US008765138B2
(12) United States Patent (10) Patent No.: US 8,765,138 B2 Stamets (45) Date of Patent: Jul. 1, 2014
(54) ANTIVIRAL AND ANTIBACTERIAL Moore et al. "Fungal Products as Food” pp. 1-17. Reprint of Moore, ACTIVITY FROMIMEDICINAL D. & Chiu, S. W. Fungal products as food. Chapter 10 in Bio MUSHROOMS Exploitation of Fkilamentous Fungi (ed S.B. Pointing & K. D. Hyde), pp. 223-251. Fungal Diversity Press: Hong Kong.* (76) Inventor: Paul Edward Stamets, Shelton, WA “Tinctura Cinchonae Compsita' from King's American Dispensa (US) tory, 1898. Retrieved from: Henriette's Herbal Homepage on May 2, 2012. Retrieved from:
U.S. Patent Jul. 1, 2014 Sheet 2 of 6 US 8,765,138 B2
** 8tect of Antibacteries compose:cis (concentration: 8% or the &:: *w:x: & 8, 8xxi & 8:8:8
US 8,765,138 B2 1. 2 ANTIVIRAL AND ANTIBACTERIAL simplex and the vesicular stomatitus virus (VSV). Wang & ACTIVITY FROMIMEDICINAL Ng (2000) isolated a novel ubiquitin-like glycoprotein from MUSHROOMS Oyster mushrooms (Pleurotus Ostreatus) that demonstrated inhibitory activity toward the HIV-1 reverse transcriptase. BACKGROUND OF THE INVENTION Arabinoxylane inhibits HIV indirectly through the enhance ment of NK cells that target the virus. Arabinoxylanes are 1. Field of the Invention created from mushroom mycelia's enzymatic conversion of The present invention relates to methods and products use rice bran (Ghoneum, M., 1998). Research by Dr. Byong Kak ful in restricting the growth, spread and survivability of Kim showed that extracts of Reishi (Ganodenna lucidum) viruses and bacteria in animals, especially humans. More 10 prevented the death of lymphocytes infected with HIV and particularly, the invention relates to methods and medicinal inhibited the replication of the virus within the mother and mushroom mycelium products for treating Herpes, Ortho daughter cells (Kim et al., 1994). In response to hot water pox, influenza, SARS, Hepatitis, Tuberculosis, Escherichia extracts of Reishi mushrooms, preserved in ethanol, Versus coli and Staphylococcus aureus and other viruses and bacte saline controls, NK cell activity was significantly augmented 18. 15 when cancer cells were co-cultured with human spleen cells. 2. Description of the Related Art (Ohmoto, 2002). A mycelial combination of 7 species grown Despite advances in modern medicine, microbes and on rice achieved a similar result, greater than any one species viruses continue to kill millions of people, stimulating the at the same dosage. As the water extract of the fruitbodies is search for new antimicrobial and antiviral agents, some of high in beta glucans while the mycelium-on-rice is low in beta which have proven to be of significant commercial value. A glucans, but is high in arabinoxylanes, two causal agents are major difficulty in the discovery of antimicrobial and antiviral identified as NK effectors. Both the extract and the heat agents is their inherent toxicity to the affected host organism. treated, freeze dried, powdered mycelium from 7 species For instance, a novel agent or treatment that kills the virus but share common activity levels of enhancing NK activity by also harms the human host is neither medically practicable 300+%. These compounds may be synergistic. This same nor commercially attractive. Hence, many new antiviral drugs 25 combination of 7 species fermented on rice had a strong effect have never made it past preliminary Screening studies as they against HIV, inhibiting replication by 99% while the water have failed to prove non-toxicity and are unsafe to consume. extract of Reishi fruitbodies was 70%, respectively. These That medicinal mushrooms have been ingested for hun results underscore that water extractions of fruitbodies and dreds, and in Some cases, thousands of years, is strong Support oral administration of myceliated rice positively influence the for their non-toxicity, making them appealing candidates in 30 immune system, activating different Subsets of immunologi the search for new antimicrobial and antiviral agents. The cell cal receptor sites. Maitake (Grifola frondosa) is currently the surface of mycelium secretes antibiotics in a kind of “sweat' subject of research in the treatment of HIV. Mizuno et al. which are known in the field as exudates or secondary (1996) noted that crude fractions from Chaga (Inonotus metabolites. These antibiotics and enzymes target distinct obliquus) showed antiviral activity against HIV. sets of microbes. Useful antibiotics isolated from mushrooms 35 Fomitopsis officinalis (Villars) Bondarzew & Singer include calvacin from the Giant Puffball (Calvatia gigantea), (Agaricum officinalis, Fomes officinalis, Fomes laricis and armilliaric acid from Honey Mushrooms (Armillaria mellea), Laricifomes officinalis) has the common names Agarikon, campestrin from Agaricus campestris (The Meadow Mush Quinine Conk, Larch Bracket Mushroom, Brown Trunk Rot, room), coprinol from Inky Caps (Coprinus species) corolin Eburiko, Adagan (ghost bread) and Taka di (tree biscuit). from Turkey Tail Mushrooms (Trametes versicolor=Coriolus 40 Once widespread throughout the temperate regions of the versicolor), cortinellin from Shiitake (Lentinula edodes), world, this perennial wood conk saprophytizes larch, Dou ganomycin from Reishi (Ganoderma lucidum) and sparassol glas fir and hemlock, preferring mature woodlands. Now from Cauliflower mushrooms (Sparassis crispa). nearly extinct in Europe and Asia, this mushroom is a resident Suzuki et al. (1990) characterized an antiviral water of the Old Growth forests of Northern California, Oregon, soluble lignin in an extract of the mycelium of Shiitake mush 45 Washington and British Columbia. Known constituents rooms (Lentinula edodes) isolated from cultures grown on include beta glucans, triterpenoids and agaricin. Forms used rice bran and Sugarcane bagasse which limited HIV replica include mushroom fruitbodies and mycelium. F officinalis tion in vitro and stimulated the proliferation of bone-marrow has traditionally been used for centuries for the treatment of cells. Clinical trials with lentinan in the treatment of HIV "coughing illnesses.” Mizuno et al. (1995) and Hanssen patients showed inhibitory activity. (Gordon et al., 1998). 50 (1996) include this mushroom in a group of polypores, the hot However, Abrams (2002) found no significant advantage in water extracts of which provide a strong host mediated using lentinan in treating AIDS patients. Another mushroom response. Agarikon was also applied topically, in a poultice, recognized for its antiviral activity is Fomes fomentarius, a as an anti-inflammatory and to treat musclefskeletal pain. hoof-shaped wood conk growing trees, which inhibited the Described by the first century Greek physician Dioscorides in tobacco mosaic virus (Aokiet al., 1993). Collins & Ng (1997) 55 Materia Medica, the first encyclopedic pharmacopoeia on the identified a polysaccharopeptide inhibiting HIV type 1 infec medicinal use of plants, in approximately 65 C.E., as a treat tion from Turkey Tail (Trametes versicolor) mushrooms ment for a wide range of illnesses, most notably consumption, while Sarkar et al. (1993) identified an antiviral substance an archaic medical term. It was not until the invention of the resident in an extract of Shiitake (Lentinula edodes) mush microscope did germ-theory suggest that infections were rooms. More recently, derivatives of the Gypsy mushroom, 60 caused by microbes. A resident on the old growth conifers, Rozites caperata, were found by Piraino & Brandt (1999) to especially spruce, hemlock, Douglas fir and on Larch, this have significant inhibition against the replication and spread amazing mushroom produces a chalky cylindrical fruitbody of varicella zoster (the shingles and chickenpox virus), that adds layers of spore-producing pores with each growth influenza A, and the respiratory syncytial virus (RSV) but not season, allowing for a rough calculation of age. Conks up to against HIV and other viruses. Eo et al. (1999) found antiviral 65 50 years have been collected, and often times they resemble a activity from the methanol-soluble fractions of Reishi mush woman, reminiscent of the Venus of Willendorf form. The rooms (Ganoderma lucidum), selectively inhibiting Herpes Haida First Peoples of the Queen Charlotte Islands, and else US 8,765,138 B2 3 4 where on the coast of British Colombia, associated this mush Stamets (2001, 2002). Besides having a direct antiviral or room, or debatably another polypore species, with the pow antimicrobial effect, mushroom derivatives can also activate erful creator spirit Raven, and as a protector of women's natural immune response, potentiating host defense, and in sexuality (Blanchette et al., 1992; Stamets, 2002). This mush effect have an indirect but significant activity against infec room was carved into animalistic forms and placed on sha tions. (Stamets, 2003). man's graves to protect them from evil spirits. Grzywnowicz As mushrooms share a more common evolutionary history (2001) described the traditional use of this mushroom by with animals than with any other kingdom, mushrooms and Polish peoples, as a treatment against coughing illnesses, humans Suffer from common pathogens in the microbial asthma, rheumatoid arthritis, bleeding, infected wounds, and world, for instance, the bacterium Staphylococcus aureus and was known for centuries as a "elixirium ad longam Vitam’’: 10 Pseudomonas flourescens. Mushrooms have a vested evolu elixir of long life. The North Coast First Peoples of North tionary interest in not being rotted by bacteria, producing western North America also discovered the use of this mush antibacterial agents to stave off infection. Work by Suay et al. room as a poultice to relieve Swellings and in teas for treating (2000) showed that various mushroom species have anti feverish illnesses. Called the Quinine Fungus in many for bacterially specific properties. Viral infections, as in viral estry manuals because of its bitter taste, this mushroom is not 15 pneumonia, can precede, for instance, bacterial infections the source of quinine, an alkaloid from the bark of the Ama from Streptococcus pneumoniae or Staphylococcus aureus, Zonian Cinchona ledgeriana tree which was widely used so the use of mushrooms having antibacterial properties can since the late 19" century to treat malaria, caused by Plas help forestall secondary infections from opportunistic patho modium falciparum. Despite the long history of use, few gens. Mushrooms having both antibacterial and antiviral modern studies have been published on its medicinally active properties are especially useful for preventing infection. Fur compounds. F officinalis merits further research as the num thermore, it is anticipated that some mushrooms will demon ber of strains is in rapid decline, especially in Europe, where strate anti-bacteriophagic properties, being dually antibacte it is on the verge of extinction (Leck, 1991). rial and antiviral. The present inventor incorrectly speculated that it is Mushrooms have within them polysaccharides, glycopro thought, but not yet proven, that Fomitopsis officinalis pro 25 teins, ergosterols, enzymes, acids and antibiotics, which indi vided an aid in preventing the Scourge of viral diseases such as vidually and in concert can mitigate viral infection. As each Smallpox among native populations of northwestern North species of mushrooms is unique, not only in its cellular archi America (Stamets 2002). Upon further investigation, the tecture, but also in its innate response to viral antagonists, inventor contacted Guujaaw (2004), President of the Haida animals, especially humans, can benefit from these antiviral People who told him “We did not have time to develop a 30 mushroom-derived agents. Since humans now face multiple defense against smallpox. Our people went from 50,000 to threats from numerous viruses, including but not limited to 500 in three years. The smallpox came from a passenger HIV, Pox (such as smallpox), West Nile virus, influenza and dropped from the ship, the Queen Charlotte. Had we known avian or bird flu viruses, coronaviruses such as SARS, hepa of a cure, we would have used it.” Moreover, tests of the titis, Lyme disease, HELA cervical virus, respiratory syncy hot-water extract from boiling this mushroom showed no 35 tial virus, hantavirus, Vesicular stomatitus, Herpes, Epstein antiviral activity with the U.S. Defense Department's Bio Barr, Varicella-Zoster, Polio, Yellow Fever, Marburg, Ebola, shield BioDefense Program whilst the water/ethanol extract VEE, Lassa and Dengue Fever, and numerous microbes from the in vitro grown mycelium originating from a tissue including Plasmodium falciparum, Bacillus anthracis, clone of this mushroom showed strong anti-pox virus activity Escherichia coli, anthrax, Mycobacterium tuberculosis, bac (U.S. patent application Ser. No. 1 1/029,861). 40 teriophages, fungi such as Candida albicans, Aspergillus, Piptoporus betulinus (Bull.:Fr.) Karst (=Polyporus betuli Fusarium, Stachybotry's and Thermoactinomycetes, as well as nus (Bull.:Fr.) Fr.) is commonly known as the Birch Polypore prions such as BSE, finding substances that afford a broad or Kanbatake. It is found throughout the birch forests of the shield of protection against multiple viruses and microbes is world, circumboreal, and is one of the most common mush difficult. Virologists are increasingly concerned about the rooms on that host. Known constituents include betulin, betu 45 threat of viral infection from animal hosts, thought to be the linic acid, agaric acid, single stranded RNA, heteroglucans, probable source of the 2003 SARS (Sudden Acute Respira and antibiotics. Forms used include mushrooms, mycelium tory Syndrome) epidemic, likely to have originated in rural on grain and fermented mycelium. Crude extracts and puri regions of China where humans and captured animals exist in fied fraction are tumor inhibiting in vitro. The novel antibi close quarters. Furthermore, the concentration of animals in otic, Piptamine, has been isolated from this fungus (Schlegel 50 factory farms wherein thousands of chickens, hogs, cows et al. 2000). Pisha et al. (1995) found, in mice studies, that and other animals are aggregated, provide a breeding envi betulinic acid, a pentacyclic triterpene, was specifically toxic ronment for contagions as well as other environmental catas to melanoma without adverse effects to the host. Farnsworth trophes. Viruses and bacteria can also breed when birds, dogs, et al. (1995) found that betulinic acid facilitated apoptosis of prairie dogs, Vermin, cats, primates, bats and other animals, melanoma. This compound has been further evaluated for the 55 including humans, have concentrated populations. These treatment or prevention of malignant melanoma. Manez et al. Sources, and more yet to be discovered, present a microbial (1997) found that selected triterpenoids reduced chronic der threat to human health. mal inflammation. Found with the famous Ice Man, the use of Smallpox is a serious acute, contagious and infectious P betulinus transcends cultures and millennia. A fungus use disease marked by fever and a distinctive progressive skin ful to stop bleeding, prevent bacterial infection, and as an 60 rash. The majority of patients with smallpox recover, but antimicrobial agent against intestinal parasites, this species is death may occur in up to 30% of cases. Many smallpox one of the most prominent and frequently encountered mush Survivors have permanent Scars over large areas of their body, room seen on birch. Capasso (1998) postulated that the Ice especially their face, and some are left blind. Occasional Man used this fungus to treat infection from intestinal para outbreaks of smallpox have occurred for thousands of years in sites (Trichuris trichiura). 65 India, western Asia and China. European colonization in both Summaries of the antiviral properties of mushrooms were the Americas and Africa was associated with extensive epi published by Suay et al. (2000), Brandt & Piraino (2000) and demics of Smallpox among native populations in the 1500s US 8,765,138 B2 5 6 and 1600s, including use as a potential biological weapon in death rate. Half the population of a community may be the United States. Smallpox was produced as a weapon by affected during an epidemic. Children are much more likely several nations well past the 1972 Bioweapons convention than adults to get sick from the flu, as are families with that prohibited such actions. School-age children—Schools are an excellent place for flu There is no specific treatment for smallpox and the only 5 viruses to infect and spread. The risk of death from influenza prevention is vaccination. In 1980, the disease was declared is highest among persons aged 65 or older, although young eradicated following worldwide vaccination programs. How children, particularly the newborn, and persons with certain ever, in the aftermath of the terrorist and anthrax attacks of chronic conditions are also at risk of death. The flu is particu 2001, the deliberate release of the smallpox virus is now larly serious because of the rapidity of outbreaks, the large regarded as a possibility and the United States is taking pre 10 number of people affected and the possibility of serious com cautions to deal with this possibility. plications such as pneumonia. The Centers for Disease Con Smallpox is classified as a Category A agent by the Centers trol and Prevention estimates that 5-20% of the population of for Disease Control and Prevention. Category A agents are the United States come down with the flu each flu season believed to pose the greatest potential threat for adverse pub (typically late fall through winter). Although most recover lic health impact and have a moderate to high potential for 15 from the illness, according to CDC estimates about 19,000 large-scale dissemination. Other Category A agents are 36,000 died from the flu and its complications each year anthrax, plague, botulism, tularemia, and viral hemorrhagic during the epidemics occurring from 1976-1999. The 1918 fevers. Even the remote potential for release of a deadly Spanish flu pandemic is estimated to have caused 20-40 mil communicable disease in an essentially non-immune popu lion deaths worldwide, including 500.00 in the United States. lation is truly frightening. The majority of the 1918 deaths were caused by secondary Orthopox (Orthopoxvirus) includes the virus that causes infections from bacteria, which exploited the scarred lung Smallpox (Variola major). Smallpox infects only humans in tissue and immune impairment. The 1957 Asian flu and the nature, although other primates have been infected in the 1968 Hong Kong flu outbreaks killed hundreds of thousands laboratory. Other members of the Orthopoxvirus genera in the United States. capable of infecting humans include monkeypox, camelpox, 25 The influenza viruses are RNA viruses belonging to the cowpox, and vaccinia. Other poxviruses capable of infecting Orthomyxoviridae family. Influenza viruses are classified humans include the Parapoxvirus pseudocowpox and Orf into types A, B and C. Type A is the most common and usually (Parapoxvirus ovis) and the Molluscipoxvirus Molluscum causes the most serious epidemics. Influenza A viruses are contagiosum. Monkeypox is a rare smallpox-like disease further divided into subtypes on the basis of two proteins encountered in villages in central and west Africa. It is trans 30 found on the Surface of the virus, hemagglutinin (H) and mitted by monkeys, primates and rodents. Camelpox is a neuraminidase (N). Influenza A viruses are found in many serious disease of camels. The genetic sequence of the cam different animals, including birds, pigs, whales and seals, elpox virus genome is most closely related to that of the with wild birds acting as the reservoir for all subtypes of Variola (smallpox) virus. Cowpox is usually contracted by influenza A viruses. The influenza A subtypes H1N1 and milking infected cows and causes ulcerating “milker's nod 35 H3N2 have circulated widely among people (the Spanish flu ules' on the hands of dairy workers. Cowpox protects against was a H1N1 virus and the Hong Kong flu was a H3N2 virus). Smallpox and was first used for vaccination against Smallpox. Type B can also cause epidemics, but generally produces a Pseudocowpox is primarily a disease of cattle. In humans it milder disease than that caused by type A. Type C viruses causes non-ulcerating “milker's nodes. Molluscum conta have never been connected with major epidemics. Yearly flu giosum causes minor warty bumps on the skin. It is trans 40 vaccines are available targeting new variant strains resulting ferred by direct contact, sometimes as a venereal disease. Orf from antigenic drift, but neither prior vaccination nor previ virus occurs worldwide and is associated with handling sheep ous infection guarantees protection from the flu since the and goats afflicted with “scabby mouth.” In humans it causes virus typically varies from year to year. a single painless lesion on the hand, forearm or face. Vaccinia, It is currently feared that a strain of avian influenza (“bird a related Orthopox of uncertain origin, has replaced cowpox 45 flu'), which naturally occurs in wildbirds and can spread to for vaccination. Other viruses of the Poxyiridae family domesticated birds, could mutate into a form easily transmis include buffalopox virus, rabbitpox virus, avipox virus, sible by human-to-human and cause a worldwide pandemic. sheep-pox virus, goatpox virus, lumpy skin disease (Neeth The H5N1 high pathogenicity avian influenza (HPAI) virus ling) virus, Swinepox virus and Yaba monkey virus. strain, which is becoming endemic in various Asian countries Poxviruses are very large rectangular viruses the size of 50 and has spread to a number of countries in the Middle East, small bacteria. They have a complex internal structure with a Africa and Europe, has particularly concerned researchers large double-stranded DNA genome enclosed within a “core” because it is spread by migratory wildfowl, because it is that is flanked by two “lateral bodies.” The surface of the virus especially virulent and has caused the death of millions of particle is covered with filamentous protein components, giv animals worldwide, because it mutates rapidly and continues ing the particles the appearance of a ball of knitting wool. The 55 to evolve and because it has spread to domesticated birds and entire virus particle is encapsulated in an envelope derived mammals including pigs and tigers and in limited circum from the host cell membranes, thereby “disguising the virus stances to humans. As influenza type A H5 hemagglutinin immunologically. Most poxviruses are host-species specific, viruses have not circulated among humans and most or all of but Vaccinia is a remarkable exception. True pox viruses are the population has no protective antibodies, there is the poten antigenically rather similar, so that infection by one elicits 60 tial that H5N1 could cause a pandemic were it to mutate to a immune protection against the others. form easily transmissible by human-to-human contact. The Influenza (“flu') is an infection of the respiratory system H5N1 avian influenza strain has caused illness in more than characterized by fever, body aches, chills, dry cough, head several hundred people in Asia and the Middle East, approxi ache, sore throat and stuffy nose. The flu, which is caused by mately half of whom have died (almost all cases are thought a variety of viruses, is notable for its ability to sweep through 65 to be the result of bird-to-human infection, but it appears there entire communities in both developed and developing coun may be rare cases of human-to-human transmission). A tries and is associated with high morbidity and a significant severe influenza pandemic could potentially result in unprec US 8,765,138 B2 7 8 edented death, social disruption and economic loss as mil adapted in humans as evidenced by the high person-to-person lions become seriously ill at the same time. transmissibility of the virus. The critical questions are SARS is a new viral illness spread mainly by close person whether there is extensive horizontal transmission between to-person contact and possibly by infected Surfaces or objects animals, and whether the jump of the virus from animals to or an airborne vector or other means. SARS is believed to human was a rare and accidental event or portends frequent have originated in rural China in November 2002. In March occurrences in the future. The answers to these questions will 2003 the alarming spread of cases caused the World Health determine whether animals are viable reservoirs for future Organization and U.S. Centers for Disease Control and Pre SARS outbreaks and whether person-to-person transmission vention to issue a global alert over cases of atypical pneumo of SARS-CoV might recur. nia that did not appear to respond to treatment. The illness was 10 named Severe Acute Respiratory Syndrome (SARS). By the With the flow of airline passengers from remote regions of third week of March 2003, researchers from several countries the world, concentrating in airports and being re-routed to had isolated a novel single-stranded RNA virus from the their destinations, the contagiousness of foreign-borne Coronavirus family (SARS-CoV) with contagiousness and viruses carried by passengers are likely to be exacerbated in high mortality rate unlike any other known human coronavi 15 these types of locations, especially within the closed com ruses. Although coronaviruses account for about thirty per partments of passenger airplanes, increasing the likelihood of cent of respiratory illnesses, most are moderate in course cross-infection. Virtually anywhere humans concentrate pro (such as common colds) with pneumonia being caused only vide opportunities for contagions to spread, whether by air or in patients with poor immune systems; SARS-CoV seemed to by physical contact. The history of viruses indicates the dan be the first Coronavirus that consistently caused severe dis gerposed by new strains for which no immunities or vaccines ease in humans. Before the outbreak was contained, it spread exist. With the increased threat of bioterrorism from weap to more than two dozen countries. By December of 2003,774 onized viruses, a readily available broad-spectrum antiviral people had died and more than 8,000 had been infected. serves the best interests of public health. World airlines were hit hard by the SARS epidemic as several carriers slashed flights and axed jobs. The tourism industry 25 BRIEF SUMMARY OF THE INVENTION suffered badly due to the fear unleashed by the outbreak, as did many other businesses and industries far from its epicen Medicinal mushrooms having unique antiviral and anti ter. In many ways SARS caused the worst economic crisis in bacterial properties are described, including mushroom spe Southeast Asia since the wave of bank failures and currency cies, mycelium, extracts and derivatives useful in preventing, devaluations that occurred there in 1988. 30 treating ameliorating, mitigating, alleviating, reducing or cur SARS causes a form of lung injury characterized by ing infection from viruses. Particularly preferred are Fomi increased permeability of the alveolar-capillary membrane, topsis, Piptoporus, Inonotus, Ganoderma, Hypsizygus, diffuse alveolar damage, the accumulation of proteinaceous Trametes and various combinations with other mushroom pulmonary edema and pulmonary failure. Symptoms species. Extracts showing target specific antiviral and anti included high fever and one or more respiratory symptoms 35 bacterial properties are disclosed, as well as methods for including, cough, shortness of breath and difficulty breathing. preparation and isolation of active fractions. In addition to fever and respiratory symptoms, SARS was Still further objects and advantages of this invention will associated with other symptoms including headache, muscu become more apparent from the following detailed descrip lar stiffness, loss of appetite, malaise, confusion, rash, diar tion and appended claims. Before explaining the disclosed rhea and low oxygen levels in the blood (hypoxia). In many 40 embodiments of the present invention in detail, it is to be cases, those symptoms were followed by pneumonia in both understood that the invention is not limited in its application lungs, sometimes requiring use of a respirator. The pathology to the details of the particular products and methods illus of SARS is not yet fully understood and the clinical symp trated, since the invention is capable of other embodiments toms are unusual. The disease was mild in children and the which will be readily apparent to those skilled in the art. Also, mortality rate in that group almost nonexistent. Persons who 45 the terminology used herein is for the purpose of description suffered from chronic disease and the elderly had a much and not of limitation. higher mortality rate. Patients who survived SARS infections recovered seemingly spontaneously while those who per BRIEF DESCRIPTION OF THE DRAWING(S) ished Succumbed to rapid respiratory decline accompanied by extensive lung tissue damage. The tissue damage appeared to 50 FIG. 1 is a chart showing the effect of antibacterial com be driven by the patients own immune system rather than the pounds (concentration: 1%) on the survival of E. coli (Es organism itself. The mechanism of SARS pathogenesis may cherichia coli) O157:H7. involve both direct viral cytocidal effects on the target cells FIG. 2 is a chart showing the effect of antibacterial com and immune-mediated mechanisms. There are no specific pounds (concentration: 10%) on the survival of E. coli O157: therapies for SARS. The use of physiologically targeted strat 55 H7. egies of mechanical ventilation and intensive care unit man FIG. 3 is a chart showing the effect of antibacterial com agement including fluid management and glucorticoids was pounds (concentration: 100%) on the survival of E. coli the only supportive therapy available. Numerous antibiotic O157:H7. therapies were tried with no clear effect. Ribavirin with or FIG. 4 is a chart showing the effect of antibacterial com without use of steroids was used in a number of patients. But, 60 pounds (Concentration: 1%) on the survival of Staphylococ in the absence of clinical indicators, its effectiveness was not CS Cai.S. proven. FIG. 5 is a chart showing the effect of antibacterial com SARS was a much more virulent strain than most coronavi pounds (Concentration: 10%) on the survival of Staphylococ ruses, leading Scientists to believe that the virus had its origins CS Cai.S. in a non-human animal, where a coronavirus can have more 65 FIG. 6 is a chart showing the effect of antibacterial com severe effects. Although this virus most likely originated from pounds (Concentration: 100%) on the survival of Staphylo a wild animal, perhaps the civet cat, the SARS virus was well COCCS CalS. US 8,765,138 B2 9 10 DETAILED DESCRIPTION OF THE INVENTION multistratosa, F unita var. prunicola, F. vinosa, F. Widdring toniae, F. Zonalis and F. Zuluensis and Laricifomes species The extracts of the mushroom mycelium of Fomitopsis including L. concavus, L. maire and L. officinalis. Piptoporus officinalis, Fomitopsis pinicola, Piptoporus betulinus, Gano species include P betulinus, P choseniae, Pelatinus, Pfax derma resinaceum, Inonotus obliquus, Hypsizygus ulmarius ineus, Phelveolus, P. maculatissimus, P. malesianus, P. para and various combinations of other species have been found by doxus, P quercinus f. monstrosa, P Soloniensis, P suberosus the present inventor to have unique antiviral properties, and Pulmi. including activity against Orthopox viruses. The mycelial products of the present invention are prefer Orthopox viruses have a notorious reputation for their sur ably grown on grains; rice is very Suitable. The mycelium viving outside of the carrier-host animal, Surviving on Sur 10 may alternatively be grown on various agricultural and for faces such as blankets, on dead skin cells, and can be readily transmitted through bodily fluids, whether they are aspirated estry products, by-products and waste products or synthetic or not. That these viruses can survive long after their host cells media and the antiviral metabolites and products harvested have died makes orthopoxes especially capable of wide using methods known to the art. Alternatively, the mycelium spread distribution. Novel antiviral agents are needed to 15 may be grown via liquid fermentation and the antiviral prod reduce the survivability of viruses beyond that of disinfec ucts harvested Subsequent to colonization. The methods for tants currently in practice. Moreover, since the entry of cultivation of mycelium that are contemplated are covered viruses are commonly through the nasal and throat cavities, or within, for example, but are not limited to, the techniques through sexual contact, contact antivirals that limit the Sur described by Stamets (1993, 2000) in Growing Gourmet and vivability of the virus, or kill the virus, and/or limit the sus Medicinal Mushrooms, and by Stamets (2005) in Mycelium ceptibility of human cells to infection by a pox virus while Running. How Mushrooms Can Help Save the World. selectively not harming healthy human cells, are needed. Although ethanol and water extracts are illustrated below, Such contact antivirals as disclosed herein could prove useful it will be obvious that the various solvents and extraction in many applications, closing some of the many vectors used methods known to the art may be utilized. The extracts may by this virus for transmission to new hosts. 25 optionally be prepared by methods including extraction with Rather than the mushrooms themselves, particularly pre water, alcohols, organic solvents and Supercritical fluids Such ferred is the live mushroom mycelium (the “vegetative' state as CO, etc. Extracts may also be prepared via steam distil of the mushroom, containing at most only primordia or young lation of Volatile components, similar to the preparation of mushrooms) and extracts thereof, particularly the cell free “essential oils” from flowers and herbs. Suitable alcohols (centrifuged) extracts. The mycelium may be cultivated, 30 include those containing from 1 to 10 carbon atoms, such as, grown or fermented on Solid, semi-solid or liquid media. for example, methanol, ethanol, isopropanol, n-propanol, Preferred derivatives include frozen, dried or freeze-dried n-butanol, 2-butanol, 2-methyl-1-propanol (t-butanol), ethyl mycelium, extracts thereof and dried, solvent-free extracts ene glycol, glycerol, etc. Suitable organic solvents include (including both "crude' extracts and cell-free centrifuged unsubstituted organic solvents containing from 1 to 16 carbon extracts). It was unexpectedly found that boiling of the mush 35 atoms such as alkanes containing from 1 to 16 carbon atoms, room in water created water extracts that showed no activity alkenes containing from 2 to 16 carbon atoms, alkynes con against pox viruses whereas the mycelium grown from a taining from 2 to 16 carbon atoms and aromatic compounds clone of the same mushroom did. containing from 5 to 14 carbon atoms, for example, benzene, Preferred antiviral species include the Fomitopsis species, cyclohexane, cyclopentane, methylcyclohexane, pentanes, particularly F officinalis and F. pinicola, Piptoporus species, 40 hexanes, heptanes, 2,2,4-trimethylpentane, toluene, Xylenes, particularly P betulinus, Ganoderma species, particularly etc., ketones containing from 3 to 13 carbonatoms such as, for Ganoderma annulare (G. annularius), G. lucidum, G. resina example, acetone, 2-butanone, 3-pentanone, 4-methyl-2-pen ceum, and G. Camosum, Hericium species, particularly H. tanone, etc., ethers containing from 2 to 15 carbonatoms Such erinaceus, Hypsizygus species, particularly, H. tessulatus and as t-butyl methyl ether, 1,4-dioxane, diethyl ether, tetrahydro H. ulmarius, Inonotus species, particularly I. obliquus, Tram 45 furan, etc., esters containing from 2 to 18 carbon atoms Such etes species, particularly Trametes versicolor and the constel as, for example, methyl formate, ethyl acetate and butyl lations of species complexes derived from them throughout acetate, nitriles containing from 2 to 12 carbonatoms such as, the evolution of taxonomic and nomenclatural history. for example acetonitrile, proprionitrile, benzonitrile, etc., Fomitopsis species include F. africana, F albomarginata amides containing from 1 to 15 carbon atoms Such as, for var. pallida, F. albomarginata var. polita, F. albomarginata 50 example, formamide, N,N-dimethylformamide, N,N-dim var. subvillosa, F. anhuiensis, F annosa f multistriata, F. ethylacetamide, amines and nitrogen-containing hetero annosa var. indica, F arbitraria, Favellanea, F. bucholtzii, F. cycles containing from 1 to 10 carbon atoms Such as pyrroli cajanderi, F. Caliginosa, F. Castanea, F. cinerea, F. concava, dine, 1-methyl-2-pyrrolidinone, pyridine, etc., halogen F. connata, F. corrugata, F. cuneata, F. cupreorosea, F. Cys Substituted organic solvents containing from 1 to 14 carbon tina, F. Cytisina, F dochmia, F, durescens, F epileucina, F. 55 atoms Such as, for example, bromotrichloromethane, carbon euosma, F, feei, F. filviseda, F hainaniana, F iberica, F. tetrachloride, chlorobenzene, chloroform, 1,2-dichloroet ibericus, F kiyosumiensis, F komatsuzaki, F. labyrinthica, F. hane, dichloromethane, 1-chlorobutane, trichloroethylene, latissima, Flignea, F. lilacinogilva, F. maackiae, F. maire, F. tetrachloroethylene, 1,2-dichlorobenzene, 1,2,4-trichlo marginata, F. mellea, F minutispora, F. nigrescens, F. nivosa, robenzene, 1,1,2-trichlorotrifluoroethane, etc., alkoxy, ary F. Odoratissima, F officinalis (Laricifomes officinalis), F. 60 loxy, cyloalkyl, aryl, alkaryl and aralkyl Substituted organic Olivacea, F. palustris, F. pinicola, F. pinicola f effusa, F. Solvents containing from 3 to 13 carbon atoms such as, for pinicola f. paludosa, F. pinicola f. resupinata, F. pseudo example, 2-butoxyethanol, 2-ethoxyethanol, ethylene glycol petchin, F. pubertatis, F quadrans, F. rhodophaea, F. rosea, dimethyl ether, 2-methoxyethanol, 2-methoxyethyl ether, F. roseozonata, F. rubidus, F. rufolaccata, F. rufopallida, F. 2-ethoxyethyl ether, etc., acids containing from 1 to 10 carbon sanmingensis, F. Scalaris, F semilaccata, F sensitiva, F. 65 atoms such as acetic acid, trifluoroacetic acid, etc., carbon spraguei, F. Stellae, F subrosea, F subungulata, F sulcata, F. disulfide, dimethyl sulfoxide (DMSO), nitromethane and sulcata, F supina, F unita, F unita var. lateritia, F unita var. combinations thereof. Extracts may also be prepared via US 8,765,138 B2 11 12 sequential extraction with any combination of the above Sol regard to Fomitopsis officinalis blends, blends consisting of vents. The extracts may be further refined by means known to 5-95% F.o. are preferred, 10-75% is more preferred and the art. 20-50% is most preferred. Preferred drying methods include freeze drying, air drying, The term “effective amount” refers to an amount sufficient spray drying and drum drying and the methods and apparatus to have antiviral activity and/or enhance a host defense for drying mycelium, extracellular metabolites, extracts and mechanism as more fully described below. This amount may derivatives disclosed in U.S. Pat. No. 4,631,837 to Magoon vary to some degree depending on the mode of administra (1986), herein incorporated by reference in its entirety. tion, but will be in the same general range. The exact effective Extracts are preferably extracted from living mycelium and amount necessary could vary from Subject to Subject, depend may be cell-free (filtered and/or centrifuged) or not. 10 ing on the species, preventative treatment or condition being The products from the culturing of the medicinal mush treated, the mode of administration, etc. The appropriate room species and mycelia, extracts and derivatives can be effective amount may be determined by one of ordinary skill deployed via several delivery systems as an effective antiviral in the art using only routine experimentation or prior knowl control, including orally-active powders, pills, capsules, teas, edge in the art in view of the present disclosure. Typical 15 therapeutic amounts of mycelium on rice (individual fungal extracts, dried extracts, Sublinguals, sprays, dispersions, solu species and/or combinations of species) are preferably 0.1-20 tions, Suspensions, emulsions, foams, syrups, lotions, oint gm./day, more preferably 0.25-10gm./day, and most prefer ments, gels, pastes, dermal patches, injectables, vaginal ably 0.5-5 gm./day. Typical therapeutic amounts of extracts creams and Suppositories. (individual fungal species and/or combinations of species) The mycelium, extracts and derivatives of Fomitopsis offi preferably deliver 0.1-20 mg. extracted materials per kg. of cinalis, Piptoporus betulinus and/or Ganoderma resinaceum body weight, more preferably 0.25-10 mg/kg. and most pref may optionally be combined with Agaricus blazei, Agaricus erably 0.5-5 mg/kg. brasiliensis, Agrocybe arvalis, Agrocybe aegerita, Auricu The antiviral extracts, mycelium and/or other derivatives laria auricula, Auricularia polytricha, Calvatia gigantean, may be incorporated into foods to produce foods with antivi Cordyceps sinensis, Flammulina populicola, Flammulina 25 ral properties, useful for protecting animals, including velutipes, Fomes fomentarius, Fomitopsis cajanderi, Fomi humans, dogs cats, horses, cows, pigs, birds, fish, insects and topsis pinicola, Ganoderma applanatum, Ganoderma cap other wild and domesticated animals, from infection. ense, Ganoderma lucidum, Ganoderma Oregonense, Gano The applicant anticipates that since DNA techniques and derma sinense, Ganoderma neojaponicum, Ganoderma other advances in taxonomy will likely result in changes in tsugae, Giganopanus giganteum, Grifolafrondosa, Hericium 30 names, the splitting of species, and even in the transfer of species to other genera, that the Polyporaceae species men abietis, Hericium erinaceus, Hericium ramosum, Hypholoma tioned in this patent application are those as understood by the capnoides, Hypholoma sublateritium, Hypsizygus tessulatus, most complete monograph on the Subject, Ryvarden & Gil Hypsizygus ulmarius, Inonotus obliquus, Inonotus dryadeus, bertson's North American Polypores, 1986 vol. I and II, Inonotus dryophilus, Lentinula edodes, Lentinus ponderosus, 35 FungiFlora, Oslo, Norway. As such, when we describe Fomi Lenzites betulina, Mycena alcalina, Phellinus linteus, Pho topsis officinalis, Piptoporus betulinus or any other mush liota adipose, Pholiota nameko, Pleurotus citrinopileatus, room species, we mean Fomitopsis officinalis sensulato, Pip Pleurotus comucopiae, Pleurotus dryinus, Pleurotus eryngii, toporus betulinus sensulato and a similar broad description Pleurotus Ostreatus, Pleurotus opuntinae, Pleurotus pulmo of any other species, each of which means that this is the narius, Pleurotus tuberregium, Polyporus sulphureus (Laeti 40 species concept as described within the broadest taxonomic porus sulphureus), Laetiporus conifericola, Polyporus hiritus, interpretation, encompassing synonyms, varieties, forms and Polyporus tuberaster, Polyporus umbellatus, (=Grifola species that have or will be split from these species since umbellata), Schizophyllum commune, Trametes versicolor original publication. As is known in the art, names change as (=Coriolus versicolor), and/or Wolfiporia cocos (=Poria new species concepts are constructed. cocos) mycelium, extracts or derivatives. 45 Fomitopsis species such as Fomitopsis officinalis, Pipto Example 1 porus species such as Piptoporus betulinus, Ganoderma spe cies Such as Ganoderma resinaceum, Inonotus species Such Tissue cultures of the mushrooms species describe herein as Inonotus obliquus, and Trametes species, such as Trametes were acquired or cloned from wild specimens by the inventor versicolor, may optionally be added to any formula or prod 50 and purified over time by Successive transfers in a clean room uct in an amount Sufficient to have the effect of preventing, laboratory using standard tissue culture techniques as treating, alleviating, mitigating, ameliorating or reducing described in Growing Gourmet and Medicinal Mushrooms infection. Stamets (1993, 2000). Fomitopsis officinalis I is a strain col The invention includes the combination of products from lected from Morton, Wash., USA. Fomitopsis officinalis X is multiple mushroom species in a form to have the accumulated 55 a strain isolated from the Hoh Rainforest, Wash., USA. Other effect of restricting the growth, spread and survivability of species were either collected or obtained from culture banks. viruses in animals, especially humans. Such forms may have The Ganoderma resinaceum utilized is a strainformerly misi the additional advantages of functioning as antibacterials, dentified as G. lucidum. Phylogenetic analysis of Ganoderma antiprotozoals, immunomodulators, nutraceuticals and/or based on nearly complete mitochondrial small-subunit ribo probiotics as well as enhancing innate immunity defense 60 Somal DNA sequences. Soon Gyu Hong and Hack Sung Jung, mechanisms and host immune response, resulting in healing. Mycologia, 96(4), 2004, pp. 742-745. Optimizing dosage is dependent upon numerous variables. Mycelial cultures were grown in sterile Petri dishes con The difference between a medicine and poison is often dos taining sterilized malt yeast rice agar. After three weeks of age. Determining the proper dose for antiviral effects will colonization in a clean room laboratory, the cultures were only require routine experimentation because the concentra 65 aseptically transferred into a 1000 ml. EBERBACHTMstirrer tions of extracts can be simply diluted or concentrated by containing 800 ml. of sterilized water. The EBERBACHTM adjusting ethanol and/or water content. In general, with container was activated using a WARINGTM blender base, US 8,765,138 B2 13 14 chopping the mycelium into thousands of fragments. This Products made from Fomitopsis officinalis, Fomitopsis pini myceliated broth was then transferred, under sterile condi cola and Piptoporus betulinus may be combined with other tions, into a sterilized glass 2000 ml. fermentation vessel mushrooms, fungi, or plant based materials to positive affect containing a 3% concentration of malt Sugar, 0.3% yeast and immunity, host defense and resistance from infectious dis 0.3% powdered rice, stir bar and 800 ml. of sterilized water. 5 eases. Grains other than rice may be additionally employed Once transferred, the fermentation flask was placed on a with similarly positive results. magnetic stir plate, and stirred at 300-400 rpm for a period of 3-4 days in front of a laminar flow hood at a temperature of Example 3 70-75° F. During that time, three-dimensional colonies of mycelium appeared, increasing in numbers and in density. 10 The general approach for determining antiviral activity and The fermentation was stopped prior to the coalescing of the toxicity as described by E. Kern for orthopoxviruses (http:// mycelium into a contiguous mycelial mat. The dissociated www.niaid-aacf.org/protocols/orthopox.htm) was utilized. fragmented mycelial mass allows for a multiple loci inocula The Selectivity Index (SI) values were determined by or tion, resulting in accelerated colonization and allowing for under the direction of Dr. Earl Kern of the USAMRIID/NIH/ the ease of further dilutions and inoculations. The fermented 15 USAID Bioshield BioDefense Program. broth was then diluted 1:10 into sterilized water, and trans An inexpensive, rapidassay Such as a CPE-inhibition assay ferred, under sterile conditions, into polypropylene incuba that is semi-automated was used initially to screen out the tion bags containing approximately 6.6 lbs or 3 kg. moistened negatives. Screening assays were conducted in low-passaged sterilized rice, adjusted to approximately 45-50% moisture human cells. Each assay system contained a positive control content. Approximately 50-100 ml. of diluted fermented fluid (CDV) and a negative control (ACV). Toxicity was deter was transferred into each of the 10 rice bags under sterile mined using both resting and proliferating human fibroblast conditions. The fresh mycelial cultures were then incubated cells. for 60-120 days in a class 100 clean room. Incubation times Screening Assay Systems for Determining Antiviral Activ are preferably 7-180 days, more preferably 30-120 days. ity Against VV and CV Once colonization was determined to be sufficient, the 25 Compounds were screened for activity against Vaccinia mycelium-colonized rice was transferred to glass containers virus (VV) and Cowpox virus (CV) using the CPE assay in for extraction. The mycelium, being delicate in nature, was HFF cells. The screening assay systems utilized were selected handled with utmost gentle care so as to not to cause cell to show specific inhibition of a biologic function, i.e., cyto damage in transfer and immediately covered with an approxi pathic effect (CPE) in susceptible human cells. In the CPE mately equal weight of 50% ethanol-water (prepared by mix 30 inhibition assay, drug is added 1 hr prior to infection so the ing equal weights of 95% (190 proof) organic ethyl alcohol assay system will have maximum sensitivity and detect and spring water), agitated, and then allowed to rest for room inhibitors of early replicative steps such as adsorption or temperature infusion-extraction for a total of 14 days. Cul penetration as well as later events. To rule out non-specific tures of Fomitopsis officinalis, Piptoporus betulinus, Gano inhibition of virus binding to cells all compounds that show derma resinaceum and the various other species were treated 35 reasonable activity in the CPE assay can be confirmed using separately in a similar fashion to the methods described a classical plaque reduction assay in which the drug is added herein. The clear fluid, the supernatant, was drawn off and 1 hr after infection. These assay systems also can be manipu decanted into 2 ounce amber bottles or other containers. lated by increasing the pre-treatment time in order to demon Dilution for bioassay was from 1:100 to 1:1000. strate antiviral activity with oligodeoxynucleotides and/or It will of course be appreciated that differing concentra 40 peptides. By delaying the time of addition of drug after infec tions and/or compositions of extracts may be easily prepared; tion, information regarding which step in the virus life cycle 3 kg. of fresh mycelium on rice for every 3000 ml. of extract. is inhibited (i.e., early VS. late functions) can be gained. or 1 g. mycelium/1 ml. extract is an example of a therapeuti Efficacy: cally useful extract. In all the assays used for primary Screening, a minimum of 45 six drug concentrations was used covering a range of 100 Example 2 ug/ml to 0.03ug/ml, in 5-fold increments. These data allowed good dose response curves. From these data, the dose that Proprietary strains of fungal species, sourced and/or origi inhibited viral replication by 50% (effective concentration nated by Stamets and Fungi Perfecti LLC, were grown under 50; ECs) was calculated using the computer software pro Class 100 clean room conditions on sterilized, certified 50 gram MacSynergy II by M. N. Prichard, K. R. Asaltine, and C. organic short grain brown rice, in accordance to methods Shipman, Jr., University of Michigan, Ann Arbor, Mich. described by Stamets (1993, 2000) in Growing Gourmet and Toxicity: Medicinal Mushrooms. The moistened rice was sterilized in The same drug concentrations used to determine efficacy high-density polypropylene bags and inoculated with myce were also used on uninfected cells in each assay to determine lium, which was fermented in liquid culture for several days. 55 toxicity of each experimental compound. The drug concen Each strain was grown to optimize the number of cell divi tration that is cytotoxic to cells as determined by their failure sions (CFU’s=colony forming units) prior to transfer into to take up a vital stain, neutral red, (cytotoxic concentration grain. Once inoculated, each strain was incubated for a dura 50; CCs) was determined as above. The neutral red uptake tion to optimize their CFU (colony forming units) maxima, assay has been found to be reliable and reproducible and and then flash frozen to -18°C. The frozen myceliated rice 60 allows quantitation of toxicity based on the number of viable was then freeze-dried in a negative pressure vacuum of 1500 cells rather than cellular metabolic activity. It is important 2000 millibars and then heated to 75° C. for 24 hours. The also to determine the toxicity of new compounds on dividing freeze-dried material was then milled to a fineness of 20-80 cells at a very early stage of testing. A cell proliferation assay standard mesh (180-850 microns). This raw material can be using HFF cells is a very sensitive assay for detecting drug filled into capsules, made into tablets, tinctures or further 65 toxicity to dividing cells and the drug concentration that used as a base for a medicinal product effective as a antimi inhibits cell growth by 50% (ICs) was calculated as crobial and/or for potentiating a host mediated response. described above. In comparison with four human diploid cell US 8,765,138 B2 15 16 lines and Vero cells, HFF cells are the most sensitive and with a 0.1% crystal violet in 3% formalin solution for 4 hr. predictive of toxicity for bone marrow cells. The stain is removed and the plates rinsed using tap water Assessment of Drug Activity: To determine if each com until all excess stain is removed. The plates are allowed to dry pound has sufficient antiviral activity that exceeds its level of for 24 hr and then read on a BioTek Multiplate Autoreader at toxicity, a selectivity index (SI) was calculated according to 5 620 nm. The ECso values are determined by comparing drug CCso/ECso. This index, also referred to as atherapeutic index, treated and untreated cells using a computer program. was used to determine if a compound warrants further study. Plague Reduction Assay Using Semi-Solid Overlay: Compounds that had an SI of 2 or more are considered active, Two days prior to use, HFF cells are plated into 6 well 10 or greater (>10) is considered very active. plates and incubated at 37° C. with 5% CO, and 90% humid Laboratory Procedures for Determining Antiviral Efficacy 10 ity. On the date of assay, the drug is made up at twice the and Toxicity desired concentration in 2xMEM and then serially diluted 1:5 Preparation of Compounds for In Vitro Testing: in 2xMEMusing 6 concentrations of drug. The initial starting as the Fungal extracts were water, ethanol and DMSO concentration is usually 200 lug/ml down to 0.06 ug/ml. The soluble, they were dissolved in tissue culture medium without virus to be used is diluted in MEM containing 10% FBS to a serum at 1 mg/ml and diluted for use as indicated below in the 15 desired concentration which will give 20-30 plaques per well. description of the assay system. Noteworthy is that the The media is then aspirated from the wells and 0.2 ml of virus extracts from the applicant's living mycelium, diluted from is added to each well in duplicate with 0.2 ml of media being 100:1 to 1,000:1, showed effectiveness against the described added to drug toxicity wells. The plates are then incubated for viruses at dosages designed for testing pure pharmaceuticals, 1 hr with shaking every 15 min. After the incubation period, underscoring that the extracts as presented are potent against an equal amount of 1% agarose will be added to an equal viruses. Volume of each drug dilution. This gives final drug concen Screening and Confirmation Assays for VV and CV trations beginning with 100 ug/ml and ending with 0.03 g/ml Preparation of Human Foreskin Fibroblast (HFF) Cells: and a final agarose overlay concentration of 0.5%. The drug/ Newborn human foreskins are obtained as soon as possible agarose mixture is applied to each well in 2ml Volume and the after circumcision and placed in minimal essential medium 25 plates are incubated for 3 days, after which the cells are (MEM) containing Vancomycin, fungizone, penicillin, and stained with a 0.01% solution of neutral red in phosphate gentamicin at the usual concentrations, for 4 hr. The medium buffered saline. After a 5-6 hr incubation period, the stain is is then removed, the foreskin minced into Small pieces and aspirated, and plaques counted using a stereomicroscope at washed repeatedly with phosphate buffered saline (PBS) 10x magnification. deficient in calcium and magnesium (PD) until red cells are 30 Screening and Confirmation Assays for Toxicity no longer present. The tissue is then trypsinized using trypsin Neutral Red Uptake Assay at 0.25% with continuous stirring for 15 min at 37° C. in a Twenty-fourh prior to assay, HFF cells are plated into 96 CO, incubator. At the end of each 15-min. period the tissue is well plates at a concentration of 2.5x10" cells per well. After allowed to settle to the bottom of the flask. The supernatant 24 hr, the media is aspirated and 125 ul of drug is added to the containing cells is poured through sterile cheesecloth into a 35 first row of wells and then diluted serially 1:5 using the flask containing MEM and 10% fetal bovine serum. The flask BioMek 2000 Laboratory Automation Workstation in a man containing the medium is kept on ice throughout the ner similar to that used in the CPE assay. After drug addition, trypsinizing procedure. After each addition of cells, the the plates are incubated for 7 days in a CO incubator at 37 C. cheesecloth is washed with a small amount of MEM contain At this time the media/drug is aspirated and 2001/well of ing serum. Fresh trypsin is added each time to the foreskin 40 0.01% neutral red in PBS is added. This is incubated in the pieces and the procedure repeated until all the tissue is CO incubator for 1 hr. The dye is aspirated and the cells are digested. The cell-containing medium is then centrifuged at washed using a Nunc Plate Washer. After removing the PBS, 1000 RPM at 4° C. for 10 min. The supernatant liquid is 200ug?well of 50% ETOH/1% glacial acetic acid (in HO) is discarded and the cells resuspended in a small amount of added. The plates are rotated for 15 min and the optical MEM with 10% FBS. The cells are then placed in an appro 45 densities read at 540 nm on a plate reader. The ECso values are priate number of 25 cm tissue culture flasks. As cells become determined by comparing drug treated and untreated cells confluent and need trypsinization, they are expanded into using a computer program. larger flasks. The cells are kept on Vancomycin and fungizone Independent cell cytotoxicity tests conducted by or under to passage four, and maintained on penicillin and gentamicin. the direction of Dr. Susan Manly and/or Dr. Samir Ross of the Cells are used only through passage 10. 50 National Center for Natural Products Research (NCNPR) at Cytopathic Effect Inhibition Assay: the University of Mississippi showed the mycelial extracts to Low passage HFF cells are seeded into 96 well tissue be non-toxic at the high levels of exposure in three human cell culture plates 24 hr prior to use at a cell concentration of culture lines. It is therefore possible that the Selectivity Index 2.5x10 cells per ml in 0.1 ml of MEM supplemented with ratios may be understated, as SI is the CC50 (cytotoxicity) 10% FBS. The cells are then incubated for 24 hr at 37° C. in 55 divided by EC (effective concentration) (the amount that lim a CO, incubator. After incubation, the medium is removed its 50% of the human cell growth rate divided by the amount and 125 ul of experimental drug is added to the first row in to kill 50% of the virus). If the SI values are understated, the triplicate wells, all other wells having 100 ul of MEM con products described herein could be loaded much higher than taining 2% FBS. The drug in the first row of wells is then that shown before evidence of cytotoxicity would be seen and diluted serially 1:5 throughout the remaining wells by trans 60 the actual antiviral activity may be much more than that ferring 25ul using the BioMek 2000 Laboratory Automation shown by cell line bioassays described herein. Furthermore, Workstation. After dilution of drug. 100 ul of the appropriate and unexpectedly, the strong antiviral activity is localized by virus concentration is added to each well, excluding cell going from ethanol as the first solvent and, after centrifuging control wells, which received 100 ul of MEM. The virus and cell-freeing, going to DMSO, samples prepared in this concentration utilized is 1000 PFU’s per well. The plates are 65 fashion showed antiviral activity whereas samples using then incubated at 37°C. in a CO incubator for 7 days. After water first followed by DMSO consistently failed to show the incubation period, media is aspirated and the cells stained activity. Hence the use of ethanol as a first step is preferred US 8,765,138 B2 17 18 over water. Note that since the living mycelium on sterilized Htu: Hypsizygus ulmarius rice has approximately 50% moisture, and hence when equal I.o. Inonotus obliquus, Quebec, Canada mass of 99% EtOH is added, the EtOH/Moisture concentra Pb.: Piptoporus betulinus, McCall, Idaho, USA tion is typically >30%, but <70% at makeup (in contrast, PhL: Phellinus inteus antibacterial activity as reported in the enclosed examples 5 P.u.: Polyporus umbellatus was preserved when water only was used). S.c.: Schizophyllum commune The influenza bioassays were conducted according to Sid T.V.: Trametes versicolor, Kamilche Pt., Washington State, well, RW and Smee, D F. In vitro and in vivo assay systems USA for study of influenza virus inhibitors, Antiviral Res., October 7 Mushroom Blend=A blend of Agaricus brasiliensis, 2000, 48(1):1-16 10 Cordyceps sinensis, Ganoderna lucidum, Grifola frondosa, All strains below were incubated for approximately two Hericium erinaceus, Polyporus umbellatus and Trametes ver months prior to extractions; some strains were incubated up to sicolor. 7 months. Activity was seen consistently within this timespan 13 Mushroom Blend=A blend of Ganoderma resinaceum, of incubation. With those strains designated as “shaken the Ganoderma applanatum, Ganoderma Oregonense, Grifola mycelium and ethanol/water were shaken and allowed to 15 frondosa, Phellinus linteus, Trametes versicolor, Fomes settle prior to decanting the extract. fomentarius, Fomitopsis officinals, Inonotus obliquus, The Fomitopsis officinalis strains and extracts described Lentinula edodes, Polyporus umbellatus, and Schizophyllum above in Example 1 were utilized. The following codes can be COU used to decipher the names of the active samples: HD: A 16 mushroom blend of: Fomitopsis officinalis, Grifola Csc: Cordyceps sinensis 20 frondosa, Inonotus obliquus, F.o. I: Fomitopsis officinalis Morton, Washington State, USA Ganoderma resinaceum, Cordyceps sinensis, Polyporus F.o. VI: Fomitopsis officinalis, Carrington Bay, Cortes Island, umbellatus, Piptoporus betulinus, Flammulina velutipes, British Columbia, Canada Agaricus brasiliensis, Phellinus linteus, Schizophyllum com F.o. X: Fomitopsis officinalis, Hoh River Valley, Washington mune, Trametes versicolor; Hericium erinaceus, Ganoderma State, USA 25 applanatum, Ganoderma Oregonense, Fomes fomentarius G. ann. Ganoderma annulare, Cortes Island, B.C., Canada and Lentinula edodes G.l.: Ganoderma lucidum HD Fraction 1: A blend of Grifola frondosa, Flammulina G. neojaponicum. Ganoderma neojaponicum velutipes, Inonotus obliquus, Ganoderna applanatum G.o.: Ganoderma Oregonense HD Fraction 2: A blend of Ganoderma resinaceum, G.r. Ganoderma resinaceum Canada 30 Cordyceps sinensis, Phellinus linteus, Ganoderma Oregon HDT-3: Mixture of Flo. I, Pb., T.V. eFSe H.e.: Hericium erinaceous HD Fraction 2: A blend of Polyporus umbellatus, Hericium H.u.: Hypsizygus ulmarius, Canada erinaceus, Piptoporus betulinus and Lentinula edodes
INFLUENZAAMDCK CELL LINE Cmpd Drug Name Virus Strain Assay/Vehicle Unit EC50 IC50 SI 1 HDT3 1x FuA Vietnam Visual Dil.
INFLUENZAAMDCK CELL LINE Cmpd Drug Name Virus Strain Assay/Vehicle Unit EC50 IC50 SI Fo-6,25x FuA Vietnam Visual Dil. O.OO34 O.O27 7.9 EtOH H3N2 1203 2004H DMSO only 24 hrs O-1 25x33 FuA Vietnam Neutral Dil. O.OO)4 O.OS 10 days (H5N1) 1203/2004H Red DMSO O-1 25x33 FuA Vietnam Neutral Dil. O.OO)4 O.O6 2O days (H5N1) 1203/2004H Red DMSO HD FuA Vietnam Neutral Dil. O.OO)4 O.O3 8 (H5N1) 203/2004H Red MEM HD FuA Vietnam Visual Dil. O.OO)4 O.04 10 (H5N1) 203/2004H MEM Gr-1 25x FuA Vietnam Neutral % O.32 3.2 10 (H5N1) 203/2004.H Red DMSO HD Fraction 1 FuA Vietnam Neutral % O.33 5.4 16 Gf, Ab, Io, Ga (H5N1) 203/2004.H Red DMSO HD Fraction 1 FuA Vietnam Visua % O.04 3.8 95 Gf, Ab, Io, Ga (H5N1) 203/2004.H DMSO HD Fraction 2 FuA Vietnam Neutral % O.OO19 -0.1 >53 Gf, Ab, Io, Ga (H5N1) 203/2004.H Red DMSO HD Fraction 2 FuA Vietnam Visua % O.04 >0.1 >25 Gl, Csc, Phl, Go (H5N1) 203/2004.H DMSO Pb-1 25x FuA Vietnam Neutral % O.O6 2.7 45 (H5N1) 203/2004.H Red DMSO Pb-1 25x FuA Vietnam Visua % O.O3 1 33 (H5N1) 203/2004.H DMSO PU 1x FuA Vietnam Neutral % O.O2 3 2O 23 days (H5N1) 203/2004.H Red DMSO PU 1x FuA Vietnam Visua % O.1 4 40 23 days (H5N1) 203/2004.H DMSO Mycena alcalina Flu A Vietnam Visua % 0.21 5.7 27 (H5N1) 203/2004.H DMSO Mycena alcalina Flu A Vietnam Visua % O.21 5.7 27 (H5N1) 203/2004H CONF DMSO Gr-1 25x FuA Vietnam Visua % O.21 5.7 27 (H5N1) 203/2004.H DMSO Gr-1 25x FuA Vietnam Visua % O.21 5.7 27 (H5N1) 203/2004H CONF DMSO Sc 25x FuA Vietnam Neutral Dil. O.OO67 >0.1 >15 (H5N1) 203/2004.H Red DMSO HD Fraction 4 FuA Vietnam Neutral Dil. O.OO86 >0.1 >12 Pu, He, Pb, Le (H5N1) 203/2004.H Red DMSO HD Fraction 4 FuA Vietnam Visual Dil. O.OO89 >0.1 >11 Pu, He, Pb, Le (H5N1) 203/2004.H DMSO G. ann. 25x FuA Vietnam Neutral Dil. O.OO61 O.066 1 EtOH only (H5N1) 203/2004.H Red DMSO 24 hrs. G. ann. 25x FuA Vietnam Visual Dil. O.OO32 O.O38 2 EtOH only (H5N1) 203/2004.H DMSO 24 hrs. Ty 25x FuA Wisconsin Visual Dil. O.OO28 O.O32 1 EtOH only (H5N1) 67/2005 Company 24 hrs. Buffer Go-4 FuA Vietnam Visual Dil. O.OO28 O.O27 O Elwah (H3N2) 203.2004H DMSO EtOH only 2 wks.
INFLUENZAARIBAVARIN CONTROL RESULTS AND ACTIVE SAMPLE COMMENTS
Control Control Control Control Control Comments on active Units EC50 EC90 IC50 SI sample 1 Ig/ml 10 >32O >32 Moderately to highly active 2 Lig/ml 2 >320.0000 >160.00 Not active by visual but slightly active by neutral US 8,765,138 B2 21 22 -continued
INFLUENZAARIBAVARIN CONTROL RESULTS AND ACTIVE SAMPLE COMMENTS
Control Control Control Control Control Comments on active Units EC50 EC90 IC50 SI ple red against IVA (H1N1). Hig hly active against IVA (H3N2). 3 g ml 1.9 active by visual but slig htly active by neutral red against IVA (H1N1). Hig hly active against IVA (H3N2). 4 g/ml 12 Hig hly active. 5 g/ml 10 Hig hly active. 6 g/ml 12 Hig hly active in trial 1, O erately active in trial 2. S orage may have CO ributed to loss of potency 7 g/ml 10 Hig hly active in trial 1, O erately active in trial 2. S orage may have CO ributed to loss of potency 8 g/ml 3.2 >100 9 g/ml 3.2 >100
INFLUENZA B, MDCK CELL LINE, ALL ACTIVE SAMPLES DILUTION DRUG UNIT Compnd Flu BVirus Name Strain ASSay Vehicle EC50 EC90 ICSO SI 1 Pb-1 25x Shanghai Neutral DMSO <0.0001 O.O6 >590 361.02 Red 2 Gr 25x Shanghai Neutral DMSO <0.0001 O.04 >400 EtOH only 361/02 Red 24 hrs 3 Gir 25x Shanghai Virus DMSO 1E-04 522 EtOH only 361/02 Yield 24 hrs 4 IO-1 25x Shanghai Neutral DMSO <0.0001 O.04 >350 EtOH only 361/02 Red 24 hrs 5 IO-1 25x Shanghai Virus DMSO 1E-04 223 EtOH only 361/02 Yield 24 hrs 6 Fo-1325x Shanghai Visual DMSO O.OOO3 O.OS 150 EtOH only 361/02 24 hrs 7 FO-1325x Shanghai Virus DMSO 1E-04 313 EtOH only 361/02 Yield 24 hrs 8 Fo-1325x Shanghai Visual- DMSO O.OOO3 O.OS 150 EtOH only 361/02 CONF 24 hrs 9 G. ann. 25x Shanghai Visual DMSO O.OOO3 O.O6 18O EtOH only 361/02 24 hrs 10 G. ann. 25x Shanghai Visual- DMSO O.OOO3 O.O6 18O EtOH only 361/02 CONF 24 hrs 11 Fo-10 25x Shanghai Virus DMSO 1E-04 171 EtOH only 361/02 Yield 24 hrs 12 Gr 25x Shanghai Neutral DMSO O.OOO4 O.OS 140 361.02 Red Pb-1 25x Shanghai Neutral DMSO O.0043 O.OS8 13 361.02 Red Gr 25x Shanghai Neutral DMSO O.OO23 0.057 25 EtOH only 361/02 Red 24 hrs Gr 25x Shanghai Visual DMSO O.OOO6 O.O47 84 EtOH only 361/02 US 8,765,138 B2 23 24 -continued
INFLUENZA B, MDCK CELL LINE, ALL ACTIVE SAMPLES DILUTION DRUG UNIT
Compnd Flu BVirus Name Strain Assay Vehicle EC50 EC90 ICSO SI
o-1 25x Shanghai Neutral DMSO O.OO21 O.057 11 EtOH only 361/02 Red 24 hrs o-1 25x Shanghai Visual DMSO O.OOO6 O.O47 S2 EtOH only 361/02 24 hrs Fo-1325x Shanghai Neutral DMSO O.OO12 O.OS4 45 EtOH only 361/02 Re 24 hrs o-1 25x Shanghai Neutral DMSO O.OO 0.057 57 361.02 Re o-1 25x Shanghai Virus DMSO O.OO 0.057 57 361.02 Yield Pb-1 25x Shanghai Neutral DMSO O.OO13 O.OS1 39 361.02 Re Pb-1 25x Shanghai Neutral DMSO O.OO24 O.OS8 24 361.02 Re Pb-1 25x Shanghai Visual DMSO O.OO15 O.O38 25 361.02 Pb-1 25x Shanghai Virus DMSO O.OO1 27 361.02 Yield Pb-1 25x Shanghai Virus DMSO O.OO15 O.O38 25 361.02 CONF Ty EtOH Shanghai Neutral DMSO O.OO12 O.042 3S 24 hrs. 361.02 Re Pll 25x Shanghai Visua DMSO O.OO11 O.O38 35 361.02 Pll 25x Shanghai Virus DMSO O.OO2 23 361.02 yield Pll 25x Shanghai Visua DMSO O.OO11 O.O38 35 361.02 CONF Sc 25x Shanghai Visua DMSO O.OO32 >0.1 >31 361.02 Sc 25x Shanghai Virus DMSO O.OO3 >31 361.02 yield Sc 25x Shanghai Visua DMSO O.OO32 >0.1 >31 361.02 CONF PL 25x Shanghai Neutral DMSO O.OO7 >0.1 >14 361.02 Red PL 25x Shanghai Visua DMSO O.OO32 >0.1 >31 361.02 PL 25x Shanghai Visua DMSO O.OO32 >0.1 >31 361.02 CONF G. neo Malaysia Visua DMSO O.OO24 O.OS 21 japonicum 2SO6, 2004 EtOH only 2 wks Hu 1X Shanghai Neutral DMSO O.OO72 O.1 14 361.02 Red Hu 1X Shanghai Visual DMSO O.OO89 O.1 11 361.02 Mycena Shanghai Visual DMSO O41 5.7 14 alcalina 361.02 HD Shanghai Neutral DMSO 0.0073 O.093 13 361.02 Red Go Malaysia Visual DMSO O.OO43 >O.OS >12 EtOH only 2506/2004 2 wks. GI34D Malaysia Visual DMSO 0.0028 O.O28 10 EtOH only 2506/2004 2 wks 3 Shanghai Visual DMSO 0.7 6 8.6 Mushroom 361.02 Blend 3 Shanghai Neutral DMSO O.3 3 10 Mushroom 361.02 Red Blend US 8,765,138 B2 25 26 -continued INFLUENZABRIBAVARIN CONTROL RESULTS AND ACTIVE SAMPLE COMMENTS INFLUENZABRIBAVARIN CONTROL RESULTS AND ACTIVE SAMPLE COMMENTS Control Control Control Control Control Comments on Onits EC50 EC90 IC50 SI active sample 5 Control Control Control Control Control Comments on Onits EC50 EC90 IC50 SI active sample 7.1 >320.0000 >45.0000 Highly active. : 7.1 >320.0000 >45.0000 Highly active. 8 g/ml 1.7 >320.0000 >190.0000 Moderately to 37.72 >8.4000 Moderately to highly active as highly active as confirmed by confirmed by 10 VYR assay. VYR assay. 9 g/ml 1.7 >320.0000 >190.0000 Moderately to g/ml 7.1 >320.0000 >45.0000 Highly active. highly active. 5 g/ml 37.72 >8.4000 Moderately to 10 g/ml 1.7 >320.0000 >190.0000 Moderately to highly active as highly active as confirmed by confirmed by VYR assay. 15 VYR assay. g/ml 1.7 >320.0000 >190.0000 Moderately to 11 g/ml 37.72 >8.4000 Moderately to highly active. highly active as g/ml 37.72 >8.4000 Moderately to confirmed by highly active as VYR assay. confirmed by 12 g/ml 7.1 >320.0000 >45.0000 Highly active. VYR assay.
HERPES SIMPLEXVIRUS Cmpd Cell Drug Name Virus Assay Line Unit EC50 EC90 CC50 SI ACVEC50 Comment Fo-1 HSV-1 CPE HFF 9/o SOI O2 >1 3.9 195 O.3 HSV 25x cells 1 and EtOH HSV only 3 2 PR weeks G. ann HSV-1 CPE HFF 9/o Sol O.8 17.8 -2S >31.2 1.2 25x cells cold Water only 24 hrs Fo-1 HSV-2 CPE HFF 9/o SOI. O.1S >1 3.9 26 O.2 HSV-1 25x cells AND EtOH HSV only 3 2 PR weeks Ty 25x HSV-2 CPE HFF 9/o Sol O.7 >5 16.8 24 O.2 Drug OOl cells residue Water stained only 24 hrs very darkly
HCV Virus, Huh 7 ET Cell Type, Drug Units Fold Dilution Primary Assay Actvty 9% Cyto High inhib. toxicity Test virus % cell Confirmatory Assay
ID Conc. control control SI EC50 EC90 ICSO C90 SISO SI90
Csc 100 85.6 11.8 25x RNA replicon Single Dose Primry Csc 100 62 S.62 25x RNA replicon Confirmatory dose respnse HCV 100 94.9 0.7 10 RNA US 8,765,138 B2 27 28 -continued HCV Virus, Huh7 ET Cell Type, Drug Units Fold Dilution Primary Assay Assay Cyto and High inhib. toxicity Assay Test virus % cell Confirmatory Assay
ID Type Conc. control control SI EC50 EC90 IC50 IC90 SISO SI90
25x replicon cold Single Water Dose 24hrs Primry 100 52.1 >100 59.6 90.9 1.14 10 RNA 25x replicon cold Confirmatory Water dose 24hrs respnse only Fo HCV 100 94.9 10 RNA 25x replicon cold Single Water Dose 24hrs Primry only Fo HCV 100 35.7 95.2 62.6 92.5 1.8 0.97 10 RNA 25x replicon cold Confirmatory Water dose 24 hrs respnse only IFN HCV 2 94.7 95.1 alpha RNA 2b replicon Single Dose Primry IFN HCV 2 O.12 O.S4 alpha RNA 2b replicon Confirmatory dose respnse Tw 100 85.6 81.6 EtOH RNA only replicon 24 Single hours Dose Primry Tw 100 S.S9 >100 >100 >100 EtOH RNA 24 replicon hours Confirmatory dose respnse IFN HCV 2 85.6 81.6 alpha RNA 2b replicon Single Dose Primry IFN HCV 2 O.O7 O.38 alpha RNA 2b replicon Confirmatory dose respnse US 8,765,138 B2 30 NaHPO.7H2O, 8.0 g NaCl and 0.2 g KCl in 1 L distilled Mushroom Extracts-% Inhibition water) to yield a suspension concentration of approximately 108 cells/ml. 7 13 HD 16 Fomitopsis Mushroom Mushroom Mushroom Treatments Bacteria officinalis Blend Blend Blend Ten fungal extracts were evaluated for their antimicrobial efficacy on the growth of E. coli O157:H7 and S. aureus for Mycobacterium 7396 63% 70-87% 63% this study. The various fungal extracts evaluated for the study tuberculosis included: ES-100 to ES-109. For each compound, different concentrations (0, 1, 10, and 100%) were prepared by diluting 10 the stock with sterile buffered peptone water. A 1-ml portion of each actively growing culture was placed into 9 ml of Top Results Against Viruses from Mushroom Extract sterile buffered peptone water containing fungal extract with Samples different pre-determined concentrations. Samples were Extract Preparation SI stored at room temperature and were drawn after 24, 48, and 15 72 hours following which microbiological analysis was per Results for Mycobacterium tuberculosis formed. All the experiments were replicated three times. Fo-1 25x EtOH only 3 weeks Active IC90 = 0.981 Experimental Design and Statistical Analysis ICSO = 0.888 The treatments were designed using a randomized com plete block design (RCBD). The organisms were treated as Example 4 blocks and within each block the effect of compound, con centration, and time of contact was evaluated on the growth Water only, room temperate, cell free, centrifuged extracts profile of the organism. The data was analyzed using analysis from live mycelium were prepared. The following codes 25 of variance (ANOVA) using the PROC GLM procedure avail define the active samples and species being employed: able in SAS software. The effect of a treatment (concentration or time of contact or compound) was deemed significant at alpha=0.05. Identification Microbiological Analysis Number Species 30 Both untreated and treated samples were analyzed to deter ES-100 F.f. mine the bacterial load prior to and after treating. Serial ES-101 G.O. dilutions were madeusing standard microbiological practices ES-102 HTU and the serial dilutions thereof (0.1 ml) were surface plated ES-103 Po. ES-104 Tw. onto blood agar. Plates were incubated at 35+2°C. for 24h 35 ES-105 F.O. X before the colonies were counted. ES-106 G.r. Results and Discussion ES-107 I.O. Effect of the Fungal Extracts on the Survival of E. coli ES-108 Pb. I O157:H7 ES-109 Tw. Fungal extracts varied significantly (P<0.05) in their anti 40 microbial effect against E. coli O157:H7. Similarly concen Note that the scales in the following charts are logarithmic tration and time of storage had a significant (P<0.05) effect on (base 10), and CFU’s are “colony forming units’. Reductions the Survival of E. coli O157:H7. FIGS 1-3 Summarize the of significance vary from ~10:1 to 10,000,000:1 over 72 effect of various fungal extract compounds on the Survival of hours of exposure of the bacteria E. coli and Staphylococcus E. coli O157:H7. In general, reductions of E. coli O157:H7 Ca,S. 45 due to fungal extract treatments decreased as follows: The basic procedure used for the E. coli and S. aureus ES-103=ES108>ES-105>ES-1OO=ES-104=ES-102>ES bioassay was: AOAC International 2000, AOAC official 101=ES-107=ES-109=ES-106. Overall the antibacterial method 96009, p. 10. In P. Cunniff (ed.), Official methods of effect increased with increase in concentration of the com analysis of AOAC International, 17th ed. AOAC Interna pound. The antibacterial activity of the compounds was maxi tional, Gaithersburg, Md. 50 mum at 100% followed by 10% and 1%. In general, ES-103, The cultures used were obtained from ATCC, E. coli O157: ES-105, and ES-108 demonstrated the maximum antibacte H7: 35150 and S. aureus: 12600. The antimicrobial efficacy rial activity on E. coli O157:H7. At the end of 72 h of storage, of the fungal extracts of live mycelium were tested on the ES-105 and ES-108 caused approximately 4-5 log reduction growth profile of E. coli O157:H7 and Staphylococcus of E. coli O157:H7 when applied at a concentration of 10% aureus. 12600. 55 and 100%. ES-103 also caused a 4 log reduction (P<0.05) of Materials and Methods E. coli O157:H7 but only when applied at 100%. Preparation of Cultures See FIG. 1, Effect of Antibacterial Compounds (Concen E. coli O157:H7 and Staphylococcus aureus. 12600 strains tration: 1%) on the survival of E. coli O157:H7 obtained from ATCC were used to generate inocula. Strains See FIG. 2, Effect of Antibacterial Compounds (Concen available as frozen (-80° C.) stock cultures in tryptic soy 60 tration: 10%) on the survival of E. coli O157:H7 broth (Becton Dickinson, Sparks, Md.) with 20% glycerol See FIG. 3. Effect of Antibacterial Compounds (Concen and were activated by inoculating both the strains in tryptic tration: 100%) on the survival of E. coli O157:H7 soy broth (TSB) and incubating at 35+2°C. for 72 h. Cultures Effect of the Fungal Extracts on the Survival of S. aureus were streaked on tryptic soy agar with 5% blood (TSA II 5% Fungal extracts varied significantly (P<0.05) in their anti SB, Becton Dickinson) and incubated at 35+2° C. for 48 h. 65 microbial effect against S. aureus. Similarly concentration Colonies from each organism were suspended in phosphate and time of storage had a significant (P<0.05) effect on the buffered saline (PBS; pH 7.4; 0.2 g KHPO, 1.5 g. survival of S. aureus. FIGS. 4-6 Summarize the effect of US 8,765,138 B2 31 32 various fungal extract compounds on the Survival of S. agaric acid and/or betulinic acid may be an intermediate in aureus. In general, reductions of S. aureus due to fungal various cellular processes or may be found to be biologically extract treatments decreased as follows: ES-103=ES108>ES incorporated into various cellular constituents. It is further 105=ES-109s-ES102>ES-101>ES-1 OO=ES-104=ES possible that such molecular matrices may serve to detoxify 107>ES=106. Overall, the antibacterial effect increased with 5 the cytotoxicity while preserving antiviral properties. How increase in concentration of the compound. The antibacterial ever, it does not appear that the antiviral properties of the activity of the compounds was maximum at 100% followed present invention may be ascribed to either agaric acid or by 10% and 1%. In general, ES-103, ES-105, ES-108 and betulinic acid and it is expected that the extracts possess novel ES-109 demonstrated the maximum antibacterial activity on antiviral and antimicrobial compounds. S. aureus. At the end of 72 h of storage ES-103, ES-105 and 10 Although ethanol was used as the organic solvent, ethanol ES-108 caused approximately 4-5 log reduction of S. aureus is clearly not the causal agent, as numerous samples of other when applied at a concentration of 100%. At the end of 72 h mushroom species showed no activity although they were of storage ES-103, ES-105, ES-108, and ES-109 caused also presented in the same form (ethanol and water) as was approximately 4-6 log reduction of S. aureus when applied at Fomitopsis officinalis. The present compositions provide anti a concentration of 10%. At the end of 48 h of storage ES-109 15 viral activity that is due to contact with mycelial components caused approximately 5-log reduction (P<0.05) of S. aureus beyond any effect due to contact with the ethanol, provide when applied at 100%; however, at the end of 72 h of storage, compositions wherein the survivability of the viruses is lim S. aureus increased by approximately 3 log CFU/ml. ited upon contact with the extract while selectively not harm See FIG. 4. Effect of Antibacterial Compounds (Concen ing healthy human cells. tration: 1%) on the Survival of Staphylococcus aureus The compositions are preferably extracted with ethanol, See FIG. 5, Effect of Antibacterial Compounds (Concen water or combinations thereof and the extracts are more pref tration: 10%) on the survival of Staphylococcus aureus erably extracted with cold, room temperature or warm sol See FIG. 6. Effect of Antibacterial Compounds (Concen vent. Not as preferred is hot or boiling solvent. tration: 100%) on the survival of Staphylococcus aureus Novel aspects of the present invention include antiviral and 25 antibacterial effect with extracts, as a topical disinfectant, i.e. CONCLUSIONS topical Surfaces, including cultures of organisms. Extracts made from the mycelium are active; extracts from Fungal extracts varied in their antibacterial effect on E. coli the mushroom are not active or not as active. O157:H7 and S. aureus. Purification of the active antivirals “appear to be extracted S. aureus was more sensitive to the fungal extracts than E. 30 with EtOH but not with HO only. Purification of the active coli O157:H7 antibacterials “appear to be extracted with HO but not with ES-105 (Fomitopsis officinalis) and ES-108 (Piptoporus EtOH. No adverse reactions from human ingestion. Water betulinus) caused approximately 4-5 log reduction of E. coli only extracts of Fo, Pb., T.V., I.o. and Po. are antibacterial; O157:H7 at the end of 72 h of storage when applied at a ethanol only extracts of F.o., Pb., T.V., G.r., G.ann., H.u., HTU concentration of 10% and 100%. 35 and I.O. are antiviral. Heat is believed to destroy most antiviral ES-103 (Pleurotus ostreatus from “bunker burlap bags”) activity; cold temperature or room temperature extraction is also caused a 4 log reduction (P<0.05) of E. coli O157:H7 but preferred. only when applied at 100%. Anti-infective agents from medicinal mushrooms and ES-103 (Pleurotus ostreatus from “bunker burlap bags”), mushroom mycelia: adjuncts to immunotherapy and potenti ES-105 (Fomitopsis officinalis) and ES-108 (Piptoporus 40 ating host defense for disease resistance. This coincides with betulinus) caused approximately 4-5 log reduction of S. the many anecdotal reports of the extracts helping fight infec aureus at the end of 72 h of storage when applied at a con tion in wounds and aiding in wound-healing. Having a treat centration of 100%. At the end of 72 h of storage ES-103, ment that is both anti-staph/anti-E. Coli and anti-viral is ES-105, ES-108, and ES-109 (Trametes versicolor) caused uniquely important. approximately 4-6 log reduction of S. aureus when applied at 45 Solving the Staph. aureus problem solves many collateral a concentration of 10%. problems in the hospital. Since so many battlefield wounds At the end of 48 hours of storage ES-109 (Trametes versi are staph-prone, and since anesthesia Suppresses the immune color) caused approximately 5-log reduction (P<0.05) of S. system, making patients more Susceptible to infection, and aureus when applied at 100%; however, at the end of 72 h of since viral infections are often complicated by Subsequent storage, S. aureus increased by approximately 3 log CFU/ml. 50 bacterial infections, fungal extracts as disclosed herein may From these data showing direct antiviral and antibacterial be particularly important for protecting citizens and soldiers. activity, it is reasonably predictable and expected that the E. coli contamination on spinach, lettuce or other crops compositions will have utility in humans in preventing, treat may re reduced, alleviated or eliminated by treatment with the ing, alleviating, ameliorating, mitigating, reducing and/or disclosed fungal extracts. The reduction in E. coli is enough to curing infection and/or symptoms from viruses, including 55 address human food concerns, including organic food pro Smallpox. ducers concerns. A food grade spray on treatment for Veg When the mycelial extracts were dried and fractionated, etables and a topical spray for food preparation Surfaces. none of the 91 fractions showed any antiviral activity at the Having an extract/compound that is dually anti-bacterial concentrations tested and yet the whole extracts continued to and antiviral is especially useful. show significant antiviral activity, repeatedly and consis 60 Topical, antibacterial effects from using water-only tently, for more than two years from creation. extracts of mycelium at room temperature. My hypothesis is GC testing of the Fomitopsis and Piptoporus extracts for this is the window in which mycelium has evolved for mil agaric acid showed no agaric acid to be present. It will be lions of years, and within this window we will find activity, noted that the activity of agaric acid does not correlate well whereas hot-water extraction is likely (not proven yet) to with the activity of the extracts in the bioassays herein. HPLC 65 destroy anti-bacterial compounds. analysis of the Fomitopsis and Piptoporus extracts showed no Since E. coli is an endospore-forming bacterium, and com betulinic acid to be present. It is, of course, possible that monly used as a Surrogate for Bacillus anthracis, aka US 8,765,138 B2 33 34 anthrax this invention anticipates that fungal preparations ferred, or an intermediate time. Extracts are preferably uti and combinations thereof found effective at reducing CFU lized when fresh as antibacterial and antiviral activity may (colony forming units) of E. coli may also prove useful at degrade with time. inhibiting the germination and growth of Bacillus anthracis, Another example of anticipated extraction is with myce thus lessening its severity of infection, or its infectivity. lium grown on rice to optimize CFU’s, immerse by equal Having a convenient, readily applied throat spray utilizing mass into 99 percent EtOH, filter, centrifuge, discard precipi the antivirally active mushroom preparations described here, tate, cell-free filter, and use. having anti-flu (including H5N1), anti-poX (Variola major), It will be understood that a supplement or extract com anti-SARS as well as antibacterially active mushroom prepa posed of ingredients from the fungi Fomitopsis officinalis, 10 Fomitopsis pinicola, Piptoporus betulinus, Ganoderma rations useful for preventing infections from TB (tuberculosis resinaceum, G. lucidum, G. annulare, Trametes versicolor, causing organisms such as Mycobacterium tuberculosis and Inonotus obliquus, Hypsizygus ulmarius, Hypsizygus tessu Mycobacterium intracellulare), can help protect passengers latus and/or other species of the genera can be used in an traveling on airplanes, trains, passenger ships, automobiles, amount Sufficient to the have the effect of preventing, treating, as well as where any groups of people congregate, from these 15 mitigating, reducing, alleviating, ameliorating or curing and other types of infectious diseases. infection from viruses or their vectors, including Cowpox, Infections from Staphylococcus aureus, particularly Variola (smallpox) and other Orthopox viruses, coronavi MRSA (Methicillin Resistant Strains of Staphylococcus ruses including SARS, HIV, influenza, avian influenza, Ven aureus) complicate recovery from Surgical operations. Hav ezuelan Equine Encephalitis, Yellow fever, West Nile, SARS, ing a topically applied anti-infective compounded with a dis Rhinovirus New World and Old World arenaviruses including infectant such as ethanol can be helpful for patient health the American hemorrhagic fevers, Lassa and lymphocytic worldwide. choriomeningitis, VEE, Hantavirus, Rift Valley fever, sandfly Another potentially useful application of this invention is fever, yellow fever, West Nile, Dengue fever, respiratory the topical application in the form of a spray upon foodstuffs, viruses, Rhinoviruses, Herpes Simplex I, Herpes Simplex II, including vegetables and meats prone to spoilage by E. coli. 25 Lyme, HELA, Epstein Barr, Ebola, Varicella-Zoster, adenovi and/or other organisms. Different than a disinfectant which ruses, Polio, Hepatitis including Hepatitis A, B and C, and/or can immediately destroy problematic bacteria, for instance, from the microbes causing Tuberculosis, pneumonia (bacte the spray envisioned within this invention has residual anti-E. rial pneumonia, viral pneumonia, and mycoplasma pneumo coli and anti-bacterial properties, so that colonies of bacteria nia), such as Plasmodium falciparum, Listeria, Pneumococ that do survive the initial exposure to a disinfectant are 30 cus, Bacillus anthracis, Escherichia coli, Mycobacterium retarded in their Subsequent growth due to the longer lasting tuberculosis, bacteriophages and fungi such as Candida albi effects of the mycelially derived spray. Similarly, the spray’s cans should be obvious to one skilled in the art and considered anti-fungal, antibacterial and anti-protozoal properties make within the scope of the invention. As the products and meth it an ideal candidate for extending shelf life of any material ods of the present invention treat both viruses and opportu that is otherwise degraded or made less useful by colonizing 35 nistic pathogenic organisms such as Mycobacterium tubercu organisms. This novelty also has applications for wound losis and other bacteria, it will be appreciated that the present healing, allowing new tissue to grow without the stifling invention is exceptionally advantageous insofar as viral infec effects of problematic bacteria Such as Staphylococcus tions can lead to bacterial infections and Vice versa. aureus. Repeated applications of such a spray combined with It will also be obvious to one skilled in the art that isolation, a disinfectant like alcohol doubly enables the usefulness of 40 fractionation, purification and/or identification of DNA, RNA this invention. and protein sequences responsible for antiviral activity and The extract may be mixed with glycerin to give fifty-fifty antiviral agents from Fomitopsis officinalis, Fomitopsis pini EtOH-glycerin, then placed under vacuum (2 C. to 10 C.) to cola, Piptoporus betulinus, Ganodemma resinaceum or the remove the alcohol and give a glycerin extract. other fungal species disclosed herein could be transferred to Similar antimicrobial/antifungal activity is expected for 45 another organism, Such as a bacterium or yeast, for the com Candida albicans, Cryptococcus neoformans, Escherichia mercial production of antiviral agents and/or its antiviral or coli, Pseudomonas aeruginosa, Mycobacterium intracellu antimicrobial active derivatives and should be considered lare and Aspergillus fumigatus, and similar antiparasitic within the scope of the invention. It is to be expected that activity is expected for Plasmodium falciparum and Leish derivative to this invention will lead to the discovery of active mania donovani. Activity is also expected against Ebola and 50 ingredients (AIs) which can be used to identify, isolate, Streptococcus pyogens. concentrate and allow for modification from Suites of fungal An anticipated method of extraction will be to take the strains in search for hyperproducers. Upon discovery of the ethanol extract and using compressed liquid carbon dioxide genes responsible for expression of AIs, these genes can be wash the EtOH extract under pressure, removing the EtOH, recopied multiple times into the DNA of yeasts, bacteria, and and then once the EtOH is removed, the liquid carbon dioxide 55 other organisms allowing for further increases in production is then evacuated. Once the liquid carbon dioxide vaporizes of a valuable medicine while lowering costs. and this liquid carbon dioxide is removed, the antivirally The publications and other materials used herein to illumi active and anti-bacterially active agents are reduced into a nate the background of the invention and in particular cases, dried form, thus allowing further potentiation and purifica to provide additional details respecting the practice, are incor tion, and this reduction becomes more useful in a wider array 60 porated by reference. of delivery systems for medicines. Methanol and acetone It should be understood the foregoing detailed description wash of mycelium by carbon dioxide may also be utilized, is for purposes of illustration rather than limitation of the optionally using a critical point dryer. Scope of protection accorded this invention, and therefore the The best solvent to use for viruses is apparently EtOH description should be considered illustrative, not exhaustive. except for Hepatitis C(HCV). The preferred extraction tem 65 The scope of protection is to be measured as broadly as the perature for antivirals is 2 C. For antibacterial and antiviral invention permits. While the invention has been described in extracts, an extraction time of 24 hours or 3 weeks is pre connection with preferred embodiments, it will be under US 8,765,138 B2 35 36 stood that there is no intention to limit the invention to those contacts the flu virus prior to the flu virus contacting said embodiments. On the contrary, it will be appreciated that cells, and wherein the composition comprises an aqueous those skilled in the art, upon attaining an understanding of the ethanol extract of Fomitopsis officinalis mycelium with a invention, may readily conceive of alterations to, modifica calculated Selectivity Index (SI-CCs/ECs) against a flu tions of, and equivalents to the preferred embodiments with- 5 virus that is a 10. out departing from the principles of the invention, and it is 10. A composition for restricting the growth, spread and intended to cover all these alternatives, modifications and Survivability of a virus comprising an aqueous ethanol Fomi equivalents. Accordingly, the scope of the present invention topsis officinalis extract, wherein the virus is an influenza should be assessed as that of the appended claims and any equivalents falling within the true spirit and scope of the 10 virus, and wherein the influenza virus is an influenza virus invention. selected from the group consisting of influenza A H5N1 and I claim: H3N2, influenza B and avian influenza, and wherein the 1. A composition for restricting the growth, spread and extract has a selectivity index (SI) against the influenza >100. Survivability of a virus comprising an aqueous ethanol extract 11. The composition of claim 10, wherein the mycelium of of a mycelium of a medicinal mushroom wherein the virus is 15 a mycelium of a Fomitopsis medicinal mushroom is selected selected from the group consisting of influenza, including from the group consisting of live mycelium, dried mycelium, influenza A H5N1 and H3N2 and influenza B, avian influenza freeze dried mycelium or a combination thereof. and Herpes Simplex I and II, wherein the medicinal mush 12. The composition of claim 10, wherein the composition room is Fomitopsis officinalis and wherein the extract has a additionally comprises a mycelial extract selected from the selectivity index (SI-CCs/ECs) against the virus >10. 2O group consisting of Ganoderma resinaceum and G. annulare 2. The composition of claim 1, wherein the mycelium is extracts, Inonotus obliquus extracts, Hypsizygus ulmarius selected from the group consisting of live mycelium, dried and H. tessulatus extracts and Trametes versicolor extracts. mycelium, freeze dried mycelium or a combination thereof. 13. The composition of claim 10, wherein the live myce 3. The composition of claim 1, wherein the aqueous etha lium is grown on a grain. nol extract is administered in a form selected from the group 25 14. A composition for restricting the growth, spread and consisting of orally-active powders, pills, capsules, teas, Survivability a virus comprising an aqueous ethano extract of extracts, dried extracts, Sublinguals, sprays, dispersions, solu a medicinal mushroom mycelium wherein the virus is tions, Suspensions, emulsions, foams, syrups, lotions, oint selected from the group consisting of influenza, wherein the ments, gels, pastes, dermal patches, injectables, vaginal influenza is influenza A H5N1 and H3N2, Influenza B, or creams and Suppositories. 30 4. The composition of claim 1, wherein the composition avian influenza, or Herpes Simplex I and/or II, wherein the additionally comprises a mycelial extract selected from the medicinal mushroom mycelium is Fomitopsis officianalis and group consisting of Ganoderma resinaceum and G. annulare wherein the extract has a selectivity index (SI) against the extracts, Inonotus obliquus extracts, Hypsizygus ulmarius virus >10 and inhibits bacteria, wherein the bacteria is and H. tessulatus extracts and Trametes versicolor extracts. 35 selected from the group consisting of Stapylococcus aureaus 5. The composition of claim 1, wherein the extract inhibits and Escheria coli, and wherein inhibition of the bacteria is bacteria selected from the group consisting of Escherichia greater than 99%. coli and Staphylococcus aureus. 15. The composition of claim 14, wherein the mycelium is 6. The composition of claim 2, wherein the live mycelium selected from the group consisting of live mycelium, dried is grown on a grain. 40 mycelium, freeze dried mycelium or a combination thereof. 7. A composition comprising an aqueous ethanol extract of 16. A composition for restricting the growth, spread and Fomitopsis officinalis mycelium wherein the extract has an Survivability of a virus comprising an aqueous ethanol extract antiviral activity Selectivity Index (SI-CCs/ECO) against of a medicinal mushroom mycelium wherein the virus is flu viruses that is a 10 and the survivability of the flu viruses is influenza, wherein the medicinal mushroom mycelium is limited upon contact with the extract of Fomitopsis officinalis. 45 Fomitopsis officinalis mycelium and wherein the aqueous 8. A composition for limiting the survivability offluviruses ethanol extract has a selectivity index (SI) against influenza upon contact with the composition while selectively not >10 and inhibits bacteria selected from the group consisting harming healthy human cells comprising an aqueous ethanol of Stapylococcus aureaus and Escheria coli, and wherein extract of live Fomitopsis officinalis mycelium wherein the inhibition of the bacteria is greater than 99%. extract has a Selectivity Index (SI-CCs/ECs) against a flu 50 17. The composition of claim 16, wherein the mycelium is virus that is a 10. selected from the group consisting of live mycelium, dried 9. A composition for limiting the susceptibility of human mycelium, freeze dried mycelium or a combination thereof. cells to infection by a flu virus, wherein the composition k k k k k