV-2) or induced by other viral infections. With respect to CS, the disclosed Spirulina extracts, for example in the form of sublingual spray formulations, can prevent or alleviate CS, which is a major factor leading to critical COVID-19 incidence, including acute respiratory distress syndrome (ARDS). Therefore, the disclosed Spirulina extracts, for example in the form of sublingual spray formulations, can provide a treatment method that is independent of mutations in SARS-CoV-2 or other viral infections and can be used as a complementary measure to administering vaccines to prevent severe COVID-19 disease and reduce ICU admissions.

[0043] The disclosed Spirulina extracts may also be used to treat other inflammatory conditions, particularly conditions associated with elevated levels of macrophage- and monocyte-induced tumor necrosis factor (TNF)-α, such as those associated with heart failure, inflammatory bowel disease (IBD) (such as Crohn's disease), thrombophilia, gingivitis, and CS associated with autoimmune-related inflammatory diseases. Specifically, the disclosed Spirulina extracts, for example in the form of a sublingual spray formulation, can reduce macrophage- and monocyte-induced TNF-α levels in patients suffering therefrom.

[0044] Figure 1is a high-level schematic diagram of a cytokine storm (CS) and its treatment by the disclosed Spirulina extract according to some embodiments of the present invention. Pathogens (e.g., viruses such as SARS-CoV-2 or influenza virus, or various bacteria) or internal factors (e.g., autoimmune reactions) can interact with monocytes and / or macrophages, induce overproduction of TNF-α, and produce health-threatening CS. It is shown herein that the presence of the disclosed Spirulina extract prevents or alleviates CS. For example, starting with the known cell signaling cascades in macrophages, application of the disclosed Spirulina extract can inhibit TNF-α by macrophages to prevent CS or alleviate its symptoms.

[0045] Two outstanding features of the disclosed Spirulina extracts prepared from Arthrospira species (e.g., A. platensis) cultured under the disclosed photosynthetic control conditions are the specific anti-TNF effect and the limited and upper range of effective concentrations (up to 10 μg / ml). These features are unique and not present in other Spirulina extracts or cyanobacteria cultured by other methods (such as low-density cultures with uniform illumination and / or cultures using daylight illumination). Among other features, the disclosed water-based extracts have upregulated bioactive compounds including c-phycocyanin, sorbitol, and adenosine derivatives, which may be contributing factors to the use of the disclosed extracts as anti-inflammatory agents.

[0046] The following results specifically illustrate these outstanding features, using LPS (lipopolysaccharide) activated macrophages and monocytes, and comparing Spirulina extracts from Arthrospira species cultured under the disclosed photosynthetic control conditions with Spirulina extracts from Arthrospira species cultured under natural light conditions. In fact, it was found that a 0.1 μg / ml concentration of the disclosed photosynthetically controlled Arthrospira species water-based extract reduced the TNF-α levels induced by macrophages and monocytes by more than 70% and 40%, respectively, and Spirulina extracts at concentration levels above 10 μg / ml lacked this activity. Therefore, it is shown that treatment with the disclosed Spirulina extract can lead to a significant reduction in COVID-CS and ARDS, and generally provide effective anti-TNF therapy.

[0047] Non-limiting examples of photosynthetic control conditions for culturing Arthrospira species include a temperature of 31±2° C., a pH of 10.8±0.2, and 700 μmol / m 2 s - 1,500 μmol / m 2 s, subranges thereof or possibly even higher irradiances. Specifically, as disclosed below, it was found that in ultra-high density cultures with densities between 3 g / l and 10 g / l and at 70 μmol / m 2s-150 μmol / m 2 The disclosed Spirulina extract is produced by culturing Arthrospira species under ultraviolet radiation intensity between 100 and 200 s. The water-based Spirulina extract can be produced by subjecting cultured Arthrospira species to water extraction and freeze-thaw cycles.

[0048] The following experimental results were achieved as described in Tzachor et al. 2021 (incorporated herein in its entirety) - the disclosed extract was compared with a "daylight extract" according to conventional practice, which represents a Spirulina extract of cyanobacteria grown under sunlight illumination. The growth conditions of the disclosed extract and the daylight extract were similar, including the irradiation intensity (750 μmol / m 2 s), except for the spectral distribution of the irradiation, including the full range of the solar spectrum for the daylight extract and red, blue, and UV LED irradiation for the disclosed extracts. Cyanobacteria (UTEX3086) were cultured at 31 ± 2°C and a pH of 10.8 ± 0.2, and both types of extracts were water extracted using physical freeze-thaw (for cell disruption).

[0049] Figure 2 A comparative metabolomics profile is provided, showing compounds that are downregulated and upregulated in the disclosed Spirulina extract according to some embodiments of the present invention compared to the "daylight" Spirulina extract grown under sunlight illumination. The extract was prepared and the metabolomics profile was obtained as described in Tzachor et al. 2021. The compounds were determined by liquid chromatography tandem mass spectrometry (LC-MS-MS). For downregulated compounds, the compounds are represented by light gray dots on the left side of the figure, and for upregulated compounds, the compounds are represented by dark gray dots on the right side of the figure. Black dots represent compounds that are similarly expressed in either extract. The compounds above the dotted line represent statistically significant differences between the two types of extracts (P value <0.05 in the T test). Compared with the daylight Spirulina extract, seven compounds were found to be significantly upregulated in the disclosed Spirulina extract, while 23 compounds were found to be significantly downregulated. Two of the upregulated compounds are sorbitol and adenosine derivatives, with 97% and 91% MS / MS spectrum similarity, respectively, compared to known databases. These bioactive compounds (with known anti-inflammatory properties) were significantly increased by 1.7 and 4.8 times (P = 0.01 and 7.8·10 -10 In addition, the C-phycocyanin (CPC) bioactive compound was found to be significantly increased by 4.7 times in the disclosed Spirulina extract. (CPC levels were measured using standard spectrophotometric methods).

[0050] Note that while phycocyanin was found in both types of extracts and is known to reduce TNF-α secretion to some extent, the known effects were much smaller and at much higher doses than those disclosed herein - indicating a synergistic effect of the multiple up-regulated compounds in inhibiting TNF-α secretion and CS, which is highlighted in the presented results. Clearly, in order to maintain this composite synergistic effect, the disclosed Spirulina extracts must be produced from biomass cultured under consistent and strictly controlled conditions (e.g., light composition, irradiation levels, temperature, pH), and regardless of external conditions, such as disclosed herein.

[0051] Figure 3A-3C Data demonstrating the anti-inflammatory activity of Spirulina extracts as measured by TNF-α and IL-6 secretion from a mouse macrophage cell line (RAW 264.7) are presented. Figure 3A Results for the Sunlight Spirulina extract are provided, and Figure 3B , Figure 3C Results for the disclosed Spirulina extracts are provided. Figure 4A and Figure 4B Data are presented demonstrating the anti-inflammatory activity of Spirulina extracts as measured by TNF-α secretion from a human monocytic cell line (THP-1). Figure 4A Results for the Sunlight Spirulina extract are provided, and Figure 4B Results for the disclosed Spirulina extracts are provided.

[0052] In both cases, data were measured using ELISA (enzyme-linked immunosorbent assay) kits as described in Tzachor et al. 2021. LPS activation of the respective cell lines is indicated by the "+" sign in the upper row of each graph ("-" data represent controls of non-activated cell lines), and TNF-α and IL-6 are indicated by the respective percentages of inhibition (in each graph, the "-" sign indicates the lack of inhibition of non-activated cell lines and an activated control). Bars represent mean ± SD (standard deviation), and **** indicates statistical significance of p < 0.001 compared to untreated LPS-activated cell lines.

[0053] Will Figure 3B and Figure 3A In comparison, the disclosed Spirulina extracts exhibited a large and significant reduction in TNF-α secretion, reaching a 70% reduction for 0.1 μg / ml extract concentration and a 50% reduction for 1 μg / ml extract concentration (see Figure 3B The prior art Spirulina extracts (herein the sunlight extracts) did not produce such a large and significant reduction in TNF-α secretion (see Figure 3ASurprisingly, a higher concentration of 10 μg / ml of the disclosed Spirulina extract did not produce such a large and significant reduction in TNF-α secretion (see Figure 3B )——Indicates that the optimal effective concentration is lower than 10μg / ml, for example at 0.1μg / ml.

[0054] It is noted that the anti-TNF-α effect exhibited by the disclosed Spirulina extract is not linearly dose-dependent, but corresponds to a non-monotonic dose-response curve (NMDRC) and is achieved by very low doses, suggesting effects on cellular endpoints such as cell proliferation and organ development through interactions with receptors.

[0055] The inventors note that these very low and specific extract concentrations enable the disclosed Spirulina extract to be administered sublingually, for example, in a sublingual spray formulation comprising the disclosed Spirulina extract and / or its fractionated compounds (and an optional pharmaceutically acceptable carrier) to produce a concentration in the blood of less than 10 μg / ml, for example 0.1 μg / ml, or 1 μg / ml or intermediate values. For example, a corresponding spray device can be configured to apply an amount of spray, for example, in one or a specified number of spray actions, that produces a blood concentration of 0.1 μg / ml - 1 μg / ml in the patient. For example, an oral spray bottle that emits 0.14 ml of spray per dose, containing, for example, a liquid having 0.4% - 4.0% (4.0 g / l-40 g / l) of the disclosed Spirulina extract, can be used twice a day (once every 12 hours) to maintain blood concentrations within the active range of 0.1 μg / ml-1 μg / ml (in adults) all day. In various embodiments, the spray device can be configured to administer a spray liquid dose of between 0.05 ml and 3 ml (or an intermediate range, such as 0.05 ml-0.5 ml, 0.1 ml-1 ml, etc.) and adjust the concentration of the active ingredient (Spirulina extract and / or compounds fractionated therefrom) accordingly.

[0056] In addition, regarding Figure 3C , noting that the disclosed Spirulina extracts are specific for TNF-α secretion and do not affect IL-6 secretion, indicating that the disclosed Spirulina extracts may provide specific inhibitors of TNF-α, rather than general inhibitors of inflammatory processes, which makes them particularly desirable for use as specific anti-CS agents. It is noted that the disclosed extracts do not exhibit off-target effects in the absence of LPS stimulation, indicating their safety.

[0057] Will Figure 4B and Figure 4AFor comparison, data are provided showing the anti-inflammatory activity of Spirulina extracts as measured by TNF-α secretion from THP-1 human monocytes. The Spirulina extracts disclosed exhibited a large and significant reduction in TNF-α secretion, approximately 40% over the entire range of extract concentrations from 0.1 μg / ml to 10 μg / ml (see Figure 4B The prior art Spirulina extracts (herein as sunlight extracts) did not produce such a large and significant reduction in TNF-α secretion (see Figure 4A ). Unlike the effect on macrophages, this effect on monocytes was constant within the concentration range.

[0058] Figure 5A , Fig. 6A and Figure 6B is a high-level schematic diagram of a culture system 100 according to some embodiments of the present invention. Figure 5B is a high-level schematic flow chart illustrating a cultivation, extraction and treatment method 300 according to some embodiments of the present invention. The cultivation system 100 is configured to cultivate algae and / or cyanobacteria at high density and under high light intensity. Figure 5A , Fig. 6A and Figure 6B The elements of can be combined in any operable combination, and the illustration of specific elements in certain figures and not in other figures is for illustrative purposes only and is non-limiting. Note that any disclosed value can be modified by ±10% of the value. The method stages can be performed according to the culture system 100 described herein, which can be optionally configured to implement the method 300. The method 300 can be implemented at least in part by at least one computer processor, such as in a controller 103 including one or more corresponding processing units. Certain embodiments include a computer program product including a computer-readable storage medium having a computer-readable program contained therein and configured to perform the relevant stages of the method 300. The method 300 can include the disclosed stages, regardless of their order.

[0059] The cultivation system 100 includes at least one first sparging unit 101 having more than one nozzle and configured to dispense a first predetermined fluid 111 (e.g., air and / or nitrogen bubbles) into a water-filled algae cultivation container 110 (e.g., a bioreactor) at a first operating flow rate to allow mixing therein (schematically indicated by arrows 118). The cultivation system 100 may also include at least one second sparging unit 102 having more than one nozzle and configured to dispense a second predetermined fluid 112 (e.g., bubbles with CO2, schematically shown, and / or dissolved phosphorus for mass transfer) into the container 110 at a second operating flow rate. Fluids leaving the container 110, such as gases from the second predetermined fluid 112, may be recycled 113 to fully utilize the remaining CO2 therein (schematically shown).

[0060] The culture system 100 may also include at least one controller 103, which communicates with the first bubbling unit 101 and the second bubbling unit 102 and is configured to control the first operating flow rate and the second operating flow rate provided thereby. The controller 103 may include one or more processing units that execute computer code. For example, at least one nozzle of the first bubbling unit 101 and / or at least one nozzle of the second bubbling unit 102 may be configured to distribute the fluid into the culture container 110 based on instructions from at least one controller 103. In some embodiments, the first operating flow rate may be based on the second operating flow rate, and / or at least one of the operating flows may be predetermined. In some embodiments, the first operating flow rate may be suitable for allowing turbulent mixing of algae in the culture container 110. In some embodiments, the second operating flow rate may be suitable for allowing mass transfer and / or assimilation of substances in the liquid in the culture container 110. Information may flow between the controller 103 and the first bubbling unit 101 and the second bubbling unit 102, as well as between the controller 103 and other elements in the system, as schematically indicated by arrows.

[0061] The second predetermined fluid 112 may include bubbles having a concentration of more than 30% CO. The source of one or more first predetermined fluids and / or one or more second predetermined fluids may be outside the cultivation system 100, for example, a geothermal power plant may provide a source of dissolved carbon and / or sulfur for the second predetermined fluid.

[0062] The first operating flow rate (e.g., 100 ml / min) of at least one nozzle of the first bubbling unit 101 may be different from the second operating flow rate (e.g., 5 ml / min) of at least one nozzle of the second bubbling unit 102. In some embodiments, at least one nozzle of the first bubbling unit 101 may have a diameter greater than about 1 mm. In some embodiments, at least one nozzle of the second bubbling unit 102 may have a diameter less than about 1 mm. In some embodiments, the nozzles of the first bubbling unit 101 and the second bubbling unit 102 may dispense the same fluid (e.g., air), and the nozzles of each bubbling unit have different diameters. The larger hole of the first bubbling unit 101 may be configured to provide a first predetermined fluid 111 with large bubbles (e.g., bubbles of air and / or nitrogen) to stir and mix 118 the suspended biomass in the container 110, while the smaller hole of the second bubbling unit 102 may be configured to provide a second predetermined fluid 112 with small bubbles (e.g., bubbles of CO2 or bubbles containing CO2) to transfer CO2 from the gas to the liquid accessible to the suspended biomass in the container 110. Advantageously, the difference in size of the delivered bubbles can prevent the bubbles of streams 111 and 112 from combining, provide simultaneous mixing 118 through the high flux of large and fast bubbles in stream 111, and provide efficient CO2 supply through the small flux of small and slow bubbles in stream 112.

[0063] The culture system 100 may further include a physical barrier 104 configured to separate a first fluid dispensed by the first bubbling unit 101 and a second fluid dispensed by the second bubbling unit 102 within the culture container 110. In some embodiments, at least one nozzle of the first bubbling unit 101 and / or the second bubbling unit 102 may be embedded in the physical barrier 104 (not shown). In some embodiments, the physical barrier 104 may be adapted to allow flow from one side of the barrier 104 (having a first fluid distribution) to the other side of the barrier 104 (having a second fluid distribution) at a predetermined (e.g., upper and lower) position of the culture container 110 so as to generate a controlled flow within the container 110.

[0064] The culture container 110 having a physical barrier 104 may include at least one light source 202 embedded in the physical barrier 104, such that the container 110 may be illuminated from the inside by the at least one light source 202 embedded in the physical barrier 104 (illumination is schematically represented by arrow 203). The culture container 110 may include more than one physical barrier 104, each physical barrier 104 including at least one light source 202, such that a modular system may be created in which algae and / or cyanobacteria grow between adjacent physical barriers 104, wherein at least one controller 103 may control illumination of all light sources 202 embedded in the physical barriers 104. In certain embodiments, the culture system 100 can be configured to achieve a very high density of cultured biomass, which produces a relatively thin illuminated zone 116 and a correspondingly small optical depth of a much thicker dark zone 117 in the container 110, while the biomass is continuously stirred 118 (e.g., by vigorous bubbling of the fluid 111 and / or the fluid 112), so that individual cells of algae and / or cyanobacteria have only a brief residence time in the illuminated zone 116 before returning to the dark zone 117. In a non-limiting example, the thickness of the illuminated zone 116 can be configured to be between 0.1 cm and 1.5 cm, depending on the density of the suspension and the illumination density, and can be controlled by the controller 103 and adjusted according to specific requirements. Therefore, the illumination 203 (and in particular its UV component) can be set at a very high level, because the short residence time prevents illumination damage to individual cells.

[0065] The culture system 100 may also include at least one sensor 105 (e.g., a temperature sensor) coupled to the controller 103 and configured to detect at least one characteristic within the culture vessel 110. For example, the at least one sensor 105 may be configured to detect any of the pH level, temperature, and pressure conditions within the culture vessel 110 and / or portions thereof. In some embodiments, the at least one sensor 105 may also be configured to detect parameters external to the culture vessel 110, such as measuring the mass flow of gaseous exhaust from the culture vessel 110 to determine the amount of a substance absorbed into the algae cells by subtracting the amount of exhaust from the amount added to the vessel (e.g., by the second bubbling unit 102).

[0066] The culture system 100 may further include at least one database 106 (and / or storage unit) configured to store algorithms for operating the controller 103, such as a database of operating rates for each nozzle and / or each bubbling unit. In some embodiments, the culture system 100 may further include a power supply 107 coupled to the controller 103 and configured to provide power to the culture system 100. The power supply 107 may be configured to supply power to the at least one first bubbling unit 101 and the at least one second bubbling unit 102, for example to operate at different rates.

[0067] The data collected by the at least one sensor 105 can be analyzed by the controller (or processor) 103 to detect whether a feature exceeds a predetermined threshold, such as a threshold value of pH level and / or temperature and / or CO2 concentration within the container 110. In the event that the condition within the culture container 110 (e.g., as detected by the sensor 105) exceeds at least one threshold value, the controller 103 can operate at least one nozzle of the first bubbling unit 101 and / or at least one nozzle of the second bubbling unit 102 at a different flow rate. For example, detection of a CO2 concentration exceeding 40% within the container 110 (or detection of a low pH level) can cause at least one nozzle of the second bubbling unit 102 to reduce the flow rate of the second bubbling unit 102 to ~2 mm / min. In some embodiments, at least one nozzle of the second bubbling unit 102 can operate only upon receiving a signal from the sensor 105 that the feature exceeds a predetermined threshold value, and not operate at a constant rate.

[0068] At least one nozzle of the first bubbling unit 101 can be configured to operate only upon receiving a signal from the sensor 105 that a characteristic exceeds a predetermined threshold, such as increasing the mixing flow 118 as the algae population density increases. At least one nozzle of the first bubbling unit 101 and / or at least one nozzle of the second bubbling unit 102 can operate continuously or possibly intermittently at a constant rate. At least one nozzle of the first bubbling unit 101 and / or at least one nozzle of the second bubbling unit 102 can operate continuously or possibly intermittently at a non-constant rate.

[0069] The culture container 110 may include a bubble column configuration having at least one first bubble unit 101 and at least one second bubble unit 102 located on the same surface of the bubble column container. The culture container 110 may have an airlift configuration having at least one second bubble unit 102 positioned at the bottom of a downcomer, which may be away from the sensor 105, so that the residence time of bubbles from the at least one second bubble unit 102 may be increased.

[0070] The culture system 100 can be configured to maintain at least 20% organic carbon within the container 110, which is calculated excluding carbon provided as CO2 bubbles. In some embodiments, at least a portion of the algae within the container 110 can include any photosynthetic microorganisms used to prepare Spirulina preparations, such as algae and / or cyanobacteria, including, for example, Arthrospira platensis, A. fusiformis, and / or A. maxima.

[0071] like Figure 5B , a method 300 for culturing algae and / or cyanobacteria in a culture system 100 may include culturing algae and / or cyanobacteria in a container having one or more light sources for emitting light in the UV spectrum (stage 301). Illumination may be provided by at least one of the light sources 202, which is configured to emit UV light in the UVA and UVB spectrums. In some embodiments, the ratio between the emission intensities of the UVA / UVB radiation may be in the range of 10-15, such as 10UBA / UVB. In certain embodiments, method 300 may be used to cultivate cyanobacteria, such as species of the genus Arthrospira, from which a Spirulina extract is produced. Method 300 may also include providing a light source having an intensity of 1,000 kJ / m 2 -10,000 kJ / m 2 UV radiation (stage 302), for example 5000 kJ / m 2 or any other intermediate value. For example, the controller 103 can be configured to control the provision of UV radiation using on / off radiation pulses. In some embodiments, each pulse can last 0.0099 seconds, and between 1-100 such pulses can be provided per second, for example, 10 times per second. Note that approximately 0.01 seconds of 1,000 kJ / m 2 The illumination produces about 10 times the intensity of sunlight UV radiation, which alters the chemical composition of the algae and / or cyanobacteria to produce the extract compositions disclosed below.

[0072] In some embodiments, the optimization of harmful UV radiation is controlled to provide and can allow to increase the amount of antiviral compounds in Arthrospira species and the spirulina extracted, while avoiding the algae or the growth rate of damage growth. In some embodiments, the on / off property provided by radiation can allow the amount of harmful radiation provided by control. In addition, the continuous mixing of suspension in the container and / or bubbling 118 (by bubbling unit 101 and / or bubbling unit 102) ensure that any individual algae or cyanobacteria cells are only exposed to intense radiation for a short time, prevent light inhibition and the damage to the cell. Controller 103 can be configured to control the degree of turbulence provided by bubbling unit 101 and / or bubbling unit 102 separately, to avoid the radiation damage (stage 302) to the cell. For example, method 300 and culture system 100 can be configured to realize required UV light regulation in the film culture system by opening and closing UV light source and / or by creating the shadow pattern of the intermittent lighting that produces algae and / or cyanobacteria. In the bubbling culture system 100, the relative speeds of the flow of the culture suspension and bubbles relative to the UV light source can be controlled to achieve a specified pattern of on / off UV exposure cycles.

[0073] Method 300 also includes gathering algae and / or cyanobacteria (stage 303), for example, implementing continuous gathering and matching the gathering rate with the growth rate. Method 300 also includes preparing extracts (stage 303) from the algae and / or cyanobacteria gathered, for example, by applying one or more freeze-thaw cycles to destroy the cell wall and enhance the extractability of the suspension. For example, the biomass gathered can be quickly frozen to -20°C, then thawed at 0°C to 4°C, until completely thawed.

[0074] Method 300 can also include extracting at least one antiviral compound from the biomass (stage 304). For example, antiviral compounds are shown to be extractable from the biomass of the species of the genus Arthrospira cultured and harvested as disclosed herein to produce spirulina antiviral extracts and / or preparations. For example, the wet biomass of spirulina can be suspended in (hot or cold) pure water to obtain a product with 10% by weight dry matter. Insoluble matter can be removed by continuous centrifugation. The supernatant containing soluble bioactive substances can be used as an antiviral extract. Spirulina antiviral extracts contain water-soluble pigments (e.g., phycocyanin), proteins, nucleic acids, polysaccharides, and ash. These compounds can be further fractionated (e.g., by chromatography and ethanol precipitation) to enhance antiviral activity. Although in some embodiments, the harvested biomass can be used directly, such as orally, the advantageous use of extracts and / or their fractionated compounds allows the use of less amounts for daily consumption, and can use sublingual / oral sprays, rather than through the digestive tract that may affect the efficacy of the active compounds.

[0075] The inventors have found that the disclosed Spirulina extract has enhanced antiviral activity against pathogenic viruses such as HSV-1, HSV-2, human cytomegalovirus, influenza virus, and COVID-19 virus relative to Spirulina extracts from cyanobacteria cultured under prior art conditions (typically including lower intensity daylight illumination and lower density of cyanobacteria in bioreactors). It was found that oral administration of Spirulina extract (e.g., 1-3 g dry weight per person per day) prevents viral infection, and alleviates symptoms of viral diseases and shortens recovery time.

[0076] Therefore, and in view of the results disclosed above, method 300 can also include treating TNF-α-related inflammation (stage 310) by applying a sublingual spray formulation containing the disclosed Spirulina extract and / or fractionated compounds at a concentration of less than 10 μg / ml (e.g., 0.1 μg / ml, 1 μg / ml, or intermediate values) to the sublingual mucosa of a patient suffering from inflammation (stage 320), for example, by applying an amount of spray (stage 330) that produces a blood concentration of 0.1 μg / ml-1 μg / ml in the patient. Treating TNF-α-related inflammation 310 can be achieved by using Spirulina extract and / or using fractionated compounds as anti-inflammatory agents to reduce macrophage and monocyte induced tumor necrosis factor TNF-α levels, for example to treat acute respiratory distress syndrome (ARDS), heart failure, Crohn's disease, thrombophilia, and / or gingivitis.

[0077] Fig. 6A and Figure 6BSchematically illustrates an embodiment of a cultivation system 100 according to some embodiments of the present invention. The cultivation system 100 may include at least one lighting unit 201 coupled to a controller 103 to illuminate a cultivation container 110. One or more lighting units 201 and the controller 103 (or another controller) may be included in a bioreactor lighting system 208 for cultivating algae and / or cyanobacteria. The distance between the cultivation container 110 and one or more lighting units 201 may be modified to control the illumination received by the cultivation container 110. For example, one or more lighting units 201 may be brought closer to the cultivation container 110 to increase the illumination of the culture therein. The distance between the cultivation container 110 and one or more lighting units 201 may be controlled by, for example, a controller 103 included in the lighting system 208. In addition to changing the distance between the lighting unit 201 and the cultivation container 110, or instead of changing the distance between the lighting unit 201 and the cultivation container 110, the illumination intensity of the light source 202 in the lighting unit 201 may be controlled. The lighting unit 201 may include at least one light source 202 (e.g., an LED) such that each light source 202 may be individually controlled by the controller 103. In some embodiments, one or more light sources 202 may be controlled to illuminate at a different intensity than another / other light sources 202. All light sources 202 may be controlled to change the intensity of illumination manually or based on a predetermined time and / or sensed conditions in the culture container 110. At least some of the light sources 202 may be configured to emit light in the UV spectrum, e.g., light within both the UVA and UVB ranges. The ratio between UVA and UVB emitted radiation (UVA / UVB ratio) may be between 10 and 15, e.g., 10, 12, 14, 15 UBA / UVB, or an intermediate value. The UV radiation may be emitted at 1,000 kJ / m 2 -10,000kJ / m 2 , for example 2000kJ / m 2 、5000kJ / m 2 、7000kJ / m 2 、9000kJ / m 2 or any other intermediate value. The controller 103 can be configured to control the provision of UV radiation using radiation pulses, for example, each pulse can last less than 0.01 seconds, for example, 0.008 seconds, 0.009 seconds, 0.0095 seconds, or 0.0099 seconds, or any intermediate value, and can provide these pulses between 1 and 100 times per second, for example 10 times per second. Note that about 0.01 seconds of 1,000 kJ / m 2The illumination produces about 10 times the intensity of daylight UV radiation, which changes the chemical composition of the algae and / or cyanobacteria to produce the extract compositions disclosed below. Some light sources 202 can be configured to emit light in the visible spectrum (e.g., wavelengths of 400nm-700nm).

[0078] The amount of light delivered to the culture container 110 can be defined as the average value of the light flux delivered to the surface of the culture container 110. The culture system 100 can be used to support ultra-high density cultures (e.g., with a biomass density of 1 g / l, 5 g / l, or up to 10 g / l or intermediate values), wherein one or more lighting units 201 are configured to have a light distribution of one or more light sources 202 that provide an average light flux per algae / cyanobacteria cell comparable to the average light flux per cell of a low-density culture that achieves a similar level of average illumination / cell. The light intensity within the culture container 110 can be measured with at least one sensor 105 and adjusted by the controller 103. For example, for ultra-high density cultures, the typical thickness of the illumination zone 116 can be in a range of, for example, between 1 mm and 5 mm, while the typical thickness of the dark zone 117 can be in a range of, for example, between 20 mm and 30 mm. The distance of the lighting unit 201 from the side of the container 110 can be adjusted relative to the primary optical thickness (or optical depth, OD) of the biomass suspension to avoid photoinhibition and / or photobleaching. For example, in an initial culture stage, when the culture density is relatively low, the lighting unit 201 may initially be kept away from the side of the container 110 to inhibit biomass growth, whereas in a later culture stage, once the OD increases, the lighting unit 201 may be moved closer to the side of the container 110 to promote biomass growth.

[0079] Continuously and / or intermittently mixing and / or stirring (schematically represented by Figure 5A The ultra-high density culture is shown in Figure 1 (indicated by arrows 118 in the figure) to allow algae and / or cyanobacteria to move between the illuminated area 116 and the dark area 117 and to prevent damage to the cells, and to produce an illumination cycle of algae / cyanobacteria (between the illuminated area 116 and the dark area 117) due to the short light path. The ultra-high density culture can be illuminated with different wavelengths because at such a biomass density, the wavelength has little effect on growth due to the short light path. This is different from the usual practice, according to which algae are illuminated with a specific wavelength (e.g., with blue light) to grow normally, because the algae may react differently to light. However, the inventors have found through experiments that illumination of any wavelength can be used for ultra-high density cultures.

[0080] The light penetration into the culture container 110 may correspond to at least one of light intensity, light wavelength, a specific algae strain, and / or algae culture density. It should be noted that the light penetration into the culture container 110 may determine the volume ratio between the illuminated area 116 and the dark area 117 within the culture container 110, and thus may affect the light intensity provided by the lighting unit 201, the gas flow rate through the first bubbling unit 101, the gas flow rate through the second bubbling unit 102, etc. - which may be adjusted and optimized separately.

[0081] In some embodiments, the culture container 110 may be illuminated by one or more lighting units 201 to provide a daily amount of more than 90% of the amount of maximum algae growth within the culture container 110. In some embodiments, the one or more lighting units 201 may be configured to illuminate the suspension in the container 110 in a non-uniform manner, for example, using a small number of high-intensity light sources 202 that are spaced apart, because the illumination of the cells is localized and temporally controlled (by agitation 118). The inventors have discovered that high-intensity intermittent lighting actually enhances the growth of algae and / or cyanobacteria compared to commonly practiced configurations with uniform distribution of low-intensity light sources.

[0082] For example, the illumination photon flux density of the at least one light source 202 may be 1200 μmol / m 2 In various embodiments, the photon flux density can be 1000 μmol / m 2 s-1500 μmol / m 2 s, or have intermediate values. In some embodiments, one or more lighting units 201 may include a plurality of lighting units 201 per m 2 At least four light sources 202. As an illustrative, non-limiting example, a light source having about 6 m 2 The illumination unit 201 with a surface area of ​​about 100 μm and a light path of about 4 cm (thickness of the illumination zone 116) may include 24 LED light sources 202, each with a power of 1200 μmol / m 2 In some embodiments, at least a portion of the cyanobacteria within container 110 includes Arthrospira species used to prepare the Spirulina extract.

[0083] The controller 103 can be configured to control the wavelength of illumination of the at least one light source 202, for example using a dedicated illumination module adapted to modify the wavelength of the emitted illumination. In some embodiments, a constant temperature of 27° C. can be maintained within the container 110. In some embodiments, the controller 103 can be configured to control the at least one light source 202 to illuminate at a wavelength of 650 nanometers. It should be noted that according to common practice, algae are illuminated with a specific wavelength (e.g., with blue light) for optimal growth, however experiments conducted by the inventors have shown that illumination with other wavelengths (e.g., with red light) can be used to obtain enhanced growth.

[0084] like Figure 6B As schematically shown in FIG. 1 , the culture system 100 can be configured to operate with a single bubbling unit. Figure 6B denoted as the third bubbling unit 211. The culture system 100 may further include one or more lighting units 201, and the third bubbling unit 211 may be configured to dispense a predetermined fluid 113 into the culture container 110. The predetermined fluid 113 may include various mixtures of one or both of the predetermined fluid 111, the predetermined fluid 112, or portions thereof. For example, the third bubbling unit 211 may include at least one nozzle for dispensing the first predetermined fluid 111 and at least one nozzle for dispensing the second predetermined fluid 112 (e.g., having different diameters), a combination of which is schematically represented in a non-limiting manner as the predetermined fluid 113. The third bubbling unit 211 may be configured to generate turbulent mixing of algae and / or cyanobacteria in the culture container 110, and thereby provide CO2 for assimilation, for example, as described in reference Figure 5A and Fig. 6A The public.

[0085] In the above description, an embodiment is an example or implementation of the present invention. The various occurrences of "an embodiment", "embodiment", "certain embodiments" or "some embodiments" do not necessarily all refer to the same embodiment. Although the various features of the present invention can be described in the context of a single embodiment, the feature can also be provided individually or in any suitable combination. On the contrary, although the present invention can be described in the context of a separate embodiment herein for the sake of clarity, the present invention can also be implemented in a single embodiment. Certain embodiments of the present invention may include features from the different embodiments disclosed above, and certain embodiments may incorporate elements from other embodiments disclosed above. The disclosure of the elements of the present invention in the context of a specific embodiment should not be construed as limiting their use only in a specific embodiment. In addition, it should be understood that the present invention can be implemented or practiced in various ways, and the present invention can be implemented in certain embodiments other than the embodiments outlined in the description above.

[0086] The present invention is not limited to those diagrams or corresponding descriptions. For example, the process does not need to pass through each illustrated box or state, or in exactly the same order as the diagram and description. Unless otherwise defined, the meaning of the technical terms and scientific terms used herein should be generally understood by those of ordinary skill in the art to which the present invention belongs. Although the present invention has been described with respect to a limited number of embodiments, these embodiments should not be interpreted as limitations on the scope of the present invention, but rather as illustrations of some preferred embodiments. Other possible changes, modifications and applications are also within the scope of the present invention. Therefore, the scope of the present invention should not be limited by what has been described so far, but by the attached claims and their legal equivalents.

Claims

1. A Spirulina extract comprising an aqueous extract of Arthrospira sp. cultured under photosynthetically controlled conditions to produce upregulated bioactive compounds including c-phycocyanin, sorbitol and adenosine derivatives, wherein the Spirulina extract has a concentration between 0.1 µg / ml and 1 μg / ml and is effective as an anti-inflammatory agent, wherein the photosynthetically controlled conditions include 31±2°C, a pH of 10.8±0.2 and a pH of 700 μmol / (m 2 s) and 1500 μmol / (m 2 s).

2. The Spirulina extract according to claim 1, wherein the photosynthetic control conditions further include in ultra-high density culture with a density between 3 g / l and 10 g / l and at 70 μmol / m 2 s - 150 μmol / m 2 Arthrospira species were cultured under UV radiation intensities between s.

3. The Spirulina extract according to claim 1 or 2, wherein the water-based extract is produced by subjecting cultured Arthrospira species to water extraction and freeze-thaw cycles.

4. The Spirulina extract according to claim 1, wherein the concentration of the Spirulina extract is 0.1 µg / ml.

5. A pharmaceutical composition comprising the fractionated compounds of the Spirulina extract according to any one of claims 1 to 4.

6. Use of the Spirulina extract or fractionated compounds thereof according to any one of claims 1 to 4 for preparing a preparation for reducing the level of tumor necrosis factor (TNF)-α induced by macrophages and monocytes.

7. The use according to claim 6, wherein the preparation is used to treat virus-induced TNF-α cytokine storm.

8. The use according to claim 6, wherein the preparation is used to treat at least one of the following: acute respiratory distress syndrome (ARDS), heart failure, Crohn's disease, thrombophilia and gingivitis.

9. A sublingual spray preparation comprising the Spirulina extract or the fractionated compounds thereof according to any one of claims 1 to 4 at a concentration of less than 10 μg / ml.

10. The sublingual spray formulation according to claim 9, further comprising a pharmaceutically acceptable carrier.

11. The sublingual spray formulation according to claim 10, having a concentration of 0.4% to 4.0% by volume of the Spirulina extract.

12. A spray device configured to administer the sublingual spray formulation according to any one of claims 9 to 11 to the sublingual mucosa of a patient suffering from inflammation.

13. The spray device according to claim 12, further configured to be capable of administering an amount of spray in one or a specified number of spray actions, the amount producing a blood concentration of 0.1 μg / ml - 1 μg / ml in the patient.

14. The spray device of claim 13, wherein the sublingual spray formulation has a concentration of 0.4%-4.0% by volume of the Spirulina extract, and the spray device is further configured to deliver between 0.05 ml and 0.5 ml per spray actuation.

15. A method for preparing a Spirulina extract, the method comprising: Cultivating Arthrospira species cyanobacteria, wherein the Arthrospira species cyanobacteria are cultivated under photosynthetic control conditions to produce up-regulated bioactive compounds including c-phycocyanin, sorbitol and adenosine derivatives, wherein the photosynthetic control conditions include 31±2° C., at a pH of 10.8±0.2 and at 700 μmol / (m 2 s) and 1500 μmol / (m 2 s) for culturing Arthrospira species; A water-based extract of cultured Arthrospira sp. cyanobacteria was prepared to produce a Spirulina extract having a concentration between 0.1 µg / ml and 1 μg / ml and effective in treating TNF-α cytokine storm.

16. The method of claim 15, wherein the photosynthetic control conditions further comprise in ultra-high density culture having a density between 3 g / l and 10 g / l and at 70 μmol / m 2 s - 150 μmol / m 2 s.

17. The method of claim 15 or 16, wherein the water-based extract is prepared by subjecting the cultured Arthrospira species to water extraction and freeze-thaw cycles.

18. Use of the Spirulina extract prepared according to any one of claims 15 to 17 for preparing a preparation for reducing the level of TNF-α induced by macrophages and monocytes.

19. Use of the Spirulina extract prepared according to any one of claims 15 to 17 for preparing a preparation for treating at least one of the following: acute respiratory distress syndrome (ARDS), heart failure, Crohn's disease, thrombophilia and gingivitis.

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