MEDICINAL AMBROSIA MARITIMA EXTRACTS

Abstract

Phytoceutical compositions from polar organic extracts of the Ambrosia maritima plants and uses thereof for treatment of coronavirus infections are described.

Claims

1. A method of treating a coronavirus infection, comprising administering an effective amount of a composition comprising a polar organic extract of a whole Ambrosia maritima plant together with a pharmaceutically acceptable carrier to a patient with a coronavirus infection.

2. The method of claim 1, wherein said effective amount is administered daily for at least 5 days.

3. The method of claim 1, wherein said polar organic extract comprises at least one sesquiterpene lactone.

4. The method of claim 1, wherein said polar organic extract comprises one or more of the following sesquiterpene lactone: parthenin, ambrosin, damsin, and neoambrosin.

5. The method of claim 1, wherein said polar organic extract comprises parthenin, ambrosin, damsin, and neoambrosin.

6. The method of claim 1, wherein said coronavirus infection is caused by a betacoronavirus.

7. The method of claim 6, wherein said betacoronavirus is selected from a group consisting of Middle East respiratory syndrome coronavirus (MERS), severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

8. The method of claim 1, wherein said coronavirus is SARS-CoV-2 and said patient is human, non-human primate, cat, dog or mink.

9. A method of treating a coronavirus infection, comprising administering an effective amount of a composition comprising sesquiterpene lactones extracted from a whole Ambrosia maritima plant together with a pharmaceutically acceptable carrier to a patient with a coronavirus infection.

10. The method of claim 9, wherein said effective amount is administered daily for at least 5 days.

11. The method of claim 9, wherein said coronavirus infection is caused by a betacoronavirus.

12. The method of claim 9, wherein said betacoronavirus is selected from a group consisting of Middle East respiratory syndrome coronavirus (MERS), severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

13. A method of preventing a coronavirus infection, comprising administering a prophylactically effective amount of a composition comprising a polar organic extract of a whole Ambrosia maritima plant together with a pharmaceutically acceptable carrier to a patient potentially exposed to a coronavirus.

14. The method of claim 13, wherein said effective amount is administered daily for at least 5 days.

15. The method of claim 13, wherein said polar organic extract comprises at least one sesquiterpene lactone.

16. The method of claim 13, wherein said organic extract comprises one or more of the following sesquiterpene lactone: parthenin, ambrosin, damsin, and neoambrosin.

17. The method of claim 13, wherein said organic extract comprises parthenin, ambrosin, damsin, and neoambrosin.

18. The method of claim 13, wherein said coronavirus infection is caused by a betacoronavirus.

19. The method of claim 18, wherein said betacoronavirus is selected from a group consisting of Middle East respiratory syndrome coronavirus (MERS), severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), and severe acute respiratory syndrome coronavirus 2 (SAR-CoV-2).

20. A method of preventing or treating a coronavirus infection in an animal, said method comprising daily administration of an effective amount of a pharmaceutical preparation comprising parthenin, ambrosin, damsin, and neoambrosin plus a pharmaceutically acceptable excipient to said animal for at least 5 days.

21. A method of treating a coronavirus infection, comprising: a) preparing a sesquiterpene lactone-containing extract comprising the steps of: i) treating a whole plant of Ambrosia maritima with a polar organic solvent in which one or more sesquiterpene lactones is soluble; ii) evaporating the polar organic solvent to produce a crude extract; iii) subjecting the crude extract to chromatography using a second organic solvent to obtain a clean extract of said one or more sesquiterpene lactones; iv) collecting fractions of the individual one or more sesquiterpene lactones to form said sesquiterpene lactone-containing extract; and, b) adding a pharmaceutically acceptable carrier to said sesquiterpene lactone-containing extract to form a pharmaceutical composition; and, c) administering an effective amount of said pharmaceutical composition to a patient with a coronavirus infection.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0080] FIG. 1A. Chemical structure of exemplary SL Ambrosin with arrows pointing to the Michael reaction points.

[0081] FIG. 1B. Chemical structure of three additional exemplary SLs found in Ambrosia plants.

[0082] FIG. 1C. Chromatographic fingerprints of sesquiterpene lactones extracted from Ambrosia maritima.

[0083] FIG. 2. Relationship between cytotoxicity (%) and concentration (μg/mL) of a purified organic extract from Ambrosia maritima.

[0084] FIG. 3A. Coronovidea family with known human pathogens.

[0085] FIG. 3B. Coronovidea family with human and animal pathogens.

DESCRIPTION OF EMBODIMENTS OF THE DISCLOSURE

[0086] The disclosure provides pharmaceutical compositions comprising plant-based compounds and novel methods pertaining to their use in coronavirus infection prevention and treatments. In particular, the polar organic extract from whole Ambrosia maritima plants in the Asteraceae family is used for prevention and treatment of various coronaviruses infections, including betacoronaviruses. One particular group of components in the organic extract, sesquiterpene lactones, have been found to interfere with viral entry and viral replication pathways. This reduces the viral load and thus infection level, enabling effective prevention and/or treatment.

[0087] Extraction Method. Briefly, the extraction uses an organic solvent such as methanol, ethanol and any carbon-based solvents, although a polar organic solvent is preferred. The whole organic extract is dried and chemically fractionated with at least one second organic solvent such as chloroform. The final step is to pool the best fractions and evaporate the second organic solvent(s). The final product can then be used in treatment.

[0088] In more detail, the extraction method of Ambrosia maritima can be as follows:

[0089] 1) Dried whole Ambrosia maritima plant is powdered using conventional means.

[0090] 2) The powdered plant is brought into contact with an organic solvent or combination of organic solvents and allowed to contact the plant matter for a predetermined amount of time for the SLs in the dried plant to move into the organic phase. This organic solvent(s) is preferably polar.

[0091] 3) The organic solvent(s) is then separated from the dried plant material.

[0092] 4) The organic extract is dried by any known means in the art.

[0093] 5) The dried extract is chemically fractionated with second organic solvent and fractions rich in the requisite SLs pooled. In some embodiments, the second organic solvent is preferably non-polar, such as chloroform.

[0094] 6) The second organic solvent (e.g. chloroform) is evaporated, leaving behind the dried, pharmaceutically active components, also referred to as a purified or clean extract.

[0095] 7) Different concentrations of the active components are used in treating various coronaviruses.

[0096] The same extraction steps can be performed for other plants in the Asteraceae family.

[0097] Other extraction methods can be employed, as suitable for organic soluble components. For example, such methods include, aqueous two-phase systems, acid/base extractions, and the like. To prepare the plant for extraction the plant is typically dried in air with no heat, then further processed by freeze thawing cycles, and/or physically lysed by freezing and thawing before extraction, and the like.

[0098] Administration Method. The active ingredient or ingredients of the organic extract of the plant material can be combined with other active ingredients before use, but preferably are used alone. The active ingredient or ingredients of the organic extract of plant material can be used as is, or can be formulated with known pharmaceutically acceptable carriers, diluents and/or excipients.

[0099] For example, gelatin capsules containing dried organic extract can be produced containing a suitable dose of the active ingredient(s). Optionally, packets containing the dried extract can be provided for mixture with e.g., hot fluids, to be taken orally. The extract can also be formulated with solid carriers for pressing into pill forms, especially with delayed release excipients for formulating once a day tablet forms or any pharmaceutical form that will be orally administered.

[0100] It may also be possible to prepare forms of the active ingredients suitable for non-oral routes of administration, such as inhalational, buccal, sublingual, nasal, suppository or parenteral dosage forms.

[0101] The above extract is significantly more stable than the natural product, even in liquid form and especially when formulated with a buffer and a chelator. In fact, stability is predicted to be improved by at least 3, 4, 5 or more orders of magnitude. It is also significantly more concentrated than the natural form, thus providing efficacy without having to consume vast quantities of plant material. Further, the dosage is much more easily controlled with concentrated, partially purified or purified material.

[0102] We also used TLC and HPLC to further purify the active ingredient(s) to be further studied and to determine their efficacy, although this work is ongoing. The removal of certain components from the organic extract, through the additional purification step can lead to fewer side effects. Ambrosia maritima is a ragweed, which is known for its allergenic effects. Thus, purification of the organic extract will lead to a reduced immune response in humans. In some embodiments, purified material from the organic extract of the Ambrosia maritima can serve to improve the ability to interrupt viral entry and replication, and increase stability of the extracted material.

[0103] Once the pharmaceutical composition is prepared, the drug is administered daily, preferably 1 to 4 times a day, and most preferably 3 times a day. The amount of purified organic extract, or active ingredients obtained therefrom, in each dose is between 250 mg to 750 mg, or about 500 mg. The length of dosing is at least 5 days, wherein preferred dosing ranges are 5-10 days, 8 to 20 days or 15 to 30 days. Prophylactical use may be longer depending on context and potential side effects, although none are yet known. Daily treatment by oral administration for 1-4 weeks is preferred, but in some cases, the length of time would be extended according to the extent of the coronavirus infection and/or the type of coronavirus.

[0104] The pharmaceutical composition can be administered before or after a patient has tested positive for a coronavirus infection. In some embodiments, the pharmaceutical composition can be administered, as a preventative treatment, to a patient exposed to a coronavirus even if the patient is not exhibiting symptoms or testing positive or a patient with no known exposure but in a high risk environment. In other embodiments, the pharmaceutical composition can be administered to a patient testing positive for a coronavirus but not exhibiting symptoms, or for a patient testing positive for a coronavirus and exhibiting symptoms.

[0105] The sesquiterpene lactones (SLs) in the present methods can be used to target a variety of coronavirus-specific pathways for entry and replication. Not all pathways involved in a coronavirus's entry into a host cell, a coronavirus infection's growth, and coronavirus infection's progression are known. Thus, the following examples are intended to be illustrative only, and not unduly limit the scope of the appended claims. Those of skill in the art should appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure herein. Further, though a betacoronavirus was utilized in the following examples, the present treatment methods are expected to be useful for treatment of most coronaviruses, either alone or in combination with other treatment modalities. In no way should the following examples be read to limit, or to define, the scope of the appended claims.

EXAMPLES

[0106] A pharmaceutical composition was prepared per the above-described methods to evaluate the ability of an Ambrosia maritima extract to treat coronavirus infections.

[0107] Unless otherwise noted, a purified polar organic extract from Ambrosia maritima was obtained by dipping the powder of the dried whole plant material into ethanol to extract the active polar compounds such as the SLs. The mixture was allowed to set at room temperature for 2 days before filtering the plant material and collecting the extract, which contained the organic solutes. The polar organic solvent was then dried off using conventional drying methods. The presence of at least four SLs, parthenin, ambrosin, damsin, and neoambrosin, were confirmed in the organic extract. These four SLs were chemically fractionated with chloroform, wherein the fractions rich in these SLs were then pooled. The chloroform was evaporated off and the fractionated material was washed with ethanol to fully rid it of chloroform. The ethanol was then removed, leaving behind the dried, pharmaceutically active components, which is referred to as “purified polar organic extract” in the following examples. This dried purified polar organic extract was suitable for use in the following cytotoxicity and in vitro examples. It was combined with dimethyl sulfoxide (DMSO) to form a 30 vol. % solution. For the in vivo “clinical” example, 500 mg of the purified polar organic extract was combined with polyethylene glycol to form a soft gelatin capsule weighing 1.22 g. This pharmaceutical composition is referred to as Composition 1 in the Examples below.

[0108] In some of the plaque reduction assays, a composition with just the fractions rich in ambrosin and damsin were prepared. The fractionation and purification steps were the same as the purified polar organic extract, and this composition was also combined with DMSO to form a 30 vol. % solution.

Cytotoxicity Assay

[0109] The cytotoxicity of the purified polar organic extract was evaluated using serial dilutions of the purified polar organic extract, from 500 μg/ml to 15.65 μg/mL, using a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

[0110] For the MTT cytotoxicity assay (IC.sub.50), samples were diluted with Dulbecco's Modified Eagle's Medium (DMEM). Stock solutions of the test compounds were prepared in 10% DMSO in ddH.sub.2O. The cytotoxic activity of the extracts was tested in Vero E6 cells by using the MMT assay in Mosmann (1983) with minor modification. Briefly, the cells were seeded in 96 well-plates (100 μL/well at a density of 3×105 cells/mL) and incubated for 24 hours at 37° C. in 5% CO.sub.2. After 24 hours, cells were treated with various concentrations of the tested compounds in triplicates. After a further 24 hours, the supernatant was discarded, and cell monolayers were washed with sterile phosphate buffer saline (PBS) 3 times. MTT solution (20 μL of 5 mg/mL stock solution) was added to each well and incubated at 37° C. for 4 hours followed by medium aspiration. In each well, the formed formazan crystals were dissolved with 200 μL of acidified isopropanol (0.04 M HCl in absolute isopropanol=0.073 mL HCL in 50 mL isopropanol). Absorbance of formazan solutions were measured at λ.sub.max=540 nm with 620 nm as a reference wavelength using a multi-well plate reader (SpectraMax M5. The percentage of cytotoxicity compared to the untreated cells was determined with Equation 1. The plot of % cytotoxicity versus sample concentration was used to calculate the concentration which exhibited 50% cytotoxicity (IC50).

[00001]$\begin{array}{cc}\%\mathrm{cytotoxicity}=\frac{\left(\mathrm{absorbance}\mathrm{of}\mathrm{cells}\mathrm{without}\mathrm{treatment}-\mathrm{absorbance}\mathrm{of}\mathrm{cells}\mathrm{without}\mathrm{treatment}\right)\times 100}{\mathrm{absorbance}\mathrm{of}\mathrm{cells}\mathrm{without}\mathrm{treatment}}& \mathrm{Equation}1\end{array}$

[0111] The results are shown in Table 1 and FIG. 2.

TABLE-US-00002 TABLE 1 Cytotoxicity results for a purified extract from Maritima Concentration of Purified polar organic extract (μg/mL) Cytotoxicity (%) 500 98.894 250 98.894 125 85.806 62.5 55.023 31.25 15.207 15.65 8.756

[0112] The cytotoxicity varied from about from 98.894% to 8.75%. As expected, the larger concentrations of the purified extract had about 100% cytotoxicity. According to the results shown in FIG. 2, the IC50 was 103.2 μg/ml in the A549 cell lines. From these results, it was determined that the suitable concentrations for the plaque reduction test were 62.5 μg/mL, 31.25 μg/mL, and 15.65 μg/mL, as each was much lower than the IC50, indicating their lack of toxicity.

Plaque Reduction Test

[0113] Plaque reduction tests were performed to measure the presently disclosed composition's ability to inhibit a COVID-19 infection.

[0114] The plaque reduction assays were carried out according to the method of Hayden et al., 1980. The sample mixtures prepared for the plaque reduction test are as follows: A ‘blank’ mixture were prepared with SARS-CoV-2 (Wuhan, CN) that was diluted to give 10.sup.5 plaque forming units (PFU)/well. This mixture was used for the control wells (for untreated virus). For the sample mixtures, 120 μL of the diluted SARS-CoV-2 was combined with 120 μL of a purified polar organic extract from Ambrosia maritima having a concentration of 62.5 μg/mL, 31.25 μg/mL, 15.65 μg/mL and 7.8 μg/mL. The cells used in the present example were Vero E6 (also called Vero C1008). A growth medium for culturing the Vero E6 cells contained DMEM, 2% fetal bovine serum (FBS) and 1% of an antibiotic solution (Gibco). Additional media used in the assay included an overlayer medium consisting of 2% agarose (autoclaved) in DMEM, and a “2× medium” consisting of DMEM 2% FBS+L-glutamine+Sodium Bicarbonate.

[0115] In more detail, Vero E6 cells (also called Vero C1008) were placed in a six well plate at a concentration of 105 cells/mL, and cultivated for 24 hours at 37° C. with 5% CO.sub.2. Meanwhile, the SARS-CoV-2 was diluted to give 105 plaque forming units (PFU)/well and mixed with a safe concentration of the purified polar organic extract to form the sample mixture and incubated for 1 hour at 37° C. After the incubation period, the growth medium was removed from the cell culture plates, and the cells were inoculated with 100 μL of the sample mixture per well.

[0116] After 1 hour of contact time for virus adsorption, the supernatant was then removed and the cells were washed with phosphate buffer solution (PBS) twice. Then, 3 mL of an overlayer medium (DMEM medium supplemented with 2% agarose) and 2X medium (1:1 ratio) were added onto the cell monolayer. Plates were left to solidify and incubated at 37° C. with 5% CO.sub.2 until formation of viral plaques (3 to 4 days).

[0117] To fix the cells, formalin (10%) was added and, after for two hours, the plates were stained with 1.5 mL of 0.1% crystal violet (Sigma Aldrich) in distilled water. The stained cells were then analyzed for plaque reduction.

[0118] Control wells using the untreated virus (blank mixture) were also prepared, incubated with Vero E6 cells, and processed in same way. The percentage of reduction in plaques formation in comparison to the control wells was recorded as: % inhibition={viral count (untreated)−viral count (treated)/viral count (untreated)}×100. The results are shown in Table 2.

TABLE-US-00003 TABLE 2 Results from Plaque reduction test Concentration of purified polar Initial Viral Viral count organic extract count after treatment Inhibition (μg/mL) (PFU/mL) (PFU/mL) (%) 62.50 100*10.sup.5  3*10.sup.5 97 31.25 25*10.sup.5 75 15.60 45*10.sup.5 55 7.80 52*10.sup.5 48 PFU = plaque forming units

[0119] The results in Table 2 show that the addition of the presently disclosed compositions decreases the viral count. As little as 7 μg/mL of the purified polar organic extract from maritima inhibited viral growth by 48%. Increasing the amounts of the purified polar organic extract decreased the viral infection. As just 62.5 μg/mL, an inhibition rate of 97% was observed, which is a high efficacy against COVID-19.

[0120] In addition to an extract with ambrosin, damsin, parthenine, and neoambrosin, a plaque reduction assay was also performed for a second sample that included just ambrosin and damsin rich fractions. This mixture also showed efficacy with great result in inhibiting viral replication. As little as 62.50 μg/mL inhibited viral growth by 97%. Thus, both of these SLs can be used alone or in combination with any of the other SLs, and decreases in the viral infection will still be seen.

Clinical Results

[0121] A double-blind control testing was performed on a group consisting of 104 consenting human subjects. The human subjects were both males and females, aged 18 to 45 with an average weight of 70-80 kg and height of 150-175 cm. Each subject had tested positive for COVID-19 using a PCR test.

[0122] The subjects were split into two sub-groups of 52 participants each. The first sub-group took a placebo. The second sub-group, also called the ‘test’ group, was treated with 500 mg of Composition 1 every 12 hours.

[0123] The test sub-group tested negative for COVID-19 after 5 days of treatment, with a cure rate of 100% after five days of treatment. It was also noted that improvements in symptoms were observed within 3 days of the first treatment with Composition 1. In contrast, all fifty-two participants in the first sub-group tested positive for COVID-19 after 5 days, and returned positive COVID-19 tests for up to 15 days.

[0124] It has been shown by the above examples that the presently described pharmaceutical compositions obtained using extracts from the martima plant can be used to treat coronavirus infections. However, this is exemplary only, and the invention can be broadly applied to any Ambrosia plant. Further, any SLs found in these plants are expected to show some level of cytotoxicity for treatment and prevention of coronavirus infections. The foregoing examples are intended to be illustrative only, and not unduly limit the scope of the appended claims.

[0125] The following references are incorporated by reference in their entirety. [0126] Tackholm, Vivi. (1974). Student's flora of Egypt. 2nd edition, Cairo University, Egypt. [0127] Sherif, A. F. and M. F. El-Sawy, (1977). Field trials of the Molluscicidal action of Ambrosia maritima (Damesisa). Bull. High Inst. Puplic Health Alex. 7:1-4. [0128] El Sawy M F, El Hamd Z M S, Loutfy N F, El Masry S and Abdel Gualil M Z (1986): J of the Egypt. Society of Parasitology, 16:1, pp 57-64. [0129] Abdallah, O. M.; Ali, A. A. and Itokawa, H. (1991). “Cytotoxic activity of Sesquiterpene lactones isolated from Ambrosia maritima”. Pharmazie, 46(6):472. [0130] Badawy, M.; Abdelgaleil, S. A. M.; Suganuma, T. and Fuji, M. (2014). “Antibacterial and biochemical activity of Pesudoguaianolide Sesquiterpene isolated from Ambrosia maritima against plant pathogenic bacteria”. Plant protect. Sci. 50 (2): 64-69. [0131] Hayden, F. G.; Cote, K. M. and Douglas Jr, R. G. (1980). “Plaque inhibition assay for drug susceptibility testing of influenza viruses”, Antimicrobial Agents and Chemo., 17 (5): 865-870. [0132] Mosmann T (December 1983). “Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays”. Journal of Immunological Methods. 65 (1-2): 55-63.