ANTI-VIRAL COMPOSITION AND METHOD FOR PREPARING THE SAME

Abstract

A composition and some methods for preparing the same are disclosed. The methods result in the extraction of a wide array of polyphenols and triterpenes. The resulting composition is a mixture rich in anti-viral, antioxidant, and anti-inflammatory ingredients that may be administered in various forms or methods of application or composition. Further, the composition of the present invention is used in the treatment or prevention of herpes simplex virus (HSV), aphthous, and other mouth ulcers.

Claims

1. A composition exhibiting anti-viral, anti-oxidant, or anti-inflammatory properties derived from sweet basil (Ocimum basilicum) for inhibiting and treating herpes simplex virus (HSV), aphthous, and other mouth ulcers.

2. A composition of claim 1, wherein the sweet basil extract comprises a therapeutically effective amount of bioactive ingredient.

3. A composition of claim 1, wherein the sweet basil extract is, or is derived from, a water extract at ambient temperature.

4. A composition of claim 1, wherein the sweet basil extract is, or is derived from, a water extract at elevated temperature and pressure.

5. A composition of claim 1 or 3, wherein the bioactive ingredient in the composition comprises at least one or a combination of glucosyringic acid, tryptophane, folinic acid, 3-ethoxy-4,5-dihydroxybenzoic acid, apocynoside, sinapic acid, rutaevin, vitamin B2, kaempferol-3-o-rutinoside, 3-hydroxy-4-methoxy-cinnamic acid, apocynoside, 4-ethyl-paeoniflorin, tamariscinoside B, 3β-hydrosantamarine-1-o-β-d-glucopyranoside, esculentoside P, esculentagenic acid, arjunglucoside I, esculentoside B or a mixture thereof.

6. A composition of claim 1 or 4, wherein the bioactive ingredient in the composition comprises of at least one or a combination of cichoriin, phenyl propionic acid, glucosyringic acid, tryptophane, 1-O-caffeoyl-β-D-glucopyranoside, apocynosideII, sinapic acid, 3-hydroxy-4-methoxy-cinnamic acid, quercetin-3-O-glucuronide 6″-methylester, roseoside 1, vitamin B2, kaempferol-3-O-rutinoside, 5,6,4′-trihydroxy-7,8,3′-trimethoxyflavone, 4-ethyl-paeoniflorin, tamariscinoside B, chicoric acid, rosmarinic acid, monocaffeoyltartaric acid (Caftaric acid), kaempferol-3-O-β-D-glucoside-7-O-α-L-arabinofuranoside, hookeroside D, kaempferol-3-O-rutinoside, salvianic acid A, salvianolic acid A, esculentoside P, esculentagenic acid, arjunglucoside or a mixture thereof.

7. A composition of any of the preceding claims, wherein the bioactive ingredients are blended to form and administer in the form or method of application or composition of a cream, lotion, liquid, emulsion, spray, serum, balm, gel, lip balm, ointment, powder, pill, tablet, consumable, ingestible, patch, powder, mouthwash, toothpaste, lozenge, mint, candy, chewing gum or similar for use in the treatment or prevention of herpes simplex virus (HSV), and/or aphthous or other mouth ulcers.

8. A method for preparing sweet basil extract or composition rich in anti-viral, anti-oxidant, and anti-inflammatory ingredients at an ambient temperature or room temperature, wherein the method comprising the steps of: a) harvesting of fresh leaves and air-drying the leaves for extraction; b) shredding or chopping the fresh leaves that are ready for extraction; c) adding the shredded leaves to a solvent and homogenizing/lightly grinding for a period of time; d) extracting bioactive ingredients using the solvent to obtain some extract rich in bioactive ingredients; e) filtering the extract; f) repeating the extraction of bioactive ingredients from the residue in the filter with additional solvent; g) combining the extracts of steps (e) and (f), and drying the extract to obtain a powdered extract.

9. A method of claim 8, wherein the sweet basil has an effective amount of bioactive ingredient.

10. A method of claim 8, wherein the sweet basil extract derived at ambient temperature comprises of an effective amount of bioactive ingredient with anti-viral, anti-oxidant, or anti-inflammatory properties.

11. A method of claim 8, wherein the bioactive ingredient in the sweet basil extract at ambient temperature comprises at least one or a combination of glucosyringic acid, tryptophane, folinic acid, 3-ethoxy-4,5-dihydroxybenzoic acid, apocynoside, sinapic acid, rutaevin, vitamin B2, kaempferol-3-o-rutinoside, 3-hydroxy-4-methoxy-cinnamic acid, apocynoside, 4-ethyl-paeoniflorin, tamariscinoside B, 3β-hydrosantamarine-1-o-β-d-glucopyranoside, esculentoside P, esculentagenic acid, arjunglucoside I, esculentoside B, stachyose, raffinose or a mixture thereof.

12. A method of claim 8, wherein the sweet basil extract is dried using a freeze dryer, spray-dryer, vacuum concentrator, dehydrator, oven, or other suitable drying methods.

13. A method for preparing sweet basil extract or composition rich in anti-viral, anti-oxidant, and anti-inflammatory ingredients at an elevated temperature and pressure, wherein the method comprising the steps of: a) harvesting of fresh leaves and air-drying the leaves for extraction; b) shredding or chopping the fresh leaves that are ready for extraction; c) adding the shredded leaves to a solvent and homogenizing/lightly grinding for a period of time; d) placing the homogenized leaves in an autoclave for a period of time; e) extracting bioactive ingredients using the solvent to obtain some extract rich in bioactive ingredients; f) filtering the extract, and g) drying the extracts to obtain a powdered extract.

14. A method of claim 13, wherein the sweet basil has an effective amount of bioactive ingredient.

15. A method of claim 13, wherein the sweet basil extract derived at elevated temperature and pressure comprises of an effective amount of bioactive ingredient with anti-viral, anti-oxidant, or anti-inflammatory properties.

16. A method of claim 13, wherein the bioactive ingredient in the sweet basil extract at elevated temperature and pressure comprises of at least one or a combination of cichoriin, phenyl propionic acid, glucosyringic acid, tryptophane, 1-O-caffeoyl-β-D-glucopyranoside, apocynosideII, sinapic acid, 3-hydroxy-4-methoxy-cinnamic acid, quercetin-3-O-glucuronide 6″-methylester, roseoside 1, vitamin B2, kaempferol-3 -O-rutinoside, 5,6,4′-trihydroxy-7,8,3′-trimethoxyflavone, 4-ethyl-paeoniflorin, tamariscinoside B, chicoric acid, rosmarinic acid, monocaffeoyltartaric acid (Caftaric acid), kaempferol-3-O-β-D-glucoside-7-O-α-L-arabinofuranoside, hookeroside D, kaempferol-3-O-rutinoside, salvianic acid A, salvianolic acid A, esculentoside P, esculentagenic acid, arjunglucoside, Fraxin, isomaltose, raffinose, stachyose or a mixture thereof.

17. A method of claim 13, wherein the sweet basil extract is dried using a freeze dryer, spray-dryer, vacuum concentrator, dehydrator, oven, or other suitable drying methods.

18. A method of any of the preceding claims, wherein the sweet basil extract derived at ambient temperature or room temperature is blended with the sweet basil extract derived at elevated temperature and pressure.

19. A method of any of the preceding claims, wherein the resultant composition is a mixture of bioactive ingredients that are blended to form and administer in the form or method of application or composition of a cream, lotion, liquid, emulsion, spray, serum, balm, gel, lip balm, ointment, powder, pill, tablet, consumable, ingestible, patch, powder, mouthwash, toothpaste, lozenge, mint, candy, chewing gum or similar for use in the treatment or prevention of herpes simplex virus (HSV), and/or aphthous or other mouth ulcers.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0028] The foregoing summary, as well as the following detailed description of the invention, is better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, exemplary constructions of the invention are shown in the drawings. However, the invention is not limited to the specific methods and structures disclosed herein. The description of a method step or a structure referenced by a numeral in a drawing is applicable to the description of that method step or structure shown by that same numeral in any subsequent drawing herein.

[0029] FIG. 1 shows a method for preparing a sweet basil extract at ambient temperature (WEAT) in one embodiment of the present invention.

[0030] FIG. 2 shows a method for preparing a sweet basil extract at elevated temperature and pressure (WEETP) in one embodiment of the present invention.

[0031] FIG. 3 shows a bar chart illustrating the relative quantification of phytochemicals present in the water extraction at ambient temperature in one embodiment of the present invention.

[0032] FIG. 4 shows a bar chart illustrating the relative quantification of phytochemicals present in the water extraction at elevated temperature and pressure (121° C., 15 psi) in one embodiment of the present invention.

[0033] FIG. 5 shows a graph illustrating the percentage cell viability of Vero cells against the water extract of sweet basil (WEAT and WEETP) after 72 hours of incubation in one embodiment of the present invention.

[0034] FIG. 6 shows a graph illustrating the percentage cell viability of HSV infected Vero cells (or percentage protection) against the water extract of sweet basil (WEAT and WEETP) after 72 hours of incubation in one embodiment of the present invention.

DETAILED DESCRIPTION OF EMBODIMENTS

[0035] The present invention is best understood by reference to the detailed figures and description set forth herein.

[0036] It is expected that the present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments are to be considered in all respects only as illustrative and not restrictive. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description. All changes that come within the meaning and range of equivalency of the claims are to be embraced within their scope.

[0037] Medicinal plants or herbs have been used for thousands of years to treat a wide array of ailments. Sweet basil (Ocimum basilicum) is one such medicinal plant. It is a popular culinary herb used in Chinese and Ayurvedic herbal systems for centuries. Sweet basil is abundant in essential oil as well as polyphenols. In addition, herbal treatments, constituting a wide array of phytochemicals, are thought to act synergistically together at multiple targets to produce a beneficial effect. Further, sweet basil has also been reported to have anti-inflammatory activity, possibly through inhibition of the Tumor necrosis factor receptor superfamily member 9 (Tnfrsf9) expression. The combination of anti-virals and anti-inflammatory preparations has produced a marked reduction in clinical signs of herpes. All of the above distinguishing features confirm sweet basil is an excellent candidate for HSV treatments, more so for drug-resistant HSV strains.

[0038] Currently, ethanol is the most commonly used bio-solvent in herb extraction. However, due to safety and environmental concerns, recent extraction techniques have primarily focused on finding solutions that minimize the use of harmful solvents while ensuring cost-effectiveness and the production of high-quality extracts through green extraction techniques. One of the best solvents to use is water. At room temperature and pressure, water has a dielectric constant of ca. 80, making it a polar solvent. However, this value can be considerably lowered under a pressurized hot water extraction technique; for example, water at 250° C. and 4 Mpa can mimic the extraction capacity of ethanol and is suitable for the extraction of low-polarity compounds.

[0039] Below are a few examples illustrating the use of sweet basil leaves in the treatment of canker sores and cold sores.

[0040] In one example, a female who had suffered from canker sores since she was in elementary school suffered from increased frequency and duration of canker sores in her thirties. At one point within the last six months of a single year, she experienced four canker sores. She observed her sores usually took approximately two weeks from the time she first noticed the tingling or discomfort indicative of a sore to completion of healing. Because of the length of time until healed, this meant she suffered from an aphthous ulcer for two months out of the six-month time period- approximately 33% of the time. These ulcers were larger and closer to her lip than she had experienced previously, limiting her eating, and in the worst cases, preventing her ability to form a smile or speak normally. Later, she began applying masticated sweet basil leaves when she felt a canker sore coming on because sweet basil was one of only two plants she had in her residence. When applied early, the masticated basil, or bolus thereof, often prevented the actualization of the ulcer. When applied after the ulcer had materialized, the basil ameliorated the pain and accelerated healing time significantly. Because she is prone to aphthous ulcers, when she accidentally bit her cheek or tongue, it almost invariably would become a canker sore. She began applying the masticated basil leaves proactively to the sites she had bitten and observed this prevented the formation of the canker sores at the trauma site. She has used the plant material effectively to accelerate healing or prevent the actualization of canker sores in at least two dozen instances.

[0041] In another example, a female in her thirties applied masticated basil leaves to the burning spot on her lip—the burning she usually felt proceeding a cold sore (also known as a fever blister). She applied it again before bed, leaving the plant matter pressed onto and over her lip overnight to attempt to hold it in place. The cold sore did not actualize. Though the plant matter dried and hardened uncomfortably and painfully to the lip and stained the lip and skin around the mouth green, evidencing the need for a more practical application method. The next time the same female felt another fever blister coming on, she applied the plant matter in the morning, again after eating meals, and before bed, leaving it on overnight as best she could. When she woke the next morning, the blister had already scabbed; within another 24 hours the scab had fallen off leaving a slightly lighter pink impression than her natural lip color. Out of three different times this female has felt an imminent fever blister and applied the plant matter, once the fever blister did not materialize; once the blister materialized but the burning was attenuated significantly and the blister healed within 2.5 days; and once the blister scabbed rapidly with the scab falling off the next day. The female reports not experiencing her first fever blister until the age of 28 or 29, and usually experiences them during high-stress periods, but has not had a recent recurrence despite describing the time as one of the most stressful of her entire life. Additionally worth noting, this female suffered from both chickenpox and shingles (HSV-3) as a child.

[0042] In both of the above examples, the individual was limited in universally treating the imminent cold sores or canker sores she felt coming on due to several factors including being unable to have the fresh plant on hand at all times - live basil plants in the United States are commonly infected with stem rot fungus (Fusarium oxysporum f. sp. basilicum) which causes the plants to die quickly and pre-cut basil leaves bought from a market have an even shorter life, often of only a 2-4 days; difficulty keeping the plant matter on the impending cold sore or canker sore site; and the physical visibility of, discomfort, and green stain resulting from, plant matter application. The use of a form or method of application including for example, but not limited to, a liquid, emulsion, spray, serum, gel, balm, lip balm, cream, lotion, ointment, pill, consumable, ingestible, patch, powder, mouthwash, toothpaste, lozenge, chewing gum, or similar other treatment containing active components of basil eliminates many of those limitations. Additionally, the WEETP extraction method described herein significantly amplifies the anti-viral components and efficacy of the plant against HSV. While the WEAT extraction method maintains a profile thought to most similarly replicate the use of the raw plant matter but allows for stabilization.

[0043] In one embodiment, the present invention relates to anti-viral compositions. In one embodiment, the present invention further relates to a process for preparing the anti-viral composition that is useful in reducing herpes simplex virus (HSV) and treating viral infections, aphthous, and other mouth ulcers. In one embodiment, the composition is based upon constituents isolated from or contained within that of sweet basil. In one embodiment, further, a method of producing plant-based formulation with anti-HSV activity is described.

[0044] In one embodiment, the method of the present invention is an effective herbal composition utilizing standard extraction methods or a green extraction technique to extract and formulate a unique herbal composition with anti-HSV, anti-oxidant and anti-inflammatory properties. The green method includes water extraction at ambient temperature (WEAT) and water extraction at elevated temperature and pressure (WEETP). Both these methods involve homogenization, filtration, and enrichment steps to optimize the specific phytochemical content of the resulting therapeutic composition. Additionally, the WEETP extraction method described significantly amplifies the anti-viral components and efficacy of the plant against HSV. While the WEAT extraction method maintains a profile thought to most similarly replicate the use of the raw plant matter but allows for stabilization.

[0045] In one embodiment, the method includes pharmacologic compositions derived from the herb Ocimum basilicum with abundant polyphenol and triterpene content. The compositions may be used topically, orally, or parenterally and exhibit strong anti-viral and anti-oxidant activities. In one embodiment, the compositions also contain anti-inflammatory ingredients. They are highly useful in treating viral infections such as HSV.

[0046] Referring to FIG. 1, a method 100 for preparing a composition or plant-based water extract in one embodiment of the present invention. At step 102, preferably the herb sweet basil (Ocimum basilicum) leaves are harvested without adding the fruits, stem, and roots. Once the leaves are harvested, they are washed and air-dried. If there is limited time for extraction, place the air-dried leaves in a plastic bag and store them in the freezer for up to 2 to 3 days for later extraction. At step 104, the leaves are shredded or chopped into pieces, and the plant material is ready for the extraction step. At step 106, the shredded leaves are added to a solvent and homogenized, then lightly ground for 15 minutes. In one embodiment, the solvent may be water. In one embodiment, the shredded leaves are added to the 2 liters of water and homogenized, then lightly ground for 15 minutes. At step 108, the bioactive ingredients are extracted using a solvent to obtain some extract rich in bioactive ingredients. The extract may be derived from a water extraction at room temperature or ambient temperature. At step 110, the extract is filtered using a filter paper placed in a funnel. At step 112, the extraction is repeated with the residue in the filter paper with additional solvent. In one embodiment, the solvent may be water. In one embodiment, the residue in the filter paper is filtered again using another 2 liters of water. At step 114, the extracts of steps 110 and 112 are combined together. At step 116, the extract is dried to obtain a powdered extract. In one embodiment, further the extract is dried or dehydrated at a maximum temperature of 50° C. (122° F.). In one embodiment, the extract is dried using a freeze dryer, spray-dryer, vacuum concentrator, dehydrator, oven, or other drying methods. Further, the extract can be stored in an air-tight container in the freezer and lasts for months.

[0047] In one aspect of the present invention, the method 100 uses a plurality of active ingredients including, but not limited to, syringic acid, tryptophane, vitamin B9, benzoic acid, apocynoside, cinnamic acid, rutaevin, vitamin B2, kaempferol, paeoniflorin, tamariscinoside, hydrosantamarine, esculentoside, esculentagenic acid, arjunglucoside, and esculentoside.

[0048] In another aspect of the present invention, the method 100 uses a plurality of active ingredients. The active ingredients of the water extract at an ambient temperature of the composition contain raffinose, stachyose, glucosyringic acid, tryptophane, folinic acid, 3-ethoxy-4,5-dihydroxybenzoic acid, apocynoside, sinapic acid, rutaevin, vitamin B2, kaempferol-3-o-rutinoside, 3-hydroxy-4-methoxy-cinnamic acid, apocynoside, 4-ethyl-paeoniflorin, tamariscinoside B, 3β-hydrosantamarine-1-o-β-d-glucopyranoside, esculentoside P, esculentagenic acid, arjunglucoside I, esculentoside B.

[0049] In one embodiment, the extract derived at ambient temperature comprising of an effective amount of sweet basil active ingredient having anti-oxidant activity. In one embodiment, the water extract at ambient temperature comprising of an effective amount of sweet basil active ingredient having anti-viral activity. In one embodiment, the extract derived at ambient temperature comprising of an effective amount of sweet basil active ingredient having anti-inflammatory activity.

[0050] Accordingly, the active ingredient of the water extract at ambient temperature comprises at least one or a combination of glucosyringic acid, tryptophane, folinic acid, 3-ethoxy-4,5-dihydroxybenzoic acid, apocynoside, sinapic acid, rutaevin, vitamin B2, kaempferol-3-o-rutinoside, 3-hydroxy-4-methoxy-cinnamic acid, apocynoside,4-ethyl-paeoniflorin, tamariscinoside B, 3β-hydrosantamarine-1-o-β-d-glucopyranoside, esculentoside P, esculentagenic acid, arjunglucoside I, esculentoside B.

[0051] Several factors are critical for the final extract, including part of the herb used, complete drying of extract at low temperature, though drying to a lesser extent may be appropriate for extracts for use in some compositions (for example, but not limited to, a cream or lotion), and storage conditions. Adherence to these factors will result in the optimal pharmacologic composition.

[0052] Referring to FIG. 2, a method 200 for preparing a composition or plant-based water extract at elevated temperature and pressure in one embodiment of the present invention. At step 202, preferably the herb sweet basil (Ocimum basilicum) leaves are harvested without adding the fruits, stem, and roots. Once the leaves are harvested, they are washed and air-dried. If there is limited time for extraction, place the air-dried leaves in a plastic bag and store them in the freezer for up to 2 to 3 days for later extraction. At step 204, the leaves are shredded or chopped into fine pieces, and the plant material is ready for the extraction step. At step 206, the shredded leaves are added to a solvent and homogenized, lightly ground for 15 minutes. In one embodiment, the solvent may be water. In one embodiment, the shredded leaves are added to the 4 liters of water and homogenized, lightly ground for 15 minutes. At step 208, place the mixture in a pressure cooker or autoclave. At step 210, the bioactive ingredients are extracted using a solvent to obtain some extract rich in bioactive ingredients. At step 212, the extract is filtered using a filter paper placed in a funnel. At step 214, the extract is dried to obtain a powdered extract. In one embodiment, further, the extract is dried or dehydrated at a maximum temperature of 50° C. (122° C.). In one embodiment, the extract is dried using a freeze dryer, spray-dryer, vacuum concentrator, dehydrator, oven, or other drying methods. Further, the extract may be stored in an air-tight container in the freezer and lasts for months.

[0053] In one aspect of the present invention, the method 200 uses a plurality of active ingredients including, but not limited to, cichoriin, propionic acid, syringic acid, tryptophane, caffeoyl-glucose, apocynoside, cinnamic acid, quercetin, roseoside, vitamin B2, flavone, kaempferol, paeoniflorin, tamariscinoside, caftaric acid, hookeroside, rosmarinic acid, salvianic acid, salvianolic acid, esculentoside, esculentagenic acid, and arjunglucoside.

[0054] In another aspect of the present invention, the method 200 uses a plurality of active ingredients. The active ingredient of the water extract of the composition at elevated temperature and pressure contains cichoriin, phenyl propionic acid, glucosyringic acid, tryptophane, 1-O-caffeoyl-β-D-glucopyranoside, apocynosideII, sinapic acid, 3-hydroxy-4-methoxy-cinnamic acid, quercetin-3-O-glucuronide 6″-methylester, roseoside 1, vitamin B2, kaempferol-3-O-rutinoside, 5,6,4′-trihydroxy-7,8,3′-trimethoxyflavone, 4-ethyl-paeoniflorin, tamariscinoside B, chicoric acid, rosmarinic acid, monocaffeoyltartaric acid (Caftaric acid), kaempferol-3-O-β-D-glucoside-7-O-α-L-arabinofuranoside, hookeroside D, kaempferol-3-O-rutinoside, salvianic acid A, salvianolic acid A, esculentoside P, esculentagenic acid, arjunglucoside, Fraxin, isomaltose, raffinose, and stachyose.

[0055] In one embodiment, the water extract derived at elevated temperature and pressure comprising of an effective amount of sweet basil active ingredient having anti-oxidant activity. In one embodiment, the water extract at elevated temperature and pressure comprising of an effective amount of sweet basil active ingredient having anti-viral activity. In one embodiment, the water extract derived at elevated temperature and pressure comprising of an effective amount of sweet basil active ingredient having anti-inflammatory activity.

[0056] Accordingly, the active ingredient of the water extract at elevated temperature and pressure comprises at least one or a combination of cichoriin, phenyl propionic acid, glucosyringic acid, tryptophane, 1-O-caffeoyl-β-D-glucopyranoside, apocynosideII, sinapic acid, 3-hydroxy-4-methoxy-cinnamic acid, quercetin-3-O-glucuronide 6″-methylester, roseoside 1, vitamin B2, kaempferol-3-O-rutinoside, 5,6,4′-trihydroxy-7,8,3′-trimethoxyflavone, 4-ethyl-paeoniflorin, tamariscinoside B, chicoric acid, rosmarinic acid, monocaffeoyltartaric acid (Caftaric acid), kaempferol-3-O-β-D-glucoside-7-O-α-L-arabinofuranoside, hookeroside D, kaempferol-3-O-rutinoside, salvianic acid A, salvianolic acid A, esculentoside P, esculentagenic acid, and arjunglucoside.

[0057] In another aspect of the present invention, the water extract composition at ambient temperature is blended with the water extract at elevated temperature and pressure. The resulting composition is a mixture of bioactive ingredients that exhibit anti-viral, anti-inflammatory, and anti-oxidant properties to treat viral infections such as HSV. These ingredients may be blended to form and administer in the form or method of application or composition of a cream, lotion, liquid, emulsion, spray, serum, balm, gel, lip balm, ointment, powder, pill, tablet, consumable, ingestible, patch, mouthwash, toothpaste, lozenge, mint, candy, chewing gum, or similar, for use in the treatment or prevention of HSV, and aphthous or other ulcers of the mouth.

[0058] Referring to FIG. 3, a bar chart 300 illustrating the relative quantification of phytochemicals present in the water extraction at ambient temperature in one embodiment of the present invention. The bar chart 300 shows the composition and the relative abundance of the bioactive ingredients. The active ingredient in the water extract of the composition at ambient temperature comprises of at least one or a combination of glucosyringic acid, tryptophane, folinic acid, 3-ethoxy-4,5-dihydroxybenzoic acid, apocynoside, sinapic acid, rutaevin, vitamin B2, kaempferol-3-o-rutinoside, 3-hydroxy-4-methoxy-cinnamic acid, apocynoside, 4-ethyl-paeoniflorin, tamariscinoside B, 3β-hydrosantamarine-1 -o-β-d-glucopyranoside, esculentoside P, esculentagenic acid, arjunglucoside I, esculentoside B, stachyose and raffinose.

[0059] Referring to FIG. 4, a bar chart 400 illustrating the relative quantification of phytochemicals present in the water extraction at elevated temperature and pressure in one embodiment of the present invention. The bar chart 400 shows the composition and the relative abundance of the bioactive ingredients. The active ingredient in the water extract of the composition at elevated temperature and pressure comprises of at least one or a combination of cichoriin, phenyl propionic acid, glucosyringic acid, tryptophane, 1-O-caffeoyl-β-D-glucopyranoside, apocynosideII, sinapic acid, 3-hydroxy-4-methoxy-cinnamic acid, quercetin-3-O-glucuronide 6″-methylester, roseoside 1, vitamin B2, kaempferol-3 -O-rutinoside, 5,6,4′-trihydroxy-7,8,3′-trimethoxyflavone, 4-ethyl-paeoniflorin, tamariscinoside B, chicoric acid, rosmarinic acid, monocaffeoyltartaric acid (Caftaric acid), kaempferol-3-β-D-glucoside-7-O-α-L-arabinofuranoside, hookeroside D, kaempferol-3-O-rutinoside, salvianic acid A, salvianolic acid A, esculentoside P, esculentagenic acid, arjunglucoside, Fraxin, isomaltose, raffinose, and stachyose.

[0060] Referring to FIGS. 5-6, the graphs illustrate the percentage cell viability of Vero cells against the water extract of sweet basil and the percentage cell viability of HSV infected Vero cells against the water extract of sweet basil after incubation in one embodiment. In one embodiment, the graph 500 shows the number of Vero cells against water extract of sweet basil in (Water extraction at ambient temperature) WEAT and the graph 600 shows the number of HSV infected Vero cells against water extract of sweet basil in (Water extraction at elevated temperature and pressure) WEETP up to a concentration of 1000 μg/ml for 72 hours. The water extract of sweet basil of the present invention showed low toxicity to Vero cells where at 250 μg/ml, the cell viability is still around 90%. The cytotoxic concentration of the compounds against Vero cell i.e., CC50 value is >1000 μg/mL while the effective concentration of compounds against HSV-1 i.e., EC50 is 735.70 μg/mL for WEETP and 812.8 μg/mL for WEAT, indicating that WEETP is more potent than WEAT in inhibiting HSV. In one embodiment, the dark line indicates the WEEPT concentration and the light line indicates the WEAT concentration.

[0061] The present invention will be explained in more detail through the examples below. The examples are presented only to illustrate the preferred embodiments of the present invention and are not intended in any way to limit the scope of the present invention.

EXAMPLE 1

Water Extraction at Ambient Temperature (WEAT)

[0062] In one embodiment, the shredded plant material is mixed with water to prepare for the first extraction at ambient temperature. A ratio of 200 ml of deionized water is added to 20 g of plant material. The mixture is then homogenized for 10 minutes and placed on a shaker for another 10 minutes. The mixture is then filtered using a Whitman No 1 filter paper. The whole process is repeated by adding another 200 ml of deionized water to the filtered residue. Both the filtrate and extract are then mixed. The extract is dried using either a freeze dryer, spray-dryer, vacuum concentrator, dehydrator, oven, or other drying methods until completely dried. 5 mg/ml of extract was then filtered using a nylon syringe filter (0.2 μm, 13 mm) before analysis by Ultra high-performance liquid chromatography (UHPLC) coupled to a Vion IMS QTOF hybrid mass spectrometer, equipped with a Lock Spray ion source used to identify the compounds.

[0063] In one embodiment, the water extract at an ambient temperature (AT) contains glucosyringic acid, tryptophane, folinic acid, 3-ethoxy-4,5-dihydroxybenzoic acid, apocynoside I, sinapic acid, rutaevin, vitamin B2, kaempferol-3-o-rutinoside, 3-hydroxy-4-methoxy-cinnamic acid, apocynoside II, 4-ethyl-paeoniflorin, tamariscinoside B, 3β-hydrosantamarine-1-o-β-d-glucopyranoside, esculentoside P, esculentagenic acid, arjunglucoside I, esculentoside B, stachyose, and raffinose.

[0064] In one embodiment, the Triterpenes such as Esculentoside P, Arjunglucoside I, Esculentagenic acid, and Esculentoside B are found abundantly in WEAT (as shown in FIG. 3). These triterpenes encompass about 45% of the bioactive ingredients in the extract. This is the first time these triterpenes are reported in the water extract of sweet basil. Several studies have indicated that Esculentoside P and Arjunglucoside I are potent anti-inflammatory ingredients. It exhibits its anti-inflammatory response by inactivating nuclear factor Kappa-B signaling pathway and phospho-c-Jun N-terminal kinase and by inhibiting nitric oxide production.

EXAMPLE 2

Water Extraction at Elevated Temperature and Pressure (WEETP)

[0065] In one embodiment, the shredded plant material is mixed with water to prepare for the second extraction. A ratio of 400 ml of deionized water is added to 20 g of plant material. The mixture is then homogenized for 10 minutes and then subjected to extraction at elevated temperature and pressure (121° C., 15 psi) using an autoclave for 15 mins. The cooled mixture is then filtered using a Whitman No 1 filter paper. The extract is dried either using a freeze dryer, spray-dryer, vacuum concentrator, dehydrator, oven, or other drying methods until completely dried. 5 mg/ml of extract was then filtered using a nylon syringe filter (0.2 μm, 13 mm) before analysis by UHPLC coupled to a Vion IMS QTOF hybrid mass spectrometer, equipped with a Lock Spray ion source used to identify the compounds.

[0066] The water extract at an elevated temperature and pressure (WEETP) contains cichoriin, phenyl propionic acid, glucosyringic acid, tryptophane, 1-O-Caffeoyl-β-D-glucopyranoside, apocynosideII, sinapic acid, 3-hydroxy-4-methoxy-cinnamic acid, quercetin-3-O-glucuronide 6″-methylester, roseoside 1, vitamin B2, kaempferol-3-O-rutinoside, 5,6,4′-trihydroxy-7,8,3′-trimethoxyflavone, 4-ethyl-paeoniflorin, tamariscinoside B, chicoric acid, rosmarinic acid, monocaffeoyltartaric acid (Caftaric acid), kaempferol-3-O-β-D-glucoside-7-O-α-L-arabinofuranoside, hookeroside D, kaempferol-3-O-rutinoside, salvianic acid A, salvianolic acid A, esculentoside P, esculentagenic acid, arjunglucoside, Fraxin, isomaltose, raffinose, and stachyose. The most abundant bioactive ingredients in WEETP are polyphenols totaling about 32% of the bioactive ingredients in the extract.

EXAMPLE 3

Total Phenol Content of Sweet Basil Water Extract

[0067] In one embodiment, the total phenolic content assay (TPC) was carried out by adding 20 μL water extract at 5 mg/ml to 100 μL Folin-Ciocalteu (1/10 diluted) followed by 80 μL sodium carbonate (7.5% m/v). The mixture was left in the dark for 15 min before absorbance was taken at 750 nm. Total phenolic content was calculated relative to the standard curve created using gallic acid and expressed as gallic acid equivalent (GAE) per 1 mg dried extract. The phenolic content of the two different approaches of water extract is described in Table 1.

[0068] The phenolic content of WEETP is ten folds higher compared to WEAT. This may be due to the lower dielectric constant of water at 121° C. and 15 psi, making it a better solvent to extract lower polarity phenolic compounds (as shown in FIG. 4).

EXAMPLE 4

Antioxidant Activity of Sweet Basil Water Extract

[0069] In one embodiment, 2,2-Diphenyl-1 -picrylhydrazyl (DPPH) free radical scavenging assay was carried out on both the water extracts. The water extracts were serially diluted from 2 mg/ml to 0.125 mg/ml. 50 μl of the extracts were added with 100 μl of DPPH (15 mM) and left in the dark for 15 minutes. A blank sample was prepared by adding extract with methanol, replacing DPPH. Negative control was prepared by replacing the extract with water. Absorbance at 550 nm was taken for all extracts. The IC.sub.50 value was calculated based on the formula given below.

[00001]${\mathrm{IC}}_{50}=\frac{\begin{array}{c}\left(\mathrm{Abs}\mathrm{negative}\mathrm{control}\right)-\\ \left(\mathrm{Abs}\mathrm{blank}\mathrm{sample}-\mathrm{Abs}\mathrm{sample}\right)\end{array}}{\left(\mathrm{Abs}\mathrm{negative}\mathrm{control}\right)}\times 100$

[0070] The IC.sub.50 value is much lower in the sweet basil WEETP, indicating a higher antioxidant capacity compared to WEAT. This may be due to the higher amounts of phenolic compounds in WEETP, as displayed in the below Table 1.

TABLE-US-00001 TABLE 1 Total Phenol content (TPC) and DPPH scavenging assay of sweet basil WEAT and WEETP TPC (μg GAE/1 mg dried Type of extract extract) DPPH (IC.sub.50, μg/ml) WEAT 4.14 ± 1.66 700.33 ± 76.79 WEETP 43.22 ± 2.21  112.33 ± 7.64 

EXAMPLE 5

Toxicity Levels of Sweet Basil Water Extract in Vitro

[0071] The maximal non-toxic dose (MNTD) is a concentration that does not show any toxicity effects on cell viability. For in vitro toxicity testing, WEAT and WEETP were tested on Vero cells (African Green Monkey Kidney cells) up to a concentration of 1000 μm/ml for 72 hours.

[0072] The cytotoxicity effect of the compounds on Vero cells was determined by using the CellTiter 96.fwdarw. Water Non-Radioactive Cell Proliferation Assay kit (Promega, USA) according to the manufacturer's instructions. This assay is developed based on the conversion of yellow-colored tetrazolium salt to the purple soluble formazan crystals by the dehydrogenase enzyme found in viable cells only. Vero cells were seeded onto 96-well culture plates at a cell density of 2×104 cells per well and allowed for overnight incubation for cell attachment. Serial dilution of the compound was prepared and added into each well to reach a final concentration of 1.95, 3.91, 7.81, 15.63, 31.25, 62.50, 125, 250, 500, 1000 μg/mL, respectively. The plate was incubated at 37° C., 5% CO2 for 72 hours. The MTS [3-(4,5-dimethylthiazol-2-yl)-5 -(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] solution was prepared and was added to each well and incubated for one hour in the dark at 37° C. The absorbance was measured at 490 nm. The maximum non-toxic dose (MNTD) and half-maximal cytotoxic concentration (CC.sub.50) were determined from the percentage of cell viability versus concentration plot as shown in FIG. 5). The CC.sub.50 of the test compound was defined as the concentration that reduced the absorbance of mock-infected cells to 50% of that of controls.

Percentage cell viability=[(OD.sub.T).sub.MOCK/(OD.sub.C).sub.MOCK]×100(%)

Where,

[0073] (OD.sub.T).sub.MOCK: absorbance of the test sample
(OD.sub.C).sub.MOCK: absorbance of cell control

TABLE-US-00002 TABLE 2 Percentage cell viability of Vero cells against WEETP and WEAT after 72 hours of incubation Average cell viability (%) Log Water extract Water extract Compound compound at elevated at ambient concentration concentration temperature & temperature (μg/mL) (μg/mL) pressure (WEETP) (WEAT) 1.95 0.29 98.07 ± 0.54 101.00 ± 1.97  3.91 0.59 98.69 ± 0.84 98.30 ± 4.97 7.81 0.89 100.05 ± 4.98  100.15 ± 4.72  15.63 1.19 99.08 ± 2.25 99.80 ± 4.85 31.25 1.49 99.45 ± 0.74 100.67 ± 5.31  62.50 1.80 97.46 ± 2.32 100.75 ± 3.33  125 2.10 94.95 ± 1.49 96.05 ± 6.12 250 2.40 89.41 ± 3.33 89.36 ± 4.06 500 2.70 76.01 ± 4.36 81.71 ± 5.06 1000 3.00 61.20 ± 0.83 81.47 ± 1.65

[0074] In one embodiment, the above table shows a percentage cell viability of Vero cells against WEETP and WEAT after 72 hours of incubation. In one embodiment, each value is the mean±S.D. of three independent experiments. In one embodiment, further referring to above Table 2 and FIG. 5, both WEETP and WEAT have a CC.sub.50 value of higher than 1000 μg/mL, indicating low toxicities in both these water extracts.

EXAMPLE 6

Anti-Viral Activity Against Herpes Simplex Virus Type-1 (HSV-1) In Vitro

[0075] Vero cells were seeded onto 96-well culture plates at a cell density of 2×104 cells per well and allowed for overnight incubation for cell attachment. Serial dilution of the water extract was prepared and added into each well to reach a final concentration of 1.95, 3.91, 7.81, 15.63, 31.25, 62.50, 125, 250, 500, 1000 μg/mL respectively. Vero cells were infected with HSV-1 at MOI (multiplicity of infection) of 2 except for the cell control. The plates were then incubated at 37° C., 5% CO.sub.2 for 72 hours. The MTS solution was prepared and was added to each well and incubated for one hour in the dark at 37° C. The absorbance was measured at 490 nm. The 50% anti-viral effective concentration (EC.sub.50) was expressed as the concentration that achieved 50% protection of virus-infected cells from the HSV-induced destruction. The percentage protection was calculated as follows:

Percentage protection=[(OD.sub.T).sub.HSV−(OC.sub.C).sub.HSV]/[OD.sub.C].sub.MOCK−(OD.sub.C).sub.HSV]×100 (%)

Where,

[0076] (OD.sub.T).sub.HSV: absorbance of the test sample
(OD.sub.C).sub.HSV: absorbance of the virus-infected control (no compound)
(OD.sub.C).sub.MOCK: absorbance of the mock-infected control

[0077] The selective index (SI) indicates the compound's safety in vitro. It is a ratio that measures the window between cytotoxicity and the anti-viral activity of the tested compound. SI was calculated as follows:

Selective index, SI=CC.sub.50/EC.sub.50

TABLE-US-00003 TABLE 3 The percentage cell viability of HSV infected Vero cells (or percentage protection) against the water extract of sweet basil (WEETP and WEAT) after 72 hours of incubation. Effective concentration (%) Water extract Water extract Compound Log compound at elevated at ambient concentration concentration temperature & temperature (μg/mL) (μg/mL) pressure (WEETP) (WEAT) 1.95 0.29  0.58 ± 0.81 −4.40 ± 4.53 3.91 0.59  0.01 ± 1.53 −5.72 ± 5.34 7.81 0.89 −0.96 ± 2.74 −2.82 ± 9.58 15.63 1.19 −1.17 ± 1.82 −2.49 ± 5.37 31.25 1.49 −2.82 ± 2.23 −0.75 ± 7.59 62.50 1.80 −1.36 ± 3.27 −4.01 ± 1.26 125 2.10 −0.80 ± 5.00 −1.77 ± 1.30 250 2.40 −0.17 ± 6.06  3.24 ± 1.25 500 2.70  39.45 ± 17.05 12.92 ± 1.02 1000 3.00 98.37 ± 2.43 57.44 14.53

[0078] Each value is the mean±S.D. of three independent experiments. Table 3 and FIG. 6, show the percentage protection against the HSV virus at different concentrations of water extract of sweet basil. For the WEETP, the EC.sub.50±735.70 μg/mL and for WEAT, the EC.sub.50=812.83 μg/mL. Almost 100% protection against the HSV is achieved at 1 mg/mL for WEETP, but only about 57% protection is achieved with WEAT at this similar concentration.

[0079] Hence, this invention provides the first evidence that sweet basil, through a safe and green water extraction method, can produce an anti-viral composition that inhibits HSV replication. Their active constituents also exhibit potent antioxidant and anti-inflammatory properties. To optimize the benefits of both these activities, the final bioactive extract may consist of a mixture of the WEAT blended with the WEETP.

[0080] The resulting composition is a mixture of anti-viral and anti-oxidant ingredients to form, for example, but not limited to, a composition of a cream, lotion, liquid, spray, serum, gel, balm, lip balm, ointment, powder, pill, tablet, consumable, ingestible, patch, powder, mouthwash, toothpaste, lozenge, mint, candy, chewing gum, or similar, to treat herpes simplex virus and/or aphthous or other ulcers of the mouth.

[0081] Advantageously, the sweet basil shows effective anti-viral activity against HSV. In one embodiment, the method provides a welcome reinforcement to the existing arsenal of increasingly resistance-prone drugs, and appeals more widely to a populous increasingly looking to non-prescription treatment options, as well as to those who do not have access to doctors and therein prescription treatments, and attenuates some of the inhibition related to seeking treatment that may be associated with the stigma or embarrassment of HSV outbreaks.

[0082] Additionally, because the therapeutic treatment window for HSV lesions is small, with treatment being most effective early on, non-prescription options allow a larger segment of the population to access effective treatment. Neurotherapeutics may mean anti-viral treatment has even greater implications for both individual and societal health than merely treating HSV lesions.

[0083] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. It should be understood that the illustrated embodiments are exemplary only and should not be taken as limiting the scope of the invention.

[0084] The foregoing description comprises illustrative embodiments of the present invention. Having thus described exemplary embodiments of the present invention, it should be noted by those skilled in the art that the within disclosures are exemplary only, and that various other alternatives, adaptations, and modifications may be made within the scope of the present invention. Merely listing or numbering the steps of a method in a certain order does not constitute any limitation on the order of the steps of that method. Many modifications and other embodiments of the invention will come to mind to one skilled in the art to which this invention pertains having the benefit of the teachings in the foregoing descriptions. Although specific terms may be employed herein, they are used only in generic and descriptive sense and not for purposes of limitation. Accordingly, the present invention is not limited to the specific embodiments illustrated herein.