CA3177111A1

CA3177111A1 - Immunomodulatory and antiviral action of boswellia gum resin extracts, derived formulations, and boswellic acids against respiratory viruses and uses thereof

Info

Publication number: CA3177111A1
Application number: CA3177111A
Authority: CA
Inventors: Sepideh BARZIN TODD; Ali RIAZI; Yuqian ZHANG; Saeed KARIMA; Abbas TAFAKHORI; Somayeh MAHMOODI BARAM; Farzad Mokhtari; Mohammad Reza SALEHI; Nasim KHAJAVI RAD
Original assignee: Individual
Current assignee: Kondor Pharma Inc
Priority date: 2020-04-30
Filing date: 2021-04-30
Publication date: 2021-11-04
Also published as: WO2021217275A1

Abstract

The present application discloses application of extracts and formulations thereof of Boswellia resin as well as purified or partly purified boswellic acids as antiviral and immunomodulatory agents against viruses that cause upper or lower respiratory diseases, such as SARS-CoV-2. The compositions claimed in this disclosure can be used alone or in combination with vaccines or other antiviral drugs as adjunctive therapy.

Description

lmmunomodulatory and antiviral action of Boswellia gum resin extracts, derived formulations, and boswellic acids against respiratory viruses and uses thereof FIELD OF INVENTION
[0001] The present application relates to immunomodulatory and antiviral actions of extracts from Boswellia oleogum resin enriched for boswellic acids as well as individual boswellic acids in pure or partly pure forms used for treatment, prevention, or amelioration of symptoms caused by respiratory virus infection.
BACKGROUND

[0002] There is an urgent need for vaccines and antiviral agents for respiratory viruses.
The pandemic spread of a virulent respiratory virus known as SARS-CoV-2/COVID-19 that emerged in 2019 resulted in large number of deaths worldwide. COVID-19 patients typically have symptoms including fever, dry cough, dyspnea, headache, hypoxemia associated with inflammation of the lungs and pneumonia. The agent causing this disease belongs to the coronavirus family that was also responsible for severe acute respiratory syndrome (SARS-Cov) and middle east respiratory syndrome (MERS-CoV) in 2003 and 2012 outbreaks, respectively.
There is a need for antiviral and/or anti-inflammatory drugs to prevent and/or treat symptoms in patients infected with respiratory viruses.

[0003] The gum resin from Boswellia sp. tree (Burseraceae family) has been used for centuries in Ayurvedic and Persian traditional medicine to treat pain and inflammation. There is a large body of evidence suggesting that boswellic acid molecules are the main anti-inflammatory compounds in Boswellia resin extracts conferring its medicinal effect(s).
Boswellic acids are believed to be the main pharmaceutical ingredients in various forms of Boswellia extracts (Ammon HP (2006) Boswellic acids in chronic inflammatory diseases. Planta Med 72(12):
1100-16).

(0004] A number of clinical studies have investigated the effects of Boswellia extracts on chronic inflammatory diseases including rheumatoid arthritis (Etzel R. (1996) Special extract of BOSWELLIA serrata (H 15) in the treatment of rheumatoid arthritis.
Phytomedicine 3(1):91-94), bronchial asthma (Gupta I, et al. (1998) Effects of Boswellia serrata gum resin in patients with bronchial asthma: results of a double-blind, placebo-controlled, 6-week clinical study. Eur J Med Res 3:511-514), ulcerative and chronic colitis (Gupta I, et al. (1997) Effects of Boswellia serrata

5 gum resin in patients with ulcerative colitis. Eur J Med Res 2(1):37-43, Gupta A, et al. (2011) Immunological adjuvant effect of Boswellia serrate (BOS 2000) on specific antibody and cellular response to ovalbumin in mice. Int Immunipharmacol, 11(8):968-975, Madisch A, et al. (2007) Boswellia serrata extract for the treatment of collagenous colitis. A double-blind, randomized, placebo-controlled, multicenter trial. Int J Colorectal Dis 22:1445-1451), Crohn's disease (Gerhardt H, et al. (2001) Therapy of active Crohn disease with Boswellia serrata extract H 15.
Gastroenterol 39:11-17, Holtmeier W, et al. (2011) Randomized, placebo-controlled, double-blind trial of Boswellia serrata in maintaining remission of Crohn's disease: good safety profile but lack of efficacy. Inflamm Bowel Dis 17(2):573-582), osteoarthritis (Sengupta K, et al. (2008) A double blind, randomized, placebo-controlled study of the efficacy and safety of 5-Loxin(R) for treatment of osteoarthritis of the knee. Arthritis Res Ther 10(4): R85; Majeed M, et al.
(2019) A pilot, randomized, double-blind, placebo-controlled trial to assess the safety and efficacy of a novel Boswellia serrata extract in the management of osteoarthritis of the knee.
Phytother Res 33(5):
1457-1468), memory, cognition, and recovery from neuronal injury (Hosseini-sharifabad M and Esfandiari E (2015) Effect of Boswellia serrata gum resin on the morphology of hippocampal CA1 pyramidal cells in aged rat. Anat Sci Int 90: 47-53; Zaker S, et al. (2015) Effect of olibanum on a rat model of Alzheimer's disease induced by intracerebroventricular injection of streptozotocin.
Physiol Pharmacol 18: 477-489; Baram SM, et al (2019) Functional improvement and immune-inflammatory cytoki nes profile of ischemic stroke patients after treatment with boswellic acids: a randomized, double-blind, placebo-controlled, pilot trial. Inflammopharmacology. 27(6): 1101-1112). The mechanism underlying the anti-inflammatory effect of boswellic acids is not entirely understood.
[0005]
Few studies of antiviral properties of Boswellia extracts have been reported.
Boswellia serrata extract (BSE) in combination with curcumin was found to inhibit mosquito-transmitted chikungunya virus (CHIKV) and vesicular stomatitis virus infections in vitro. Treatment with BSE was shown to block the entry of CHIKV Env-pseudotyped lentiviral vectors, and suppressed CHIKV infection. Furthermore, vesicular stomatitis virus particles and viral infections were also reduced, thereby demonstrating the antiviral activity of the combination therapy (von Rhein C, et al. (2016) Curcumin and Boswellia serrata gum resin extract inhibit chikungunya and vesicular stomatitis virus infections in vitro. Antiviral Res. 125:51-57.). In another study, Goswami et al (2018) (Goswami D, et al. (2018) Boswellia serrata oleo-gum-resin and 13-boswellic acid inhibits HSV-1 infection in vitro through modulation of NF-KB and p38 MAP
kinase signaling.
Phytomed. 51: 94-103) showed that BSE and 13-boswellic acid efficiently inhibited wild-type and a clinical isolate of herpes simplex virus (HSV-1) infection of green monkey kidney cells through modulation of NF-OB and p38 MAP kinase signaling. NF-OB is required for viral replication and its suppression inhibits viral RNA replication. The study further demonstrated reduction of TNFa, IL-6, and IL-1p pro-inflammatory cytokines after HSV-1 infected mouse macrophages were treated with BSE or 13-boswellic acid (Goswami D, et al. (2018) Boswellia serrata oleo-gum-resin and p-boswellic acid inhibits HSV-1 infection in vitro through modulation of NF-KB and p38 MAP
kinase signaling. Phytomed. 51: 94-103).

[0006] A subgroup of patients infected with coronaviruses such as SARS-CoV show an acute severe systemic inflammatory response called cytokine release syndrome or hyperinflammation associated with significant increase in cytokines including IL-1p, IL-6, and TNF-a (Tisonick JR, et al. (2012) Into the eye of the cytokine storm. Micro Mol Biol Rev: 16-32).
SUMMARY OF THE INVENTION

[0007] Considering the inhibitory effect of Boswellia extracts on pro-inflammatory cytokines such as IL-6, IL-1 and TNF-a, we investigated the use of Boswellia extracts and/or formulation derivatives can be used as therapies for patients presenting hyperinflammation, and for antiviral and immunomodulatory effects, including against coronaviruses.
The present application discloses application of extracts of Boswellia oleo-gum resin, various formulations thereof, or purified or partly purified boswellic acids or their derivatives as dietary supplement or medicinal agent against viruses that cause upper or lower respiratory illnesses. It also discloses the results of investigatory experiments and trials.

[0008] In accordance with the present invention, there is provided an antiviral and immunomodulatory composition comprising Boswellia resin extract enriched for at least one boswellic acid selected from the following group: AKBA, 11-KBA, a-BA, and 13-BA. In an aspect of this invention, there is provided a formulation comprising the composition dissolved in at least one of: coconut oil, and medium chain triglycerides (MCI) containing either C8, C10, or C12 triglycerides. In another aspect of the invention, there is provided a formulation comprising the composition dissolved in at least one or more organic solvents. In another aspect of the invention, the organic solvents include at least one of: Omega 3 polyunsaturated fatty acids, phosphatidylcholin, olive oil, flaxseed oil, walnut oil, ethanol, methanol, ethyl acetate, hexane, and dimethylsulfoxide,. In another aspect of the invention, the extract is prepared from Boswellia serrata or Boswellia carterii or Boswellia papyrifera or Boswellia frereana.
In another aspect of the invention, the composition or formulation is combined with other antiviral or immunomodulatory natural health products, dietary supplements, or drugs for adjunctive therapy.
In yet another aspect of the invention, the composition or formulation is used as an antiviral agent to modulate the level of NE-KB in a mammal. In another aspect, the mammal is a human. Another aspect of the invention is the use of the composition or the formulations as an immunomodulatory agent to modulate the level of IL-la, IL-1p, TNF-a, and IL-6 in a mammal. In another aspect, the mammal is a human. Another aspect of the invention is the use of the composition or formulations to improve immune system health by promoting the proliferation of lymphocytes and the lowering of the neutrophil to lymphocyte ratio (NLR). In another aspect, there is provided the use of the composition or the formulations to prevent or alleviate severe inflammation or cytokine storm/hyperinflammation caused by respiratory viruses. In an aspect, the respiratory virus is influenza. IN another aspect, the respiratory virus is a coronavirus. Another aspect of the invention is delivery of the composition or the formulations through the pulmonary system using a nebulizer or inhaler. In still another aspect, there is delivery of the composition or the formulations, comprising oral administration or other parenteral routes of administration.
In accordance with the present invention, there is provided an antiviral and immunomodulatory composition comprising: a Boswellic acid selected from the following group:
AKBA, 11-KBA, cc-BA, and p-BA, acetyl-a-BA and acetyl--BA. In an aspect of the invention, there is provided a formulation comprising the composition dissolved in at least one of: coconut oil, and medium chain triglycerides (MCT) containing either C8, C10, or C12 triglycerides. In another aspect, the composition is dissolved in at least one of more organic solvents. In still another aspect, the organic solvents include at least one of: Omega 3 polyunsaturated fatty acids, phosphatidylcholin, olive oil, flaxseed oil, walnut oil, ethanol, methanol, ethyl acetate, hexane,and dimethylsulfoxide.
In another aspect, the formulations are prepared from Boswellia serrata or Boswellia carterii or Boswellia papyrifera or Boswellia frereana. In another aspect the composition or the formulations are combined with other antiviral or immunomodulatory natural health products, dietary supplements, or drugs for adjunctive therapy. Another aspect of the invention is the use of the composition or the formulations as an antiviral agent to modulate the level of NF-KB in a mammal.
Another aspect of the invention is such use where the mammal is a human.
Another aspect of the invention is the use of the composition or the formulations as an immunomodulatory agent to modulate the level of IL-1a, IL-113, TNF-a, and IL-6 in a mammal. In another aspect, the mammal is a human. Another aspect of the invention is the use of the composition or the formulations to improve immune system health by promoting the proliferation of lymphocytes and the lowering of the neutrophil to lymphocyte ratio (NLR). Another aspect is the use of the composition or the formulations to prevent or alleviate severe inflammation or cytokine storm/hyperinflammation caused by respiratory viruses. In another aspect, the respiratory virus is influenza. In another aspect, the respiratory virus is a coronavirus. In still another aspect, there is provided a method for delivery of boswellic acids, where the boswellic acids are dissolved in a solvent, comprising delivery through the pulmonary system using a nebulizer or inhaler. In another aspect, there is provided a method for delivery of boswellic acids, where the boswellic acids are dissolved in a solvent, comprising oral administration or other parenteral routes of administration.

[0009] In accordance with the present invention, there is provided an antiviral and immunomodulatory composition comprising Boswellia resin extract enriched for at least the four boswellic acids AKBA, 11-KBA, a-BA, and 13-BA. In an aspect of this invention, there is provided a formulation comprising the composition of dissolved in at least one of:
coconut oil, and medium chain triglycerides (MCT) containing either 08, 010, or C12 triglycerides. In another aspect, the Boswellia resin extract is dissolved in at least one of more organic solvents.
In another aspect, organic solvents include at least one of: Omega 3 polyunsaturated fatty acids, phosphatidylcholin, olive oil, flaxseed oil, walnut oil, ethanol, methanol, ethyl acetate, hexane, and dimethylsulfoxide.
In another aspect, the exract is prepared from Boswellia serrata or Boswellia carterii or Boswellia papyrifera or Boswellia frereana. In another aspect, the composition or the formulation is combined with other antiviral or immunomodulatory natural health products, dietary supplements, or drugs for adjunctive therapy. Another aspect is the use of the composition or the formulations as an antiviral agent to modulate the level of NF-KB in a mammal. In another aspect, the mammal is a human. Another aspect is the use of the composition or the formulations as an immunomodulatory agent to modulate the level of IL-la, IL-1f3, TN F-a, and IL-6 in a mammal. In an aspect, the mammal is a human. Another aspect is the use of the composition or the formulations to improve immune system health by promoting the proliferation of lymphocytes and the lowering of the neutrophil to lymphocyte ratio (NLR). Another aspect is the use of the composition or the formulations to prevent or alleviate severe inflammation or cytokine storm/hyperinflammation caused by respiratory viruses. In another aspect, the respiratory virus is influenza. In another aspect, the respiratory virus is a coronavirus. In another aspect, there is provided a method for delivery of the composition or the formulations comprising delivery through the pulmonary system using a nebulizer or inhaler. In another aspect, there is provided a method for delivery of the composition or the formulations comprising oral administration or other parenteral routes of administration.

[0010] Other features and advantages of the present application will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating embodiments of the application, are given by way of illustration only and the scope of the claims should not be limited by these embodiments, but should be given the broadest interpretation consistent with the description as a whole.
BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The embodiments of the application will now be described in greater detail with reference to the attached drawings in which:

[0012] Figure 1 shows the molecular structure of main bioactive boswellic acids found in Boswellia resin extract.

[0013] Figure 2a depicts HPLC chromatograms of an exemplary Boswellia extract representing 11-KBA, and AKBA.

[0014] Figure 2b depicts HPLC chromatograms of an exemplary Boswellia extract representing a-BA, and f3-BA.

[0015] Figure 3 demonstrates the impact of treatment of human epithelial cells (L-132, ATCC CCL5) cells) with Boswellia extract preparations on Coronavirus-HuCoV-229E proliferation as represented by the number of viral plaques. Also it demonstrates the result of MIT cell viability assay after lung cells were exposed to HuCoV-229E virus and treated with purified boswellic acids.

[0016] Figure 4 demonstrates the level of proinflammatory cytokine IL-1a and IL-13 in the peripheral blood of elderly individuals taking Boswellia serrata extract enriched for boswellic acids for one month.

[0017] Figure 5 demonstrates the level of proinflammatory cytokine IL-6 and TN F-a in the peripheral blood of elderly individuals taking Boswellia serrata extract enriched for boswellic acids for one month.

[0018] Figure 6 demonstrates length of hospitalization, blood oxygen saturation, and neutrophil to lymphocyte (NLR) ratio of COVID-19 patients treated with liquid formulation containing Boswellia extract compared to placebo on day 14 post treatment.

[0019] Figure 7 contains Table 1, which summarizes the patient outcome and blood biomarker analysis of COVID-19 patients treated with a formulation consisting Boswellia extract dissolved in MCI oil compared to placebo.

[0020] Figure 8 contains Table 2, which demonstrates the PCR test results of patients who tested positive for Covid-19 participating in a study at the onset, day 0 and on day 14.
DETAILED DESCRIPTION

[0021] The present disclosure relates to compositions consisting of Boswellia resin extracts, formulation derivatives thereof, and/or individual boswellic acids with application as an antiviral composition, agent or pharmaceutical for infections caused by respiratory viruses.
Accordingly, the present application includes a composition for use as a dietary supplement, agent, drug or a combination in adjunctive therapy.

[0022] Turning to Figure 1, the molecular structure of six boswellic acids are illustrated:
11-keto-b-boswellic acid (KBA) (also denoted 11-KBA). 3-0-acetyl-11-ketao-b-boswellic acid (AKBA), a-boswellic acid (also denoted a-BA), 13-boswellic acid (also denoted 13-BA), 3-acetyl-a-boswellic acid, and 3-acetyl-13-boswellic acid. These are the main bioactive boswellic acids found in boswellia resin extract.

[0023] Figures 2a and 2b depict HPLC chromatograms of an exemplary Boswellia extract representing 11-KBA, AKBA, a-BA, and 8-BA. Figure 2a shows HPC chromatograms of a typical Boswellia extract representing 11-KBA and AKBA, while Figure 2b shows HPC
chromatograms of a typical Boswellia extract representing a-BA, and 8-BA.

[0024] As described below, the inventors have investigated and found that purified or partly purified boswellic acids, and specifically boswelia resin extract prepared to increase the concentration of 11-KBA, AKBA, a-BA, and 13-BA, has antiviral and immunomodulatory effects.
We have also found that Boswellia extracts promote the lowering of the neutrophil to lymphocyte ratio (NLR). Our investigations, including clinical trials, indicate that Boswellia resin extract with increased concentration of 11-KBA, AKBA, a-BA, and 13-BA, are useful to prevent or alleviate severe inflammation caused by respiratory viruses, such as coronaviruses as well as influenza.
This includes the prevention or treatment of symptoms associated with respiratory viruses including viruses from Coronaviridae (e.g. SARS-CoV-1, SARS-CoV-2, MERS), Orthomyxoviridae (e.g. Influenza A H1N1, H5N1, H7N 1), Parayxoviridae (e.g.
RSV), Picornaviridae (e.g. Rhinovirus), Adenoviridae (e.g. adenovirus) and Parvoviridae (e.g. HBoV).

[0025] The Boswellia acids and/or Boswellia resin extracts are usually prepared, and can be administered, dissolved in a solvent. Suitable solvents include coconut oil, medium chain triglycerides (MCT) containing either C8, C10, or C12 triglycerides, or organic solvents including Omega 3 polyunsaturated fatty acids, phosphatidylcholin, olive oil, flaxseed oil, walnut oil, ethanol, methanol, ethyl acetate, hexane, dimethylsulfoxide, Combinations of these solvents can also be used.

[0026] The boswellic acids and/or extracts can be used in combination with other plant extracts or drugs in a formulae or composition. In some embodiments, the compositions of the application further comprise one or more bioactive agents. In some embodiments, the individual boswellic acids are used in combination with other plant extracts or drugs in a formulae or composition. In some embodiments, the one or more bioactive agents are selected from ginsenosides, curcumin, crocin, bacosides, and astaxanthin.

[0027] In some embodiments, the composition is purified or partly purified AKBA molecule or a modification thereof as new chemical entity when AKBA is the precursor molecule. In some embodiments, the composition is purified or partly purified 11-KBA molecule or a modification thereof as new chemical entity when 1 1- KBA is the precursor molecule.

[0028] In some embodiments, the composition is purified or partly purified a-BA molecule or a modification thereof as new chemical entity when a-BA is the precursor molecule. In some embodiments, the composition is purified or partly purified p-BA molecule or modification thereof as new chemical entity when p-BA is the precursor molecule.

[0029] In some embodiments, the composition is purified or partly purified 3-acetyl-a-boswellic acid molecule or modification thereof as new chemical entity when 3-acetyl-a-boswellic acid is the precursor molecule. In some embodiments, the composition is purified or partly purified 3-acetyl-p-boswellic acid molecule or a modification thereof as new chemical entity when 3-acetyl--boswellic acid acid is the precursor molecule.

[0030] In some embodiments, the one or more of boswellic acids or derivatives may be conjugated to a carrier molecule such polyethylene glycol and/or formulated with various chemicals (liposome or nanoparticle encapsulation) and/or excipients for delivery to maximize bioavailability.

[0031] In some embodiments, the Boswellia resin extract or boswellic acids may be obtained from any of the following species: Boswellia serrata or Boswellia carterii or Boswellia sacra or Boswellia papyrifera or Boswellia frereana.

[0032] In some embodiments, the composition is formulated as oral capsule in the form of dry or softgel capsule, spray, liquid, cream or gel. In some embodiments, the composition is formulated for delivery through oral administration. In some embodiments, the composition is formulated for delivery via nebulization through nasal spray or inhaler devices for pulmonary delivery system. In some embodiments, the composition is formulated as a liquid solution for injection. In some embodiments, the composition is formulated as a cream for transdermal delivery.

[0033] It is believed that Boswellia acids or extracts may be usefully applied in mammals.
In some embodiments, and in some of the tests described below, the mammal is human. In some embodiments, the subject is a companion animal or livestock. In some embodiments, the subject is a canine, feline, equine or bovine.
EXAMPLES

[0034] The following non-limiting examples are illustrative of the present application:
Example 1:

[0035] METHODS

[0036] Preparation of boswellic acid (BA) solution enriched for AKBA, 11-KBA, a-BA, and

[0037] An ethanol extract (dry powder) from Boswellia serrata (BSE) 90% enriched for boswellic acids was obtained from a supplier. The following procedures were performed to prepare BA solution with a final concentration of 100-500 lag of each of four boswellic acids, AKBA, 11-KBA, a-BA, and r3-BA. First BSE was dissolved in 99.99% Ethanol at 20 mg/ml and thoroughly mixed. The solution was incubated at 55 C for 2 hours with vigorous shaking every 15 min. The solution was diluted to 50% ethanol using phosphate buffered saline (PBS). The mixture was placed at room temperature for 15 min and the precipitates were removed by centrifugation.
Finally, the solution was filter sterilized using 0.45-micron filter and stored at -20 C until use.

[0038] Determination of the level of boswellic acids

[0039] High performance liquid chromatography (HPLC) was performed using an HPLC
system (Ultimate 3000TM, Thermo ScientificTm). Column used as solid phase was XBridgeTM C18 (4.6 mm x 100 mm, particle size 3.51Jm). For measuring the amount of AKBA and 11-KBA, standards and test samples were dissolved in 5 mg/mL DMSO. Serial dilutions were made in methanol and samples were sterilized using 0.45 pm syringe filters. Before applying the samples to the column, 50 pL of sample was mixed with 50 pL of mobile phase (acetonitrile and water at 90:10 %V/V). Samples were run at a flow rate of 1 mUminute and peaks were detected at 260 nm. For measuring a-BA and p-BA samples were dissolved in methanol and ran on the column as mentioned above. Peaks were detected at 210 nm.

[0040] Coronavirus plaque assay

[0041] The human coronavirus used in this study was the 229E
strain (ATCC #VR-740).
L-132 cell line. (ATCC #CCL-5) was used to grow the virus and perform the plaque assay. Cells were grown in MEM (GIBCO-BRL Cat # 41600-016) in the presence of L-glutamine, antibiotics, and 10% FBS in 75 cm2 flasks at 37 C. Confluent monolayers of cells were trypsinized and dispensed into 12-well plates. The cells were dispensed at a density to allow for the formation of confluent monolayers within 48-72 hours. The growth medium from each plate was aspirated and 50-100 pL of the Boswellia extract test solution (10 mg/ml) was added and the cells were incubated for 30 minutes with occasional shaking at 33 C. Different concentration of coronavirus 229E was added and the cells were incubated for 1 hour at 33 C to allow the virus absorption to the cell monolayer. Each dilution was titrated in triplicate. Each monolayer was overlaid with 2 mL
of an overlay medium containing supplemented MEM, 2% FBS, DEAE-Dextran, 5'bromo-2'deoxyuridine, 26 mM MgCl2 and purified agar (Oxoid L28). The ratio of the agar and the supplemented medium was 1:1. Once the overlay had solidified, the plates were held for 6 days in a 5% CO2 atmosphere at 33 C. Cells were then fixed and stained with 0.5%
crystal violet solution in 25% methanol.

[0042] MTT cell viability assay

[0043] M RC-5 cells were seeded at 20,000 cells per well in a 96 well plate and incubated for 24 hours at 37 C. Adequate concentration of coronavirus 229E was added to each well in serum free media. Cells were then treated with various concentration of BSE
extract or the purified boswellic acids and incubated for 36-48 hours at 33 C. After the incubation period, MTT labeling reagent (SigmaTM, M2128-1G) was added and the plate was incubated for additional 3 hours.

Finally, MTT solvent was added to each well and the light absorbance were measured at OD=
590 nm as an indicator of cell viability.

[0044] RESULTS & DISCUSSION

[0045] Our assay demonstrated that the number of plaques in cells treated with Boswellia extract was drastically reduced and/or undetectable at the virus concentrations used.

[0046] Please see Figure 3, which shows the impact of treatment of human epithelial cells (L-132, ATCC CCL5) cells) with Boswellia extract preparations on Coronavirus-HuCoV-229E
proliferation as represented by the number of viral plaques. In panel A, Cl represents control (100 pl of solvent), C2 represents control (50 pl of solvent), T1 represents test (100 pl of Boswellia extract), 12 represents test (50 pl of Boswellia extract). Also, V1 and V2 represent two different doses of the virus used: V1 represents 10^6 plaque forming units, while V2 represents 10^5 plaque forming units. Also, Figure 3 panel B demonstrates the result of MTT cell viability assay after lung cells were exposed to HuCoV-229E virus and treated with purified boswellic acids. In Figure 3 panel B, A represents untreated control cells (untreated with virus), B represents the cells that are treated with the virus, C represents cells treated with virus and AKBA, D
represents cells treated with virus and a-BA, E represents cells treated with virus and 13-BA, F
represents cells treated with virus and acetyl-a-BA, and G represents cells treated with virus and acetyl-13-BA. The data was analyzed using a student-T test and values below 0.05 were considered significant. There is a significant improvement in the viability of cells treated with acetyl-a-BA. Acetyl-p-BA and p-BA also showed improvement in cell viability.

[0047] Furthermore, our MTT cell viability assay demonstrated that 3-acetyl-a-boswellic acid significantly improved the cell viability and reduced the cytotoxic effect of the coronavirus. 3-BA and acetyl--BA also showed a trend in lowering the effect of the coronavirus on the cells tested in this assay. These indicate that the antiviral effect seen with the Boswellia extract is either through one or a combination of boswellic acids present in the extract. The reduction in the number of plaques strongly indicates that the replication of the virus is compromised, and that modulation of NF-KB pathway (as an important central protein for viral replication) by boswellic acids is playing a major role in inhibition of coronaviral replication and might be an effective treatment for patients infected with coronaviruses such SARS-CoV-2.

Example 2:

[0048] METHODS

[0049] Preparation of Boswellia extract enriched for AKBA + 11-KBA

[0050] BSE containing a minimum of 8% AKBA + 11-KBA (I
nflawellTm) was prepared. An adequate amount of BSE was dissolved in DMSO for 2 hours and filtered through a 0.45-micron filter. AKBA fraction was identified and collected using preparative H PLC.
The purified AKBA was lyophilized and added back to the original BSE to standardize the level to at least 8% AKBA + 11-KBA (w/w). The level of BAs in the final extract was confirmed using HPLC.

[0051] Pilot clinical trial to show immunomodulatory effect of Boswellia extract

[0052] A pilot clinical trial was conducted on patients with people suffering from dementia ranging from 65 to 80 years of age. The patients received Boswellia extract or placebo capsules (1200 mg per day) for 1 month. Blood samples from both groups were collected at baseline, month 1 and month 6 (end of study). Plasma levels of inflammation markers I L1-a , IL-1 [3, IL-6, and TNF-a were analyzed and compared at baseline versus month 1. The results, showing the level of the four proinflammatory cytokines is reduced in the peripheral blood of elderly individuals taking Boswellia serrata extract enriched for boswellic acids for one month, as seen in Figures 4 and 5.

[0053] Figures 4 and 5 show the results of the trial showing the plasma level of proinflammatory cytokines IL-la, IL-43, TN F-a, and IL-6 in individuals taking enriched Boswellia extract (BA). The plasma levels of IL-la (Figure 4, panel A), IL-1f3 (Figure 4, panel B), TNF-a (Figure 5, panel C), and IL-6 (Figure 5, panel D) were measured in individuals taking either placebo or 1200 mg daily of Boswellia extract enriched for boswellic acids. The level of cytokines at the onset and after one-month of treatment is shown. The cytokine levels were drastically reduced in individuals taking the Boswellia extract compared with the placebo group. A linear mixed model was run on the cytokine levels in the two groups. The model included treatment (drug, placebo), time (before, after), a treatment by time interaction term. The results were reported as adjusted mean scores and p-values of p<0.05 was considered statistically significant as indicated with asterisks.

[0054] RESULTS & DISCUSSION

[0055] Analysis of inflammation biomarkers in human subjects taking AKBA-enriched Boswellia capsules demonstrated significant reduction in pro-inflammatory cytokines, IL-113, IL-6, and TNF-a in the peripheral blood. The patients taking Boswellia extract tolerated well the dose of the product used with no serious adverse events. Overall, these results indicate that Boswellia extract may suppress hyperinflammation (also known as cytokine release storm) associated with respiratory viruses.
Example 3

[0056] METHODS

[0057] Preparation of boswellia liquid formulation for human COVID-19 clinical trial

[0058] BSE containing a minimum of 8% AKBA + 11-KBA was prepared.
An adequate amount of BSE was dissolved in coconut oil (rich in 08 and C10 medium chain triglycerides) for 2 hours with shaking. Soy lecithin, propolis, and honey were used as emulsifier and sweeteners.
The placebo was prepared similarly using all the ingredients excluding the BSE.

[0059] Pilot clinical trial of COVID-19 patients to determine the efficacy of Boswellia formulation on COVI D-19 patients.

[0060] A randomized double-blind clinical trial was conducted on patients accepted to hospital with mild to moderate COVI D-19 symptoms. The inclusion criteria were (i) testing positive for SARS-Cov-2 using a polymerase chain reaction (PCR) test, and (ii) having oxygen saturation level of between 87 to 92. Patients with heart disease or other types of respiratory disease such as COPD and Asthma were excluded. Patients were divided into Boswellia (drug) and placebo groups and treated for 14 days. All patients remained on the standard medications (such as remdesivir) used for managing COVID-19. Clinical data were collected at day 0 and day 14.

[0061] RESULTS AND DISCUSSION

[0062] As shown in the table in Figure 7, analysis of data collected from the COVID-19 patient cohort in our study showed a significantly shorter hospitalization, higher oxygen saturation level, as well as a significantly lower neutrophil to lymphocyte ratio (NLR) in the patients treated with the Boswellia formulation. The higher oxygen saturation level in the drug treatment group is indicative of the positive effect of the active compounds in the Boswellia formulation used. The reduction of NLR in the drug treatment group represents an increase in lymphocyte proliferation and improved ability of the body to mount immune response against SARS-Cov-2.
Furthermore, our cytokine analyses showed significant reduction in IL-6, a cytokine that is highly elevated in the blood of COVID-19 patients with lung hyperinflammation.

[0063] Furthermore, treatment with the Boswellia formulation reduced the number of patients that were testing positive for the virus by 44%. As shown in the table in Figure 8, a statistically significant number of patients who took the Boswellia formulation as outlined above showed significant improvements in negative PCR test results over a 14 day period compared with patients taking a placebo. Overall the results of this clinical trial indicated that Boswellia and its active compounds (boswellic acids) would be useful, alone or in combination with other antiviral drugs or vaccines, for the treatment of COVID-19 patients.

Claims

Claims:

1. An antiviral and immunomodulatory composition comprising:
Boswellia resin extract enriched for at least one boswellic acid selected from the following group: AKBA, 11-KBA, a-BA, and p-BA.

2. A formulation comprising the composition of claim 1 dissolved in at least one of: coconut oil, and medium chain triglycerides (MCT) containing either or combination of C8, C10, or C12 tri glycerides.

3. A formulation comprising the composition of claim 1 dissolved in at least one or more organic solvents.

4. The formulation of claim 3 where the organic solvents include at least one of: Omega 3 polyunsaturated fatty acids, phosphatidylcholin, olive oil, flaxseed oil, walnut oil, ethanol, methanol, ethyl acetate, hexane, and dimethylsulfoxide,.

5. The composition of claim 1, when the extract is prepared from Boswellia serrata or Boswellia carterii or Boswellia papyrifera or Boswellia frereana

6. The composition of claim 1 or the formulations in claims 2, 3 or 4 combined with other antiviral or immunomodulatory natural health products, dietary supplements, or drugs for adjunctive therapy.

7. The use of the composition of claim 1 or the formulations in claims 2, 3 or 4 as an antiviral agent to modulate the level of NF-KB in a mammal.

8. The use of claim 7, where the mammal is a human.

9. The use of the composition of claim 1 or the formulations in claims 2, 3 or 4 as an immunomodulatory agent to modulate the level of IL-la, IL-1p, TN F-a, and IL-6 in a mammal.

10. The use of claim 9 where the mammal is a human.

11. The use of the composition of claim 1 or the formulations in claims 2, 3 or 4 to improve immune system health by promoting the proliferation of lymphocytes and the lowering of the neutrophil to lymphocyte ratio (NLR).

12. The use of the composition of claim 1 or the formulations in claims 2, 3 or 4 to prevent or alleviate severe inflammation or cytokine storm/hyperinflammation caused by respiratory viruses.

13. The use of claim 12 where the respiratory virus is influenza.

14. The use of claim 12 where the respiratory virus is a coronavirus.

15. A method for delivery of the composition of claim 1 or the formulations in claims 2, 3 or 4, comprising delivery through the pulmonary system using a nebulizer or inhaler.

16. A method for delivery of the composition of claim 1 or the formulations in claims 2, 3 or 4, comprising oral administration or other parenteral routes of administration.

17. An antiviral and immunomodulatory composition comprising:
A boswellic acid selected from the following group: AKBA, 11-KBA, a-BA, and f3-BA, acetyl-a-BA and acetyl-p-BA.

18. A formulation comprising the composition of claim 17 dissolved in at least one of: coconut oil, and medium chain triglycerides (MCT) containing either C8, C10, or C12 triglycerides.

19. A formulation comprising the composition of claim 17, further comprising the boswellic acid being dissolved in at least one of more organic solvents.

20. The formulation of claim 19 where the organic solvents include at least one of: Omega 3 polyunsaturated fatty acids, phosphatidylcholin, olive oil, flaxseed oil, walnut oil, ethanol, methanol, ethyl acetate, hexane,and dimethylsulfoxide,.

21. The composition of claim 17, when the boswellic acids are prepared from Boswellia serrata or Boswellia carterii or Boswellia papyrifera or Boswellia frereana.

22. The composition of claim 17 or the formulations in claims 18, 19 or 20 combined with other antiviral or immunomodulatory natural health products, dietary supplements, or drugs for adjunctive therapy.

23. The use of the composition of claim 17 or the formulations in claims 18, 19 or 20 as an antiviral agent to modulate the level of N F-KB in a mammal.

24. The use of claim 23, where the mammal is a human.

25. The use of the composition of claim 17 or the formulations in claims 18, 19 or 20 as an immunomodulatory agent to modulate the level of IL-la, I L-1f3, TNF-a, and IL-6 in a mammal.

26. The use of claim 25 where the mammal is a human.

27. The use of the composition of claim 17 or the formulations in claims 18, 19 or 20 to improve immune system health by promoting the proliferation of lymphocytes and the lowering of the neutrophil to lymphocyte ratio (NLR).

28. The use of the composition of claim 17 or the formulations in claims 18, 19 or 20 to prevent or alleviate severe inflammation or cytokine storm/hyperinflammation caused by respiratory viruses.

29. The use of claim 28 where the respiratory virus is influenza.

30. The use of claim 28 where the respiratory virus is a coronavirus.

31. A method for delivery of boswellic acids, where the boswellic acids are dissolved in a solvent, comprising delivery through the pulmonary system using a nebulizer or inhaler.

32. A method for delivery of boswellic acids, where the boswellic acids are dissolved in a solvent, comprising oral administration or other parenteral routes of administration.

33. An antiviral and immunomodulatory composition comprising:
Boswellia resin extract enriched for at least the four boswellic acids AKBA, KBA, a-BA, and f3-BA.

34. A formulation comprising the composition of claim 33 dissolved in at least one of: coconut oil, and medium chain triglycerides (MCT) containing either C8, C10, or C12 triglycerides.

35. A formulation comprising the composition of claim 33 dissolved in at least one of more organic solvents.

36. The formulation of claim 35 where the organic solvents include at least one of: Omega 3 polyunsaturated fatty acids, phosphatidylcholin, olive oil, flaxseed oil, walnut oil, ethanol, methanol, ethyl acetate, hexane, and dimethylsulfoxide.

37. The formulation in claim 33, when the extract is prepared from Boswellia serrata or Boswellia carterii or Boswellia papyrifera or Boswellia frereana.

38. The composition in claim 33 or the formulations in claims 34, 35 or 36 combined with other antiviral or immunomodulatory natural health products, dietary supplements, or drugs for adjunctive therapy.

39. The use of the composition in claim 33 or the formulations in claims 34, 35 or 36 as an antiviral agent to modulate the level of N F-KB in a mammal.

40. The use of claim 39, where the mammal is a human.

41. The use of the composition in claim 33 or the formulations in claims 34, 35 or 36 as an immunomodulatory agent to modulate the level of IL-1a, IL-113, TNF-a, and IL-6 in a mammal.

42. The use of claim 41 where the mammal is a human.

43. The use of the composition in claim 33 or the formulations in claims 34, 35 or 36 to improve immune system health by promoting the proliferation of lymphocytes and the lowering of the neutrophil to lymphocyte ratio (NLR).

44. The use of the composition in claim 33 or the formulations in claims 34, 35 or 36 to prevent or alleviate severe inflammation or cytokine storm/hyperinflammation caused by respiratory viruses.

45. The use of claim 44 where the respiratory virus is influenza.

46. The use of claim 44 where the respiratory virus is a coronavirus.

47. A method for delivery of the composition in claim 33 or the formulations in claims 34, 35 or 36, comprising delivery through the pulmonary system using a nebulizer or inhaler.

48. A method for delivery of the composition in claim 33 or the formulations in claims 34, 35 or 36, comprising oral administration or other parenteral routes of administration.