WO2021236023A1

WO2021236023A1 - Fermented - concentrated extraction method of houttuynia cordata for medical using

Info

Publication number: WO2021236023A1
Application number: PCT/TH2020/000057
Authority: WO
Inventors: Udom RINCOME; Somsak RINCOME
Original assignee: Rincome Udom
Priority date: 2020-05-18
Filing date: 2020-08-11
Publication date: 2021-11-25
Also published as: CN115697365A

Abstract

Methods of fermentation and extraction of concentrated herbs in order to get more active pharmaceutical ingredients has been done by taking none defected and clean Plukow leaf in clean water, rinse thoroughly and let drain. Then put it into the fermentation tank. Weigh sugar cane powder, add starters namely Saccharomyces cerevisiae, Lactobacillus casei (GenBank: AF320255.1), Lactobacillus plantarum (GenBank: AF320256.1) in another tank, add clean water to sufficient 1,000 litres, stir thoroughly, then pour the mixture to the first fermentation tank to flood over the Plukow leaf. Close the lid tightly and expose the fermentation tank in the sun light for 1 month. Remove the leaves from the fermented water, dry in a hot air oven at 100° (degrees Celsius) then grind into fine powder. Mix the fine powder with fermented water in ratio of 1:2 weight by weight in roasting tank, ignite the head of the gas stove to heat the wet powder to reach 100-150° while the roasting tank rotates slowly for 1 hour. After that, dries the powder in hot air oven for 100° for 8-12 hours, then mix the powder with the leftover fermented water as the same procedure mentioned above until fermented water in the tank exhausted. The concentrated Plukow extracts with more quercetin and beta glucan will be obtained. This concentrated herb extracts can be used for active pharmaceutical ingredient in medical herb or food supplement.

Description

TITLE OF INVENTION

Fermented-concentrated extraction method of Houttuynia cordata for medical using TECHNICAL FIELD

Chemistry related to Houttuynia cordata fermented and concentrated extraction method and immunology.

BACKGROUND ART

Biological fermentation has been known and widely used in foreign countries since 1965. In Thailand, knowhow about effective microorganism (EM) for biological fermentation relating to agriculture has been distributed to farmers since 1976. Farmers widely applied biological fermentation throughout the country, as well as applied to ferment many herbs for health benefits. Traditional method of biological fermentation is by using various vegetables and herbs to ferment together in culture media in the same container. Such fermentation is using molasses from a distillery, sugar cane juice or honey as culture media in air restriction container. The bubbles resulting from the fermentation process can be discharge through the vent. Fermentation takes about 1 month or until no more bubbles come out, is considered the end of the fermentation process. Farmers therefore poured out fermented water for future use. This process may not be appropriate and cannot determine as working pattern in an industrial system that requires to obtain active pharmaceutical ingredients (API) with high specificity and concentration that can be used for production and commercial benefits.

The inventor found that the herb named Houttuynia cordata which local named as Plukow, Kaotong, Kaow tong or common names are Chinese Lizard tail, Fishwart, Heartleaf, Houttuynia, Chamelon plant, used in this embodiment, has many useful phytochemicals such as quercetin, that has good antioxidant properties and can induce superoxide dismutase enzyme, that acts as antioxidant in the body, more active too.

There are also many reports showing that Plukow has many interesting pharmacological effects such as anti-mutagenic effect, anti-cancer effect, effect on promoting the work of the body, anti-obesity activity, liver protection, antiviral, antibacterial, eliminate free radicals, anti leukemia and good for the treatment of sinusitis.

According to the method of the inventor, by biological fermentation of fresh leaf of Plukow in the fermentation tank, the concentrated of certain active pharmaceutical ingredients can be obtained. This method is done by using 2-10% concentration of sugar cane (crushed into powder), as a culture media, in the air restriction tank at 25-40° (degrees Celsius). The microorganisms will grow and propagate in the fermentation tank. These microorganisms will decompose the cell walls of the Plukow, affecting the release of many active substances inside the Plukow cell to dissolved into the fermented Plukow water continuously. Sufficient fermenting time, such as 1 month, will allow the bubbles or gas, from fermenting process, to vent through the venting channel until all gone. The tank will still be closed tightly and keep on fermenting for another 3-12 months more. After that, removes the Plukow leaves from the fermentation tank and dries in a hot air oven at a temperature of 100° for 8-10 hours. Grind the dried Plukow leaves to get the fine -powdered.

Mix the remaining fermented water from the Plukow fermentation tank together with the dried Plukow fine -powdered in a ratio of 2:1 weight by weight in a spherical mixing machine which acting as a roasting tank. Turn on the motor to rotate the spherical mixing machine. While the machine is spinning, ignite a gas stove to burn the outside metal plate of the roasting tank area to conduct the heat from the outside tank area into the wet Plukow powder inside. By doing this, the temperature of wet Plukow fined-powder in the mixing machine will reach 100-150° within a short time period gradually during the spinning time, repeatedly for 1 hour. The mixture of fermenting-extracted Plukow leaves will be stimulated by high temperature, without burning, for several short period of time and leads to stimulate the interaction between a mixture of phytochemicals and enzymes that occurs in the fermentation process, causing the increasing of quercetin, contained in fermented Plukow extracts, dramatically. Then dried the Plukow powder in hot air oven at temperature of 100° for 8-12 hours.

Repeat the above process of mixing the remaining fermented water from the Plukow fermentation tank together with the dried Plukow fine -powdered in a ratio of 2:1 weight by weight in a spherical mixing machine, until the Plukow powder is dried, again and again until the fermented water is all gone.

The final product can increase the amount of quercetin for at least 2,400 times and increase at least 9 times of beta glucan higher than those found in fresh Plukow leaf. This final product can be utilized in pharmaceutical and medical purposes as immune system modulation, prevention and treatment of many diseases, both in human and animals.

In addition, intensive fermentation of Plukow extraction with the aforemention process will get a final product that contains dead cells of probiotics. Yeast, Saccharomyces cerevisiae, will also be added in as an active pharmaceutical ingredient from fermented Plukow as mention above. The substance named beta glucan, which is a component of yeast cell wall structure, is also produced. This beta glucan has a unique molecular structure which can help to release more qualify stem cell from bone marrow into bloodstream of human and animal while taking it. Then stem cell, the healing system of the body, migrate to restore or repair damage cell and tissue to resume normal function.

The laboratory experiment with mice found that when injected dead cells of Lactobacillus or polynucleotides of 2 subspecies of Lactobacillus, found in fermented Plukow, into mice subcutaneously for 10 days, the bacteria namely Lactobacillus easel (GenBank: AF320255.1). (http : // www .nebi .nlm .nih. gov/nuccore/AF3202551 and Lactobacillus plantarum (GenBank: AF320256.1) ( http://ncbi.nlm.nih.gov/nuccore/AF3202561 can boost immune system of mice dramatically. Causing the white blood cells of mice to destroy 5 pathogens that cause disease in humans more efficiently as following.

1. Burkholderia pseudomallei - This pathogen causes melioidosis, which can be found in human and many mammals such as goats, sheep, pigs, cows and buffaloes. The disease is found in Northern Australia, South East Asia and Southern China and Taiwan. Especially in Thailand, there are 2,000-3,000 patients per year, average 4.4 per 100,000 people. The highest incident of the disease has been found in the Northeast region, especially in the rainy season. This disease is a public health problem in Thailand. Due to the high mortality rate especially patient with bloodstream infections which has a mortality rate of around 60%. And often found severe symptoms of death in people with chronic diseases such as diabetes and kidney disease. There are often recurrent symptoms in the case of short-term treatment and immune-compromised patients, the changing from normal of eating habit and lifestyles will increase the severity of melioidosis among these people. Results from our mice immunity test, it turns out that white blood cells can be able to kill Burkhoderia pseudomallei up to 95.45%.

2. Shigella flexneri - This gram-negative pathogen causes dysenteria which has both defecation with or without blood. In Thailand, there are the reports of 3.4 Shigella-induced dysentery patients per 100,000 population. Results from mice immunity test turns out that white blood cells of mice can kill 100% of Shigella flexneri.

3. Staphylococcus aureus - The endemic pathogen of skin and nasal cavity and also the important pathogenic bacteria in food. Infections caused by this pathogen in many organs such as skin ulcer, hair gland, sebaceous gland, subcutaneous fat, infectious arthritis and bloodstream infection, is the main cause of patient’s hospitalization, 3 times longer compare to other types of infection and the death rate is 5 times higher. This pathogen can grow in the condition with or without air, causing food poisoning by consuming food that contains toxic substances which are heat-resistant toxins. Contamination of less than 1 microgram of these toxic substances in food can cause illness with nausea, vomiting, dizziness, abdominal cramping and fatique. Staphylococcus aureus is the highest cause of food poisoning in Thailand, approximately 8.25%. This pathogen has been resisted to penicillin, only one year after the use of penicillin, in the year 1946. In addition, the emerging problem in medical community in these days is the multidrug resistant organism (MDROs) that are resisted to 2 or more groups of drugs. And also there are pathogens that are resisted to all drugs used (Pan-drug resistant organisms), which are gram-positive bacteria named Methicillin-resistant Staphylococcus aureus (M.R.S.A.) is the big problem that medical community around the world is facing right now. According to data from our mice experiments taking the Plukow extract, the result showed that 99.46% of Staphylococcus aureus can be killed by mice white blood cell. As well as the inventor experience from using biological fermented Plukow powder with chronic skin infection patient who had been treated in the hospital for more than 10 years and had never been healed, the infection wounds was getting better and could be healed within 2 months.

It may be a new way of using Plukow, according to this embodiment, to treat drug-resistant infections with mainstream medicine strategy in the future more effective. Salmonella group B - Salmonella is a gram-negative bacteria. It is the most common cause of food poisoning. A small amount of bacteria in food can cause illness. It can grow at temperature between 8-45° in foods with acid-base between 4-9. Animal-derived foods such as raw or uncooked meat or carcasses, duck, raw eggs, products that contain raw eggs, raw or unsterilized milk and dairy products such as butter, ice cream, cheese and some vegetables can transmit this disease from animals to humans. The use of dirty water in agriculture or to wash fresh food can also cause contamination. Salmonella is the second leading cause of food poisoning in Thailand, after Staphylococcus aureus , with a prevalence of approximately 5.08%. In addition, Salmonella is also a problem in the chicken export industry. Thai frozen pork exported aboard many times in the past was canceled. Due to the detection of Salmonella contamination in frozen chicken and pork, meat products had been rejected causing damage to exporters. If the chickens and pigs are raised with food mix with an appropriate amount of biological fermented Plukow in order to build immunity against Salmonella with standard methods for raising chickens and pigs, the amount of antibiotics used in the farm can be reduced. The use of antibiotic in the farm, even though within the minimum effective dose, to prevent animal infection which will remain some residue left over, can be replace by using biological fermented Plukow. The biological fermented Plukow powder will stimulate immunity in chickens and pigs. With less amounts of using, there is no toxic residue in the dissected meat. The mice immunity test in our experiment showed that the white blood cells of mice can kill 99.82% of Sallmonella group B.

5. Group A streptococci - This pathogen cause the inflammation of throat or strep throat, tonsillitis and may affect the larynx too. Strep throat is a common cause of sore throat in pediatric patients (37%) and may cause rheumatic fever if there is not enough treatment. Rheumatic fever is an inflammation of many organ systems including heart disease, polyarthritis, Sydenham’s chorea, subcutaneous nodules and skin (erythrema marginatum). Group A streptococci is one of the important necrotizing or carnivorous bacteria (fascillitis) in Thailand. About 15.5 per 100,000 population cases of meat-eating bacteria in both children and adults has been reported with a mortality rate of approximately 17-49%. From our mice immunity testing in the laboratory, it turned out that the white blood cells of mice can kill 99.83% of Group A streptococci.

To study the efficiency of DNA and dead cell of Lactobacillus spp. , using Lactobacillus plantarum and Lactobacillus casei extracted DNA from Plukow biological fermented juice extract and heated kill Lactobacillus spp. at 100° for 30 minutes. Four mice were given 250 mg. of DNA subcutaneously. Checked antibody for IL - 2, IL -12, and IF -g by ELISA after 10 days later. The immune stimulation of death cell of L. casei and L. plantarum is fast and can stimulate antibody for pathogen.

DNA fragment of L. casei can promote White Blood Cells to destroy various pathogenic bacteria and stimulate the mice to produce TNF - Alpha . L. casei (fragmented DNA) can stimulate immune system (e.g. IgM, IgGl, IgG2a, IgG2b) of mice within 10 days (titer 12,800 - 204,800)

Bactericidal effect of stimulated mice serum can kill 5 pathogens as in table 1.

Table 1.

Lactobacillus microorganisms, both Lactobacillus casei and Lactobacillus plantarum, isolated from the biological fermented Plukow although in dead cell by boiling water for 30 minutes, or using RNA fragment called polynucleotides, can adjust the immune system in mice mentioned above as well.

The microorganism, yeast called Saccharomyces cerevisiae, contains 30-35% of beta glucan, which is complex sugar molecules, as its cell wall structure. This embodiment invention of biological fermented process, according to the said method, can raise beta glucan up to 9 times higher than in fresh Plukow leaf. Beta glucan can stimulate bone marrow to produce and release stem cell into bloodstream continuously by the mechanism that beta glucan binds into the receptor of macrophage called dectin-1 and encourage the macrophage to produce MMP-9 (Matrixmetalloproteinase-9) enzyme, which has the ability to degrade SDF-1/ CXCR4 axis protein that attach stem cell to the bone marrow. More stem cell will be released from bone marrow into bloodstream.

Many medical science research and development papers reported that stem cell is the authentic restoring and regenerative unit of the whole body. Stem cell is a reserve cell of the body and responses for restoring damaged cells, tissues or organs and repairs malfunction of all types of tissues to work properly all the time and every day too. Therefore, if there are enough stem cells in the bloodstream, the body can be able to set balance working, without any degenerative diseases occur. Stem cell medicine tells us that Alzheimer's disease and all type of degenerative diseases are caused by the lower number of stem cells in the blood stream. When using natural substances from Saccharomyces cerevisiae as a mixture of the recipe of the drug, beta glucan which is the main component of the yeast cell wall can support bone marrow of the body to release stem cells into the bloodstream. Therefor slowdown the degeneration of the body or can restore the body back to normal function, especially in case of enough nutritions in the body. Biological fermented Plukow extract, according to this invention, combinds with mainstream medicine principles can cure about 70 degenerative diseases.

The inventor has been developed biological fermented Plukow method for more than 20 years and the experiences from field data has been recorded, such as 1. The use of biological fermented Plukow extract, a. as liquid form by using 5-10 ml. 2 times a day for 1 month or b. as powder form by using 200-400 mg. 2 times a day for 1 month

The patients were all over Thailand and nearby countries. The number of cases that were healing or getting better from the field records showed that Cardiovascular disease 158 cases Diabetes 141 cases

Osteoporousis and osteoarthritis 102 cases Dizziness with fatique 68 cases Autoimmune diseases 64 cases Respiratory diseases 60 cases

Senile disease that cause dementia, wrinkle or hard skin 58 cases

Hyperlipidemia and obstruction from fat in the blood vessel 54 cases

Gastric ulcer and GERD 52 cases

Headache and migraine disease 45 cases

Herniated nerve 42 cases

Blurred vision caused by diabetes 37 cases

Cancer in various organs 35 cases

Chronic kidney disease 22 cases

Gout 14 cases

Amnesia disease 10 cases

Viral infection diseases 9 cases

Prostate enlargement 7 cases

Upper respiratory infection with chronic inflammation 6 cases

Thalassemia anemia 6 cases

Irritable bowel disease 5 cases

Effective for 1,500 cases of intractable diseases 2. The inventor donated biological fermented Plukow powdered 10,000 capsules, each capsule contains 220 mg. of biological fermented Plukow, to Sierra Leone for Ebola infection patients on 3 Aug. 2014 through the Chancellor of University of Sierra Leone. In which a Thai person who works in Sierra Leone was the coordinator, also a doctor who worked at the Central Hospital in the capital, Freetown of Sierra Leone, received some Plukow capsule to use with the Ebola patients to treat the disease. Some Plukow capsules were distributed to villagers who were infected with Ebola and some capsules were sent for testing at temporary drug testing center of the United Nation in Guinea along with donating drugs from another 20 countries at that time. The results showed that Plukow capsules could help people with Ebola who lived in a village near the Thai coordinator, mentioned above, after taking Plukow capsules containing 220 mg. of Plukow powder. The infected villagers recovered from illness within only 3 days. Just like the Ebola infected patients that the doctor treated with Plukow capsule in the Central Hospital in Freetown, Sierra Leone capital, and the testing of UN testing center in Guinea, the result also showed the same effective of biological fermented Plukow for Ebola, a severe viral hemorrhagic, infection too.

From the information in item 1 and 2 above, the inventor is confident in the properties of biological fermented Plukow according to this embodiment can better strengthen the immune system, eliminate pathogens and restore, repair and stimulate cell activities of the body. According to the inventor and the test team in mice and experiences in applying to treat the infection, the above Ebola virus, as well as various diseases such as a severe type of hemorrhagic fever, patients were also getting better. The properties of biological fermented Plukow, according to this embodiment, can trigger bone marrow to release stem cell in amount up to the severity or inflammation of disease. When certain stem cell that circulates in bloodstream, migrates to damaged tissue or inflammatory cell, it will creep out of the capillary and moves to cell or damaged tissue then differentiate to be target cell or tissue to restore that cells or differentiate to be a natural killer cell or other cell that the body needs to get rid of virus or repair the damage cell. If the symptom of the disease is more severe inflame then the stem cell can detect better and accelerate more stem cell release from bone marrow. In addition, the experiment found that biological fermented Plukow extracted according to this embodiment has the ability to reduce cytokines and enzymes which show inflammation in the body, such as TNF-a and IL-Ib, IL-6 and iNOS. A lot of quercetin in biological fermented Plukow extract according to this invention, has an antioxidant property and also induce the activity of superoxide dismutase enzyme, the strongest antioxidant of the body. Quercetin and superoxide dismutase help to strengthen the antioxidant function and reduce inflammation of each other. It can reduce mild and severe inflammation tissue during illness. Less inflammation area will be masked, while the more inflammation area will be detected by stem cell. More stem cell will migrate to that inflammation area. By this process the body will be able to use stem cells that release from the bone marrow, which is the reaction of beta-glucan in the fermented Plukow according to this invention, with maximum efficiency.

Concentrated extract of biological fermented Plukow powder according to this invention when tested with induced-diabetic rats, by streptozotocin together with high-fat feeding, it appeared that biological fermented Plukow powder increased serum insulin hormone and lowered blood sugar level week by week and also increased the level of glucose tolerance. All mice in the experiment showed significant reduction of diabetic symptoms. Figure 1 shows the mice could maintain the body weight by reducing water intake and food intake.

The HFD, HFD-STZ and HC abbreviation in figure 1-8 is stand for high food diet, HFD-STZ abbreviation existed in figure 1-8 is stand for high food diet with streptozotocin and HC existed in figure 1-8 is stand for biological fermented Plukow or houttuynia cordata extract. The mice receiving the biological fermented Plukow powder could use sugar as a source of energy for cells and helped the pancreas to increase the number of beta cells in term of Islet of Langerhans in figure 2.

The outcome of fasting blood glucose(FBG) is better than using metformin, the standard diabetic drugs, as in figure 3, in the same dose.

The biological fermented Plukow has anti-inflammatory effect of pancreas by reducing inflammation and enzymes related to the inflammatory process in the pancreas, such as TNF-a, IL-6, IL-Ib, COX-2 and iNOS as shown in figure (4), (5), (6), (7) and (8)

In addition, the biological fermented Plukow powder can also increase the sensitivity of insulin hormone in resistant fat cells. Therefore, the biological fermented Plukow powder, according to this embodiment, also has an antidiabetic property.

In summary, the biological fermented Plukow powder, according to this embodiment, can give more useful active pharmaceutical ingredients for human and animal. This embodiment can yield up to 9 times of beta glucan and up to 31,798 milligrams of quercetin per kilogram of Plukowas as in figure 9, while natural Plukow contains only 12.67 milligrams of quercetin as in figure 10, or up to 2,509 times of quercetin has been produced more than natural source, for the same amount of Plukow. Then this embodiment is suitable for processing Plukow herbal formula as herbal medicine, food supplement and cosmetic in the future.

TEST REPORT

DISCLOSURE OF INVENTION

Intensive herbal biological fermentation process in order to get more active pharmaceutical ingredients able to be use to stimulate the body’s immune system of human, animal and plants can be done by weigh fresh Plukow leaf, without defects, rinse thoroughly, let it drain and then put into the fermentation tank. Weigh the sugar cane powder into another tank, dissolve with clean water, then pour the sugar cane mixture into the fermentation tank. Close the lid tightly and left to expose in the sun light for 1 - 12 months. Remove Plukow leaves from the fermentation tank and dry in hot air oven at 100° for 8-12 hours until completely dry. Grind the dry leaves to fine powder.

Mix the powder with the fermented water from the fermentation tank in a ratio of 1:2 weight by weight. Mix thoroughly in a roasting tank. Turn on the motor of the roasting tank and ignite the head of gas stove to raise the outside of a roasting tank temperature to 100- 150°. The temperature of wet Plukow powder, next to the inner side of a roasting tank, will reach near 150° in a short time while the tank is rotating slowly for 1 hour. After that, dry Plukow powder in hot air oven at 100° for 8-12 hours to obtain more concentrated Plukow extract.

Repeat the above procedure by mixing the concentrated Plukow powder with the fermented water left in the fermentation tank, 1:2 weight by weight ratio, until all the fermented water is gone. The final concentrated Plukow powder will contain more quercetin and can be used as ingredient in better herbal formula as well.

The purpose of this fermented herb extracts is to produce a concentrated biological fermented Plukow powder with more concentration of phytochemicals, especially quercetin and beta glucan which will increase more than occurring in natural herb, to be use better in restoring various degenerative diseases and infectious diseases of human and animals. And to be used to reduce or replace the use of antibiotics and also use as a complementary medicine.

MODE FOR CARRYING OUT THE INVENTION

Concentrated herb extracts from Plukow according to this invention, 100% weight by weight consists of starters which are Saccharomyces cerevisiae, Lactobacillus casei (GenBank: AF320255.1) and Lactobacillus plantarum (GenBank: AF320256.1) mixed with ingredients as follows:

- Complete, no defect and clean Plukow leaves 5.0 - 20.0% by weight - Clean water 15.0 - 70.0% by weight

- Sugar cane powder 4.0 - 10.0% by weight

The best ratio of ingredients in making fermented extracts is composed of starters, Saccharomyces cerevisiae, Lactobacillus casei (GenBank:AF320255.1) and Lactobacillus plantarum (GenBank: AF320256.1) mix with the ratio of the following ingredients:

- Complete, no defect and clean Plukow leaves 20.0% by weight

- Clean water 70.0% by weight

Sugar cane powder 10.0% by weight

PRODUCTION PROCESS WITH THE FOLLOWING STEPS:

1. Weigh complete, no defect and clean Plukow leaves, put in a 1,000 litres fermented tank, opaque white plastic and food grade,

2. Weigh 70 kilograms sugar cane powder, put in another tank, add clean water, mix thoroughly until all the sugar cane is dissolved to get 7% sugar solution. Add starters, Saccharomyces cerevisiae, Lactobacillus casei (GenBank: AF320255.1) and Lactobacillus plantarum

(GenBank: AF320256.1)

3. Put the mixture in item 2 into the tank item 1 until nearly filled the tank. Close the tank tightly, leave in a limited air condition for 1 month with a little pressure vent from the fermenting tank.

4. Still close the lid of the tank tightly and set at temperature 40 ° or set to expose to the sun light for 3-12 months.

5. Remove Plukow leaves from fermenting tank and dry in hot air oven at 100° and grind into fine powder.

6. Mix the fine powder from step 5 with fermented water from fermented tank in a ratio of 1:2 weight by weight, in a roasting tank. Ignite the gas stove while turn on a motor to rotate the roasting tank, leading the inner part of roasting tank to reach 100-150°. The wet Plukow will contact 100-150° temperature shortly many times during 1 hour of roasting.

7. Dry Plukow powder in hot air oven at 100° for 8-12 hours. 8. Put the leftover fermented water into dried powder from step 7 in a ratio of 2:1 weight by weight and run the same process in roasting tank for 1 hour again (as step 6).

9. Dry the powder in hot air oven at 100° for 8-12 hours as in step 7.

10. Repeat step 8 and 9 until the leftover fermented water is all gone. Then we can get required concentrated Plukow powder.

11. The final Plukow powder can be used as a high concentration of active pharmaceutical ingredients for medical herb and food supplement. BEST MODE FOR CARRYING OUT THE INVENTION

As described in the topic of the full disclosure of the invention.

Claims

Claims:

1. Herbal formula to stimulate immune system of the body, in which the herbal powder has preparation process consist of starters, Saccharomyces cerevisiae, Lactobacillus casei (GenBank: AF320255.1) and Lactobacillus plantarum (GenBank: AF320256.1) and

- No defected Plukow leaves 5.0 - 20.0% by weight

- Sugar cane powder 4.0 - 10.0% by weight

- Clean water 15.0 - 70.0% by weight

2. Herbal formula to stimulate the body's immunity according to claim 1, Plukow powder preparation components consist of

- No defected Plukow leaves 20.0% by weight

- Sugar cane powder 10.0% by weight

- Clean water 70.0% by weight

3. Formula of production process, herbal formula to stimulate the body's immunity in accordance with claims 1-2 or any of the following with the following steps a. Bring no defected fresh Plukow leaves in a certained weight, wash thoroughly, allow to drain. Put in a white plastic, food grade, fermentation tank. b. Weigh sugar cane powder into another tank, then add starters, Saccharomyces cerevisiae, Lactobacillus casei (GenBank: AF320255.1) and Lactobacillus plantarum (GenBank: AF320256.1), add clean water to sufficient quantity of 1,000 litres. c. Pour the mixture from the second tank into the first tank to cover the herbs.

Close the tank lid to have enough air inlet to prevent high pressure in the fermentation tank. Leave for 1 month until no more bubbles come out. Then close the tank lid tightly and place it outdoor where it can be exposed to the sun light for 3-12 months. d. Dry Plukow fermented leaves at 100⁰ (degree Celcius) in hot air oven for 8-12 hours then grind it into fine powder. e. Mix the powder with fermented water in a ratio of 1:2 weight by weight, then mix in the roasting tank. Ignite the gas stove while turn on a motor to rotate the roasting tank leading the inner skin of roasting tank to reach 100-150⁰ and the wet Plukow could reach 100-150⁰ shortly many times during 1 hour of roasting. f. Dry Plukow powder in hot air oven at 100⁰ for 8-12 hours. g. Repeat step e. and step f. by putting the leftover fermented water to dried powder in a ratio of 2: 1 weight by weight and run the same process in roasting tank for 1 hour again until run out of fermented water from the tank. Then we can get required Plukow powder. h. The final concentrated Plukow powder can be used as high concentration of active pharmaceutical ingredients for medical herb and food supplement.

4. The embodiment as claim in item 3 as a mixture of drugs or dietary supplements, 10 to 100 milligrams per 1 kg of body weight per day, can be used to prevent and treat infection caused by pathogens such as bacteria Burkholderia pseudomallei, Shigella flexneri, Staphylococcus aureus, Sallmonella dysentery, Group A streptococci and used to prevent and treat common cold caused by rhinoviruses and other viral infection e.g. influenza, chikungunya, measles, chickenpox, smallpox, mumps, herpes simplex, herpes zoster, hepatitis A, hepatitis B, hepatitis C, dengue fever, HIV, Ebola, SARS-CoV-2.

5. The embodiment as claim in item 3 as a mixture of drugs or dietary supplements, 10 to 100 milligrams per 1 kg of body weight per day, can be used to prevent and treat degenerative diseases e.g. type 1 and 2 diabetes including blurred vision caused by diabetes, cardiovascular disease, osteoporosis and osteoarthritis, chronic dizziness and fatigue, peptic ulcers, GERD, thalassemia, headache and migraine, herniated disc disease, lung cancer, breast cancer, leukemia, chronic kidney disease, gout, prostatic hyperplasia, menopause syndrome, Parkinson’s disease, Rheumatoid arthritis, schizophrenia paranoid, dementia, Alzheimer’s disease, etc.