HK1223030B - Reaction platform and method for making pollen based materials in combination with beeswax and uses thereof - Google Patents

Description

Reaction platform and method for preparing pollen-based substances in combination with beeswax and their uses

Field of the Invention

Methods and reaction platforms for making pollen-based products are described. Cosmetic and nutritional formulations suitable for administration to an individual are described, comprising pollen-based substances, including polypeptides, amino acids, fatty acid triglycerides, and flavonoids, in combination with beeswax.

background

Honeycomb in its natural state contains interesting chemistry, including the release of valuable bioactive components of pollen that are transported to the hive through the action of certain enzymes. However, using honeycomb raw materials or mixtures presents problems, including component separation, contamination with microbial flora, and/or exposure to pesticides used to eliminate pests (e.g., mites). Therefore, a method is needed to efficiently utilize streamlined production using synergistic combinations of desired honeycomb components.

The inventors (Weir, formerly O'Brien) have demonstrated that plants possess internal genetic mechanisms to control the process and progression of apoptosis, also known as programmed cell death (PCD). In one example, chromosome condensation, a hallmark of PCD in mammalian cells, can be reversible in plant cells during the early stages of apoptosis (O'Brien et al., The Plant Journal (1998) 13(6):803-814).

Pollen has a hard shell called sporopollenin, which is very resistant to chemical degradation. Furthermore, pollen has proteins on its surface that are known to cause allergic reactions. In one method, a reaction is used to "explode" or "burst" pollen grains under pressure, followed by the use of proteases to hydrolyze and inactivate the proteins that cause allergic reactions.

In another possible two-step reaction, pollen can be manipulated to initiate germination and thereby more gently and naturally release the bioactive contents of the pollen.

Therefore, if a method could be found to first open or germinate the pollen grains to biologically release the beneficial components and then add other honeycomb components, this would mimic the natural process and provide a new way to obtain nutritional or cosmetic products.

Atopic dermatitis (commonly known as eczema) is a complex skin disease characterized by itching, impaired epidermal barrier function, and immunoglobulin sensitization to a variety of food and environmental allergens (Sohn, A. et al., "Eczema," Mt. Sinai J. Med. (2011) 78: 730-739). The skin inflammation manifests as erythema, which may include scaling and crusting (Krafchik, B.R., "Eczema," Paediatr. Child Health (2000) 5: 101-105). It usually occurs due to the interaction of genes and the environment. Patients with eczema are at higher risk of developing skin infections. Skin care products, including moisturizing creams, are being used to treat dry, scaly skin (Loden, M. et al., "A double-blind study comparing the effect of glycerine and urea on dry, eczematous skin in atopic patients," Acta Derm. Venereol. (2002) 82:45-47). The effectiveness of these creams has been shown to have a positive impact on the quality of life of patients who tried them (Eberlein, B. et al., "Adjuvant treatment of atopic eczema: assessment of an emollient containing N-palmitoylethanolamine (ATOPA study)," J. Eur. Acad. Dermatol. Venereol. (2008) 22:73-82).

If a method could be found to use plant-based substances to influence or control immune or PCD signaling pathways in mammalian cells, this would be a valuable contribution to the fields of nutrition and medicine.

Furthermore, if a method could be found to use plant-based substances, including pollen-based extracts, to treat inflammatory or skin conditions, this would be a valuable contribution to the fields of nutrition, cosmetics, and medicine.

SUMMARY OF THE INVENTION

Pollen-based extracts comprise germinated pollen, honeycomb components, and optionally enzyme-containing materials (including honeydew or plant meal).

Aqueous skin creams contain pollen-based extracts and one or more of glycerin, natural oils, lubricants, preservatives, vitamins, fragrances, emulsifiers, or waxes. Skin creams can be used to treat skin conditions or inflammation.

A method of treating eczema or psoriasis is provided, the method comprising: administering to an individual in need of such treatment a therapeutically effective amount of an aqueous skin cream. The skin cream can be applied topically to the skin.

A two-stage reaction platform for producing pollen-based extract materials is provided, comprising the following steps: a first stage comprising opening and/or germinating pollen grains, reacting the treated pollen grains with one or more honeycomb components selected from beeswax, honey, or an enzyme-containing substance, and stirring to form a jelly; and a second stage comprising heating the jelly in a sealed container to produce an extract. The extract may be a fermented extract. It should be understood that the extract may be further fermentable by adding one or more components.

Brief introduction of the attached figure

FIG1 depicts fatty acid analysis of opened and fermented pollen prepared according to one embodiment of the present invention.

2 depicts fatty acid analysis of opened and fermented pollen (from both the supernatant and sediment of the reaction) prepared according to one embodiment of the present invention.

FIG3A depicts the treatment of HL-60 cells with a coconut water based extract (as a non-fermented "water" extract) having very potent cytoprotective activity in an MTT assay.

FIG3B depicts the treatment of HL-60 cells with a coconut water based extract (as a 2-stage "ethanol" extract of the fermentation) that has very strong cytoprotective activity and promotes cell proliferation in the MTT assay.

Figure 4 depicts the treatment of HL-60 cells with a coconut water based extract (as a fermented 2-stage "ethanol" extract) containing Tamarillo powder that has very strong cytoprotective activity and promotes cell proliferation in an MTT assay.

Figure 5 depicts gel electrophoresis (SDS-PAGE) analysis of opened and fermented pollen prepared according to one embodiment of the present invention. Lanes are labeled as follows: DE2-DE9 (12.5 μl loading), protein marker, and DE2-DE7 (2.5 μl loading), as described below.

6 depicts RP-UHPLC and negative ion electrospray ionization (ESI) analysis of opened and fermented pollen prepared according to one embodiment of the present invention (Example 2A), showing the products of a 2-stage fermentation reaction up to 72 hours.

Figure 7A shows a photograph of a hand of a human patient with severe eczema before treatment.

FIG. 7B shows a photograph of the eczema patient of FIG. 7A after 4 weeks of topical treatment with the fermented pollen-based cream of Example 6B.

Figure 8A shows a photograph of an arm of a human patient with severe eczema before treatment.

FIG8B shows a photograph of the eczema patient of FIG8A after 4 weeks of topical treatment with the fermented pollen-based cream of Example 6B.

FIG9A shows a photograph of a leg of a human patient with psoriatic lesions before treatment.

FIG. 9B shows a photograph of the psoriasis patient of FIG. 9A after 4 weeks of topical treatment with the fermented pollen-based cream of Example 6D.

10 depicts a bar graph showing the Score of Severity of Atopic Dermatitis (SCORAD) index for a clinical treatment group (n=20) that received a skin cream containing a fermented pollen-based extract prepared according to one embodiment of the present invention (Example 6B).

11 depicts a bar graph showing the Dermatology Life Quality Index (DLQI) for a clinical treatment group (n=20) that received a skin cream containing a fermented pollen-based extract prepared according to one embodiment of the present invention (Example 6B).

Detailed description

Below, a detailed description of one or more embodiments of the present invention is provided, along with accompanying tables and figures that illustrate the principles of the invention. Thus, this detailed description illustrates the invention by way of example and not limitation. This description will clearly enable one skilled in the art to make and use the invention, and describes several embodiments, modifications, variations, alternatives, and uses of the invention, including what we presently believe to be the best mode of carrying out the invention. It should be clearly understood that routine variations and modifications may be made to the described invention, and such variations and modifications are clearly within the spirit and scope of the invention.

In other words, the present invention is described in conjunction with these embodiments, but the invention is not limited to any embodiment. The scope of the present invention is limited solely by the claims, and the invention encompasses numerous alternatives, modifications, and equivalents. The following description provides numerous specific details to provide a thorough understanding of the present invention. These details are provided for illustrative purposes only, and the present invention may be practiced according to the claims with or without some or all of these specific details.

A safe and effective pollen-based skin cream has been provided which comprises beeswax and optionally an enzyme which can be administered to an individual in a therapeutically effective amount for the treatment of eczema or psoriasis.The pollen-based skin cream can be applied topically to the skin.

Pollen has a hard shell called sporopollenin, which is very resistant to chemical degradation. In addition, there are proteins on the surface of pollen that cause known allergic reactions. In one method, a reaction is used to make the pollen grains "explode" or "break" under pressure (a common non-biological method), and then proteases are used to hydrolyze and inactivate the proteins that cause allergic reactions. Optionally, peptidases can then be used to produce peptides from the resulting mixture or soup.

Instead of trying to make pollen grains explode (which does have some effect), attempts are made to manipulate or treat pollen grains to initiate germination and thereby release their contents naturally, thereby optimizing biological activity and providing a much more targeted product. This can be considered a "biological" approach to opening the pollen. If the pollen grains explode, the compounds released are more responsive to stress (which can be beneficial for some medical conditions), but if they are released gently through germination, they promote cell renewal and repair (as discussed previously). By utilizing a biological germination pathway, viable pollen grains begin germination while damaged or dead pollen grains remain and can be filtered out. Therefore, by removing dead pollen grains, this approach is also an improvement.

In one embodiment, it has been discovered that a two-step process can be used to produce pollen-based extract materials, the process comprising biologically opening and/or germinating pollen grains, and reacting the treated pollen grains with one or more honeycomb components (e.g., beeswax, propolis, etc.).

Development of fermentation methods and opening of pollen grains.

First, in a sealed stainless steel bottle, a small amount of dry pollen (25g) was mixed with white sugar (5g) and active yeast (5g) in water (500mL). At ambient temperature, after about 72 hours, the resulting mixture fermented and produced alcohol and the accompanying alcohol smell.

Subsequent experiments were conducted using honeydew (isolated from the New Zealand beech scale insect Ultracoelostoma assimile (Maskell)) and raw honey (manuka, honeysuckle, clover) from beech trees in southern New Zealand (Nothofagus spp.). In New Zealand, this insect is primarily found in the South Island and is most common on black beech (N. solandrisolandri) and mountain beech (N. s. cliffortioides), but generally any beech will suffice (Gaze et al., N.Z.J.Ecol. (1983) 6:33-37). In these experiments, fermentation occurred within approximately 24 hours at higher temperatures (37°C, 42°C, or 65°C), but took 48 hours at 25°C. Approximately 66% of the pollen grains were open. Furthermore, the honeydew did not produce an alcoholic odor, but instead produced a very golden, oily broth.

Following fermentation and pollen grain opening, fatty acid (FA) content analysis was performed as shown in Figures 1 and 2 (NZLabs, Auckland, New Zealand). Exemplary filtration of the sediment after fermentation is described in Preparation Examples A and B below.

The FA analysis results clearly showed that the fatty acid content was maintained and protected in the opened pollen grains. The key thing demonstrated by this experiment was that fatty acids were released from the pollen, but in the unexploded pollen grains, there were no fatty acids in the analyzed soup.

Development of reaction platform

After discovering a method to effectively open the pollen grains, the reaction of the pollen grain contents with other components from the honeycomb (eg, beeswax, propolis, etc.) was performed. The method was developed through some preparation examples as described below.

It has been discovered that honeydew (from southern beech forests) containing insect enzymes performs better than raw honey. Honeydew is a substance extracted by scale insects that feed on the sap of beech trees. This honeydew is a honey substance, but contains enzymes from the scale insects and their gut flora, including fructophilic lactic acid bacteria. This discovery led to the conclusion that honeydew contains enzymes from scale insects and various bacteria, which are also present in some of the raw honey tested. Without being bound by theory, it is believed that honeydew enzymes may be similar to those in bee saliva and therefore also contribute to the second stage of the method.

A description of the components used in the representative two-step process in various useful forms follows.

Raw pollen: contains fatty acids, peptides, EPA, DHA, long-chain alkanes, hormones, vitamins, phytohormones, cellulose, lignin and flavonoids.

Raw Beeswax: Contains pheromones, phytic acid, chitin and fatty acids.

Raw honey: Contains naturally occurring bacteria such as lactic acid bacteria.

Honeydew: Contains potassium, sugars, insect enzymes, and fructophilic lactic acid bacteria.

Pineapple Powder: Contains bromelain, homologous serine proteases, and peptidases; contains potassium.

Alternative plant powders containing flavonoids and/or enzymes: tamarillo (antioxidant and enzymes), blackcurrant (antioxidant/cell repair/brain function), kawa kawa (bladder and digestive health/antioxidant/anti-inflammatory/detox/acne), propolis (acne), pine bark (estrogen regulating and sunscreen), acai berry, pomegranate, cherry (sleep aid/melatonin), kiwi, papaya, and feijoa. Freeze-dried plant powders can be prepared as follows. A pulp of the desired plant (e.g., pineapple, tamarillo, kava, etc.) is prepared. The resulting pulp is frozen to -20°C for 48 hours and then freeze-dried. Alternatively, useful freeze-dried plant powders are commercially available from Alaron GMP Manufacturers (Alaron Products, Nelson, New Zealand). Alternatively, spray-dried or even fresh plant sources can be used in the reaction, for example, initially using the stems and fruit of a pineapple before obtaining the freeze-dried powder.

Other useful plant components may include, but are not limited to: apple pulp or peel; avocado pulp or peel; beech bark or leaves; blueberries; blackberries; acai berries, ginger; grape skins; sesame oil; rimu bark or leaves; turmeric; onions; oranges; kiwi pulp, peel or seeds; kauri bark or leaves; kohe kohe; mango; manuka oil or leaves; noni; olive waste, peel or oil; grapeseed oil, argan oil, jojoba oil and pohutakawa.

Coconut milk (i.e. coconut water): Contains hormones and nutrients. Coconut water/coconut milk is obtained by purchasing fresh coconuts and draining the fresh coconut milk just before use. Commercially available coconut milk can be used but cannot be pasteurized.

Coconut Oil: Contains peptides, fatty acids, EPA, DHA, and especially myristic acid. A useful brand is Home Essentials brand, 100%.

Mussel Oil: Contains peptides, fatty acids, EPA and DHA.

Certain honeycomb components (e.g., honey, honeydew, pollen, beeswax, raw propolis) were sourced from Scott Apiaries and sold as Hanmer Bees, Hanmer Springs, North Canterbury, New Zealand. Propolis for use as an aqueous extract can be prepared by treating 25 g propolis/50 mL water in a dark, sealed glass container with gentle rotation at 28° C. for 5 days.

Glycerol (ie, glycerol) can be purchased from Sigma Aldrich (St. Louis, Missouri).

The water can be filtered sterile rainwater, distilled water, deionized water, RO water, etc.

Preparation Example A (including pineapple powder)

The following ingredients were mixed: raw pollen (50 g), raw beeswax (25 g), pineapple powder (12.5 g), raw honey (12.5 g), glycerin (12.5 g), and water (1000 mL). After mixing, the reaction mixture in a stainless steel container was heated to 60° C. and stirred for 15 minutes. At this point, the steel container was sealed in an oxygen-free environment and pressurized to 120 psi. The container was stored at 60° C. for 24 hours. During this period, the pressure in the container increased, and the mixture became bubbly and exothermic. The reaction naturally ceased within 16-24 hours. The solution was then filtered to remove sediment. This reaction product extract was heated, poured into a cream formulation (e.g., Example 3), and manually whipped using a whisk until smooth (i.e., having a consistency of equal viscosity).

The reaction yields a reaction product solution that still has protease activity, peptides, and triglycerides.

In this manner, the reaction product was formulated at 5% by weight in a commercially available aqueous cream (see, e.g., Experimental Aqueous Cream Formulation, Example 3). This formulated reaction product was tested on subjects with various skin diseases. This reaction product was effective on senile plaques, crusted eczema, psoriasis, herpes, molluscum, and acne. However, when used on inflammatory eczema or lupus, the reaction product caused irritation.

Preparation Example B (comprising pineapple powder and tamarillo powder)

The following ingredients were mixed: raw pollen (50 g), raw beeswax (25 g), pineapple powder (6.25 g), tamarillo powder (6.25 g), mussel oil (25 g), honey (12.5 g), glycerin (12.5 g), and water (1000 mL). After mixing, the reaction mixture in a stainless steel container was heated to 60° C. and stirred for 15 minutes. At this point, the steel container was sealed in an oxygen-free environment and pressurized to 120 psi. The container was stored at 37° C. for 24 hours. During this period, the pressure in the container increased, and the mixture became bubbly and exothermic. The reaction stopped naturally within 16-24 hours. The solution was then filtered to remove sediment.

In this experiment, the temperature was lowered to protect the plant powder. This method produces peptides, esterified fatty acids, and esterified flavonoids. This lower temperature also protects the vitamins and amino acids from the pollen from thermal denaturation. It is also contemplated that other oils may be used in place of mussel oil, including but not limited to a combination of mussel oil/coconut oil (1:1 weight/weight).

To the extent that the above reactions occur and/or undergo fermentation, the resulting pollen-based material is considered fermentable.

In one embodiment, the pollen opening and fermentation reaction can be carried out as described in a sealed steel container or other suitable pressure vessel. In an alternative embodiment, the pollen opening and fermentation reaction can be carried out aerobically as described in a suitable plastic or glass container or reaction vessel.

One problem at this point is that all other competitors and the methods described herein involve cracking the pollen grain to release the contents therein (abiotic methods). Most desirable would be a biological method to produce those bioactive components in an enhanced bioactive state, as when the pollen grain begins to germinate. Here, the contents are made bioavailable for optimal germination during the osmotic process.

The plant cell germination process is equivalent to embryogenesis in humans and is therefore optimal for cell regeneration, division, wound repair, etc.

Coconut milk (also known as coconut water) is known to contain all the nutrients needed for embryo formation, as well as plant hormones that stimulate cell division and growth, including indoleacetic acid, gibberellins, and auxins. Coconut milk also contains the potassium required for germination through osmosis. One study found that coconut milk (now called coconut water) can be used to make plant cells omnipotent and grow entirely new plants from single cells. This was done using Eustoma species, none of which were previously able to regenerate because they were unaffected by normal hormonal stimulants (O'Brien et al., Plant Cell Tissue and Organ Culture (1993) 33:31-37).

Instead of cracking pollen grains, a biological approach is presented herein to trigger germination and thereby activate it, i.e., produce enhanced biological activity. Without being bound by theory, it is believed that if the pollen grains germinate, enzymes in the honeydew and/or from the pollen itself can provide the protease/peptidase enzymatic reactions that other components (e.g., lactic acid bacteria or pineapple powder) can provide.

Based on the osmotic uptake of potassium, pollen grains will begin to germinate. Honey/honeydew and pineapple both contain enough potassium to trigger this reaction if used in a reaction platform, especially in warmer temperatures. Coconut milk is also high in potassium, so the combination of potassium and plant hormones leads to triggering germination.

By using coconut water, the product is infused with the minerals and growth hormones found in coconut water. These components work synergistically with the pollen to promote cell renewal and repair, unlike stressors that rupture or damage the pollen.

The above method can be further understood with reference to the following examples.

Example 1 (Reaction Platform)

The following ingredients are mixed: raw pollen (50 g), coconut milk (200 ml), and honeydew (12.5 g). These three ingredients are heated to 30°C and stirred for 30 minutes to stimulate germination. Next, the following ingredients are added: melted raw beeswax (25 g), glycerin (12.5 g), and water (1000 mL). Optional ingredients may include: coconut oil, pineapple powder, or other plant powders (as listed above). Depending on which components are added and the final desired medical or nutritional treatment, the reaction is carried out at 25°C, 30°C, 37°C, 42°C, or 65°C. The reaction platform can be carried out anaerobically or aerobically.

Example 2 (Two-stage reaction platform)

All reactions performed below were incubated anaerobicly at 37°C for 72 hours. Optionally, beeswax can be used at 37°C, resulting in a more anaerobic reaction because the beeswax forms a solid wax layer on top of the other components. If the reaction is continued for 7 days, the beeswax is enzymatically digested and becomes part of the solution (i.e., self-emulsifying).

Example 2A (Eczema/Psoriasis)

Element Weight (g) pollen 50g beeswax 25g coconut oil 25g glycerin 12.5g honeydew 12.5g coconut water 200ml

(Stage 1) Pollen was soaked in coconut water at 25°C for 30 minutes. Simultaneously, beeswax was melted in a stainless steel bowl placed in boiling water. Once the beeswax was melted, it was stirred vigorously for 5 minutes and the bowl was placed in an incubator at 65°C, causing the beeswax to remain liquid.

After soaking for 60 minutes, glycerin is then stirred into the pollen mixture, followed by honeydew. The mixture is then stirred at 25°C for 30 minutes, completing Part 1. Next, coconut oil is stirred into the melted beeswax, completing Part 2.

The pollen mixture (Part 1) is then stirred into the beeswax mixture (Part 2) and the entire mixture is stirred vigorously by hand for about 15 minutes until a thick gel forms (end of Stage 1). Note that stirring by hand is laborious and magnetic stirring on a hot plate may be helpful.

(Stage 2) This colloidal mixture was then placed into a stainless steel container with a screw-seal type lid and incubated at 37°C for at least 72 hours, which produced the isolated 2-stage extract (End of Stage 2).

In a preferred embodiment, plant powders (or other useful plant parts) such as tamarillo, kava, etc., can be added after the gel is formed, and the entire mixture is then subjected to heating under closed conditions and/or fermentation. In an alternative embodiment, if any other ingredients are desired (e.g., pineapple powder, fruit powder, etc.), these ingredients are added after the gel is formed, when the mixture is cooled. In other words, tamarillo, kava, etc. can be added after the second stage is completed.

In another embodiment, other ingredients (eg, pineapple powder, tamarillo, kava, fruit powder, etc.) may be added and heated after Stage 2 is complete.

It should be understood that the fermentation process in stage 2 can be carried out for about 24 to 168 hours. In a preferred embodiment, the incubation is carried out at 37° C. for about 24 to 72 hours. In a more preferred embodiment, the incubation is carried out at 37° C. for about 72 hours.

In another alternative embodiment, increasing the coconut water content in stage 1 can reduce manufacturing costs.

In another alternative embodiment, the molten beeswax (or the components of Part 2) can be maintained at a lower temperature (e.g., 42° C. or 37° C.) to protect other more sensitive components (e.g., amino acids, peptides, or fatty acids). In this embodiment, the beeswax self-emulsifies into the final product mixture.

Example 2B (age spots/actinic keratosis)

Element Weight (g) pollen 50g beeswax 25g coconut oil 25g glycerin 12.5g honeydew 12.5g coconut water 200ml pineapple powder 12.5g

Example 2C (Antimicrobial Agents and Wound Healing)

Element Weight (g) pollen 50g beeswax 25g coconut oil 25g glycerin 12.5g honeydew 12.5g coconut water 200ml Propolis 5ml*

*Added at the end of the reaction. Propolis was prepared as 25g/50ml water.

Example 2D (Antiviral - Herpes, Herpes Zoster, Molluscum, Chickenpox)

Element Weight (g) pollen 50g beeswax 25g coconut oil 25g glycerin 12.5g honeydew 12.5g coconut water 200ml Kava powder 12.5g Propolis 5ml*

*Added at the end of the reaction. Propolis was prepared as 25g/50ml water.

In Examples 2E, 2F, and 2G below, beeswax was omitted so the solution was more liquid (less viscous) after the addition of the oil, and the reaction took an additional 24-48 hours to complete.

Example 2E (Makeup - Beauty/Anti-Aging)

Element Weight (g) pollen 50g coconut oil 25g glycerin 12.5g honeydew 12.5g coconut water 180ml Seaweed water 20ml*

*Microalgae were grown to exponential phase, centrifuged and the slurry added to the reaction vessel before fermentation at approximately 30°C.

Optionally, 12.5 g of blackcurrant powder was added to this example as an antioxidant.

Example 2F (Core Recipe - Optional Tamarillo)

Element Weight (g) pollen 50g coconut oil 25g glycerin 12.5g honeydew 12.5g coconut water 200ml Tamarillo powder (optional) 12.5g

Example 2G (Cosmetic-Beauty/Estrogen)

Element Weight (g) pollen 50g coconut oil 25g glycerin 12.5g honeydew 12.5g coconut water 180ml Seaweed water 20ml* 12.5g

*Grow microalgae to exponential phase, centrifuge and add to slurry.

^a Pine bark powder is prepared by hot water extraction of pine bark followed by freeze drying.

Chemical analysis

Reaction samples prepared according to the principles of the present invention (i.e., Example 2A or 2B) were analyzed to assess how the chemical composition of these mixtures changed during incubation. Phytochemical analysis was performed by liquid chromatography-high resolution mass spectrometry (LC-HRMS) and protein/peptide composition was assessed by SDS polyacrylamide gel electrophoresis (SDS-PAGE) using standard methods.

After incubation for 0 hours (starting material) to 72 hours in the second stage fermentation reaction, the sample was thoroughly mixed and an aliquot (1.0-1.5 g) was extracted with ethanol. For incubations according to Example 2B, pineapple powder was added to starting materials DE5 and DE7 (Example 2A with or without beeswax, respectively) after time 0. The resulting extracts were analyzed by LC-MS using reverse phase ultra-high performance liquid chromatography (RP-UHPLC) and negative ion electrospray ionization (ESI).

In Figure 5, the samples tested are as follows:

DE2: Extract sample 2A, without beeswax, 72 hours

DE3: Extract sample 2A, 72 hours

DE4: Extract sample 2B, without beeswax, 24 hours

DE5: Extract Sample 2A, 0 hours (Starting Material – Stage 1)

DE6: Extract sample 2B, 24 hours

DE7: Extract Sample 2A, without beeswax, 0 hours (Starting Material – Stage 1)

DE8: Extract sample 2B, without beeswax, 72 hours

DE9: Extract sample 2B, 72 hours

A 12.5% reducing SDS-PAGE gel was used, labeled Biorad #161-0373, and the stain type was AllBlue (Bio-Rad Lab., Hercules, California).

In general, Figure 5 shows that as part of a 2-stage process, larger molecular weight proteins are degraded or digested into smaller molecular weight protein components.

Samples DE5 and DE7 are starting materials for a 2-stage process (stage 1), which show that a large number of proteins in the samples have high molecular weights (>250 kD) - so much so that many of them do not enter the gel. Samples DE2 and DE3 show a prominent band between 50-75 kD (which is also present in lower amounts in the remaining samples). Another band at approximately 25 kD appears in samples DE4, DE6, and DE8, and to a lesser extent in DE9.

Samples DE4, DE6 and DE8 showed significant staining at molecular weights less than 15 kD. Samples DE2, DE3 and DE9 showed proteins/peptides in this range to a lesser extent.

Thus, the size distribution of proteins over time in the incubated samples was significantly smaller compared to the starting materials (DE5 and DE7), whether with or without pineapple starch enzyme, or with or without beeswax.

In Figure 6, the progress of the second stage of incubation is observed and the reaction products are analyzed. The samples tested are as follows:

DE5: Extract sample 2A, 0 hours (starting material – stage 1), as described above

DE6’: Extract sample 2A, 24 hours

DE9': Extract sample 2A, 48 hours

DE11': Extract sample 2A, 72 hours

The process of the second stage fermentation reaction has shown the pattern that is determined as dicaffeoylquinic acid isomer and is considered to the compound of dicaffeoyl tartrate or ether compound.As shown in Figure 6, after 72 hours, observe and form the multiple isomer of phenolic compound dicaffeoylquinic acid, it is indicator or the mark of the second stage reaction.Think that also multiple dicaffeoyl tartrate or ether isomer (for example, the tetramethyl ether of caffeoyl moiety) has been formed.These samples demonstrate and contain a large amount of caffeoylquinic acid isomer and the dicaffeoyl tartrate or ether compound (that is, chicoric acid derivative, comprises methyl ether or ester) that are considered.

Thus, while not wanting to be bound by theory, peptides are produced in a 2-stage process according to a "lipophilization" or "lipolyzation" process, in which proteins are digested, phenolic compounds are isomerized or methylated, and fatty acids can participate in esterification reactions to produce useful and beneficial new products. In another embodiment, it is contemplated that hydroxylation and/or acetylation of flavonoids may occur along with fatty acids. In another embodiment, it is contemplated that hydrolysis of tannins or glycosylation of aglucans may occur during the 2-stage fermentation reaction. In yet another embodiment, it is contemplated that hydroxylation of fatty acids may occur during the 2-stage fermentation reaction. In fact, it was observed by HPLC analysis that the liquid composition of the 2-stage reaction product produced an increased lipid content.

A lubricating cream was produced as follows.

Example 3 (Experimental aqueous cream preparation)

Element Weight/weight (%) Cetearyl Alcohol 8.1% White soft paraffin 15.0% liquid paraffin 6.0% Sodium lauryl sulfate 0.9% Phenoxyethanol 1.0% purified water 70.0%

An aqueous cream without the extract was used as a placebo, so the premise was that the extract had to be clinically significantly better than the aqueous cream as the current nonsteroidal anti-eczema treatment.

Accordingly, the use of parabens significantly inhibited the efficacy of the extract in some human volunteers. In some human volunteers, phenoxyethanol did not completely inhibit but still reduced the efficacy of the extract, which is assumed to be due to its bactericidal properties. Similar results were found with methylparaben, with a significant reduction in the efficacy of the extract.

Next, a batch of aqueous creams was prepared using Geogard 221, which contains 8% dehydrated acetic acid and 87% benzyl alcohol (available from Lonza, Allendale, New Jersey), as an antibacterial preservative to replace phenoxyethanol and/or parabens. When tested in several human volunteers, this formulation provided the full efficacy of the extract. Thus, it was found that the type of preservative is important and can be considered an additive with more "natural product" similarity.

Example 4 (natural-based cream preparation)

Element Weight/weight (%) Cetearyl Alcohol 8.1% Cetomacrogol* 15.0% glycerin 2.5% Crodamol (Coconut Oil) 2.0% beeswax 2.0% Geogard** 0.5% purified water 70.0%

*Cetomacrogol 1000, a PEG polymer

**Geogard 221

Thus, a useful aqueous cream formulation has been discovered which is very stable and does not require the use of bactericidal preservatives. This material is a beautiful ointment that can be applied to human skin.

The Phase 2 extract of Example 2A is added to the base cream of Example 4 in an amount of 5% by weight based on the total weight of the formulation. A useful amount of the Phase 2 extract can be from about 0.5% to about 10% by weight based on the total weight of the formulation. A preferred range of the Phase 2 extract can be from about 0.5% to about 5% by weight based on the total weight of the formulation.

In addition, essential oils or fragrances were added to the base cream of Example 4, wherein the amount of citrus oil extract added was 0.4 wt % based on the total weight of the formulation. The amount of one or more essential oils used can be from about 0.4 wt % to about 1 wt % based on the total weight of the formulation.

Example 5 (Testing of a base cream containing the extract of Example 2A)

The base cream of Example 4 (Table 1) containing 5 wt% of the extract of Example 2A and 0.4 wt% of the citrus oil extract was tested using human volunteers as follows.

Table 1

This formulation cleared eczema and psoriasis on the volunteers as shown in Table 1. Subjects 1, 3, 4, 5, 8, 9, and 10 did not use the cream for one week and then tried the cream of Example 6 (below) prepared for clinical trials with equivalent results to Example 4.

Example 6A (Skin Cream Formulation)

Skin cream formulations were prepared as follows.

**Geogard 221

Olivem 1000, available from B&T Srl (Milan, Italy), is a nonionic, non-ethoxylated self-emulsifying system derived from olive oil for use in oil-in-water creams and lotions.

Initial trials using Example 6A in human volunteers as in Example 5 gave similar results for eczema and psoriasis, among others.

Example 6B (Skin Cream Formulation)

In another embodiment, a skin cream formulation is prepared.

¹ Lipidated fatty acids and peptides, as shown in Figures 1, 2, and 5 described herein.

The final formulation is an off-white viscous cream with a pH of 4.0-5.0. Microbiological properties: Aerobic plate count: (30°C) 377 cfu/g; yeast and mold: <1 cfu/g. The cream can be applied topically to human skin by hand.

In one embodiment, about 1% to about 10% by weight of the Phase 2 extract may be used. In another embodiment, about 70% to about 90% by weight of water may be used. Increasing the water content and/or alternatively increasing the coconut water content in Phase 1 (as described above) may reduce manufacturing costs.

The cream formulation of Example 6B was tested in clinical trials as described below.

Example 6C (Clinical Study - Eczema)

The purpose of this study was to determine the effectiveness of Example 6B in treating mild to moderate eczema, including reducing the appearance of lesions, reducing symptoms of itching and scaling, and reducing redness.

Clinical study design

This study was designed as an open-label, adaptive design pilot study.

Otherwise healthy subjects aged 18-70 years with mild to moderate eczema were included in the study. A total of n = 40 subjects were screened, of which n = 21 were randomized; however, only n = 20 subjects completed the study. Other standard inclusion/exclusion criteria were used. Adverse events were monitored. Initial IRB approval of the protocol was granted by the MaGil IRB (Rockville, MD). All recruitment materials were approved by the IRB before use.

For each subject, the study duration was up to 5 weeks, consisting of 5 visits, with a trial period of 30 days (approximately 4 weeks).

V1: Screening Visit – 7 days

V2: Day 0 – Baseline

V3: Day 7

V4: Day 15

V5: Day 30 – End of study

Subjects were asked to undergo a 7-day wash-off of any and all eczema treatments.

Dermatological assessment. A dermatological examination was performed by a qualified practitioner. This assessment included the Score of Severity of Atopic Dermatitis (SCORAD), which evaluates and grades the nature of the lesions. Subjects also completed the Dermatology Life Quality Index (DLQI), which is a self-assessment of each subject's own skin condition.

Photographs. Subjects were photographed for inspection and evaluation of skin lesions, identified by anatomical location and size.

Allocation Procedure. At this visit, allocations were as follows:

Subjects were given a one-week supply of the study product (cream of Example 6B) and instructed to apply twice daily. Subjects were given a daily dosing log.

Instructions: Instruct patients to apply the cream topically to the affected area twice daily. That is, patients with eczema are instructed to apply the cream topically to a given lesion twice daily.

Dosage: Based on the clinical trials presented herein, an average of 2.46 g of the cream of Example 6B was used per patient per day and an average of 1.52 g of the cream of Example 6B was used per lesion.

Data Analysis: All descriptive and inferential analyses for all endpoints were performed using Social Sciences statistical software (SPSS version 19).

The primary objective was to assess the reduction in lesion appearance by examining photographs of skin lesions (Figures 7A-7B and 8A-8B). The secondary objective was to assess the reduction in pruritus, scaling, and redness by various dermatological assessments, as shown in Table 1A.

Table 1A

Visual Analog Scale (VAS) symptom scores for itching, scaling, and redness. Subjects were also asked to undergo a dermatological assessment of itching, scaling, and redness of their eczema skin lesions. Analysis showed that the mean VAS symptom scores for itching, scaling, and redness decreased significantly from week 1 to weeks 2, 3, and 4 (p-value ≤ 0.05 for all time points).

According to Table 1A, from week 1 to week 4, the pruritus (VAS) symptom score decreased from baseline to -30.27%.

According to Table 1A, from week 1 to week 4, the scaling (VAS) symptom score decreased from baseline to -30.53%.

According to Table 1A, from week 1 to week 4, the redness (VAS) symptom score decreased from baseline to -33.95%.

A statistically significant change in the severity of eczema was found from baseline to day 30 (p-value ≤ 0.05). Overall, moderate eczema was reported as decreasing in severity, and 19.44% of lesions had resolved by day 30. It was noted that the majority of moderate to severe lesions also decreased (based on lesion counts and assessment of mild, moderate, or severe classification). See Table 1A.

It was also observed that the overall mean size (length x width) of eczematous skin lesions was significantly reduced after treatment with the topical cream. For example, the mean length decreased by -23.8% (from 6.3 cm to 4.8 cm), while the mean width decreased by -22.8% (from 3.9 cm to 3.0 cm).

SCORAD is a clinical tool used to assess the extent and severity of eczema (scoring system for atopic dermatitis). Dermatologists use this tool before and after treatment to determine whether treatment is effective.

The Score of Severity of Atopic Dermatitis (SCORAD) index is used as a standardized rating scale for eczema. This combined scoring index was developed in 1993 by the European Task Force on Atopic Dermatitis. It has been tested for authenticity and reliability and has shown altered sensitivity in trials of topical steroids and UV-A therapy. It combines the assessment of disease severity using a rule of 9, utilizing six clinical features of disease intensity (assessed at a single representative site) plus a visual analogue score for pruritus and sleep loss. The index has been shown to be consistent with an overall assessment of disease severity and consistent with a variety of circulating factors thought to reflect disease activity in atopic dermatitis.

According to Table 1A, within-subject analysis showed that the mean SCORAD index scores of subjects in the treatment group had a statistically significant decrease from baseline to Day 7 (p-value = 0.049) and Day 30 (p-value = 0.004). In addition, a consistent decrease from baseline to all time points was noted, with the greatest decrease (-49.11%) found on Day 30. Figure 10 shows the SCORAD results.

Participants completed the Dermatology Life Quality Index (DLQI), a 10-question self-assessment of each subject's own skin condition. The DLQI asks patients how they perceive the impact of their skin disease on different health aspects related to their quality of life over the past week.

According to Table 1A, the subjects' mean Dermatology Life Quality Index (DLQI) scores showed a continuous decrease from baseline to all time points, with the largest decrease at Day 30 (-48.03%). A statistically significant change was observed from baseline to Day 30 (p-value ≤ 0.05). Figure 11 shows the DLQI results.

The subjects' vital scores from day 0 to day 30 were also assessed. It was observed that there were no statistically significant changes in the subjects' body temperature, systolic blood pressure, diastolic blood pressure, pulse rate, and respiratory rate from baseline to any time point, although they showed a trend of increase or decrease. On the other hand, a significant but smaller increase in body weight was observed from baseline to day 15 (p≤0.05). According to Table 1A, compared to baseline, on day 15, the weight gain was +0.81% (about 1.2 pounds); by day 30, the weight gain was +0.97% (about 1.5 pounds). From the nutritional point of view of patients suffering from various conditions or diseases described herein, such results may be valuable.

There were no adverse events or serious adverse events reported for this study.

In summary, the results of the clinical study indicate that the product of Example 6B is effective in treating mild to moderate atopic dermatitis (also known as eczema), as evidenced by a reduction in the size of skin lesions and other signs of the disease (e.g., itching, scaling, and redness).

It is expected that topical application of skin creams will successfully treat symptoms associated with eczema, psoriasis, and related inflammatory conditions. Creams are typically applied topically to the skin in a manner that substantially covers the affected surface area. Creams can be formulated as cosmetic, pharmaceutical, or nutritional compositions that include a pharmaceutically or nutritionally acceptable carrier, respectively.

In human subjects, a useful therapeutic dose of the skin cream (Example 6B) can range from, but is not limited to, about 1 g to about 5 g. Another narrower suitable dose range is from about 1 g to about 2 g, for example, 1-2 g/lesion. For application to human skin, another useful therapeutic dose range is from about 0.5 g/lesion to about 1.5 g/lesion.

For example, in human patients, the skin cream (Examples 6A-6D) can be provided at a daily dose of about 1 g to about 5 g. Another narrower suitable dosage range is about 1 g to about 2 g per day. Another narrower suitable dosage range is about 1 g to about 3 g per day.

Example 6D (psoriasis)

A test cream was prepared by replacing the 2-stage extract (Example 2B) with Extract 2A in Example 6C.

A psoriasis patient with scaly flare-ups on the legs was treated with the psoriasis test cream (Figure 9A).Topical application of the cream (as shown above in Example 6C) over 4 weeks provided noticeable improvement by reducing crusting and scaling (Figure 9B).

It is also contemplated that the 2-stage reaction platform described herein can be used to produce oral formulations based on a combination of pollen-based reaction products and honeycomb components. Such a product can include a 2-stage reaction mixture that has been freeze-dried or lyophilized and then appropriately formulated for oral administration.

Example 7 (Cell Protection Test)

The MTT assay has been adapted to measure whether the extract can protect cells from apoptosis (programmed cell death), which is achieved by,

1. Oxidative stress using hydrogen peroxide, or

2. DNA damage using etoposide (chemotherapy drug).

The MTT assay is traditionally used to measure the cytotoxicity of compounds/extracts. This is achieved by measuring cell proliferation via the reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to formazan in the mitochondria of living cells. If cells are stressed or dying, they cannot undergo this reaction, resulting in a low OD (optical density). If cells function well in the presence of an extract, cell proliferation occurs. Therefore, the OD is directly correlated with the number of viable (living) cells.

The higher the OD, the more viable cells are present in the shown Figures 3A, 3B and 4. None of the extracts showed cytotoxicity as the bars were the same height as in the cell only treatment.

The traditional MTT assay was modified to measure the cytoprotective properties of the extract. This provides a measure of whether the extract can protect cells from stress. HL-60 cells were tested. As shown in Figure 3A, the experimental premise is as follows:

1. Cells only – Grow cells for 48 hours only;

2. Cells + hydrogen peroxide. Grow the cells for 24 hours, then add hydrogen peroxide (a pro-oxidant) for another 24 hours. Hydrogen peroxide is toxic to cells, thus inhibiting growth or even killing them.

3. Cells + Etoposide. The cells were grown for 24 hours, and then etoposide (a chemotherapy drug) was added for another 24 hours. Etoposide is very toxic to cells, killing them by damaging their DNA and preventing them from dividing, thus preventing them from proliferating. Thus, etoposide leads to inhibition of cell growth and ultimately cell death.

4. Extract only: Incubate cells with the extract for 48 hours. If the extract is toxic to the cells, the OD will be lower than the OD of the cell-only treatment.

5. Extract + hydrogen peroxide. Add the extract to the cells for 24 hours, then add hydrogen peroxide. If the extract is able to protect the mitochondria from oxidative stress (i.e., free radicals) from hydrogen peroxide, the cells will continue to grow; and

6. Extract + etoposide. Add the extract to the cells for 24 hours, then add etoposide. If the extract protects DNA from the etoposide chemotherapy, the cells will continue to grow.

Effective extracts (e.g., tamarillo, apple peel, and kava) can be used to assess biological activity. For example, a tamarillo extract can be prepared and reacted as described above by adding tamarillo powder just before sealing the container (before Stage 2), or it can be left unreacted and added after the reaction is complete (after Stage 2, or by omitting Stage 2 entirely). Biological activity appears to be enhanced or even altered by reaction with other components because the lipids in the reaction mixture synergistically enhance biological activity by acting as lipid membrane carriers and/or by esterifying flavonoids. In the following examples, the "water" sample indicates that the tamarillo extract or the coconut water extract without tamarillo (as in Example 2F) was not reacted or fermented in Stage 2, while the "ethanol" sample indicates that the tamarillo extract or the coconut water extract without tamarillo (as in Example 2F) was reacted and fermented in Stage 2.

Therefore, an MTT assay was performed as a standard method, using Example 2F (diluted 1:16) without tamarillo as the "water" extract, meaning that in this case, no second-stage fermentation was performed. This extract demonstrated strong efficacy, as it was non-cytotoxic and protected cells from DNA damage and free radical stress. FIG3A shows that the coconut water-based extract (without tamarillo) as the "water" extract had very strong cytoprotective activity, and FIG3B shows that the coconut water-based extract (without tamarillo) as the "ethanol" extract was even more effective in promoting cell proliferation.

Next, the same experimental procedure was performed using the above-described tamarillo extract (Example 2F). The tamarillo "water" extract itself has been shown to partially reduce cell growth and possess strong cytoprotective bioactivity against hydrogen peroxide and etoposide. When incorporated into a fermentation reaction to provide the tamarillo "ethanol" extract, significant cell proliferation and cytoprotection were observed, as shown in Figure 4 (i.e., protection provided by "Tam"). Thus, the tamarillo bioactivity was synergistically altered by the reaction.

The extracts of the present invention tested in this example show strong efficacy because they are non-cytotoxic and protect cells from DNA damage and free radical stress. It was noted that both hydrogen peroxide and etoposide induce apoptosis. This is how chemotherapy works - it induces DNA damage and then cell death. The results of these experiments show that the extracts of the present invention have strong cytoprotective activity against oxidative stress and against DNA-damaging chemotherapy. Therefore, these results demonstrate that these extracts protect against apoptosis induction.

Example 8 (Immunological test)

Cytokine expression was measured by flow cytometry using standard methods using dual-tag antibodies for IL-10 and TNFα (BioActives Research New Zealand Ltd., Auckland, N.Z.). For this experiment, cells were treated as follows:

1. Cells only – Grow cells for 48 hours only;

2. (Control) Cells + lipopolysaccharide (LPS). Grow cells for 24 hours, then add LPS (a pro-inflammatory bacterial toxin) for another 24 hours.

3. Extract only: cells were incubated with the extract for 48 hours; and

4. Extract + LPS: Add the extract to the cells for 24 hours, then add LPS.

Table 2

TNF is a pro-inflammatory cytokine. Increased TNF indicates inflammation. LPS (lipopolysaccharide) is a bacterial toxin that was used to induce inflammation in this study (TNF levels ranged from an average of 19.9 to 31.7).

IL-10 is an anti-inflammatory cytokine involved in reducing allergic reactions such as eczema. By increasing IL-10, TNF, along with other inflammatory cytokines, is reduced. LPS also triggers an increase in TNF, leading to a cascade that reduces IL-10. As shown in Table 2, in the presence of LPS, the coconut and tamarillo extracts prepared according to the present invention maintained TNF expression at normal levels while simultaneously increasing IL-10 to combat pro-inflammatory attacks.

Example 9 (Acne Cream Formulation)

In another embodiment, a cream formulation is prepared for treating acne.

^2. Does not contain beeswax.

Omit the beeswax as it is not beneficial in acne skin treatments.

Optionally, up to 2.0% by weight of the total composition of salicylic acid may be added. In one embodiment, 0.5% by weight of salicylic acid is added.

Podocarpus extract is obtained from the bark of a native species.

It is expected that topical application of acne creams will successfully treat the symptoms associated with acne and related inflammatory conditions. Creams are typically applied topically to the skin in a manner that substantially covers the affected surface area. Creams can be formulated as cosmetic, pharmaceutical, or nutritional compositions that include a pharmaceutically or nutritionally acceptable carrier, respectively.

Cosmetics (or cosmeceuticals) of the present invention or nutritional composition can be used in combination with an acceptable carrier in nutrition. In this preparation, active ingredient can constitute 1 % by weight -99 % by weight, or alternatively, 0.1 % by weight -99.9 % by weight. Or, active ingredient can be in the scope of about 5 % by weight -about 75 % by weight or about 10 % by weight -about 75 % by weight." acceptable carrier in nutrition " represents any carrier, diluent or excipient compatible with other ingredients of preparation and harmless to user. Useful excipient includes microcrystalline cellulose, magnesium stearate, calcium stearate, any acceptable sugar (for example, mannitol, xylitol), and for cosmetic purposes, oil base is preferred.

The topical pharmaceutical compositions of the present invention may be administered in combination with a pharmaceutically acceptable carrier. In such a formulation, the active ingredient may constitute 1% to 99% by weight, or alternatively, 0.1% to 99.9% by weight. "Pharmaceutically acceptable carrier" means any carrier, diluent, or excipient that is compatible with the other ingredients of the formulation and is not harmful to the user.

According to certain embodiments, the topical pharmaceutical compositions disclosed herein can be provided in the form of an ointment, cream, lotion, gel, or other transdermal delivery system, as described in L. V. Allen, Jr. et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, 9th ed., pp. 272-293 (Philadelphia, Pennsylvania: Lippincott Williams & Wilkins, 2011), which is incorporated herein by reference.

As used herein, ointment refers to a semisolid preparation comprising an ointment base into which one or more active ingredients are incorporated or fused (i.e., melted together with the other components of the formulation and then cooled with continuous stirring to form a solidified preparation). The ointment base can be in the form of an oily or hydrocarbon base (e.g., petrolatum or a petrolatum/wax combination); an absorbent base that allows the incorporation of an aqueous solution, resulting in the formation of a water-in-oil emulsion (e.g., hydrophilic petrolatum) or a water-in-oil emulsion that allows the incorporation of additional amounts of aqueous solution (e.g., lanolin); a water-removable base that is an oil-in-water emulsion that can be diluted with water or an aqueous solution (e.g., hydrophilic ointment, USP); or a water-soluble base that does not contain an oily component (e.g., a polyethylene glycol (PEG) formulation that combines PEG with an average molecular weight of less than 600 and PEG with an average molecular weight of greater than 1000), etc.

As used herein, cream refers to a semisolid product comprising one or more active, fermented pollen-based substances, or medicaments, dissolved or dispersed in a water-in-oil emulsion or an oil-in-water emulsion or another type of water-washable base. Generally speaking, creams are distinguished from ointments by their ease of application/spreading on, for example, a skin surface and their ease of removal from the treated surface.

As used herein, lotion refers to a suspension of solid materials in an aqueous vehicle. Generally, lotions have a non-greasy character and spread better over large areas of skin than ointments, creams, and gels.

As used herein, gel refers to a semisolid system comprising a dispersion of small molecules and/or macromolecules in an aqueous carrier, which is rendered jelly-like by the addition of a gelling agent. Suitable gelling agents include, but are not limited to, synthetic macromolecules (e.g., carbomer polymers), cellulose derivatives (e.g., carboxymethyl cellulose and/or hydroxypropyl methyl cellulose), and natural gums (e.g., xanthan gum, carrageenan, etc.). Gel preparations can be in the form of a single-phase gel, in which the active or pharmaceutical ingredient is uniformly dispersed throughout the liquid carrier without visible boundaries, or a two-phase gel, in which small, independent particles of a flocculant or active or pharmaceutical ingredient are dispersed within the liquid carrier.

Transdermal preparations can be formed from ointments, creams, or gels that have been combined with penetration enhancers and are designed to deliver active or pharmaceutical ingredients systemically. Penetration enhancers include, for example, dimethyl sulfoxide, ethanol, propylene glycol, glycerol, PEG, urea, dimethylacetamide, sodium lauryl sulfate, poloxamers, Spans, Tweens, lecithin, and/or terpenes, among others.

Other semisolid forms suitable for use as cosmetic and/or topical pharmaceutical compositions include pastes (preparations containing a larger portion of solid matter, making them harder than ointments) and glycerol gelatins (plastic masses containing gelatin, glycerin, water and an active or pharmaceutical ingredient).

In other embodiments, the topical and/or cosmetic compositions can be prepared according to the dosage forms described in Sample Preparation of Pharmaceutical Dosage Forms, ed. B. Nickerson (New York: Springer, 2011), which is incorporated herein by reference.

In addition, shampoos and other types of cleaning products are also contemplated.In addition, topical sprays are also contemplated, including sprays for one or more applications to, for example, the skin, back, neck, arms, legs, and trunk.

For oral administration, the fermented pollen-based extract or its solid formulation (e.g., lyophilized powder) can be combined with one or more solid inactive ingredients to prepare tablets, capsules, pills, powders, granules, or other suitable dosage forms. For example, the active agent can be combined with at least one excipient, such as a filler, binder, wetting agent, disintegrant, solution retarder, absorption accelerator, wetting agent, adsorbent, or lubricant. Other useful excipients include magnesium stearate, calcium stearate, mannitol, xylitol, sweeteners, starch, carboxymethyl cellulose, microcrystalline cellulose, silica, gelatin, silicon dioxide, etc.

Route of administration

The fermented pollen-based extract can be administered by any route, including, but not limited to, oral, sublingual, buccal, ocular, pulmonary, rectal, and parenteral administration, or as an oral or nasal spray (e.g., mist vapor, liquid droplets, or solid particle inhalation). Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, intravaginal, intravesical (e.g., to the bladder), intradermal, transdermal, topical, topical spray, or subcutaneous administration.

Treatment can be carried out for the desired length in a single uninterrupted session or in discrete sessions. The treating physician will know how to increase, decrease, or interrupt treatment based on the patient's response. According to one embodiment, treatment is carried out for about 4 to about 5 weeks. The treatment schedule may be repeated as needed.

In addition, it is believed that this technology and method can be applied to other reaction platforms. The above examples describe such a technology platform, wherein different starting materials can be added depending on the desired end product. By changing the settings, such as aerobic/anaerobic, temperature and pressure, different end product compounds are obtained. The end products produced by the reaction platform are similar to those produced in nature. These compounds mimic those that control immune and cell death signaling pathways in nature, which can be used to treat a range of immune and apoptosis related diseases.

In an alternative embodiment, the technology platform may comprise a bioreactor that utilizes naturally occurring chemical reactions to produce modified or novel bioactive compounds that modulate mammalian cell signaling. These chemical reactions can be applied to any plant, microbial, marine, or insect-derived extract to produce bioactive components that directly interact with human signaling pathways to modulate immune and cell death responses.

Although the present invention has been described in the foregoing specification with respect to certain embodiments and many details have been mentioned for the purpose of illustration, it will be apparent to those skilled in the art that the present invention may have additional embodiments and that some of the details described herein may be varied considerably without departing from the basic principles of the invention.

In the context of describing the present invention (particularly in the context of claims), the use of quantifiers should be interpreted as including both singular and plural numbers, unless otherwise stated in this article or the context clearly negates. The enumeration of numerical ranges herein is only intended to be a shorthand method of expression for referring to each individual numerical value falling within the scope, and unless otherwise stated herein, each individual numerical value is included in this specification as it is individually quoted herein. All methods described herein can be carried out in any suitable order, unless otherwise stated herein or the context clearly negates. The use of any and all embodiments provided herein, or exemplary terms (e.g., "such as") is only intended to better illustrate the present invention and not limit the scope of the present invention as originally claimed. The terms in this specification should not be interpreted as indicating any unclaimed element necessary for implementing the present invention.

All references cited herein are hereby incorporated by reference in their entirety. The present invention may be embodied in other specific forms without departing from its spirit or essential attributes and, therefore, reference should be made to the appended claims rather than to the foregoing description as indicating the scope of the invention.

Claims (19)

1. A method for manufacturing a fermented pollen-based composition, the method comprising the steps of: (a) Soaking dried pollen grains in coconut water at ambient temperature to provide soaked pollen; (b) Treat the soaked pollen with glycerol and honeydew at ambient temperature to provide a germinating pollen mixture; (c) Beeswax that has been melted at 65°C and remains liquid; (d) Treat the melted beeswax with an equal weight of coconut oil to provide a beeswax mixture; (e) Add the germinating pollen mixture to the beeswax mixture; (f) Stirring the combined mixture to form a gel; and (g) The gelatinous substance is incubated in a sealed container at 37°C for 72 hours to produce a fermented pollen-based composition. 2. The method of claim 1, wherein the soaked pollen is germinated. 3. The method of claim 1, wherein the weight of the pollen is twice the weight of the beeswax, and wherein the weight of the glycerin and honeydew is half the weight of the beeswax. 4. The method of claim 1, wherein the enzyme-containing component is added before or after step (g). 5. The method of claim 4, wherein the enzyme-containing component is selected from: pineapple powder, tree tomato powder, kava powder, kiwi powder, honey, other honeydew, and mixtures thereof. 6. The method of claim 1 or 4, further comprising step (h): adding a substance selected from propolis, pine bark powder or mixtures thereof to terminate fermentation. 7. A fermented pollen-based composition prepared according to the method of claim 1. 8. The fermented pollen-based composition of claim 7, comprising one or more peptides and one or more fatty acids. 9. The fermented pollen-based composition of claim 8, further comprising at least one dicaffeoylquinic acid. 10. The use of a fermented pollen-based composition comprising one or more peptides, one or more fatty acids, and at least one dicaffeoylquinic acid, manufactured according to claim 1, in the preparation of a skin cream for treating or preventing skin conditions in an individual. The skin cream contains 1-10% by weight of a fermented pollen-based composition; The skin cream is applied topically to the individual who requires this treatment in a therapeutically effective amount; The symptoms of the skin condition are reduced, and the skin condition is selected from: eczema, psoriasis, actinic keratosis (AK), age spots, molluscum contagiosum, and lupus. 11. The use as claimed in claim 10, wherein the individual is a person. 12. The use according to claim 11, wherein the skin condition is eczema and the skin cream is applied to one or more skin lesions in an amount of 0.5g to 3g per day. 13. The use of claim 12, wherein the number and/or size of the one or more skin lesions is reduced by at least 20%. 14. The use as claimed in claim 12, wherein the number and/or size of the one or more skin lesions is reduced by at least 15%. 15. The use according to claim 12, wherein the atopic dermatitis severity score (SCORAD) is reduced by at least 40%. 16. The use as claimed in claim 12, wherein the average Dermatology Quality of Life (DLQI) score is reduced by at least 40%. 17. A topical skin cream composition, said topical skin cream composition comprising: The fermented pollen-based composition manufactured according to the method of claim 1 comprises one or more peptides, one or more fatty acids and at least one dicaffeoylquinic acid, wherein the fermented pollen-based composition is present in an amount of 1-10% by weight based on the total weight of the topical skin cream composition. An emulsified oil containing cetyl oleate and sorbitol oleate, wherein the presence of the emulsified oil is 7% by weight based on the total weight of the topical skin cream composition; At least one plant oil component, present in an amount of 5-7% by weight based on the total weight of the topical skin cream composition; Glycerin, present in an amount of 2-4% by weight based on the total weight of the topical skin cream composition; and Water, which is present in an amount of 70% by weight based on the total weight of the topical skin cream composition. 18. The topical skin cream of claim 17, further comprising at least one vitamin selected from vitamin A, vitamin C, vitamin E, and mixtures thereof, wherein the at least one vitamin is present in an amount of 1-2 by weight based on the total weight of the topical skin cream composition. 19. The topical skin cream of claim 17, wherein the at least one plant oil component is selected from: shea butter, safflower oil, macadamia nut oil, hazelnut oil, blackcurrant oil, kiwi seed oil, grape seed oil, argan oil, jojoba oil, and mixtures thereof.