Plant-based extract complex composition having the properties for supporting the relief of musculoskeletal pain, antibacterial and anti-inflammatory and process of manufacturing the same

A plant-based extract complex composition having the properties for supporting the relief of musculoskeletal pain, antibacterial and anti-inflammatory is obtained from the process of forming a homogeneous mixing mixture by mixing in a specific order into a container including (A) a ginseng extract solution having a first percentage (%) by weight, (B) a medicinal herbs extract solution having a second percentage (%) by weight, (C) a plant-derived nanovesicles ingredient component having a third percentage (%) by weight, (D), a plant-derived essential oils ingredient having a fifth percentage (%) by weight (E) a methyl salicylate component having a fourth percentage (%) by weight, (F) an excipients component having a sixth percentage (%) by weight, and (G) water to create a mixture.

1. 11. A process of manufacturing a plant-based extract complex composition having the properties for supporting the relief of musculoskeletal pain, antibacterial and anti-inflammatory comprising: (i) preparing material including: (A) a ginseng extract solution having a first percentage (%) by weight, (B) a medicinal herbs extract solution having a second percentage (%) by weight, (C) a plant-derived nanovesicles ingredient having a third percentage (%) by weight, (D) a plant-derived essential oils ingredient having a fifth percentage (%) by weight, (E) a methyl salicylate component having a fourth percentage (%) by weight, (F) an excipients component having a sixth percentage (%) by weight, and (G) water; wherein the total percentage by weight of the composition is determined by the sum of the first to sixth percentages plus the percentage of water to make up 100% by weight of the plant-based extract complex composition; wherein the ginseng extract solution is extracted from parts of ginseng, including roots, stems, leaves, and flowers that have been cleaned, then crushed or chopped and soaked in solvent or saline solution; the roots have a minimum age of 5 years old; stems, leaves, and flowers of ginseng have a minimum age of 3 years old; the ginseng parts are selected from one or more parts of plants belonging to the genus Panax, the Panax species being selected from the group consisting of: Panax notoginseng, Panax bipinnatifidus, Korean ginseng, Panax Ginseng, Panax japonicus, American ginseng, Panax quonquefolius, Ngoc Linh ginseng, Panax vietnamensis, Panax wangianus, Panax zingiberebsis, Panax pseudoginseing, and Panax stipuleanatus; wherein the medicinal herbs extract solution is prepared by performing steps (a1) to (a3): (a1) collecting and pre-processing material from medicine herbals (a)-(l) including removing damaged parts, washing, and drying to 8%-15% moisture, chopping and stored in separate instruments; wherein material from medicine herbals (a)-(l) include: (a) the flower of Carhamus tinctorius L., (b) the resin from the fruit and stem of Daemonorops draco (Willd.) Blume, (c) the roots and tubers of Coptis chinensis Franch., (d) the roots and tubers of Reynoutria japonica Houtt., (e) the bark of Carhamus tinctorius L., (f) the tubers of Zingiber officinale Rosc., (g) the fruits of Illicium verum Hook. f. Illicium verum, (h) the whole plant of Piper sarmentosum, (i) the leaves of Artemisia vulgaris, (j) the tubers of Codonopsis pilosula Nannf, (k) the flower buds of Flos caryophylatac, and (l) the seeds of Brassica alba; (a2) creating a medicine herbals mixture by performing steps (a′) to (b′): (a′) collecting and pre-processing material from medicine herbals (m)-(n) including removing damaged parts, washing, drying overnight exposed to dew and drying to 8%-15% moisture, chopping and stored in separate instruments; wherein the material from medicine herbals (m)-(n) include: (m) Salvia miltiorrhiza and (n) Achyranthes bidentate uses the roots; and (b′) mixing the material from medicine herbals (a)-(l) prepared in step (a1) and the material from medicine herbals (m)-(n) prepared in step (a′) to obtain the medicine herbals mixture; and (a3) soaking the medicine herbals mixture with 70% ethanol solvent in a ratio of 18:70 w/v at room temperature, stirring at a frequency of 200-250 rpm for 10 days to obtain a mixture 1, filtering the mixture 1 to obtain a filtrate 1 and residue 1; soaking the residue 1 with 70% ethanol solvent in a ratio of 18:70 w/v, stirring at a frequency of 200-250 rpm for 10 days to obtain a mixture 2, filtering the mixture 2 to obtain a filtrate 2 and residue 2; soaking the residue 2 with 70% ethanol solvent in a ratio of 18:70 w/v, stirring at a frequency of 200-250 rpm for 10 days to obtain a mixture 3, filtering the mixture 3 to obtain a filtrate 3 and a residue 3; mixing the filtrate 1 with the filtrate 2, and filtrate 3 to obtain a filtrate mixture, removing the solvent from the filtrate mixture by vacuum evaporation at 40° C.-45° C. to obtain the medicinal herbs extract solution; wherein the plant-derived nanovesicles ingredient is prepared by mixing a first nanovesicles ingredient with a second nanovesicles ingredient in a ratio of 2:3 w/w; the first nanovesicles ingredient is prepared by performing steps (b1) to (b8): (b1) collecting the material from plants in the following percentage (%) by weight including 2 parts of Ilicium verum Hook. f. Illicium verum, 3 parts of Artemisia vulgaris, and 3 parts of Piper sarmentosum to obtain a temporary mixture; (b2) washing the temporary mixture three times with deionized water at 20° C. 25° C.; (b3) pureeing the washed temporary mixture with phosphate buffer solution (PBS) in a ratio of 1:3 w/v at a speed of 7,000-8,000 rpm for 15 minutes to obtain a first temporary solution; (b4) filtering the first temporary solution by a filter membrane with a diameter 0.20-0.22 μm to obtain a second temporary solution; (b5) centrifuging the second temporary solution by ultracentrifugation at 120,000×g for 100 min at 4° C. to obtain a temporary residue; (b6) dissolving the temporary residue in phosphate buffer solution (PBS), transferring to a 45% sucrose gradient solution, and ultracentrifuging at 130,000×g for 100 min to obtain a third temporary solution; (b7) washing the third temporary solution with PBS and centrifuging at 150.000×g for 60 min at 4° C. to obtain a fourth temporary solution; and (b8) filtering the fourth temporary solution through a filter membrane with a diameter 0.20-0.22 μm to obtain the first nanovesicles ingredient; the second nanovesicles ingredient is prepared by performing steps (c1) to (c9): (c1) collecting a fruit mixture in the following percentage (%) by weight including 3 parts of dragon fruit (Hylocereus undatus, Hylocereus costaricensis, Hylocereus megalanthus, Hylocereus undatus costaricensis), 3 parts of avocado (Persea americana), and 1 part of watermelon (Citrullus lanatus); (c2) washing the fruit mixture three times with deionized water at 20° C.-25° C.; (c3) pureeing the washed fruit mixture with phosphate buffer solution (PBS) in a ratio of 1:1 (g/mL) at a speed of 7,000-8,000 rpm for 15 minutes to obtain a first foundation solution; (c4) filtering the first foundation solution by a filter membrane with a diameter 0.20-0.22 μm to obtain a second foundation solution; (c5) centrifuging the second foundation solution by ultracentrifugation at 100,000×g for 60 min to obtain a residue; (c6) dissolving the residue in phosphate buffer solution (PBS), transferring to a 45% sucrose gradient solution, and ultracentrifuging at 130,000×g for 100 min to obtain a third foundation solution; (c7) stirring the third foundation solution with a 10% polyethylene glycol-8000 (PEG8000) solution in a ratio of 1:1 v/v, and incubating for 8-10 hours at 4° C., then centrifuging at 110,000×g for 40 minutes at 4° C. to obtain a precipitate; (c8) dissolving the precipitate in phosphate buffer solution (PBS) in a ratio of 1:2 w/v to obtain a foundation solution; and (c9) filtering the foundation solution by a tangential flow filtration (TFF) to obtain the second nanovesicles ingredient; wherein the technical specifications related to TFF include a molecular size of 500 kDa, and filtering at a flow rate of 20 mL/min with the transmembrane pressure maintained at 2 bar; wherein the plant-derived essential oils ingredient is prepared by mixing an essential oil of cajeput leaf, an essential oil of camphor leaf, an essential oil of cinnamon leaf, an essential oil of eucalyptus leaf, an essential oil of rosemary leaf, an essential oil of peppermint leaf, an essential oil of perilla leaf, and an essential oil of ginger rhizome n the respective mass ratios of (1-2):2:1:(1-2):2:1:(1-2):1; wherein each plant-derived essential oils ingredient is extracted by performing steps (d1) to (d3): (d1) collecting and pre-processing material from plant individually including removing damaged parts, washing, chopping, and soaking with an enzyme solution in a ratio of 1:2 w/v for 30 minutes, and filtering to obtain an enzyme-treated plant material; in which the enzyme solution is prepared by: creating an enzyme composition by mixing a cellulase with hemicellulase, and xylanase in a ratio of 1:1:1 w/w/w; and dissolving the enzyme composition with water in a ratio of 1:15000 w/v; (d2) grinding a mixture of 1 part the enzyme-treated plant material with 7 parts saturated salt solution, let stand after grinding for 1 hour with microwave assistance at a power of 300-500 W, then steam distillation for 3 hours to obtain a crude essential oil; and (d3) removing water from the crude essential oil by adding Na2SO4 salt to crystallize at −15° C., and filtering to obtain the plant-derived essential oils ingredient wherein the methyl salicylate component is synthesized by: dissolving 1.5 g of salicylic acid (10 mmol) with 4.35 mL of 10% methanol in a 250 mL three-necked flask, adding 0.27 mL of 10% H2SO4, and refluxing at 82° C.-83° C. for 6 hours; evaporating by rotary vacuum evaporation of the mixture after reaction at 62° C.-65° C. to remove the solvent, cooling the mixture after evaporation and putting it in a decanter; coating the rotary vacuum evaporation flask with saturated NaHCO3 solution put it in the decanter, adding 7 mL of saturated NaHCO3 to the decanter, gently shaking the decanter then let it stand to obtain a lower solution layer; washing the lower solution layer three times with saturated NaHCO3 solution, wherein each time washing with saturated NaHCO3 solution is 7 mL; washing the lower solution layer three times with distilled water, wherein each time washing with distilled water is 12 mL; adding 0.5 g of anhydrous Na2SO4 into the reaction mixture, filtering, and distilling fractional to obtain the methyl salicylate component at temperature of 220° C.; wherein the excipients component is selected from the group consisting of ethanol, polysorbate 60, sorbitan oleate, triethanolamine, carbomer or combinations thereof; (ii) obtaining a plant-based extract complex composition by mixing in a specific order into a container including (A) a ginseng extract solution having a first percentage (%) by weight, (B) a medicinal herbs extract solution having a second percentage (%) by weight, (C) a plant-derived nanovesicles ingredient having a third percentage (%) by weight, (D) a plant-derived essential oils ingredient having a fifth percentage (%) by weight, (E) a methyl salicylate component having a fourth percentage (%) by weight, (F) an excipients component having a sixth percentage (%) by weight, and (G) water to create a mixture; wherein after each addition of the mixing ingredients to the mixture is stirred until the mixture is homogeneous; and (iii) filling and packaging.
2. 22. The process of claim 1, wherein the properties for supporting the relief of musculoskeletal pain, antibacterial and anti-inflammatory depend on the percentage (%) by weight of each ingredients (A)-(G) including a first formula, a second formula, a third formula, and a fourth formula; wherein the third formula is stronger than the first formula, the first formula is stronger than the second formula, and the second formula is stronger than the fourth formula; in which the comparative factor is properties for supporting the relief of musculoskeletal pain, antibacterial and anti-inflammatory.
3. 33. The process of claim 2, wherein the first formula comprises: (A) the ginseng extract solution having 0.01%-0.5% by weight; (B) the medicinal herbs extract solution having 12%-55% by weight; (C) the plant-derived nanovesicles ingredient having 5%-10% by weight; (D) the plant-derived essential oils ingredient having 10%-28% by weight; (E) the methyl salicylate component having 0.001%-0.8% by weight; (F) the excipient component having 0.001%-5% by weight; and (G) the remainder is the water.
4. 44. The process of claim 2, wherein the second formula comprises: (A) the ginseng extract solution having 0.01%-0.5% by weight; (B) the medicinal herbs extract solution having 0.1%-12% by weight; (C) the plant-derived nanovesicles ingredient having 1%-5% by weight; (D) the plant-derived essential oils ingredient having 28%-48% by weight; (E) the methyl salicylate component having 0.8%-10% by weight; (F) the excipient component having 0.001%-5% by weight; and (G) the remainder is the water.
5. 55. The process of claim 2, wherein the third formula comprises: (A) the ginseng extract solution having 0.01%-0.5% by weight; (B) the medicinal herbs extract solution having 12%-55% by weight; (C) the plant-derived nanovesicles ingredient having 5%-10% by weight; (D) the plant-derived essential oils ingredient having 28%-48% by weight; (E) the methyl salicylate component having 0.8%-10% by weight; (F) the excipient component having 0.001%-5% by weight; and (G) the remainder is the water.
6. 66. The process of claim 2, wherein the fourth formula comprises: (A) the ginseng extract solution having 0.01%-0.5% by weight; (B) the medicinal herbs extract solution having 0.1%-12% by weight; (C) the plant-derived nanovesicles ingredient having 1%-5% by weight; (D) the plant-derived essential oils ingredient having 28%-48% by weight; (E) the methyl salicylate component having 0.001%-0.8% by weight; (F) the excipient component having 0.001%-5% by weight; and (G) the remainder is the water.

FIELD OF THE INVENTION
The invention pertains to the field of health care product manufacturing, particularly to a plant-based extract complex composition effective in reducing symptoms the relief of musculoskeletal pain, supporting treatment of respiratory diseases. More specifically, the invention relates to a method for producing a plant-based extract complex composition having the properties for supporting the relief of musculoskeletal pain, antibacterial and anti-inflammatory and process of manufacturing the same.
BACKGROUND ART
Natural compounds are extracted from various sources, among which herbs are the most common. Due to their biodiversity, crude extracts from herbs often contain bioactive compounds such as tannins, flavonoids, alkaloids, and phenolics, which have been widely used as sources of natural medicines and health care products. In recent years, medicinal plants have attracted significant attention as a source of secondary metabolites for screening in the treatment of chemotherapeutic drug resistance, using in vitro, in vivo, and in silico approaches. Drug development through phytopharmaceutical techniques offers an efficient, cost-effective, and safe alternative to conventional methods such as animal cell culture and microbial fermentation. Therefore, plant-derived natural compounds have the potential to provide patients with faster and more accessible therapeutic options.
Extraction is the preliminary step to separate the desired natural products from the raw material. Different extraction methods are used based on the components and compounds in the plant extract. Distillation, sublimation, and solvent extraction are the most common methods used to extract the different active ingredients in plants. Some techniques with unique special mechanisms are created to ensure better extraction efficiency. Ultrasonic extraction, microwave extraction, supercritical fluid extraction, and combined extraction with the help of ultrasound waves are advanced extraction techniques.
Nanovesicles derived from natural products are gaining attention as innovative therapeutic agents due to their biocompatibility, low immunogenicity, and ability to transport bioactive molecules such as proteins, lipids, and nucleic acids. Plant-derived nanovesicles exhibit structural similarities to mammalian exosomes, making them suitable for drug delivery, microbiome-targeted therapy, and regenerative medicine. Nanovesicles' Potential applications include treating cancer, inflammation, and metabolic disorders. Additionally, nanovesicles have applications in cosmetics, agriculture, and the food industry.
Inflammation is the body's defense response to harmful stimuli such as allergens and/or tissue damage; on the other hand, the uncontrolled inflammatory response is the main cause of a variety of disorders, including allergies, cardiovascular dysfunction, metabolic syndrome, cancer, and autoimmune diseases that impose a huge economic burden on individuals and therefore on society. There are many different drugs to control and prevent inflammation. However, some drugs will cause side effects; the goal is to use effective doses with the least side effects. Therefore, it is necessary to apply anti-inflammatory factors from natural extracts to enhance pharmacological effects and reduce the level of unwanted side effects to the lowest level.
It can be seen that the above technical solutions all meet the set purposes and requirements. However, the above inventions are still limited in that the compound components are very volatile, easily oxidized, and evaporated within a few hours, as well as the ability to penetrate the capillaries under the skin to help reduce pain, muscle stiffness due to heavy activities or sports, especially effective in physical therapy support. In addition, these preparations need to be controlled for the ability to cause skin irritation, not poisoning and odor for users when they are in working outdoors for a long time.
Therefore, it is necessary to provide a plant-based extract complex composition containing stable, non-volatile, easy-to-use, and multifunctional active ingredients, which can be used as a massage medicine to support the relief of joint pain and inflammation, as well as antibacterial and antiviral products used externally on the skin or through the nose such as herbal inhalation products.
It is also necessary to provide a plant-based extract complex composition having the relief of musculoskeletal pain, antibacterial and anti-inflammatory does not cause any negative effects on the user's health and does not cause skin irritation.
Finally, it is also necessary to provide a plant-based extract complex composition having affordable and easy to produce, taking advantage of available raw materials, easy to collect, non-toxic, environmentally friendly but still ensuring the quality and effectiveness of the relief of musculoskeletal pain, antibacterial and anti-inflammatory.
This invention provides solutions to achieve the above goals.
SUMMARY OF THE INVENTION
Accordingly, the first aspect of the invention is to provide a plant-based extract complex composition having the properties for supporting the relief of musculoskeletal pain, antibacterial and anti-inflammatory is obtained from the process of forming a homogeneous mixture by mixing in a specific order into a container including (A) a ginseng extract solution having a first percentage (%) by weight, (B) a medicinal herbs extract solution having a second percentage (%) by weight, (C) a plant-derived nanovesicles ingredient having a third percentage (%) by weight, (D) a plant-derived essential oils ingredient having a fourth fifth percentage (%) by weight, (E) a methyl salicylate component having a fifth percentage (%) by weight, (F) an excipients component having a sixth percentage (%) by weight, and (G) water to create a mixture; wherein after each addition of the mixing ingredients to the mixture is stirred until the mixture is homogeneous; wherein after each addition of the mixing component to the container, the complex is stirred until complex is homogeneous; wherein the percentage (%) by weight is determined by the sum from the first percentage (%) to the sixth percentage (%) plus the percentage (%) of the water to make up 100%.
The second aspect of the invention is to provide a plant-based extract complex composition having the properties for supporting the relief of musculoskeletal pain, antibacterial and anti-inflammatory depend on the percentage (%) by weight of each ingredients (A)-(G) including a first formula, a second formula, a third formula, and a fourth formula; wherein the third formula is stronger than the first formula, the first formula is stronger than the second formula, and the second formula is stronger than the fourth formula; in which the comparative factor is properties for supporting the relief of musculoskeletal pain, antibacterial and anti-inflammatory. These four formulas can be used as massage balm, massage gel, inhalation oil, liniment oil, aromatherapy products, medicinal products such as pain relief, expectorant products, and skin care products, etc.
The third aspect of the invention is to provide a plant-based extract complex composition having the properties for supporting the relief of musculoskeletal pain, antibacterial and anti-inflammatory according to the first formula comprises: (A) the ginseng extract solution having 0.01%-0.5% by weight; (B) the medicinal herbs extract solution having 12%-55% by weight; (C) the plant-derived nanovesicles ingredient having 5%-10% by weight; (D) the plant-derived essential oils ingredient having 10%-28% by weight; (E) the methyl salicylate component having 0.001%-0.8% by weight; (F) the excipient component having 0.001%-5% by weight; and (G) the remainder is the water.
The fourth aspect of the invention is to provide a plant-based extract complex composition having the properties for supporting the relief of musculoskeletal pain, antibacterial and anti-inflammatory according to the second formula comprises: (A) the ginseng extract solution having 0.01%-0.5% by weight; (B) the medicinal herbs extract solution having 0.1%-12% by weight; (C) the plant-derived nanovesicles ingredient having 1%-5% by weight; (D) the plant-derived essential oils ingredient having 28%-48% by weight; (E) the methyl salicylate component having 0.8%-10% by weight; (F) the excipient component having 0.001%-5% by weight; and (G) the remainder is the water.
Another objective of the present invention is to provide a plant-based extract complex composition having the properties for supporting the relief of musculoskeletal pain, antibacterial and anti-inflammatory according to the third formula comprises: (A) the ginseng extract solution having 0.01%-0.5% by weight; (B) the medicinal herbs extract solution having 12%-55% by weight; (C) the plant-derived nanovesicles ingredient having 5%-10% by weight; (D) the plant-derived essential oils ingredient having 28%-48% by weight; (E) the methyl salicylate component having 0.8%-10% by weight; (F) the excipient component having 0.001%-5% by weight; and (G) the remainder is the water.
Another objective of the present invention is to provide a plant-based extract complex composition having the properties for supporting the relief of musculoskeletal pain, antibacterial and anti-inflammatory according to the fourth formula comprises: (A) the ginseng extract solution having 0.01%-0.5% by weight; (B) the medicinal herbs extract solution having 0.1%-12% by weight; (C) the plant-derived nanovesicles ingredient having 1%-5% by weight; (D) the plant-derived essential oils ingredient having 28%-48% by weight; (E) the methyl salicylate component having 0.001%-0.8% by weight; (F) the excipient component having 0.001%-5% by weight; and (G) the remainder is the water.
Another objective of the present invention is to provide a process of manufacturing a plant-based extract complex composition having the properties for supporting the relief of musculoskeletal pain, antibacterial and anti-inflammatory comprising:

    
    
        (i) preparing material including: (A) a ginseng extract solution having a first percentage (%) by weight, (B) a medicinal herbs extract solution having a second percentage (%) by weight, (C) a plant-derived nanovesicles ingredient having a third percentage (%) by weight, (D) a plant-derived essential oils ingredient having a fourth percentage (%) by weight, (E) a methyl salicylate component having a fifth percentage (%) by weight, (F) an excipients component having a sixth percentage (%) by weight, and (G) water;
        (ii) obtaining a plant-based extract complex composition by mixing in a specific order into a container including (A) a ginseng extract solution having a first percentage (%) by weight, (B) a medicinal herbs extract solution having a second percentage (%) by weight, (C) a plant-derived nanovesicles ingredient having a third percentage (%) by weight, (D) a plant-derived essential oils ingredient having a fourth percentage (%) by weight, (E) a methyl salicylate component having a fifth percentage (%) by weight, (F) an excipients component having a sixth percentage (%) by weight, and (G) water to create a mixture;
        wherein after each addition of the mixing ingredients to the mixture is stirred until the mixture is homogeneous; and
        wherein the percentage (%) by weight is determined by the sum from the first percentage (%) to the sixth percentage (%) plus the percentage (%) of the water to make up 100%; and
        (iii) filling and packaging.
    
    


Finally, another objective of the present invention is to provide a method for supporting the relief of musculoskeletal pain, antibacterial and anti-inflammatory comprising topically applying a plant-based extract complex composition to the skin at the pain location of a subject in need thereof, wherein the plant-based extract complex composition is obtained from the process of forming a homogeneous mixture by mixing in a specific order into a container including (A) a ginseng extract solution having a first percentage (%) by weight, (B) a medicinal herbs extract solution having a second percentage (%) by weight, (C) a plant-derived nanovesicles ingredient having a third percentage (%) by weight, (D) a plant-derived essential oils ingredient having a fifth percentage (%) by weight, (E) a methyl salicylate component having a fourth percentage (%) by weight, (F) an excipients component having a sixth percentage (%) by weight, and (G) water to create a mixture; wherein after each addition of the mixing ingredients to the mixture is stirred until the mixture is homogeneous; wherein the percentage (%) by weight is determined by the sum from the first percentage (%) to the sixth percentage (%) plus the percentage (%) of the water to make up 100%.
These and other advantages of the present invention will no doubt become obvious to those of ordinary skill in the art after having read the following detailed description of the preferred embodiments, which are illustrated in the various drawing Figures.



BRIEF DESCRIPTION OF THE DRAWING
The accompanying drawings, which are incorporated in and form a part of this specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention.
FIG. 1 is a flowchart illustrating a process of manufacturing plant-based extract complex composition having the properties for supporting the relief of musculoskeletal pain, antibacterial and anti-inflammatory in accordance with an embodiment of the present invention.



DETAILED DESCRIPTION OF THE INVENTION
Reference will now be made in detail to the preferred embodiments of the invention, examples of which are illustrated in the accompanying drawings. While the invention will be described in conjunction with the preferred embodiments, it will be understood that they are not intended to limit the invention to these embodiments. On the contrary, the invention is intended to cover alternatives, modifications, and equivalents, which may be included within the spirit and scope of the invention as defined by the appended claims. Furthermore, in the following detailed description of the present invention, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, it will be obvious to one of ordinary skills in the art that the present invention may be practiced without these specific details. In other instances, well-known methods, procedures, components, and circuits have not been described in detail so as not to unnecessarily obscure aspects of the present invention.
In the embodiment of the present invention, percent mass or percentage (%) by weight=(mass of solute/mass of solution)×100%. The unit of mass is usually grams, or kilograms. Mass percent is also known as the correct percentage by weight or w/w %. It should also be noted that the molar mass is also within the meaning of the invention. Molar mass is the total mass of all atoms in a mole of compound. Total all volume percentages add up to 100%.
According to the embodiment of the invention, a plant-based extract complex composition having the properties for supporting the relief of musculoskeletal pain, antibacterial and anti-inflammatory 100 (“composition 100”) is obtained from the process of forming a homogeneous mixture by mixing in a specific order into a container including the mixing ingredients Z1 to Z7 are listed in Tables 1 below: a ginseng extract solution having a first percentage (%) by weight, a medicinal herbs extract solution having a second percentage (%) by weight, a plant-derived nanovesicles ingredient having a third percentage (%) by weight, a plant-derived essential oils ingredient having a fourth percentage (%) by weight, a methyl salicylate component having a fifth percentage (%) by weight, an excipients component having a sixth percentage (%) by weight, and water to create a mixture; wherein after each addition of the mixing ingredients to the mixture is stirred until the mixture is homogeneous; wherein the percentage (%) by weight is determined by the sum from the first percentage (%) to the sixth percentage (%) plus the percentage (%) of the water to make up 100%. It should be noted that the mixing ingredients Z1 to Z7 are not mixed in the specific order described, the final product will not support the relief of musculoskeletal pain, antibacterial and anti-inflammatory. Within the scope of the present invention, the term “homogeneous mixture” includes the following meanings:

    
    
        (a) A homogeneous mixture is a solution that completely dissolves in a specific order the ingredients (Z1) to (Z7) having the correct percentage (%) by weight;
        (b) A homogeneous mixture acts as a reactant, allowing the addition of ingredients to contribute their chemical and physical properties to create a new composition; and
        (c) A homogeneous mixture chemically bonds with other complementary ingredients including but not limited to ionization reactions, covalent reactions, reducing reactions, replacement reactions, and rearrangement reactions to form a new chemical composition.
    
    


According to the embodiment of the invention, the properties for supporting the relief of musculoskeletal pain, antibacterial and anti-inflammatory depend on the percentage (%) by weight of each ingredient Z1-Z7 including a first formula, a second formula, a third formula, and a fourth formula; wherein the third formula is stronger than the first formula, the first formula is stronger than the second formula, and the second formula is stronger than the fourth formula; in which the comparative factor is properties for supporting the relief of musculoskeletal pain, antibacterial and anti-inflammatory.







TABLE 1







The mixing ingredients Z1 to Z7 to create the composition 100 according


to the embodiment of the invention











Percentage (%) by weight














first
second
third
fourth 


Note
The ingredient
formula
formula
formula
formula





Z1
The ginseng
0.01-0.5 
0.01-0.5 
0.01-0.5 
0.01-0.5 



extract







solution






Z2
The medicinal
12-55
0.1-12
12-55
0.1-12 



herbs extract







solution






Z3
The plant-
 5-10
1-5
 5-10
1-5



derived







nanovesicles







ingredient






Z4
The plant-
10-28
28-48
28-48
28-48



derived







essential oils







ingredient






Z5
The methyl
0.001-0.8 
0.8-10 
0.8-10 
0.001-0.8 



salicylate







component






Z6
The excipients
0.001-5   
0.001-5   
0.001-5   
0.001-5   



component






Z7
The water
rest
rest
rest
rest









Next, referring to FIG. 1, a process 200 for manufacturing a composition 100 in accordance to an exemplary embodiment of the present invention is illustrated.
At step 201, all ingredients Z1 to Z7 are carefully prepared and stored in separate containers.
According to the embodiment of the invention, the ginseng extract solution (Z1) is extracted from parts of ginseng, including roots, stems, leaves, and flowers that have been cleaned, then crushed or chopped and soaked in solvent or saline solution. According to the preferred embodiment of the present invention, the roots have a minimum age of 5 years old; stems, leaves, and flowers of ginseng have a minimum age of 3 years old. The ginseng parts are selected from one or more parts in the genus Panax of the including Panax notoginseng, Panax bipinnatifidus, Korean ginseng, Panax japonicus, American ginseng, Panax quonquefolius, Panax vietnamensis, Panax wangianus, Panax zingiberebsis, Panax pseudoginseing, and Panax stipuleanatus. 
According to the embodiment of the invention, the medicinal herbs extract solution (Z2) is prepared by performing steps (a1) to (a3):

    
    
        (a1) collecting and pre-processing material from medicine herbals (a)-(l) are listed in Tables 2 below including removing damaged parts, washing, and drying to 8%-15% moisture, chopping and stored in separate instruments;
        (a2) creating a medicine herbals mixture by performing steps (a′) to (b′):
        
            (a′) collecting and pre-processing material from medicine herbals (m)-(n) are listed in Tables 2 below, including removing damaged parts, washing, drying overnight exposed to dew and drying to 8%-15% moisture, chopping and stored in separate instruments; wherein the material from medicine herbals (m)-(n) include: (m) Salvia miltiorrhiza, and (n) Achyranthes bidentate uses the roots; and
            (b′) mixing the material from medicine herbals (a)-(l) prepared in step (a1) and the material from medicine herbals (i)-(n) prepared in step (a′) to obtain the medicine herbals mixture; and
        
        
        (a3) soaking the medicine herbals mixture with 70% ethanol solvent in a ratio of 18:70 w/v at room temperature, stirring at a frequency of 200-250 rpm for 10 days to obtain a mixture 1, filtering the mixture 1 to obtain a filtrate 1 and residue 1; soaking the residue 1 with 70% ethanol solvent in a ratio of 18:70 w/v, stirring at a frequency of 200-250 rpm for 10 days to obtain a mixture 2, filtering the mixture 2 to obtain a filtrate 2 and residue 2; soaking the residue 2 with 70% ethanol solvent in a ratio of 18:70 w/v, stirring at a frequency of 200-250 rpm for 10 days to obtain a mixture 3, filtering the mixture 3 to obtain a filtrate 3 and a residue 3; mixing the filtrate 1 with the filtrate 2, and filtrate 3 to obtain a filtrate mixture, removing the solvent from the filtrate mixture by vacuum evaporation at 40° C.-45° C. to obtain the medicinal herbs extract solution.
    
    









TABLE 2







The medicinal herbs ingredients (a)-(l) to create the medicinal herbs


extract solution according to the embodiment of the invention















Scientific
Part





Note
Name of
name
used
Compound
Indications
Distribution





(a)
The
Carhamus
flower
Carthamin
Activating blood
Ha Giang,



safflower
tinctorius L

Hydroxysafflor
circulation
Northern






Flavonoid

midland and






(Kaempferol)

mountainous








region of








Vietnam


(b)
The
Daemonorops
The
Dracoresin
Activating blood
Indonexia



Dragon's
draco (Willd.)
resin
Acid benzoic
circulation




Blood
Blume
from
Essential oil







the








fruit








and








stem





(c)
The
Coptis
The
Berberin
Antibacterial
Lao Cai



Chinese
chinensis
roots

and anti-
(Sapa),



goldthread
Franch.
and

inflammatory
Hoang Lien





tubers


Son








mountain








range,








Northwestern








region of








Vietnam


(d)
The
Reynoutria
The
Anthraglycosid
Musculoskeletal
Lao Cai, Yen



Japanese
japonica
roots
Polydatin,
pain
Bai, Son La,



knotweed
Houtt.
and
resveratrol

Dien Bien,





tubers


and Hoa








Binh








provinces


(e)
The
Cinnamomum
The
Cinnamaldehyde
Musculoskeletal
Cao Bang,



cinnamon
loureiroi
bark
Coumarin
pain
Yen Bai,




Nees



Quang Ninh,








Thanh Hoa,








Nghe An,








and Thai








Nguyen








provinces


(f)
The ginger
Zingiber
The
Gingerol
Cold hands and
In Vietnam,




officinale
tubers
Shogaol
feet, Painful
ginger is




Rosc.

Zingerone
numbness and
cultivated







weakness,
across all







Arthralgia
provinces







syndrome
nationwide,








from








mountainous








regions to








the plains


(g)
The star
Illicium
the
Anethole
Painful
Cao Bang,



anise
verum Hook.
fruits

numbness
Lang Son,




f.


associated with
Tuyen







Bi syndrome
Quang, Bac







and arthritis
Kan, Ha








Giang, and








Thai Nguyen








provinces


(h)
The piper
Piper
all
Beta-
Treatment of
The plains



lolot
sarmentosum
parts
caryophyllene
disorders
and midland





of the
Benzyl acetate
caused by wind,
regions of





plant
alkaloid:
cold, and
Vietnam






piperine,
dampness,







piperidine,
leading to







piplartine
numbness and







flavonoid:
paralysis of the







quercetin,
hands and feet







kaempferol,








apigenin




(i)
The
Artemisia
the
Essential oil:
Pain relief for
Northern



mugwort
vulgar
leaves
Monoterpen,
musculoskeletal
mountainous






Sesquiterpen
pain, sciatica,
provinces






lacton, và Este
and arthritis;
such as Lao






dehydromatricaria
Supporting
Cai, Lai






Flavonoid
cerebral blood
Chau, Yen







circulation
Bai Cao








Bang, Lang








Son, and Ha








Giang


(j)
The
Codonopsis
the
Saponin
Enhancing red
Lam Dong,



codonopsis
pilosula
tubers
Polysaccharide
blood cell
Quang Nam,




Nannf


production,
Kon Tum, Da







promoting blood
Nang, Ha







nourishment,
Giang, Lang







and improving
Son, Lai







anemia
Chau, Lao







conditions
Cai, and Cao








Bang








provinces


(k)
The clove
Flos
flower
Eugenol
Musculoskeletal
The




caryophylatac
bud
beta-
pain
Moluccas






caryophyllene

Islands








region of








Indonesia,








Asia, and








Africa


(l)
The white
Brassica alba
seeds
Myrosin,
Anti-
Vietnam



mustard


sinapine,
inflammatory,




seed


sinalbin, sinigrin,
reduces







fatty oils,
swelling, treats







saponins
musculoskeletal








pain



(m)
The Dan
Salvia
the
Danshensu (3-
Antithrombotic
China



Shen
miltiorrhiza
roots
(3,4-








dihydroxyphenyl)








lactic acid)




(n)
The goat's
Achyranthes
the
Saponin
Treatment of
China



head
bidentata
roots
triterpenoid
arthritis and








lower back pain









According to the embodiment of the invention, the plant-derived nanovesicles ingredient (Z3) is prepared by mixing a first nanovesicles ingredient with a second nanovesicles ingredient in a ratio of 2:3 w/w.
According to the embodiment of the invention, the first nanovesicles ingredient is prepared by performing steps (b1) to (b8):

    
    
        (b1) collecting the material from plants in the following percentage (%) by weight including 2 parts of Illicium verum Hook. f., 3 parts of Artemisia vulgaris, and 3 parts of Piper sarmentosum to obtain a temporary mixture;
        (b2) washing the temporary mixture three times with deionized water at 20° C.-25° C.;
        (b3) pureeing the washed temporary mixture with phosphate buffer solution (PBS) in a ratio of 1:3 w/v at a speed of 7,000-8,000 rpm for 15 minutes to obtain a first temporary solution;
        (b4) filtering the first temporary solution by a filter membrane with a diameter 0.20-0.22 μm to obtain a second temporary solution;
        (b5) centrifuging the second temporary solution by ultracentrifugation at 120,000×g for 100 min at 4° C. to obtain a temporary residue;
        (b6) dissolving the temporary residue in phosphate buffer solution (PBS), transferring to a 45% sucrose gradient solution, and ultracentrifuging at 130,000×g for 100 min to obtain a third temporary solution;
        (b7) washing the third temporary solution with PBS and centrifuging at 150.000×g for 60 min at 4° C. to obtain a fourth temporary solution; and
        (b8) filtering the fourth temporary solution through a filter membrane with a diameter 0.20-0.22 μm to obtain the first nanovesicles ingredient.
    
    


According to the embodiment of the invention, the second nanovesicles ingredient is prepared by performing steps (c1) to (c9):

    
    
        (c1) collecting a fruit mixture in the following percentage (%) by weight including 3 parts of dragon fruit (Hylocereus undatus, Hylocereus costaricensis, Hylocereus megalanthus, Hylocereus undatus costaricensis), 3 parts of avocado (Persea americana), and 1 part of watermelon (Citrullus lanatus);
        (c2) washing the fruit mixture three times with deionized water at 20° C.-25° C.;
        (c3) pureeing the washed fruit mixture with phosphate buffer solution (PBS) in a ratio of 1:1 (g/mL) at a speed of 7,000-8,000 rpm for 15 minutes to obtain a first foundation solution;
        (c4) filtering the first foundation solution by a filter membrane with a diameter 0.20-0.22 μm to obtain a second foundation solution;
        (c5) centrifuging the second foundation solution by ultracentrifugation at 100,000×g for 60 min to obtain a residue;
        (c6) dissolving the residue in phosphate buffer solution (PBS), transferring to a 45% sucrose gradient solution, and ultracentrifuging at 130,000×g for 100 min to obtain a third foundation solution;
        (c7) stirring the third foundation solution with a 10% polyethylene glycol-8000 (PEG8000) solution in a ratio of 1:1 v/v, and incubating for 8-10 hours at 4° C., then centrifuging at 110,000×g for 40 minutes at 4° C. to obtain a precipitate;
        (c8) dissolving the precipitate in phosphate buffer solution (PBS) in a ratio of 1:2 w/v to obtain a foundation solution; and
        (c9) filtering the foundation solution by a tangential flow filtration (TFF) to obtain the second nanovesicles ingredient; wherein the technical specifications related to TFF include a molecular size of 500 kDa, and filtering at a flow rate of 20 mL/min with the transmembrane pressure maintained at 2 bar.
    
    


According to the embodiment of the invention, the plant-derived essential oils ingredient (Z4) is prepared by mixing in the following percentage (%) by weight including the essential oils ingredients (a′)-(h′) are listed in Tables 3 below, including: 1-2 parts of an essential oil of cajeput leaf, 2 parts of an essential oil of camphor leaf, 1 part of an essential oil of cinnamon leaf, 1-2 parts of an essential oil of eucalyptus leaf, 2 parts of an essential oil of rosemary leaf, 1 part of an essential oil of peppermint leaf, 1-2 parts of an essential oil of perilla seed, and 1 part of an essential oil of ginger rhizome;







TABLE 3







The plants selected as raw materials to create the plant-derived essential oils


ingredient (Z4) according to the embodiment of the invention















Part




Note
Name of
Scientific name
used
Indications
Distribution





(a′)
The
Melaleuca
leaf
Supporting the
Thua Thien Hue,



essential oil
cajuputi

treatment of
Quang Binh,



of cajeput


respiratory diseases
Quang Tri, Long



leaf


and enhancing the
An, Dong Thap,






immune system
and Tay Ninh







provinces


(b′)
The
Cinnamomum
leaf
Analgesic, anti-
Ha Giang, Tuyen



essential oil
camphora

inflammatory,
Quang, Phu Tho,



of camphor


antibacterial, and
Cao Bang, and



leaf


respiratory-supporting
Lang Son






effects
provinces


(c′)
The
Cinnamomum
leaf
Antibacterial, anti-
Thanh Hoa, Nghe



essential oil
verum

inflammatory, and
An, Khanh Hoa,



of cinnamon


stress-relieving
Tay Ninh, Ba Ria-



leaf


effects
Vung Tau, and







Kien Giang (Phu







Quoc) provinces


(d′)
The essential
Eucalyptus
leaf
Supporting respiratory
Central Vietnam



oil of
globulus Labill.

health, relieving
and the Central



eucalyptus


stress, and enhancing
Highlands



leaf


immunity



(e′)
The
Salvia
leaf
Enhancing memory,
Central Vietnam



essential oil
rosmarinus

relieving stress,
and Southern



of rosemary


supporting the
Vietnam



leaf


respiratory system,







improving oral health,







promoting skin care,







and stimulating hair







growth



(f′)
The
Mentha
leaf
Supporting digestion,
Lao Cai, Nghe An,



essential oil
arvensis L

relieving pain, cooling
Lai Chau, Yen Bai



of


the body, and
provinces



peppermint


promoting relaxation




leaf






(g′)
The
Perilla
seed
Anti-allergic, anti-
Vietnam



essential oil
frutescens (L.)

inflammatory, anti-




of perilla
Britt.

cancer, stress-




seed


relieving, weight







management-







supporting, and skin-







improving effects



(h′)
The
Zingiber
rhizome
Supporting digestion,
In Vietnam, ginger



essential oil
officinale Rosc.

relieving nausea and
is cultivated



of ginger


pain, exhibiting anti-
across all



rhizome


inflammatory effects,
provinces






and promoting blood
nationwide, from






circulation
mountainous







regions to the







plains









According to the preferred embodiment of the present invention, each essential oils ingredient (a′)-(h′) is extracted by performing steps (d1) to (d3):

    
    
        (d1) collecting and pre-processing material from plant individually including removing damaged parts, washing, chopping, and soaking with an enzyme solution in a ratio of 1:2 w/v for 30 minutes, and filtering to obtain an enzyme-treated plant material;
        
            in which the enzyme solution is prepared by:
            
                creating an enzyme composition by mixing a cellulase with hemicellulase, and xylanase in a ratio of 1:1:1 w/w/w; and
                dissolving the enzyme composition with water in a ratio of 1:15000 w/v;
            
            
        
        
        (d2) grinding a mixture of 1 part the enzyme-treated plant material with 7 parts saturated salt solution, let stand after grinding for 1 hour with microwave assistance at a power of 300-500 W, then steam distillation for 3 hours to obtain a crude essential oil; and
        (d3) removing water from the crude essential oil by adding Na2SO4 salt to crystallize at −15° C., and filtering to obtain the plant-derived essential oils ingredient.
    
    


According to the embodiment of the invention, the methyl salicylate component (Z5) is synthesized by:

    
    
        dissolving 1.5 g of salicylic acid (10 mmol) with 4.35 mL of 10% methanol in a 250 mL three-necked flask, adding 0.27 mL of 10% H2SO4, and refluxing at 82° C.-83° C. for 6 hours;
        evaporating by rotary vacuum evaporation of the mixture after reaction at 62° C.-65° C. to remove the solvent, cooling the mixture after evaporation and putting it in a decanter;
        coating the rotary vacuum evaporation flask with saturated NaHCO3 solution put it in the decanter, adding 7 mL of saturated NaHCO3 to the decanter, gently shaking the decanter then let it stand to obtain a lower solution layer;
        washing the lower solution layer three times with saturated NaHCO3 solution, wherein each time washing with saturated NaHCO3 solution is 7 mL;
        washing the lower solution layer three times with distilled water, wherein each time washing with distilled water is 12 mL; and
        adding 0.5 g of anhydrous Na2SO4 into the reaction mixture, filtering, and distilling fractional to obtain the methyl salicylate component at temperature of 220° C.
    
    


According to the embodiment of the invention, the excipients component (Z6) is selected from the group consisting of ethanol, polysorbate 60, sorbitan oleate, triethanolamine, carbomer or combinations thereof. These molecules can be used in native form or with chemical modifications. Such components may be used individually or in combination.
Still with FIG. 1, at step 102, obtaining a plant-based extract complex composition by mixing in a specific order into a container the ingredients (Z1)-(Z7) including: a ginseng extract solution having a first percentage (%) by weight, a medicinal herbs extract solution having a second percentage (%) by weight, a plant-derived nanovesicles ingredient having a third percentage (%) by weight, a plant-derived essential oils ingredient having a fourth percentage (%) by weight, a methyl salicylate component having a fifth percentage (%) by weight, an excipients component having a sixth percentage (%) by weight, and (G) water to create a mixture;

    
    
        wherein after each addition of the mixing ingredients to the mixture is stirred until the mixture is homogeneous; and
        wherein the percentage (%) by weight is determined by the sum from the first percentage (%) to the sixth percentage (%) and plus the percentage (%) of solvent is the water to make up 100%.
    
    


It is also noted that the terminology “admixed” in step 202 used in the present invention means that foundation mixture is added or reacted with or dissolved homogeneously to the plurality of mixing ingredients (Z1-Z7) using stirrers such as magnetic stirrers. Step 202 is performed by a magnetic stirrer. Magnetic stirrer has been known in previous art so the description of the structure and its operating principle will not be described in detail in the invention.
According to the embodiment of the invention, the properties for supporting the relief of musculoskeletal pain, antibacterial and anti-inflammatory depend on the percentage (%) by weight of each ingredients (Z1)-(Z7) mixed in step 202, the composition 100 is obtained from the process 200 including a first formula, a second formula, a third formula, and a fourth formula are listed in Tables 1; wherein the third formula is stronger than the first formula, the first formula is stronger than the second formula, and the second formula is stronger than the fourth formula; in which the comparative factor is properties for supporting the relief of musculoskeletal pain, antibacterial and anti-inflammatory.
According to the embodiment of the invention, the composition 100 according to the first formula comprises: (Z1) the ginseng extract solution having 0.01%-0.5% by weight; (Z2) the medicinal herbs extract solution having 12%-55% by weight; (Z3) the plant-derived nanovesicles ingredient having 5%-10% by weight; (Z4) the plant-derived essential oils ingredient having 10%-28% by weight; (Z5) the methyl salicylate component having 0.001%-0.8% by weight; (Z6) the excipient component having 0.001%-5% by weight; and (Z7) the remainder is the water.
According to the embodiment of the invention, the composition 100 according to the second formula comprises: (Z1) the ginseng extract solution having 0.01%-0.5% by weight; (Z2) the medicinal herbs extract solution having 0.1%-12% by weight; (Z3) the plant-derived nanovesicles ingredient having 1%-5% by weight; (Z4) the plant-derived essential oils ingredient having 28%-48% by weight; (Z5) the methyl salicylate component having 0.8%-10% by weight; (Z6) the excipient component having 0.001%-5% by weight; and (Z7) the remainder is the water.
According to the embodiment of the invention, the composition 100 according to the third formula comprises: (Z1) the ginseng extract solution having 0.01%-0.5% by weight; (Z2) the medicinal herbs extract solution having 12%-55% by weight; (Z3) the plant-derived nanovesicles ingredient having 5%-10% by weight; (Z4) the plant-derived essential oils ingredient having 28%-48% by weight; (Z5) the methyl salicylate component having 0.8%-10% by weight; (Z6) the excipient component having 0.001%-5% by weight; and (Z7) the remainder is the water.
According to the embodiment of the invention, the composition 100 according to the fourth formula comprises: (Z1) the ginseng extract solution having 0.01%-0.5% by weight; (Z2) the medicinal herbs extract solution having 0.1%-12% by weight; (Z3) the plant-derived nanovesicles ingredient having 1%-5% by weight; (Z4) the plant-derived essential oils ingredient having 28%-48% by weight; (Z5) the methyl salicylate component having 0.001%-0.8% by weight; (Z6) the excipient component having 0.001%-5% by weight; and (Z7) the remainder is the water.
Finally, at step 203, filling and packaging the composition 100.
According to the embodiment of the invention, the composition 100 is obtained from the process 200 is not limited to the dosage form, it can be in the form of a solution, a cream, a paste, a gel, a foam, a solid, or a powder, depending on the dosage form thereof.
According to the embodiment of the invention, a method for supporting the relief of musculoskeletal pain, antibacterial and anti-inflammatory comprising topically applying the composition 100 to the skin at the pain location of the subject in need thereof, wherein the composition 100 is obtained from the process 200 has been described above.
EXAMPLES
The following experimental section is provided purely by way of illustration and is not intended to limit the scope of the invention as defined in the appended claims. In the following experimental section, reference is made to the appended drawings, wherein:
The example 1-4: modulation 100 g the composition 100 is created by process 200, including four examples listed in Table 4 below.







TABLE 4







components of the composition 100 according four examples in


accordance with exemplary embodiment of the present invention.













Weight (g)
















Example
Example
Example
Example












No.
Components
1
2
3
4















Z1
The ginseng extract solution
0.1
0.1
0.1
0.1


Z2
The medicinal herbs extract solution
45
12
45
12


Z3
The plant-derived nanovesicles
5
2
5
2



component

















Z4
Plant-derived
essential oil of
1
1
4
4



essential oils
cajeput leaf







component
essential oil of
1
1
2
2




camphor leaf








essential oil of
1
1
2
2




cinnamon leaf








essential oil of
1
1
4
4




eucalyptus leaf








essential oil of
1
1
2
2




rosemary leaf








essential oil of
1
1
2
2




peppermint leaf








essential oil of
1
1
4
4




perilla leaf








essential oil of
1
1
2
2




ginger rhizome
















Z5
The methyl salicylate component
0.3
0.9
0.9
0.3













Z6
The excipients
ethanol
0.05
0.001
0.001
1.4



component
polysorbate 60
0.001
1
1
0.9




sorbitan oleate
0.001
1
1
0.9




triethanolamine
0.001
1
1
0.2




carbomer
0.001
1
1
0.001












Z7
Water
rest
rest
rest
rest









The composition 100 according to examples 1-4 is not limited to the dosage form, it can be in the form of a solution, a cream, a paste, a gel, a foam, a solid, or a powder. According to the preferred embodiment of the present invention, composition 100 according to examples 1-3 is used to support the symptoms of inflammation, bone, and joint pain; and composition 100 according to example 4 is preferably in liquid form for external use, nasal decongestant, respiratory support or steam inhalation or as an ingredient for relaxing massage.
The results of the analysis of the bactericidal and safety properties of the composition 100 according to examples 1-4 were tested for toxicity at a dose of 0.01 mL/20 body weight, applied to the back skin for 30 days in mice, showing no effect on the parameters of red blood cells, AST, ALT, and creatinine. The composition of 100 increased the number of leukocytes but did not affect the ratio of MID cells, monocytes, and granulocytes compared to the control group using a solvent. At the same time, there was no difference in the microstructural characteristics of the liver and kidney of mice after using preparation 100 compared to the control group using solvent.
The composition 100, according to examples 1-4, was tested for biological activity including an antioxidant test by DPPH free radical scavenging and anti-lipid peroxidation ability, and antibacterial activity tests listed in Table 5 below.







TABLE 5







Results of biological activity testing of the composition 100 


according to the embodiment of the present invention









No.
Biological activity testing
The results





1
The DPPH free radical scavenging
(+)


2
The anti-lipid peroxidation ability
(+)


3
The antibacterial activity
Staphylococcus aureus, 




Escherichia coli,




Candida albicans, 




Pseudomona saeruginosa, 




Bacillus subtilis





wherein (+) is a positive sample; it has DPPH free radical scavenging and anti-lipid peroxidation ability greater than 50%.






The example 5 evaluating the skin irritation of the composition 100, which is obtained from the example 3 in accordance with an exemplary embodiment of the present invention.
Test subjects: the experiment was performed on New Zealand White, white fur. Rabbits met the experimental requirements: healthy adult (9-10 weeks old), regardless of gender, healthy skin and no skin diseases. Total of 03 rabbits was used for experimental (weight: 2.0-2.2 kg/rabbit). Rabbits were kept separately to avoid possible cross-infection through the respiratory tract and contact; They were acclimatized raised in experimental conditions for 5 days prior to the research; room temperature 25° C.-30° C., light guaranteed 12 hours of darkness, 12 hours of light every day. Rabbits were fed according to the food standards for research animals, water (boiled and cooled) drink freely. total of 03 rabbits was used for experimental
Animal preparation: 24 hours before the experiment, rabbits were shaved to remove the fur in the back and hips. Divide the shaved skin into two sections, choosing each section to be about 6 cm2 (2.5 cm×2.5 cm).
Experimental design: Conduct a drug test with 3 rabbits; each rabbit is used to apply 0.5 g of the composition 100, and the skin without drug application is used as a control. Prepare non-irritating gauze with dimensions of 2.5 cm×2.5 cm. Place the composition 100 on the gauze, with a dose of 0.5 g, spread evenly over the surface of the gauze. Place the gauze on the rabbit skin (1 gauze/rabbit). Fix the gauze with non-irritating adhesive tape. Mark the location of the test sample with a marker. After 4 hours of fixing the gauze on the rabbit skin, remove the gauze and tape and clean with distilled water. Observe, evaluate, and score the erythema and oedema indices on the skin where the composition 100 was applied compared to the adjacent skin where the composition 100 was not applied was described and scored according to the scoring system in Table 6 at 1 hour, 24 hours, 48 hours, and 72 hours after cleaning the composition 100. If there is any damage, monitor the rabbit for 14 days to assess the recovery ability. When the damage has recovered, stop monitoring.







TABLE 6







Scoring of erythema and oedema









Rating


Reaction
Points










erythema








No erythema
0


Very slight erythema (barely perceptible)
1


Well defined erythema
2


Moderate to severe erythema
3


Severe erythema
4







oedema








No oedema
0


Very slight oedema (barely perceptible)
1


Well defined oedema (edges of area raised)
2


Moderate to severe oedema (edges of area raised~1 mm)
3


Severe oedema (edges of area raised more than 1 mm 
4


and extending outside the application area)










Evaluation of results: For each rabbit, the irritation score was calculated by dividing the sum of the two levels of erythema and edema by the number of observations. The irritation score of the composition 100 is taken as the average of the irritation score of the tested rabbits. The irritation score is compared with the levels prescribed in Table 7 to determine the skin irritation potential of the test sample on rabbits.







TABLE 7







Classification of skin irritation levels on rabbits










Irritation category
Mean score







Negligible Irritation
    0-0.5



Slight Irritation
>0.5-2.0



Moderate Irritation
>2.0-5.0



Severe Irritation
>5.0-8.0










The Result







TABLE 8







Skin irritation level of the composition 100 on rabbit skin









Time












1 hour
24 hours
48 hours
72 hours
















erythema
oedema
erythema
oedema
erythema
oedema
erythema
oedema























Rabbit
CP
ÐC
CP
ÐC
CP
ÐC
CP
ÐC
CP
ÐC
CP
ÐC
CP
ÐC
CP
ÐC


























1
1
0
1
0
1
0
1
0
0
0
0
0
0
0
0
0


2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0


3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0


Mean
0.3
0
0.3
0
0.3
0
0.3
0
0
0
0
0
0
0
0
0





Note:


CP: applied area the composition 100; ÐC: Control area






Based on Table 8 shows that: at 1 hour after removing the composition 100, the total irritation score was 0.3+0.3=0.6, classified as slight irritation. At 24 hours: The total irritation score was 0.6, classified as slight irritation. At 48 hours: The total irritation score was 0.0, classified as negligible irritation. At 72 hours: The total irritation score was 0.0, classified as negligible irritation. The skin used as a control showed that at all time points, there were no signs of irritation.
At the time of the experiments, all rabbits were healthy, had normal function, and showed no signs of itchy rash or scratching at the site where the medicated gauze was placed. Rabbits ate, drank, lived, slept, etc. normally. There were no digestive disorders (such as diarrhea, increased salivation, etc.) or disorders of animal activities and motor behavior (tremors, convulsions, sleep disorders, coma). Compared with the classification of irritation levels on rabbit skin: The composition 100 of the preparation was classified as insignificantly irritating.
The example 6 evaluating the anti-thrombotic effect of the composition 100, which is obtained from the example 3 in accordance with an exemplary embodiment of the present invention.
Test subjects: the experiment was performed on healthy adult Wistar white rats; the quantity used was 30 rats, both male and female, weighing 180-210 g/rats. They were nurtured to stabilize for 1 week before conducting the experiment at room temperature (27° C.-28° C.). They were fed with synthetic pellets for mice, and the drinking water was tap water.
Experimental design: The white rats were randomly divided into 3 plots of 10 rats each.

    
    
        Plot 1: (biology control): no impact;
        Plot 2: apply voltaren 0.2 g/l rat foot; and
        Plot 3: apply the composition 100 0.2 g/l rat foot.
    
    


The rats were treated with the drug 5 times for 3 consecutive days. The first day, 1 hour after applying the test samples, inflammation was induced by injecting 0.25 ml/mouse of 1% carrageenin (dissolved in saline) into the right hind paw of the mouse.
Measure the volume of the rat (up to the ankle joint) using a plethysmometer at the following times: before induce inflammation (V0), 1 hour after induce inflammation (V1), 2 hours (V2), 4 hours (V3) and 6 hours (V4), 24 hours (V5), 30 hours (V6) and 48 hours (V7).
The paw edema volume results are calculated using Fontaine's formula.

    
    
        The increase in paw edema volume of each rat was calculated according to the formula:
    
    





 
  
   Δ
   ⁢
   V
   ⁢
      
   %
   ⁢
      
   hour
  
  =
  
   
    
     
      V
      t
     
     -
     
      V
      0
     
    
    
     V
     0
    
   
   ×
   1
   ⁢
   0
   ⁢
   0
  
 



wherein: ΔV % is the increase in paw edema volume;

    
    
        V0 is the increase in paw edema volume before induce inflammation;
        Vt is the increase in paw edema volume after induce inflammation;
        The anti-inflammatory effect of the test samples is evaluated by the ability to inhibit edema response (1%) according to the formula:
    
    





 
  
   I
   ⁢
      
   %
  
  =
  
   
    
     
      Δ
      ⁢
      
       V
       c
      
      ⁢
         
      %
     
     -
     
      Δ
      ⁢
      
       V
       t
      
      ⁢
         
      %
     
    
    
     Δ
     ⁢
     
      V
      0
     
     ⁢
        
     %
    
   
   ×
   1
   ⁢
   0
   ⁢
   0
  
 



    
    
        wherein: ΔV % is average increase in edema volume of the rat's paw in the control group
        
            ΔVt% is average increase in edema volume of the rat's paw in the apply the composition 100
        
        
    
    


Measure the thickness of the rat's paw: one technician holds the mouse still. Another person places a ruler on the thickest part of the rat's paw. When the two edges of the ruler touch the rat's paw, the electronic screen will display the measurement in mm at the following times: before Induce inflammation (V0); 1 hour after Induce inflammation (V1), 2 hours (V2), 4 hours (V3) and 6 hours (V4), 24 hours (V5), 30 hours (V6) and 48 hours (V7).

    
    
        The calculation of the % increase in thickness is similar to the paw edema volume according to the formula:
    
    





 
  
   Δ
   ⁢
   D
   ⁢
      
   %
  
  =
  
   
    
     
      D
      t
     
     -
     
      D
      0
     
    
    
     D
     0
    
   
   ×
   1
   ⁢
   0
   ⁢
   0
  
 



    
    
        
        
            wherein: ΔD % is the increase in thickness;
            
                D0 is the increase in thickness of the rat's paw before induce inflammation;
                Dt is the increase in thickness of the rat's paw after induce inflammation;
            
            
        
        
        The anti-inflammatory effect of the test samples is evaluated by the ability to inhibit edema response (1%) according to the formula:
    
    





 
  
   I
   ⁢
      
   %
  
  =
  
   
    
     
      Δ
      ⁢
      
       D
       c
      
      ⁢
         
      %
     
     -
     
      Δ
      ⁢
      
       D
       t
      
      ⁢
         
      %
     
    
    
     Δ
     ⁢
     
      D
      0
     
     ⁢
        
     %
    
   
   ×
   1
   ⁢
   0
   ⁢
   0
  
 



    
    
        
        
            wherein: ΔD % is average increase in thickness of the rat's paw in the control group
            
                ΔVt % is average increase in thickness of the rat's paw in the apply the composition 100
            
            
        
        
    
    


The Result
The Edema Volume of the Rat's Paw







TABLE 9







Anti-inflammatory effect of the composition 100 on inducing paw


edema in rat model through the edema volume of the rat's paw index











edema (%)

















after 1
after 2
after 4
after 6
after 24
after 30
after 48


Plot
n
hour
hours
hours
hours
hours
hours
hours


















Plot 1: biology
10
14.56
35.82
47.86
27.88
13.96
16.68
15.64


control










Plot 2: apply
10
4.86
6.95
22.84
8.94
5.87
4.82
2.43


voltaren 0.2 g/l










rat foot










Plot 3: apply the
10
12.45
14.44
25.78
11.79
7.98
6.83
4.96


composition 100










0.2 g/l rat









Based on Table 9 shows that: The increase in paw edema volume at the following times after inducing inflammation in the test samples-use group was significantly lower than that of the biological control. Specifically, after 48 hours of the test samples use, it was found that the group applying the composition 100 and the group applying voltaren at a concentration of 0.2 g/l rat paw had an increase in mouse paw volume of 4.96% and 2.43%, significantly lower than the biological control group not using the drug at 15.64%.
The Thickness of the Rat's Paw







TABLE 10







Anti-inflammatory effect of the composition 100 on inducing paw


edema in rat model through the thickness of the rat's paw











thickness (%)

















after 1
after 2
after 4
after 6
after 24
after 30
after 48


Plot
n
hour
hours
hours
hours
hours
hours
hours


















Plot 1: biology
10
85.67
97.53
105.27
90.41
69.14
60.26
30.65


control










Plot 2: apply
10
57.74
61.68
68.34
58.29
49.48
39.88
15.67


voltaren 0.2 g/l










rat foot










Plot 3: apply the
10
81.65
83.76
90.16
81.33
58.34
46.13
17.62


composition 100










0.2 g/1 rat









Based on Table 10 shows that: The increase in thickness of the rat's paw at the following times after inducing inflammation was the smallest in the Voltaren group (15.67%) and the composition 100 group (17.62%), which was significantly lower than the biological control group (30.65%). The composition 100 group had a change in the increase in paw thickness.
In short, both indexes of paw edema and thickness of the rat's paw gave similar results: reduced paw edema volume and thickness of the rat's paw in the group applying the composition 100 and the group applying voltaren. Thus, composition 100 has an anti-inflammatory effect on the carrageen-induced inflammation model of the white rat paw.
The example 7 evaluating the pain relief effect of the composition 100, which is obtained from the example 3 in accordance with an exemplary embodiment of the present invention.
Test subjects: the experiment was performed on healthy adult (Swiss albino) white mice, the quantity used was 40 rats, both male and female, 5-6 weeks old, weighing 20+2 grams. They were nurtured to stabilize for 1 week before conducting the experiment at room temperature (27° C.-28° C.). They were fed with synthetic pellets for mice, and the drinking water was tap water.
Experimental design: The white mice were randomly divided into 4 plots of 10 rats each.

    
    
        Plot 1: (biology control): do not apply to the mouse's feet;
        Plot 2: apply salonpas gel to the entire soles of the mouse's feet;
        Plot 3: apply voltaren to the entire soles of the mouse's feet; and
        Plot 4: apply the composition 100 to the entire soles of the mouse's feet.
    
    


After 30 minutes from the time of application, the mice were measured for pain response using the hot plate method.
The measurement method is as follows: Place the mouse on the hot plate, which is always maintained at 56° C. by a thermostat. The reaction time to thermal stimulation was calculated from the time the mouse was placed on the hot plate until the mouse had a reflex to licking and/or shaking its hind paw. Removed mice that respond too quickly (before 8 seconds) or too slowly (after 30 seconds). Comparison of response times to thermal stimuli before and after the test samples application and comparison between plots of mice.
The Result







TABLE 11







Effect of the composition 100 on temperature


reaction time of white mice













Pain 





response





time



Plot
n
(seconds)







Plot 1 biology control
10
4.24



Plot 2 (Salonpas gel)
10
5.37



Plot 3 (Voltaren)
10
5.21



Plot 4 (the composition 100)
10
6.46










Based on Table 11 shows that: all control samples showed the effect of prolonging the time to the appearance of pain response compared with the control plot. The time to appearance of pain response of plot biology control was 4.24 seconds, while the plot using the composition 100 had the effect of prolonging the time to appearance of pain response by 6.46 seconds, more than the control samples salonpas gel was 5.37 seconds and voltaren 5.21 was seconds. Thus, composition 100 had the effect of reducing pain in white mice using the hot plate method.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “includes” and/or “including,” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, element components, and/or groups thereof.
While the preferred embodiment to the invention had been described, it will be understood that those skilled in the art, both now and in the future, may make various improvements and enhancements which fall within the scope of the claims which follow. These claims should be construed to maintain the proper protection for the invention first described.
The description of the present invention has been presented for purposes of illustration and description but is not intended to be exhaustive or limited to the invention in the form disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the invention. The embodiment was chosen and described in order to best explain the principles of the invention and the practical application and to enable others of ordinary skill in the art to understand the invention for various embodiments with various modifications as are suited to the particular use contemplated.
The flow diagrams depicted herein are just one example. There may be many variations to this diagram or the steps (or operations) described therein without departing from the spirit of the invention. For instance, the steps may be performed in a differing order, or steps may be added, deleted, or modified. All of these variations are considered a part of the claimed invention.
While the preferred embodiment to the invention had been described, it will be understood that those skilled in the art, both now and in the future, may make various improvements and enhancements which fall within the scope of the claims which follow. These claims should be construed to maintain the proper protection for the invention first described.
The foregoing description details certain embodiments of the invention. It will be appreciated, however, that no matter how detailed the foregoing appears in text, the invention can be practiced in many ways. As is also stated above, it should be noted that the use of particular terminology when describing certain features or aspects of the invention should not be taken to imply that the terminology is being re-defined herein to be restricted to including any specific characteristics of the features or aspects of the invention with which that terminology is associated. The scope of the invention should therefore be construed in accordance with the appended claims and any equivalents thereof.