JP2003500452A - Pharmaceutical formulations of bioactive substances extracted from natural resources - Google Patents

Abstract

  (57) [Summary] The present invention relates to a method for extracting and purifying a bioactive substance from various plants and herbs. More specifically, the invention relates to the use of supercritical fluid extraction and / or fluorocarbon solvent extraction to provide birch root, bilsonima sp. A method for extracting and isolating biologically active substances from plants and herbs, such as Repens, Brucella species, Turnera species, Peregian species, Hemia salicifolia, Cidium species, Enterrobium species, Cichopetarum oracoides, Lilliosma obata and Chaunochiton cappelleri. . The invention further relates to the separation of bioactive substances contained in the extract using packed column supercritical fluid chromatography or HPLC with or without gas in the mobile phase with a moderator. The invention also relates to pharmaceutical preparations and dietary supplements, which may be prepared from the extracted bioactive substance, and to the use of such pharmaceutical preparations and dietary supplements for treating various human diseases.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a method for extracting and purifying bioactive substances derived from various plants and grasses. More specifically, the invention relates to a method of extracting and separating bioactive substances from various plants and grasses using supercritical fluid extraction and / or fluorocarbon solvent extraction. The invention further relates to the separation of bioactive substances contained in the extract using packed column supercritical fluid chromatography. The present invention also relates to formulations, pharmaceutical formulations and dietary supplements that may be prepared with the extracted bioactive agents, and the use of such pharmaceutical formulations and dietary supplements for treating various human diseases. 2. Background Description Throughout history, humans ingest and otherwise consume a wide variety of vegetation and herbs and extracts of such vegetation and herbs to relieve tingling and pain, improve immunity to infections, It has been used to treat various illnesses or even induce relief of relaxation and stress.

One of the plants commonly ingested by people in the South Pacific for inducing relaxation is the roots of Kava. (K. Schubel, J. So
c. Chem. Ind. , 43, 766, 1924; G. Va
n Veen, Rec. Trav. Chim. , 58, 52, 193
9). Hippopotamus roots consist of dried rhizomes and / or shoots of Piper methysticum Forst (Pepperceae). Hippopotamus roots are most typically taken by drinking a soaked water known as a drinking hippo (the hippo root is mixed with water and crushed).

The first attempts to identify active compounds in hippo roots were made over 100 years ago. Such efforts have identified a kava lactone known as kabapyrone. To date, more than 10 kava lactones and 4 other substances have been identified in the roots of hippopotamus, including: kabain, dihydrokabaine (also known as marindinine), methistine, dihydromethystisine, yangonin, and desmethoxyyangonin. Is mentioned. (V. Lebot, M. Merling and L. Lindstrom, Kava the Pacific Drug, Yele University Press, New Haven, C.
T, 1992). Such compounds are neutral, specifically substituted d-
It is a nitrogen-poor compound that may be referred to as a lactone and a substituted a-pyrone. The lactone ring is substituted with a methoxy group at the C3 position, the difference in the compounds is
As shown in FIG. 24, it is in the degree of unsaturation (eg, Yangonin, Desmethoxyyangonin, Cabaine and Metistisine) or the substitution of benzene (eg, Dihydrocabaine and Dihydromethistine).

The particular kava lactones in hippo root extracts vary according to their origin. Different species of kavalactone have been found to have different physiological effects in vivo depending on their molecular structure. All-natural cabalactone is C3 and C
It contains an enol double bond between 4. Yangonin's dienolide appears to be pharmaceutically inactive. For enolides, the effective optimum changes as a function of hydrogenation of the double-bonded C7. For example, kabain has the most potent effect as a local anesthetic, dihydromethystisine as a convulsant, and dihydrokabine as a necrosis enhancer. (R. Hansel, Chara
cteriation and Physiologic Active
ty of Some Kava Constituents Pacific
Science, July 1968, Vol. XXII, pp29
3-313).

Moreover, certain kavalactones currently depend on whether the roots and stems of the plant, in addition to the rhizome, are included in the extract. High quality birch root extract is sold based on the total content of birch lactones rather than analysis of the individual lactones it contains. The concentration range of the total level of kava lactone in the birch root extract used is generally in the range of 30 to 55% by weight, for example in Germany.

Although many types of cabalactone have been identified, there is no convenient and efficient method for both root extraction and separation of the individual extracted lactones. Traditional extraction method (
For example, steam distillation) typically mixed 100 grams of roots with a suitable amount of distilled water to produce a slurry with a volume of approximately 200 mL. (A. R. Furgiu
ele, W.E. J. Kinnard, M .; D. Aceto and J
． P. Buckley, J.M. Pharmaceutical Sci. , 54, 248, 1965). The slurry was steam distilled and the first 100 mL of distillate was collected, filtered and lyophilized. The yield of each extraction was about 50 mg. As another method, liquid-solid extraction at room temperature has been reported, and the slurry was thoroughly mixed in a Waring mixer for 15 minutes. The mixture was then filtered and freeze dried. The filtrate was sometimes continuously extracted with chloroform instead of freeze-drying.
By this purification operation, impurities were basically removed from the aqueous layer. The yield of extract in such a method varied depending on the solvent and the method used.

Since kava lactone is easily dissolved in ethanol, a hippo root extract is generally produced in recent years using ethanol as a solvent. The extractable substances are
It is in the form of a yellowish brown paste or powder, which is then examined to ensure the proper concentration of kavalactone.

A plant commonly ingested in Mexico and other Latin American countries is Byrsonima crassifolia.
) (Nanche). The medical significance of this tropical tree native to Mexico is 1
It has been historically documented since the 6th century. Traditional healers use this plant to treat digestive disorders, especially diarrhea and dysentery.

To date, about 21 chemicals have been extracted from the dried leaves and bark of this tree, including β-sitosterol and betulin (triterpenes), pipecolic acid and proline (amino acids), and catechin and Examples include quercetin (flavonoid). (Bejar E. et al., Constit
uents of Bilsonima crassifolia and their
spasmogenic activity, Int. J. Pharm
acog. , 33: 1: 25-32, 1995). The discovery of pipecolic acid
It is a rare compound in nature and is significant because it is an important intermediate product of many pharmaceutical preparations showing therapeutic effects on stroke, Parkinson's disease, Alzheimer's disease and other neurological and vascular diseases. . Prior to the discovery of pipecolic acid in Bilsonima clasifolia, formulations containing pipecolic acid were derived from various cultivated microorganisms.

Traditional healers prepared an aqueous solution of Bilsonima as tea. It was recently discovered that the aqueous extract of Bilsonima contains only catechins. However,
The use of methanol to extract bioactives from Birsonima allows the isolation of a wide variety of triterpenes, amino acids and flavonoids.

Plants in the genera Ascurus and Kuratagas are known to contain bioactive substances that affect the cardiovascular system. For example, Kuratagas Oxyacanta, C.I. Azaloulas, C.I. Monogina, C.I. Pentalina, C.I. Rabigatta and C
． Nigra's crude drug formulations have been used in European herbal medicine for this purpose for centuries. Kuratagas pinnatifida has long been used in Chinese medicine for similar purposes. Similarly, the use of Ascurus hippocastanum in Europe for the treatment of cardiovascular disease has been well documented. The effect is due to escin, a mixture of triterpene glycosides, which has anti-exudative and vascular tightening effects. While such European and Asian species have been the subject of extensive research, the common new species endemic to the New World have been widely ignored.
Known as the English "California Chestnut" and the Spanish "berruco", Ascurus California was used by aboriginals and early California settlers for a variety of purposes. The dried bark of this tree was used for toothache, fresh seeds were eaten after leaching bitterness components, untreated fruits were used for the treatment of hemorrhoids, as a fish venom, and as an abortant.

Analysis of Ascurus californica seeds by several groups shows the presence of a number of known bioactive compounds, which are the proteins β-methylalanine, phenylalanine, isohomoleucine, isohomo-6-hydroxyleucine,
Mino-4-methyl-hex-trans-4-enoic acid and gamma-glutamyl-
2-A-hex-4-enoic acid; benzoid arbutin and hydroquinone; flavonoids epicatechin; and coumarin eleuceroside B-1; and the carbohydrate quebrachitol. This chemical feature is characterized by the European A. It is different from Hippocastanum.

Extracts of Cratagas spp. And Ascurus spp. Are generally prepared using various solvents, such as methanol, ethanol or acetone. The extract is obtained from leaves and flowers of Cratagas sp., Seeds of Ascrus sp., Leaves and bark.

The plant known as jojoba, Simmondsia chinensis, is native to the southwestern United States and the desert region of Mexico. Jojoba has a unique wax ester oil that is 50-60% of the seed weight. This oil is currently used in cosmetics and lubricants. The rest of the seeds contain about 25% crude protein after degreasing, but not as much as oil. The defatted flour contains sugars and 11-15% of the natural product's unique groups.

One of the natural products contained in jojoba, Simmondsin, has been shown to be an effective hunger satiety agent by reducing food intake in mice, rats and chickens. (Cokearea et al., Ind. Crops Prod., 4: 91-96, 1995). Simmondsin has also been shown to be a useful weight loss agent for dogs. See U.S. Patent No. 5,962,043. However, jojoba flour also contains other anti-nutritional elements such as trypsin inhibitors, polyphenols, bitterness, non-nutritive proteins, and non-digestible jojoba oil.

Methods have been described for removing the so-called “toxic” ingredients from jojoba seed meal in order to make it fit the animal's mouth as a diet. US Pat. No. 5,672,37
No. 1 (dOsterlynck), US Pat. No. 4,209,534 (Ban
igan et al. ), And U.S. Pat. No. 4,148,928 (Sodin
See i). Also, a solvent is used to extract Simmondsin from jojoba flour (US Pat. No. 6,007,823).

Phaffia paniculata, commonly called Brazilian carrot, is an Amalathaseaae (Am) that grows in parts of Brazil, Paraguay, Uruguay and Argentina.
It is a plant of the family Aranthaceae). Although all parts of the plant are used in folk remedies, it is the roots that are considered medically most valuable. Traditionally, this plant has been used to treat diabetes, rheumatism, ulcers, leukemias and other cancers and has been used as a tranquilizer, general tonic and aphrodisiac.

Recent studies have revealed that this plant has biological activity as an anti-allergic agent, analgesic agent, anti-inflammatory agent, and anti-cancer agent, has a weak CNS inhibitory effect, and reduces vascular permeability. Was done. The plant has also been shown to be non-toxic to humans.

The plant extracts include allantoin, daucosterol, b-ecdysone, fafinic acid, fafosides A, B, C, D, E and F, polypodin B, β-sitosterol, stigmasterol, and stigma. Sterol-3-O-β-D
-Has been shown to contain glycosides.

Commonly referred to as Damiana, Hierva del Benado and other names, C. typhia and other C. cinerea species are small perennial herbs ranging from California to South America. This plant is used as an aphrodisiac as well as a sexual tonic, sputum, diuretic, antidiabetic, to enhance conception, to treat semen leak, orchitis, nephritis, chronic cough, stimulant, digestive agent. It has been used as a drug and laxative before the Columbus era. Clinical tests of various Trunella preparations have shown cytotoxic and antihyperglycemic effects. It has been found that extracts of this plant are not mutagenic.

[0021] Crunella species are arbutin, caffeine, gonzalitocin, β-sitosterol, acetovanillin-like benzenoid compounds, hexacosan-1-ol, tetraphyllin B, N-triacontane, tricosan-2-one, and 1-8-. It is known to contain aromatic oils containing cineole, paracymene, a-pinene, b-pinene and three sesquiterpenes.

Roots of plants of the genus Perezia produce perezone (2- (1,5-dimethyl-4-hexenyl) -3-hydroxymethyl-p-benzoquinone). Peresone is a sesquiterpene benzoquinone that exhibits redox properties. Certain plants of the genus Perezia have been used as laxatives in Mexican folk remedies.

Studies on the effect of perezone on electron transfer in biological membranes have shown that perezone, unlike rotenone, amital and antimycin A, inhibits mitochondrial electron transfer in rat liver mitochondria. (Car
abez A. et al. , Effect of peresone, a sesquiterpene benzoquinone, on electron transfer in biological membranes, Arch Biochem. Bioph
ys. , 260 (1): 293-300, 1988, Jan. ). Coenzyme Q
It was shown that peresone enhances the low respiration of rat liver mitochondria with 10 (CoQ) removed.

Heimia salicifolia was used as a traditional medicine in the United States to treat inflammation. In a recent study, two alkaloids from Hemia salicifolia, cryogenin and nesodin, were found to be more than twice as potent as aspirin as inhibitors of prostaglandin synthetase prepared from bovine seminal vesicles. It was

Hemia salicifolia shoots growing in vitro are active in alkaloid biosynthesis, and include biphenylquinolizidine lactone vertin, ritrin and riforin, the ester alkaloids demethoxyabresorin and epidemethoxyabresorin, It has been found that phenylquinolizidinol, demethyllasbin-I and demethyllasbin-II are obtained. (
Rother A. , Phenyl- and biphenyl-quinolizidines of Hemia salicifolia grown in vitro. Nat. Prod. ; 4
8 (1): 33-41, 1985, Jan-Feb). Hemia salicifolia seedlings 5-10 days old are also used to extract bioactive species. Rothe
r, et al., two isomers with different carbon configurations of the bridge head,
2-Hydroxy-4- (3-hydroxy-4-methoxyphenyl) quinolizidine has been isolated. Radioactive dilution was used to isolate 2-keto-4- (3-hydroxy-4-methoxyphenyl) quinolizidine from seedlings.

Although such plant species and a number of other plant species are known for various therapeutic and curative effects, when multiple plants are combined, such plants have not yet been described. It has the additional advantage and synergistic effect. The bioactive substances that make such plants medicinal effective are generally extracted with solvents and / or water. This technique has several drawbacks such as the cost of the solvent, the costs associated with safe disposal, and the removal of the solvent from the extract.

Moreover, herbal chemistry is complex, and herbs are responsible for their pharmacological actions on a number of different molecular moieties, often more than one compound. Many solvents have only limited efficacy for certain classes of compounds, resulting in poor extraction. This type of extraction generally yields low concentrations of bioactive material, requiring multiple extractions with different solvents to isolate different materials.

[0028] One extraction method that removes multiple bioactive agents at high concentrations is desirable. Separation methods that allow effective separation of substances to obtain purified, therapeutically useful amounts of bioactive substances are also desirable. Such a method provides new extracts from known plant species, the ability to isolate useful amounts of specific bioactive substances, new uses of extracts derived from known plant species and more efficient extraction. Will do.

Supercritical fluid extraction and supercritical fluid chromatography have been used in chemical technology for many years. Cases such as carbon dioxide and propane have proven to have excellent solvation properties when pressurized, especially above their critical point. This so-called supercritical region occurs when the gas is heated to a point above which it normally becomes a liquid and, at the same time, above the boiling point, which is significantly lower, to prevent liquefaction. This "supercritical fluid" is neither a liquid nor a gas, but exhibits both properties. In particular, supercritical fluids have excellent solvation properties with high selectivity for specific analytes. pressure,
This selectivity can be further adjusted by changing the temperature and using a gas mixture.

Lopez and Benedicto used supercritical CO ₂ to extract kava lactones from kava herbs. (V. Lopez-Avila and J. Benedicto, J. High Resolute Chromatog.
r. , 20: 555, 1997). In each extraction, satisfy the cartridge 10mL 2.5 grams birch herbaceous, 250 atm, the kinetics extraction for 60 minutes at 60 ° C., and extracted with CO ₂ was adjusted with pure CO ₂ and 15% ethanol. The extracted sample was collected in a vial maintained at 22 ° C., which was previously filled with 4 mL of ethanol. Recovery was less than 25% when using pure CO ₂ as the extraction fluid, but was greater than 90% (compared to solid-liquid extraction) when using 15% ethanol adjusted CO ₂ . Each of the kava lactones extracted via GC / MS was identified. Supercritical fluid extraction was not only highly effective, but few were extracted with it.

While CO ₂ has been found to be generally effective in extracting kavalactones, CO ₂ acts as an extraction medium only at extreme pressures, typically thousands of pounds per inch. This factor contributes to the high cost of the equipment and the inherent risks associated with extreme pressure vessels. Various types of chromatography have been used to separate and determine the major components of birch extracts. Nakayama et al. Used thin layer chromatography to separate and quantify the six capabalactones (RL Young, JW Hylin, DL Plucknett, Y. Kawano, and R. T.). . Nakayama, Phyto
Chemistry, 5, 795, 1966). Later, Gracza
Separated the mixture of kavalactones using normal phase high pressure liquid chromatography (HPLC) (L. Gracza and P. Ruff, J. Chr.
omatogr. , 486, 193, 1980). Haberlein et al. Also used normal phase HPLC to separate and quantify a series of kavalactones (H. Har.
berlein, G .; Boonen, and M.D. A. Beck, P
lanta Med. , 63, 63, 1997; Boonen, M
． A. Beck, and H.M. Harberlein, J .; Chr
omatogr. , B, 702, 240, 1997). Reversed phase HPLC was used to separate the kavalactone, but most of the isolate was poor. (R
． M. Smith, H.M. Thaklar, T .; A. Arowol
o and A. A. Shafi, J .; Chromatogr. , 2
83, 303, 1984). Recently, Shao et al. Used reverse-phase HPLC with an atmospheric pressure chemical ionization mass spectrometer in positive ion format to separate and identify specific kavalactones. Baseline separation of 6 lactones was achieved within less than 36 minutes (Y. Shao, K. He, B. Zheng and Q. Zheng, J. Chromatogr., A, 825: 1, 1998).

Improvements to this field, although some such methods have been found to be quite effective in identifying, obtaining, separating and isolating various kavalactones Is necessary. Furthermore, there is a lack of methods for conveniently, accurately obtaining, separating and isolating different species of bioactive substances from other plant species. Moreover, in today's health conscious society there is a need for new applications of substances of natural origin and methods of obtaining such therapeutically useful substances.

[0033]

[Outline of the Invention]

The present invention overcomes the problems and shortcomings associated with current strategies and designs and provides a novel method for extracting, separating and isolating bioactive agents from natural sources. The invention further relates to the novel use of such extracts.

Further, one embodiment of the present invention provides a bioactive agent comprising the steps of using supercritical fluid extraction (SFE) or near critical extraction (NCE) for preparative and / or commercial scale extraction. The method is directed to said preparative and / or commercial scale extraction. SF
E or NCE may be achieved by CO ₂ that is adjusted with CO ₂ or other various volatiles. SFE or NCE may also be accomplished as a batch extraction, continuous cascade extraction, or countercurrent solvent extraction.

Another embodiment is the above preparation of a bioactive agent comprising coupling SFE or NCE with supercritical fluid chromatography with or without a modifier for preparative and / or commercial scale processing. Directed to methods for commercial and / or commercial scale processing. In this embodiment, isopropylamine may be used as a modifier in SFC.

Another embodiment of the invention is the use of a dense gas in the supercritical, near-critical or subcritical state with or without a modifier for preparative and / or commercial scale extraction. Is directed to said method for the preparation and / or commercial scale treatment of bioactive substances comprising. The dense gas may be any non-chlorinated fluorocarbon solvent and the modifier may be any other volatile material. The extraction may be carried out under a pressure of 0-10 bar or under supercritical or near-critical fluid conditions. Dense gas extraction may also be accomplished as batch extraction, continuous cascade extraction, or countercurrent solvent extraction.

Another embodiment of the invention is directed to a method of separating a bioactive agent comprising the step of SFC. The process using SFC preferably uses HH ₂ and / or in SFC separation.
Or the use of C4 columns alone or in combination.

Another embodiment of the invention is the pharmaceutical formulation of the extract of Birsonima sp. Recovered by supercritical fluid extraction and / or dense gas or by various organic solvents and / or water and the need for treatment. A method of administering a therapeutically effective amount of such a formulation to healthy patients. The Bilsonima spp. Extract is used alone or, with beneficial effects, in combination with similarly prepared Scidium spp. And Enterolobium spp. Extracts. The composition comprises extracts or isolated products of Ascurus californica and Kuratagas mexicana, alone or in combination with each other,
Alternatively, it may be included in combination with extracts from various Brucella species.

Another embodiment of the invention is directed to the extraction of Simmondsin compounds from jojoba and the use of such compounds as human weight loss agents. Another embodiment of the invention is a muirapuama (a natural drug from various species, including Chicopetalum oracoides, Liriosma ovata and Chaunotitone caprelli), for use as a health tonic and to support sexual function. Formulations and compositions, with or without, are combinations of phytochemicals extracted from Crunella spp. And Phaffia spp.

Another embodiment of the invention is directed to formulations and compositions for use as non-steroidal anti-inflammatory drugs (NSAIDs), comprising a combination of phytochemicals, eg, extracted from Hemia salicifolia. .

Other embodiments and advantages of the invention are set forth in part in the description below,
It will also be apparent, in part, from this description, and may be taught from practice of the invention.

[0042]

DESCRIPTION OF THE INVENTION

As embodied and broadly described herein, the present invention is directed to methods of isolating and purifying bioactive agents from a variety of natural sources. The invention is further directed to pharmaceutical formulations and nutraceuticals that may be prepared from bioactive agents, and the use of such pharmaceutical formulations and dietary supplements for treating various human conditions.

1. Supercritical fluid extraction When an extremely large pressure is applied to a gas, the gas changes state and becomes a liquid. However, above a certain pressure (critical pressure) and a certain temperature (critical temperature), the gas can be further pressurized without liquefying. This combination of pressure and temperature is known as the critical point above which the gas becomes a supercritical fluid. A gas in a supercritical fluid state has a diffusivity of gas, but has a solvating power for liquids. The supercritical fluid may be pressurized to achieve a density close to 1.0 kg / l, similar to many liquids.
A further property of supercritical fluids is that, for any solute, the solvation power is a complex function of fluid density. As a result, supercritical fluids are often used to selectively extract or separate certain compounds from a mixture by changing the fluid density through changing pressure and temperature.

Carbon dioxide is a volatile material commonly used for supercritical fluid extraction.
At temperatures above 39 ° C (its critical temperature) and pressures between 200 and 600 bar, C
O ₂ extracts caffeine from coffee and tea and flavors and flavor oils from certain plants and spices (US Pat. Nos. 5,512,285 and 5,120).
, 558), and certain plants and herbs, pharmacologically active ingredients can be extracted. Depending on the temperature and pressure used, whether the temperature and pressure change during extraction, and the extraction method, supercritical fluid extraction can be used to selectively remove or isolate different substances from plant species. it can.

In one embodiment of the present invention, Piper methisticum, Vilsonia, Ascurus californica, Kuratagas mexicana, Simmondsia quinensis, Fifa, Brucella, Crunella, and Hemia salicifora, Sidium, Enterolobium. CO ₂ supercritical fluid extraction is used to extract bioactive substances from a variety of natural sources, including seeds, Cichopetarum oracoides, Liriosma ovata, and Chaunochiton caprelli.

Supercritical fluid extraction can be applied to the quantification of roots, leaves, bark or other parts of plants or grasses containing bioactive substances, or combinations thereof. Generally, the particular part (s) or part (s) are crushed to form a powder or paste. One or more temperatures, preferably at least 45 ° C., and at least two pressures,
Preferably at a pressure between and up to 400 and 600 bar between the minimum 200 and 400 bar, it may be extracted powder or paste by CO _2. During extraction, by using a number of temperature more pressure and one than one, it is possible to extract a variety of biologically active substances which may be soluble in CO ₂ at a specific pressure and temperature levels.

In a further embodiment, the extraction is a mixture of CO ₂ and at least one other volatile substance, such as butane, propane, ethanol, hexane, or other suitable volatile substance known to those skilled in the art. May be done in. The gases may be used in any optimal ratio to each other. In a preferred embodiment, extraction is 17: performed by combining CO ₂ and ethanol in a ratio of 3.

In another embodiment, the plant or herb is ground, softened in liquid, or mixed with a solvent, and then the solvated mixture is extracted with the supercritical fluid CO ₂ . See, eg, US Pat. No. 4,985,265. Under the great pressure of supercritical fluid extraction, the co-solvent mixture with CO ₂ leaves a liquid single-phase state.
This type of liquid-liquid extraction improves elution of specific analytes. A wide variety of solvents suitable for solvating various biologically active substances in natural sources are used, including alcohols, weak acids, ketones, chlorine derivatives, hydrocarbons, fluorinated hydrocarbons, acetates, ethers, or the like. However, the present invention is not limited to this.

The extraction of the present invention is carried out for at least 5 minutes, preferably 30 minutes, more preferably 60 minutes, during which the extracted sample is collected in a collection container, preferably a solid phase trap filled with C18. To be done. After the extraction is completed, for the solvent suitable for solvating the extracted bioactive substance, for example, kavalactone, the trap is rinsed with 50/50 ethanol / methanol chloride to recover most of the sample in the trap. A similar method of the present invention is outlined in Examples 1 and 2 below, where seven different kava lactones were extracted from the roots of hippopotamus. The method of the present invention is one at a time
It can be used to extract bioactive substances from one natural source or from multiple natural sources in a single extraction.

The supercritical fluid extraction of the present invention is performed as a batch extraction, a continuous cascade extraction,
It can be performed as a countercurrent solvent extraction, or a combination thereof. Most supercritical fluid extractions in the field of natural products involve the construction of equipment which is a batch loading system. In such a system, the extraction vessel is loaded with raw materials and the pressure and temperature are raised to the desired supercritical processing range. After the extraction is complete, reduce the pressure and temperature,
The container can be opened and the natural resources consumed can be removed, after which the process can be repeated. To date, this method has proved economically valuable unless, for example, carried out on a sufficiently large scale (eg decaffeination of coffee) or the value of the target compound is sufficiently high. Not not. In continuous cascade extraction, multiple vessels are sequentially brought online in a continuous fashion, and a supercritical fluid passes from vessel to vessel to collect a particular target compound in each vessel. See U.S. Pat. No. 5,120,558; also Stahl et al. , Dense Gases for Extraction and Ref for extraction and purification.
ining) (Springer-Verlag, Berlin, 1988)
See also). This method allows a CO ₂ fluid containing a small amount of analyte from a partially extracted container to dissolve more analyte from a new container that is sequentially introduced, thus averaging the CO ₂ fluid. It is advantageous in that the average loading rate of CO ₂ is increased because the loading rate is effectively increased and thus the analyte extraction per hour is increased.

In the countercurrent extraction process of the present invention, bioactives are extracted from plants and herbs and concentrated in a series of countercurrent mechanical presses. See U.S. Patent No. 6,013,304. The press may be held at elevated pressure and elevated temperature to facilitate supercritical fluid extraction as outlined above. As the supercritical fluid becomes more highly concentrated in its analyte contents via continuous pressurization, the fluid containing the analyte is recirculated to the original pressure, further analyte is extracted, and Will be concentrated.

2. High Density Gas Extraction Due to its non-flammable nature, contrary to propane or butane, and its excellent solvation properties for a wide range of target analytes, CO ₂ is among the most common volatiles in supercritical fluid extraction technology. Has become. However, CO ₂ is only effective as an extraction medium at extreme pressures. This creates the high cost of the equipment for performing the extraction and the inherent risks associated with extreme pressure vessels. Moreover,
Since scaling up such devices is prohibitive, they tend to remain small. Furthermore, the supercritical CO ₂ extraction system operates at temperatures above 39 ° C. Keeping unstable natural materials at such temperatures for an extended period of time during processing can result in thermally or enzymatically induced damage.

Recently, non-chlorinated fluorocarbon solvents have been disclosed for extracting flavors and flavors from natural sources. See U.S. Pat. No. 5,512,285. In one embodiment of the invention trifluoromethane, difluoromethane, fluoromethane, pentafluoroethane, 1,1,1-trifluoroethane, 1,1-
Difluoroethane, 1,1,1,2,2,3,3-heptafluoropropane, 1
, 1,1,3,3,3-hexafluoropropane, 1,1,1,2,2-pentafluoropropane, 1,1,1,2,2,3-hexafluoropropane, 1,1
, 2,2,3,3-hexafluoropropane, 1,1,1,2,3,3-hexafluoropropane, and 1,1,1,2-tetrafluoroethane, but not limited to Chlorinated fluorocarbon solvents may be used. The solvent used in the present invention may be a mixture of these solvents, the boiling point of the mixture may be adjusted to a specific step, or the selective elution of a specific bioactive substance may be promoted. The solvent may be further prepared by mixing with another volatile material such as, for example, butane, hexane, ethanol, or other suitable material.
In a preferred embodiment, the non-fluorinated carbon solvent used for extraction is tetrafluoroethane, preferably 1,1,1,2-tetrafluoroethane. In a more preferred embodiment, the tetrafluoroethane is not so prepared.

In the process of the present invention, ground or ground natural resources such as vegetation and / or herbs are contacted with a non-chlorinated fluorocarbon solvent so as to charge the solvent with the analyte. The charged solvent is recovered and transferred to isolate the sample. In one embodiment of the invention, the extraction vessel is sealed, evacuated and then the herb or plant is contacted with the non-chlorinated fluorocarbon solvent in the vessel. The resulting mixture of solvent and natural resource is maintained under pressure so that the solvent is charged with the analyte in intimate contact with the natural resource. This type of extraction may be performed in any extractor that is optionally sealed and evacuated. The extractor may be made of stainless steel, thick-walled glass, or any other non-reactive material capable of withstanding elevated or decreased pressure. The extraction may be performed at any suitable temperature, preferably room temperature or lower.

In another embodiment, the extraction is conducted as a supercritical fluid extraction at elevated pressure and various temperatures. Higher pressures and temperatures may be used to properly elute the desired analytes, particularly when the fluorinated hydrocarbon solvent is prepared with another volatile material having a higher boiling point.

In another embodiment, the plant or herb may be crushed, soaked, or mixed with a solvent, then the solvated mixture with a fluorinated hydrocarbon solvent or a conditioned fluorinated hydrocarbon solvent. You may extract. This type of liquid-liquid extraction improves the elution of specific analytes. A wide variety of solvents suitable for solvating various biologically active substances in natural sources may be used, including alcohols, weak acids, ketones, chlorine derivatives, hydrocarbons, fluorinated hydrocarbons, acetates. , Ether or combinations thereof, but is not limited thereto.

The extraction may be carried out as a batch, continuous cascade extraction or as countercurrent solvent extraction. In a preferred embodiment of the invention, the bioactive substance is extracted from plants and / or herbs with a fluorocarbon solvent in a continuous cascade extraction. The extractor may be in communication with the evaporator. During evaporation of the solvent from the eluted analyte, the solvent can be intermittently passed from the reactor to the evaporator to maintain a liquid level and gas filled headspace in the evaporator. Evaporation of the solvent may be accomplished by drawing gaseous solvent from the headspace of the evaporator. The aspirated gaseous solvent may be transferred to a compressor or similar device to reliquefy the solvent, thereby economically recycling the solvent.

In another embodiment, the evaporator may have one or more heat sources to control the temperature of the evaporator during evaporation of the solvent. In a further embodiment, the heat source may be thermostatically controlled to provide a constant evaporation temperature. Since non-chlorinated fluorinated hydrocarbons generally boil and fly before the desired analyte, it is not necessary to raise the distillation temperature of the solution during the solvent recovery step of the process. The extract produced in this way contains very low levels of solvent residues.

The vapor pressure of most fluorocarbon solvents is greater than atmospheric pressure at room temperature. For example, the vapor pressure of 1,1,1,2-tetrafluoroethane is 5.6 bar at 20 ° C. In a preferred embodiment, the extraction is therefore carried out at a pressure of 0-10 bar, preferably 3.5-6 bar. Most of these solvents must be operated in equipment that can tolerate such pressures, but this equipment is part of the cost of the equivalent equipment needed to operate supercritical CO _2. And the degree or inherent risk of sophistication in a manufacturing plant for manipulating liquefied hydrocarbon gas under pressure is small.

3. Supercritical Fluid Chromatography Following the extraction method, once the bioactive material is recovered as an extract from plants and grasses, the material may be extracted using various techniques, such as gas chromatography (GC) or high pressure liquid chromatography (HPLC). It can be separated and isolated.
However, gas chromatography is not scalable to provide a method to isolate each substitution on a large scale. On the other hand, liquid chromatography has a disadvantage of using a large amount of solvent.

In one embodiment of the invention, packed column supercritical fluid chromatography is used to separate bioactive substances in extracts obtained from various natural sources. Bioactive substances that can be separated from such sources include terpenes, terpenoids, flavones and flavonoids, steroids, sterols, saponins and sapogenins, alkanes, alkaloids, amines, amino acids, aldehydes, alcohols, fatty acids, lipids, lignans, phenols. , Pyrone, butenolide, lactone, chalcone, ketone, benzene, cyclohexane, glucoside, glycoside, cyanidin, furan, phorbol, quinone and phloroglucinol.
The present invention is also applicable to the recovery of high molecular weight bioactive substances such as proteins, peptides, enzymes, polysaccharides and carbohydrates. Sources from which bioactive substances can be isolated include hippopotamus Kava (eg Kava root
), Byrsonima, Asculus Aesculus (eg, A. californica), Crataegus mexicana, Jojoba, Phaffia, Alternella Pfaffia, Alternanthera (eg, A. repens A. repensera, Brucella). Turnera, Perezia, Perezia, Heimia (eg H. salicifolia H. salicifolia
), Sidium Psidium, Enterolobium Enterlobium, Chicopetalum Ptyc
hopetalum (eg, P. olacoides), Liriosma (
For example, L. L. ovata) and Chaunochiton (eg C.
Various plant species of C. kappleri) are included, but are not limited to. Plants from which extracts can be prepared according to the invention and from which natural substances can be isolated include the following higher plants: Acanthopanax
, Acanthopsis, Acanthopsis, Acanthosicyos, Acanthus, Acanthus, Achyranthes, Acokanthera
, Aconitum Aconitum, Acorus Acorus, Acronychia Acronychia, Actaea Actaea, Actinidia Actinidia, Adenia Adenia, Adhatda Adh
atoda, Aegle Aegle, Aesculus Aesculus, Aframon Aframomum, Agatake Agastache, Agathoma, Alchemilla Alchemilla, Aleurites, Allium, Aloe Aloe, Alonsoa Alonsoa
, Aloysia, Alloyton Alphitonia, Alpinia Alpinia, Alternantera Alternate, Amaranthus Amaranthus, Amomon Amo
mum, Amphipterygium, Amyris Amyris, Anxusa Anchusa, Ancistrocladus, Anemopsis Ane
mopsis, Angelica Angelica, Annoma Annona, Anonidium
, Antimis Anthemis, Antidesma Antidesma, Apium Apium, Araria Aralia, Aristolochia Aristolochia, Artemisia Artemisia, Artocarpus, Asarum Asarum, Asclepias Asclepias
, Asimina Asimina, Aspalanthus Aspalanthus, Asparagus Asparagu
Aspidosperma, Astragalus, Astragalus, Astronium, Astropa Atropa, Avena Avena, Azadirachta Azad
irachta, Azara, Baccaria Baccharis, Bacopa Bacopa, Balantiches Balanites, Bambusa Bambusa, Barrelaria Barleria, Barosma Barosma, Bauhinia, Bauhinia, Belamcanda, Belamcanda, Benincasa B
enincasa, Berberis, Berchemia, Berchemia, Bixa,
Bocconia Bocconia, Borago Borago, Boronia Boronia, Boswellia Bos
wellia, Brosimmann Brosimum, Brucea Brucea, Brunferushia Brunfe
lsia, Bryonia, Buddleja, Brunesia, Bulnesia, Bupleurum, Brucella Bursera, Brissonima Byrsonima, Calamenta Calamitha, Karea Calea, Calophyllum Calophyllum, Camellia
Camellia, Camptotheca, Cananga Cananga, Canalium Cana
rium, Canella Canella, Capparis Capparis, Capsicum Capsicum, Carthamus Carthamus, Calum Carum, Cassia Cassia, Cassine Cassine, Castanospermum Castanospermum, Catalpa Catalpa, Catacata Catapea Ciapeia Cayapeonia Cayapeonia, Cayapeonia Cayapeonia, Cayapeonia Cayapeonia St. Lantas Centranthus, Cephaelis, Kirant Dendron Chiranthod
endron, Chondrodendron, Chrysophyllu chrysophyllu
m, Shimicifuga Cimicifuga, Cinchona Cinchona, Cinnamomum
, Cistus Cistus, Citrus Citrus, Clausena Clausena, Kunikas Cn
icus, Coccoloba, Codonopsis, Coffea
, Coix Coix, Cola Cola, Coleus Coleus, Colleia Colletia, Converetum Combretum, Comiphora Commiphora, Cordia Cordia, Coria Coriaria, Correa Correa, Corridalis Corydalis, Costas Costas
Crataegus, Croton, Croton, Cryptolepis
, Cudrania, Cudrania, Cuminan Cuminum, Cuphea Cuphea, Cucurma Cucurma, Cyclanthera, Cymbopogon,
Cinara Cynara, Cynoglossum, Cyperus Cyperus, Cyrtocarpa, Delbergia Dalbergia, Delea Dalea, Danae Danae,
Daphne, Datuna, Datura, Daucus, Decadon,
Dendrocalamus, Dendropocax, Dendropanax, Deppea Deppea, Derris Derris, Desmon Desmos, Dichrostachy
s, Dictamnus, Digitalis Digitalis, Dillenia,
Dioscorea Dioscorea, Dioscoreo Films Dioscoreophyllum, Diosma Diosma, Diosphyros Diospyros, Dorimis Drimys, Zboisia Duboi
sia, Duguetia, Dysoxylum, Echinacea
a, Ecipipta Eclipta, Eretia Ehretia, Ekbergia Ekebergia, Eleagnus Eleagnus, Eretalia Elettaria, Eleutherococcus Eleuthe
rococcus, Enceria Encelia, Entandrophragma,
Ephedra, Epimedium Epimedium, Eriobotrya, Erodium Erodium, Eryngium, Erythrochit
on, Erythroxylum, Escorzia, Escholzia, Esenbeckia, Euclea Euclea, Eucomia, Eucommia, Eugesia
Euodia, Eupatorium, Fabiana Fabiana, Ferula Ferul
A, Fevillea Fevillea, Fitnia Fittonia, Flinderusia Flinder
sia, Foenicolum Foeniculum, Galesia, Galfimia Galph
imia, garcinia garcinia, gouge caucia Gaudichaudia, caulseria
Gaultheria, Gelsemium, Gentiana, Geranium G
eranium, Giganto Korla Gigantochloa, Gingko Gingko, Glotidione Glo
chidion, Glopeosperman Gloeospermum, Greiwa Grewia, Greia Greia, Guaiacum Guaiacum, Guimenema Gymn
ema, Haematoxylum, Hamamelis, Hamelia Hame
lia, Harpagophytum Harpagophytum, Hauya Hauya, Heimia Heimia, Helleborus, Hieracium, Hierochuroe Hierochlo
e, Hilleria Hilleria, Hippophae Hippophae, Houttuynia,
Hovenia Hovenia, Humuls Humulus, Huperzia Huperzia, Hula Hura,
Hybanthus Hybanthus, Hydnocarpus Hydnocarpus, Hydnophytum Hydnop
hytum, Hydrastis Hydrastis, Hydrocotyl Hydrocotyle, Hymenaea Hymenaea, Hyoscumus Hyoscamus, Hypericum Hypericum, Hyptis
Hyptis, Hissopus Hyssopus, Iboza, Igiosperman Idiosperm
um, Ilex Ilex, Ilycium Illicium, Indigo ferra Indigofera, Inga Inga, Inula Inula, Iochroma Iochroma, Ilecine Iresine,
Iris lris, Jacaranda, Jatropha Jatropha, Juniperus Juniperus, Justicia Justicia, Kadsura Kadsura, Camepferia Kaempferia, Lactuca Lactuca, Lagokirs Lagochilus, Larea Larre
a, Laurus Laurus, Labandra Lavandula, Lausonia Lawsonia, Leonurus Leonurus, Leucas Leucas, Ligusticum Ligusticum, Lindera Li
ndera, Lippia Lippia, Liriosoma Liriosma, Litosea Litsea, Lobelia Lobelia, Ronchocarpus Lonchocarpus, Ronicera Lonicera, Lysium Lycium, Macfadyena Macfadyena, Mackulla Ma
clura, Mangifera Mangifera, Mansoa Mansoa, Marc Gravia Marcgr
avia, Marbium, Martinella Martinella, Matricaria Matri
caria, Maytenus, Medicago, Medicago, Melissa Melissa, Mentha Mentha, Mimosa Mimosa, Mimusops, Mitragyna
, Montanoa, Morkillia, Morkillia, Mouriri Mouriri, Mucuna Mucuna, Muchisia Mutisia, Milika Myrica, Myristica Myristica, Nardostachys, Nepeta Orepoa Opotea, Ocotea, Orea Olea, Ochotea, Olea, Olea Panax, Papavel Papaver, Pappa Pappea, Parthenium Parthenium, Passiflora Passiflora, Paulinia Paullinia, Pelargoni
um, Penstemon Penstemon, Perezia Perezia, Perilla Perilla, Persear Persea, Petiveria Petiveria, Petroselinum Petroselinum, Peuse Danum Peucerana Pilacarina Pycarina, Phylanta Pycarpus, Pycarpus Pycharta, Phylanta Phycarantha
nellia, Pipper Piper, Piqueria Piqueria, Pitecellobium Pithecellobiu
m, Pit Sporum Pittosporum, Plectranthus Plectranthus, Pleoropetalum Pleuropetalum, Porphyrum Podophyllum, Pogostemon Pogost
emon, Polygala Polygala, Polygonum Polygonum, Polymnia Polymnia, Sapotal Psacalium, Phycotoria Psychotria, Terrigota Pterygota, Cicopetalum Ptychopetalia, Querica Quara, Punicnja Pearica, Punicara Quatia Quara, Pycnantia Puma
Rauvolfia, Remania, Reneamania, Reneumia, Reum Rheum, Rolinia Rollinia, Rolipa Rorippa, Los Marinas Rosm
arinus, Rudbeckia, Ruelia Ruellia, Rumex Rumex, Ruscus Ruscus, Ruta Ruta, Saccharum, Salix Salix, Salvia Salvia, Sambucus Sambucus, Sanguinaria Sanguinaria, Sapium Sapium, Sassafras Sassafras, Sassafras, Sassafras
celetium, Schizandra Schizandra, Securidaca, Securinega Se
curinega, Serenoa Serenoa, Simmondsia Simmondsia, Smilux Smi
lax, Stachytarpheta, Stachytarpheta, Stachys Stachys, Staurogin
Staurogyne, Stelecocarpus Stelechocarpus, Stephania Stephania, Sterconia Steculia, Stevia Stevia, Strophanthus Strophanthu
s, Strychnos Strychnos, Symphytum Symphytum, Syzygiu
m, Tabebuia Tabebuia, Taberna Emantana Tabernaemontana, Tabernante Tabernanthe, Tanacetum Tanacetum, Taxus Taxus, Tecoma Tecoma
, Terminalia Terminalia, Teucrium, Taumatococcus Th
aumatococcus, Tribulus Tribulus, Trifolium Trifolium, Trigonella Trigonella, Triprilaris Triplaris, Triumefetta Triumfetta, Crunella Turnera, Tussilago Tussilago, Urchia Virga Uriagne Uria Urna Uia Una, Unu Urania Una, Urania Tyrannora
um, Valeriana Valeriana, Valesia Vallesia, Bang Area Vangueria, Vanilla Vanilla, Verozia Velozia, Bepris Vepris, Velvascum Ver
bascum, verbena Verbena, Vetiveria vetiveria, villa la Virola, viscum viscum, vismere vismia, vitex vitex, boacanga voacanga, walburgia warburgia, withania withania, zanthoxyram zanthoxyl
um, Zingiber, Zingiber, Zigyphus, and Zygophyl
lum.

In addition to the genus of higher plants listed above, biological resources such as algae, bacteria, fungi, lichens, mosses, and marine organisms such as corals, sponges, echinoderms or other Compounds can be recovered from biological sources such as invertebrates or vertebrates.

Various stationary phases, pressures, temperatures and concentrations of modifiers can be applied to optimize the separation. To illustrate the packed column supercritical fluid chromatographic separations of the present invention in Examples 3-10 below, the separation of extracted cabalactone is used. The present invention provides SFC compliance with both semi-preparative and preparative scale fractions that ultimately provide isolation of each substitution in the analyte mixture in milligram quantities. Moreover, in accordance with the present invention, a device for separation of analytes can be assembled to connect an extractor and an evaporator to extract and separate specific bioactive components from selected plants and herbs, It is possible to create a series of continuous working steps for isolation.

4. Therapeutic botanical extracts and their use i) Bilsonima extract In one embodiment of the invention, an extract of Bilsonima spp., Such as Bilsonima clasifolia, containing various triterpenes, amino acids and / or flavonoids is prepared. To be done. Such an extract may be prepared using water, for example a non-aqueous solvent such as methanol, ethanol or ethyl acetate, a mixture of water and a non-aqueous solvent, or using one of the extraction methods described above. Good. Bilsonima extracts include, but are not limited to, for example, gastrointestinal disorders (eg, diarrhea, Crohn's disease, irritable bowel syndrome) and nervous and vascular diseases such as stroke, Parkinson's disease and Alzheimer's disease. . It may be administered to humans in therapeutic amounts to treat various ailments.

Extracts of Bilsonima may further be combined with extracts from other plant species including, but not limited to, Sidium species such as Sidium guayaba, and Enterolobium species such as Enterolobium cyclocarpam. Extracts of such other species may be prepared by any known method or the methods described above.

In a further embodiment of the invention, an extract of Bilsonima spp. And / or other extract to be combined with the Bilsonima extract is isolated in order to isolate the bioactive substance for treating a particular disease. You may. For example, pipecolic acid may be isolated from leaves and bark extracts of Bilsonima clasiforiae. Separations may be performed using any separation technique known to one of ordinary skill in the art or packed column supercritical fluid chromatography separation methods as described herein. Once isolated, the pipecolic acid is administered in therapeutic amounts in combination with itself or with other bioactive substances from Bilsonima or extracts of other plants to treat nervous and vascular diseases. You may.

In a still further embodiment, the Bilsonima spp. Extract is alone or in combination with other plant or herbaceous origin and extracts, or the Bilsonima spp. Isolated bioactive substance is alone or in other plants. Alternatively, it may be combined with other bioactive substances of herbs and other inactive ingredients or medicinal ingredients to be administered to a patient to form capsules, tablets, pastilles or elixirs. ii) Extracts of North American varieties of Ascurus spp. and Kuratagas spp. In one embodiment of the invention, extracts of Asculus spp., such as Ascrus californica, containing escin and various triterpene glycosides, and various flavonoids and oligomers of An extract of a Cratagas species such as Kuratagas mexicana containing procyanidins is prepared. Such an extract may be water, for example
It may be prepared using a non-aqueous solvent such as methanol, ethanol or ethyl acetate, a mixture of water and a non-aqueous solvent, or using one of the extraction methods described above. The extracts of Ascurus californica and Kuratagas mexicana, alone or in combination, may be administered to humans in therapeutic amounts to treat a variety of conditions including but not limited to cardiovascular diseases. . Such an extract may be given as a dietary supplement to provide a protective effect on the heart and blood vessels, especially in the case of cardiovascular lesions induced by cardiac ischemia and life-threatening reperfusion. See U.S. Pat. No. 5,925,355.

Extracts of Ascurus californica and Kuratagas mexicana may further be combined with extracts derived from other plant species, including but not limited to Brucella species such as Brucella microfila. . Extracts of such other species may be prepared by any method known to those of skill in the art or by any of the methods described above.

In a further embodiment of the invention, the extract of Ascrus californica and Kuratagas mexicana spp. And / or the other extract to be mixed with either or both of the extracts of Ascurus and Kuratagas is It may be separated to isolate a particular bioactive agent for treating the disease. For example, Askrus
Hydroquinone may be isolated from California. Separations may be performed using any technique known to one of ordinary skill in the art or packed column supercritical fluid chromatography separation methods as described herein. Once separated, the hydroquinoic acid can be used by itself or in Ascurus californica, Kuratagas mexicana,
Alternatively, it may be administered in a therapeutic amount for treating cardiovascular diseases in combination with bioactive substances derived from extracts of other plants.

In yet a further embodiment, the extract of Ascrus spp. And Clatagas spp. Alone or in combination with other herbaceous or herbaceous origin and extracts, or the isolated bioactive substance of Ascrus spp. It may be made into capsules, tablets, pastilles or elixirs alone or in combination with other vegetation or herb bioactives in combination with other inactive or pharmaceutical ingredients to be administered to a patient. iii) Extract of jojoba In one embodiment of the invention water, for example a non-aqueous solvent such as methanol, ethanol or ethyl acetate, a mixture of water and a non-aqueous solvent, water and a non-aqueous solvent in sequence, or Using one of the extraction methods described above, an extract of Simmondsia quinensis (also known as S. californica and jojoba) is prepared, particularly an extract of defatted jojoba powder containing Simmondsin. The Simmondsia extract may be administered to humans in therapeutic amounts as hunger satiety and weight loss agents.

The Simmondsia extract may be further combined with extracts derived from other plants. Extracts of other species may be prepared by any method known to those of skill in the art or by the methods described above. In a preferred embodiment, Simmondsin is separated from other substances in Simmondsia extract. Separations may be performed using any separation technique known to one of ordinary skill in the art or packed column supercritical fluid chromatography separation methods as described herein. Once isolated, Simmondstin may be administered in therapeutic amounts as a hunger satiety or weight loss agent in combination with itself or with other bioactive agents derived from Simmondsia or other plant extracts. . Therapeutically effective doses to fill hunger are 5-5 / kg body weight
It is between 00 mg, preferably between 10 and 250 mg per kg body weight, more preferably between 20 and 100 mg per kg body weight, and even more preferably between 25 and 50 mg per kg body weight.

In yet a further embodiment, the extract of Simmondsia spp. Alone or in combination with other plant or herbaceous origin and extracts, or the isolated bioactive substance of Simmondsia spp. Alone or in other plants Alternatively, it may be combined with a herb bioactive substance and combined with other inactive ingredients or medicinal ingredients to be administered to a patient to form capsules, tablets, pastilles or elixirs. iv) Extracts of Crunella spp. and Phaffia spp. In one embodiment of the invention, an extract of a Trnella spp. such as C. jiffusa and a Phaffia sp. Is prepared. Such an extract may be prepared using water, for example a non-aqueous solvent such as methanol, ethanol or ethyl acetate, a mixture of water and a non-aqueous solvent, or using one of the extraction methods described above. Good. Extracts of Crunella spp. And Phaffia spp. Alone or in combination with another include, for example, various cancers such as diabetes, rheumatism, ulcers, leukemias, chronic cough, orchitis, and semen leak,
It may be administered to humans in therapeutic amounts to treat various illnesses including, but not limited to. Such extracts may be administered as a dietary supplement or health tonic to boost energies, aid digestion, and enhance conception.

Extracts of Crunella spp. And Phaffia spp. Also include other plants including, but not limited to, muirapuama (natural drugs from various species, including Chicopetalum oracoides, Liriosma ovata and Chaunotitone cappelli). It may be combined with an extract derived from the seed. Extracts of such other species may be prepared by any method known to those of skill in the art or by any of the methods described above.

In a further embodiment of the invention, an extract of Crunella spp. And Phaffia spp., And / or
Or other extracts to be mixed with extracts of Crunella spp. And / or Phaffia spp. May be separated to isolate specific bioactive substances for treating specific diseases. For example, β-sitosterol may be isolated from extracts of C. typhi and / or Phaffia paniculata. Separations may be performed using any separation technique known to one of ordinary skill in the art or packed column supercritical fluid chromatography separation methods as described herein. Once isolated, β-sitosterol supports either or both of male and / or female sexual function in combination with itself or with other bioactive substances derived from vine, Phaffia, or other extracts. May be administered in a therapeutic amount as a health tonic.

In yet a further embodiment, the extracts of Crunella spp. And Phaffia spp. Alone or in combination with other plant or herbaceous origin and extracts, or the isolated biologically active substances of Crunella spp. And Phaffia spp. It may be made into capsules, tablets, pastilles or elixirs, alone or in combination with other plant or herb bioactives, in combination with other inactive or pharmaceutical ingredients to be administered to a patient. v) Hemia spp. Extract In one embodiment of the invention, an extract of Hemia spp., such as Hemia salicifolia, is prepared containing various alkaloids and quinones. Such an extract may be prepared using water, for example a non-aqueous solvent such as methanol, ethanol or ethyl acetate, a mixture of water and a non-aqueous solvent, or using one of the extraction methods described above. Good. The Hemia spp. Extract may be administered to humans alone or in combination in therapeutic amounts to treat a variety of conditions including, but not limited to, inflammation of the joints and muscles. In a preferred embodiment, the Hemia spp. Extract is combined and administered in a therapeutic amount as a non-steroidal anti-inflammatory drug (NSAID).

The extract of Heimia species may be further combined with extracts from other plant species. Extracts of such other species may be prepared by any method known to those of skill in the art or by any of the methods described above.

In a further embodiment of the invention, the extract of Heimia spp., And / or the other extract to be mixed with the extract of Hemia spp. Is a specific bioactive substance for treating a specific disease. May be separated to isolate For example, the arkanoids cryogenin and nesodin may be isolated from Heimia salicifria. Separations may be performed using any separation technique known to one of ordinary skill in the art or packed column supercritical fluid chromatography separation methods as described herein. Once isolated, cryogenin and nesogin treat inflammation in joints, muscles or other tissues either by themselves or in combination with other bioactive substances derived from extracts of Hemia salicifolia or other plants. May be administered in a therapeutic amount.

In a further embodiment, the extract of Heimia spp. Alone or in combination with other herbs or herbaceous origin and extracts, or the isolated bioactive substance of Hemia spp. Alone or in other plants Alternatively, it may be combined with other bioactive substances of herbs and other inactive ingredients or medicinal ingredients to be administered to a patient to form capsules, tablets, pastilles or elixirs.

The following experiments are provided to illustrate embodiments of the invention, but they should not be considered as limiting the scope of the invention.

[0080]

【Example】

Example 1 . Using extraction vessel of a supercritical fluid extraction -CO ₂ extracted 3 mL, it was supercritical fluid extraction of kavalactones described in Example 1 to 2 (SFE). Each extract contained 0.5 grams of finely ground birch root. Various experimental conditions, as described below, were used to determine the conditions that maximize the recovery of kavalactone. 60 with liquid CO ₂ flow rate of 2 mL / min
The SFE method was performed for 1 minute.

Supercritical fluid extraction was performed at 350 atm and 450 atm. A solid phase trap filled with C18 was used to collect the extracted sample. Trap temperature is + 10 ℃
Set to. 50% ethanol / CH ₂ Cl ₂ after each supercritical fluid extraction is completed
The trap was rinsed with 10 mL of / 50% mixture. Then, the extraction volume was adjusted to 25 mL with CH ₂ Cl ₂ .

[0082]   Cabalactone standard-Because there is no standard for pure kavalactone, it is
The birch root extract and the SFE extract obtained by the crushing method were compared. For this purpose
25 mL of 50/50 CH as extraction solvent₂Cl₂/ 30 minutes in MeOH,
0.5 grams of hippo root was sonicated. Then filter the extract with 2 μm filter paper
did. Then connected to a Hewlett-Packard Model 5972 mass spectrometer
Hewlett Packard Model 5890 Gas Chromatographic Extraction
The thing was analyzed. Ultrasonic crushing extraction is 100% of total cabalactone in root sample
Assumed to bring income. CO₂CH for recovery of kava lactone using SFE₂Cl ₂ / Comparison with recovery of kava lactone using MeOH sonication.

Columns 1 and 2 of Table 1 show SFE recovery of different kava lactones from hippo roots using different SFE conditions. This recovery is expressed as a percentage of the recovery obtained by conventional sonication methods. Peak identification was performed by comparing the three strongest ions in the mass spectrum of each peak, and Vi
orica Lopez-Avila et al. (V. Lopez-Avila an
d Benedicto, J .; High Resolut. Chroma
togr. , 20, 555, 1997).

[0084]

[Table 1] Table 1 Recovery ratio of various kava lactones from hippo root using supercritical fluid extraction * ------------------------- --------------------------------------------- Compound 350 atm 450 atm 350 atm 450 atm 60 ° C 60 ° C 60 ° C 60 ° C 100% CO ₂ 100% CO ₂ 85% CO ₂ 85% CO ₂ 15% EtOH 15% EtOH ---------------- -------------------------------------------------- ---- 7,8-Dihydrocabain 92.9% (7) 97.5% (6) 92.7% (2) 91.1% (5) -------------------- -------------------------------------------------- Kabain 93.6% (5) 100.0% (4) 102.9% (4) 107.0% (4) ----------------------------- ----------------------------------------- 5,6-Dihydrokabine 86.1% ( 8) 80.3% (5) 74.0% (8) 79.9% (7) --------------------------------- ------------------------------------- 5,6,7,8-Tetrahydroangonin 97.9% (5) 92.9% (8) 96.4% (4) 106.0% (6) ------------------------------------------------ -------------------------------------- J Hydromethistisine 93.2% (8) 88.4% (6) 95.3% (8) 104.1% (4) -------------------------- -------------------------------------------- Yangonin 84.7% (11) 67.6% (12) 72.5% (9) 84.1% (9) ----------------------------------- ----------------------------------- Metistisine 95.9% (7) 66.2% (10) 111.1% (7 ) 137.6% (12) -------------------------------------------- -------------------------- * = 50/50% based on comparison of SFE with extraction by CH ₂ Cl ₂ / MeOH sonication Recovery () =% RSD for 3 repeated extractions.

Example 2 CO ₂ Extraction with Ethanol Another supercritical fluid extraction was performed to investigate the efficiency of obtaining cabalactone with a mixture of CO ₂ and ethanol. In this experiment, 350 bar and 450
An extracted solution of 85% CO ₂ and 15% ethanol at atmospheric pressure was used. The temperature of the trap was kept at 60 ° C. Table 1 shows that some species of kava lactones were extracted more efficiently by SFE than by sonication, especially caabain and methystisine. Table 2 shows the retention time, molecular weight (MW), and the three strongest ions in the mass spectrometric analysis of kavalactone isolated via SFE as described above.

[0086]

[Table 2] Table 2 3 found in electron impact mass spectrum of root extract of kava lactone by SFE
One of the strongest ions ---------------------------------------------- ------------------------ No. Compound name Retention time Molecular weight Three strongest ions (m / z) ----------------------------------- ----------------------------------- 1. 7,8-Dihydrokabine 27.85 232 127 (100), 91, 117 2. Cabaine 29.05 230 98 (100), 68, 69 3. 5,6-Dihydrokabaine 29.85 228 228 (100), 157, 69 4. 5,6,7,8-Tetrahydroangonine 30.71 262 121 ( 100), 147, 262 5. Dihydromethystisine 31.96 276 135 (100), 276, 136 6. Yangonin 32.95 258 258 (100), 187, 230 7. Metistisine 33.16 274 148 (100), 135, 274 ---- -------------------------------------------------- ----------------

FIG. 1 shows the separation by gas chromatography / mass spectrometry (GC / MS) of kavalactone extracted with SFE. 2-8 show the mass spectra of each kavalactone listed in Table 2. Table 3 shows the retention times and the four strongest ions for the mass spectra of the other peaks eluting before the main kavalactone.

[0088]

Table 3 Table 3 The three strongest ions in the electron impact mass spectrum of the peak eluted before the major cabalactone extracted by SFE. -------------------------------------------------- --No. Compound name Retention time 4 strongest ions (m / z) ------------------------------------ ---------------------------------- 1. Unknown 19.40 91 (100), 65, 188, 97 2. Unknown 22.01 186 (100), 95, 128, 155 3. Unknown 23.01 121 (100), 218, 77, 78 4. Unknown 24.35 135 (100), 77, 232, 136 5. Unknown 26.93 135 (100), 230, 115, 128 ------------------------------------ ----------------------------------

9 to 13 show the other major peaks (tR = eluted) that eluted before the cabalactone.
19.40, 22.1, 23.01, 24.35 and 26.93 minutes). The peak of tR-23.1 is considered to be a spike (ghost peak) in MS. Figure 14 shows GC of kavalactone extracted by ultrasonic crushing.
/ MS chromatogram is shown.

To compare the absolute weights of extracts obtained by SFE and sonication, 0.5 grams of hippo root were extracted by SFE and sonication, as discussed above.
Each extract was then transferred to a container of known weight. The solvent of each extract was evaporated under a stream of nitrogen. Table 4 shows the weights and ratios of specimens extracted from 0.5 g of hippo root using SFE and sonication.

[0091]

[Table 4] Table 4 Extracted from hippo root by supercritical fluid extraction and solid-liquid extraction (ultrasonic crushing) ---------- ------------------------------------------------ Extraction Sample Extracted weight (g) Weight (g) Sample weight% ------------------------------- --------------------------------------- SFE, 350 bar, 60 ° C 85 / 15CO ₂ / EtOH 1.0 0.0716 7.16 2 mL / min ------------------------------------ ---------------------------------- SFE, 350 bar, 60 ° C 85/15 CO ₂ / EtOH 0.5 0.038 7.6 2mL / min ------------------------------------------ ----------------------------- Ultrasonic disruption, 15mL 50/50 CH ₂ Cl ₂ / MeOH 0.5 0.039 7 . 8 30 minutes ---------------------------------------------- ------------------------

Such a result indicates that more than 90% of the measured cabalactone is pure C
Although extracted via O _2, we show that a more complete extraction of kavalactone is found with 85% CO ₂ and 15% ethanol as supercritical fluid.

Example 3 Separation of Kabalactone Using Supercritical Fluid Chromatography The following separations were performed using a Hewlett Packard Model G1205A Supercritical Fluid Chromatograph (SFC) system equipped with a variable UV detector.

The detection wavelength was set to 254 nm. Various column and chromatographic conditions were applied to determine the most favorable separation of cabalactone. 15 to 17 show the results of an experiment in which a kavalactone extract was subjected to SFC using NH ₂ , DIOL and CN columns. Chromatographic conditions for the NH ₂ column are: column material: NH ₂ with water; trade name: Altec Sphenosorb; length: 25 cm; inner diameter: 4
． 6 mm ID; pressure: 125 atm; temperature: 60 ° C .; flow rate: liquid CO ₂ 2 mL /
It was a minute.

Modulator programming was started with 2/98% MeOH / CO ₂ and held for 3 minutes, then ramped up to 10/90% MeOH / CO _{2 in} 0.4% minutes. D
Chromatographic conditions for the IOL column are: Column material: DIOL; Trade name: Vydac Model Supleco
Length: 25 cm; inner diameter: 4.6 mm ID; pressure: 125 atm; temperature: 60 ° C .;
Flow rate: ₂ mL / min with liquid CO ₂ . Regulator programming is 2/98%
Starting with MeOH / CO ₂ and hold for 3 minutes, then 0.4% for 10 /
Raised to 90% MeOH / CO ₂ . Chromatographic conditions for the cyano (CN) column are: Column material: CN; Trade name: Alltech; Length: 25 cm;
Inner diameter: 4.6 mm ID; pressure: 275 atm; temperature: 60 ° C .; flow rate: ₂ mL / min with liquid CO ₂ .

The modifier programming was 2/98% MeOH / C at a rate of 0.4%.
Started with O ₂ . As can be seen with reference to Figures 15-17, the best separation of kavalactone is N
Obtained on H ₂ column. Both the CN and DIOL columns provided some separation, but co-elution of the components was observed.

Example 4 Optimization of CN column conditions Figure 18 shows the SFC of Cabalactone by SFC at high temperature (80 ° C) using a CN column.
The result of having separated FE extract is shown. Other chromatographic conditions are the same as those described for CN above. The selectivity of the column at 80 ° C has changed,
A portion of the lactone co-eluted at ° C was separated. In addition, some lactone co-eluted at 80 ° C was previously separated at 60 ° C.

Separation of kava lactone was carried out under the same conditions using a CN column at low temperature (40 ° C.).
Did not improve. In addition, the concentration of the regulator (programming of the regulator is 2/98
% MeOH / CO ₂ and hold for 3 min, then 0.5% min 10
/ 90% MeOH / CO ₂ ) and pressure (125 atm) changes as shown in FIG.
The selectivity of the column was unchanged as shown in 9.

Example 5 Optimization of NH ₂ column conditions Then, under various conditions, NH ₂ chromatography columns were used to convert S
The SFC separation of FE extract was optimized. Figures 20 and 21 are 40 ° C and 80, respectively.
9 shows separation of kavalactone extract by NH ₂ column at 0 ° C. Chromatographic conditions were: pressure 125 atm, and flow rate, liquid CO ₂ 2 mL / min.
Modulator programming was started with 2/98% MeOH / CO ₂ and held for 3 minutes, then ramped up to 10/90% MeOH / CO _{2 in} 0.4% minutes. Figure 2
As shown at 0, temperature separation reduced the selectivity of the column and the lactone co-eluted. At higher temperatures, better solutions were obtained for the components eluted at 23.39 and 23.97 min (Figure 21) compared to the separation obtained at 60 ° C (Figure 15).

FIG. 22 shows the results of separating the same birch root on an NH ₂ column using the same conditions as described in FIG. 21, except the pressure was increased to 275 atm. Co-elution of several components was observed. The modifier concentration was then varied to optimize the elution time of the sample. For this experiment, modifier programming was 7/93% Me
Start with OH / CO ₂ and hold for 3 minutes then 10/90 at 0.2% / min
The same pressure (125 atm), temperature (80 ° C.), flow rate ( ₂ mL / min with liquid CO ₂ ) and NH ₂ column were used, with the exception of increasing to% MeOH / CO ₂ . Figure 2
A separation similar to 1 was obtained. However, the analysis time was reduced from 24 minutes to 11 minutes.

The seven peaks obtained using the NH ₂ column are kava lactones identified using supercritical fluid extraction of hippo root and GC / MS. The area percentage of each peak in the chromatogram is as follows: 3, 9.5, 50, 6.9, 5.
8, 4.0 and 20%.

Example 6 . All CO ₂ and supercritical fluid extraction as described by CO ₂ that is adjusted with 15% ethanol SFE experiment kavalactones, Isco equipped with ACCUTRAP ^TM and variable flow restrictor - sysplex (Isco-Suprex) (Nebraska Lincoln, VA) Prep Master. For each extraction, 0.5 grams of pre-ground powder of birch root was used. Flow rate, ₂ mL of liquid CO 2 /
Extraction was performed for 60 minutes. Two pressures (350 and 450 atm) were used for extraction at 60 ° C. The extracted sample was recovered using a solid phase trap filled with C18. The temperature of the trap is +10 when pure CO ₂ is used as the extraction fluid.
The temperature of the trap was set to 60 ° C when 15% ethanol-adjusted CO ₂ was used. After the end of each extraction, the trap was rinsed with 10 mL or a 50/50 mixture of ethanol / CH ₂ Cl ₂ . Extraction volume of 25 m with CH ₂ Cl ₂
Adjusted to L.

Since there were no criteria to determine the extraction efficiency of kava lactone, all SFE extracts were compared to liquid-solid extraction (LSE) of hippo root via ultrasonic disruption method. Sonicate 0.5 g of hippo root in 10 mL of 50/50% MeOH / CH ₂ Cl _{2 for} 60 minutes at room temperature using an ultrasonic bath from Fisher Scientific (Pittsburgh, PA). LSE was performed by crushing. Then, the supernatant was filtered through a nylon Acrodisc filter having a germane 0.45 μm. The final volume was adjusted to 50 mL and analyzed by GC / MS. This extraction was assumed to recover 100% of all kavalactone from the roots.

To obtain all SFC separations, a Hewlett Packard G1205A Supercritical Fluid Chromatography (SFC) system equipped with a high pressure flow cell and equipped with a variable UV detector was used. Lactone detection was monitored at 254 nm.
The same equipment was used for semi-preparative scale separations, except that the maximum flow rate (4 mL / min) was used.

Table 5 lists the columns used in this test and the corresponding manufacturers.
For semi-preparative scale separation, the column was 250 x 10 mm and dp = 5 μm, while for analytical scale testing, a 250 x 4.6 mm, dp = 5 μm column was employed. Seven capabalactones were identified in the supercritical extract using GC / MS (Hewlett Packard 5890 Gas Chromatography with 5971A mass selective detector and 7673 autosampler, Wilmington, Del.).
All GC separations were obtained on a 30 mx 0.25 mm ID x 25 μm dpDB-5 (J & W Scientific, Forson, Calif.) Fused silica capillary column. The temperature of the column was kept at 50 ° C for 3 minutes and then programmed to 280 ° C at a rate of 10 ° C / min.

[0106]

[Table 5] Table 5                   Column used in this test *     ------------------------------------------------       Column supplier     ------------------------------------------------       Spherisorb NH2 All Tech       Ultima Cyan Alltech       SUPERCOSIL LC-DIOL SUPERCO       C4 Protein Vatic       Diphenyl batik     ------------------------------------------------       * 250 x 4.6 mm, 5 Fdp

HPLC grade methanol and ethanol were purchased from EM Sciences (Gibbstown, NJ). For the supercritical fluid extraction test and the supercritical fluid chromatography test, SFE / SFC grade CO ₂ was used, and Air Products and Chemical Co. (Allentown, PA).

Various conditions were used to obtain a quantitative extraction of kava lactones from hippo roots. To determine the extraction efficiency of kava lactones from hippo root, pure CO ₂ and 15
% Were investigated two pressure with both CO ₂ prepared in ethanol. In order to determine the extraction efficiency of each lactone, 50/50% CH ₂ Cl ₂ / Me was used as an extraction solvent.
The same amount of birch roots was extracted using OH via solid-liquid extraction (sonication). It was assumed that the liquid-solid extraction results were recovered with a yield of 100%. Table 6 shows some SFE
The relative extraction efficiency of each kava lactone extracted from the hippo root under the conditions is shown. The results show that most kava lactones can be extracted by, for example, nitrogen, hydrogen, or preferably a near-critical or supercritical gas such as butane, propane or freon. 9 with pure CO ₂ at 350 atm 60 ° C
An efficiency of over 0% was obtained. CO ₂ adjusted with 15% ethanol can be used to obtain even higher extraction efficiency of kava lactones from hippo roots. However, the extraction efficiency using a pure CO ₂ as an extraction fluid was less than 25%. This may be because the sample size used in the extraction was too large compared to the results, or the differences may reflect the different trapping methods used in the two tests.

[0109]

[Table 6] Table 6 Recovery ratio of kava lactones from hippo roots using SFE * ---------------------------------------- Compound 350 atm 60 ° C 450 atm 60 ° C 350 Atmosphere 60 ° C 450 Atmosphere 60 ° C 100% CO ₂ 100% CO ₂ 85 / 15CO ₂ / EtOH 85 / 15CO ₂ / EtOH ----------------------- ----------------------------------------------- 7,8 -Dihydrokabine 92.9 (7) 97.5 (6) 92.7 (2) 91.1 (5) ------------------------------- --------------------------------------- Kabain 93.6 (5) 100.0 (40) 102.9 ( 4) 107.0 (4) -------------------------------------------- -------------------------- 5,6-Dihydrocabain 86.1 (8) 80.3 (5) 74.0 (8) 79.9 (7)- -------------------------------------------------- ------------------ Dihydromethystisine 93.2 (8) 88.4 (6) 95.3 (8) 104.1 (4) ------------ -------------------------------------------------- -------- Yangonin 84.7 (11) 67.6 (12) 72.5 (9) 84.1 (9) ------------------------- --------------------------------------------- Metistisine 95.9 (7) 66.2 (10) 111.1 (7) 137.6 (12) --------------------------------- ------------------------------------- 5,6,6,8-Tetrahydroangonin 97.9 ( 5) 92.9 (8) 96.4 (4) 106.0 (6) ------------------------------------ ---------------------------------- * =% recovery is 50/50 CH ₂ Cl ₂ / MeOH ultrasonic waves Based on comparison with extraction by crushing. All extractions were performed at a flow rate of 2 mL / min for 60 minutes. () = RSD for 3 repeated extractions.

Example 7 Supercritical Fluid Chromatography-NH ₂ Column for Kavalactone In the second part of this study, different columns with the same diameter, different particle sizes (eg different stationary phases) were used to optimize the SFC separation of kavalactone. , And chromatographic conditions were investigated. Efficient analytical separations with supercritical fluids would be advantageous in preparation for future scale-up work to isolate large quantities of each cabalactone.

FIG. 25 shows the separation of the kavalactone extract using an Alltech Superisov NH ₂ column. Separation using a gradient of CO ₂ adjusted with methanol gives 60
It was obtained at a constant temperature of 125 ° C. and 125 ° C. Initial concentration of methanol in CO ₂ is 2%
Hold it constant for 3 minutes and then at a rate of 0.4% / min up to 10% MeOH
Increased. A separation of all kava lactones was obtained. However, the sixth peak eluted as a shoulder in front of the last major peak. Low and high temperatures were tried to improve the latter elution separation. When the column temperature was lowered to 40 ° C, several peaks co-eluted. Raising the column temperature to 80 ° C. not only provided a baseline separation (FIG. 26) for most cabalactone, but also between peaks 6 (tR
= 23.4 minutes) A high resolution was obtained. Increasing the pressure from 125 atm to 275 atm yielded cabalactone which eluted only four peaks. Peak 3 is 1
Eluted as the two main peaks, but peaks 1 and 2, peaks 4 and 5, and peak 6
And 7 seemed to co-elut.

Next, the concentration gradient of the modifier was changed at 60 ° C. in order to obtain not only better separation but also analysis in a shorter time. For this purpose, the initial modifier concentration was increased to 7%. After 3 minutes, adjust the concentration of the modifier at a rate of 0.2% / minute to 10
Increased to%. FIG. 27 shows the separation obtained. As observed, increasing the initial modifier concentration not only provided a faster separation (12 min vs. 25 min analysis time), but with higher resolution of the last two peaks. Separation was also provided.

Example 8 . Cavalactone supercritical fluid chromatography-C4 protein column 125 atm, 70 ° C., ₂ mL / min with liquid CO ₂ , and modifier program (99
1% CO ₂ / MeOH for 4 minutes, then 0.1% / min 97 /
3% CO ₂ / MeOH increased to) using, C4 from Vachikku (Vatic)
Most of the lactone co-eluted by the protein column. Separation was steadily improved by increasing the oven temperature to 80, 90 and 100 ° C. with the modifier program. FIG. 28 shows that the first mobile phase was 100 ° C. and 98.5 / 1.
． Shows the separation of kavalactones extract with 5% CO ₂ / MeOH. Increasing oven temperature provided baseline separation of most peaks. Increasing the density by increasing the column back pressure resulted in several peaks of co-elution. Lowering the initial modifier concentration and the gradient of modifier concentration did not improve separation at higher pressures. Methanol was mixed with CO ₂ before mixing with CO ₂ .
The addition of 1% isopropylamine (as the second modifier) not only improved the separation of the lactone, but also reduced the analysis time. Isopropylamine provides additional selectivity to obtain better separation (Figure 29).

Example 9 Kabalactone SFC-CN, DIOL and Diphenyl Columns Figure 30 shows the separation of kavalactones on a Vatic diphenyl column.

Unlike the other separations, the initial back pressure on the column was set at 125 atmospheres for 3 minutes and 5
The pressure was increased to 195 atm / min. The first mobile phase is 98/2% CO ₂
/ A MeOH, and elevated at a rate of 0.1% / min to 93/7% CO ₂ / MeOH. Separation was obtained at 80 ° C. and a flow rate of 2 mL / min. Only 5 peaks were observed. Peaks 2 and 3 co-eluted as did peaks 6 and 7.

[0116] Figure 31 is 125 atm at 60 ° C., 98 / starting at 2% CO ₂ / MeOH, 3 minutes keeping with a rate of subsequent 0.4% / min 90/10% CO ₂ / Figure 6 shows SFC separation of kavalactone using Altima CN column from Alltech in a modifier program up to MeOH. As observed, most of the sample co-eluted. Separation could not be improved by increasing or decreasing either temperature, pressure or modifier concentration. It is believed that the CN column does not have sufficient selectivity to fractionate all components.

Separation of the same extract from Supleco with a Spelcosil DIOL column was obtained. Chromatography conditions are 125 atm, 60 ° C
And a mobile phase composition starting with 98/2% CO ₂ / MeOH and held for 3 minutes and then increasing at 0.4% / min to 90/10% CO ₂ / MeOH. The separation was similar to that obtained via the CN column. Again, the temperature and composition of the modifier did not have a significant effect on the separation.

The results obtained from the evaluation of such a column show that the NH ₂ column and the protein C
It was shown that four columns provided a (most) baseline separation of all kavalactones. However, most of the peaks appeared to be better fractionated on the Protein C4 column compared to the NH ₂ column. Therefore, the protein C4 stationary phase was used for the semi-preparative separations.

Example 10 Semi-preparative separations Semi-preparative separations consisted of a single protein C4 column (250 x 100 mm).
, 5 μm dp). The parameters were changed to optimize the separation. The optimized separation employed 125 atm, 80 ° C., and a flow rate of 4 mL / min with a CO ₂ concentration gradient adjusted with methanol (FIG. 32). The results showed that the single protein C4 column was not efficient enough to separate all the kavalactones in the semipreparative mode. Then two semi-preparative protein C4 columns were connected in series to obtain a separation. FIG. 33 shows the separation of the kavalactone extract (injection volume 5 mg) using the conditions described above. As observed, baseline separation of most kavalactones was achieved on a semi-preparative scale using two columns connected in series.

Extraction of different cabalactones with efficiencies above 90% was obtained using pure CO ₂ . However, using CO ₂ conditioned with 15% ethanol,
Higher extraction efficiency was obtained. Also, using methanol-adjusted supercritical CO ₂ ,
Separation of different kava lactones from birch root extract was performed. The results showed that different cabalactone separations could be obtained using analytical scale amino and protein C4 columns. A semi-preparative separation of kavalactone was performed using two protein C4 columns connected in series. A baseline separation of most components was obtained.

From considering the specification and practicing the invention disclosed herein, other embodiments and uses of the invention will be apparent to those skilled in the art. U.S. Patent Provisional Applications, Application Nos. 60 / 102,912, 60 / 122,526, and 6th
No. 0 / 136,409, and all US and foreign patents and patent applications, including US patent application Ser. Nos. 09 / 408,922 and 09 / 518,191, incorporated herein by reference. All cited references are specifically and entirely incorporated herein by reference. The specification and examples are intended to be considered merely exemplary, with the true scope and spirit of the invention as set forth in the following claims.

[Brief description of drawings]

FIG. 1 Gas chromatography of cabalactone extracted using SFE /
The separation by mass spectrometer (GC / MS) is shown.

FIG. 2 is a mass spectrum of Kavalactone 1 listed in Table 2.

FIG. 3A is a mass spectrum of Kavalactone 2 listed in Table 2. FIG. 3B is a mass spectrum of Kavalactone 2 listed in Table 2.

FIG. 4 is a mass spectrum of Kavalactone 3 listed in Table 2.

FIG. 5 is a mass spectrum of Kavalactone 4 listed in Table 2.

FIG. 6 is a mass spectrum of Kavalactone 5 listed in Table 2.

FIG. 7 is a mass spectrum of Cabalactone 6 listed in Table 2.

FIG. 8 is a mass spectrum of Kavalactone 7 listed in Table 2.

FIG. 9 is a spectrum (tR = 19.40 min) of the other major peaks eluted before kavalactone.

FIG. 10 is the spectrum (tR = 22.1 min) of the other major peaks eluting before kavalactone.

FIG. 11 is the spectrum (tR = 23.01 min) of the other major peaks that eluted before kavalactone.

FIG. 12 is a spectrum (tR = 24.35 min) of the other major peaks eluted before kavalactone.

FIG. 13 is a spectrum (tR = 26.93 min) of the other major peaks eluted before kavalactone.

FIG. 14 is a GC / MS chromatogram of cabalactone extracted by ultrasonic disruption.

FIG. 15 shows the results of an experiment in which a kavalactone extract was the subject of an NH ₂ column.

FIG. 16 is the result of an experiment in which a kavalactone extract was the subject of a DIOL column.

FIG. 17 shows the results of an experiment in which a kavalactone extract was the subject of a CN column.

FIG. 18: Results of an experiment in which the kavalactone extract was separated by SFC at elevated temperature (80 ° C.) using a CN column, all other conditions of chromatography are the same as described for CN above. .

FIG. 19 is a result showing that pressure (125 atm) did not change the selectivity of the column.

FIG. 20 shows the separation of kavalactone extract on NH ₂ column at 40 ° C. and 80 ° C., respectively.

FIG. 21 shows separation of kavalactone extract on NH ₂ column at 40 ° C. and 80 ° C., respectively.

FIG. 22 shows the results of separating the same birch root extract on an NH ₂ column using the same conditions as described in FIG. 21, except the pressure was increased to 275 atm.

FIG. 23. Modifier program started with 7/93% MeOH / CO ₂ .
Same pressure (125 atm), temperature (80 ° C.), flow rate (2 mL / min liquid, held for 3 minutes, then increased to 0.2% / min to 10/90% CO ₂ / MeOH. The results using CO ₂ ) and a column (NH ₂ ) are shown.

FIG. 24A is the chemical structure of one of the seven identified kava lactones. FIG. 24B is another chemical structure of the identified kava lactone. Figure 2
4C is another chemical structure of the identified kava lactone. FIG. 24D is another chemical structure of the identified kavalactone. FIG. 24E is another chemical structure of the identified kava lactone. FIG. 24F is another chemical structure of the identified kavalactone. FIG. 24G is another chemical structure of the identified kavalactone.

FIG. 25 SFC separation of kava lactone extract on column NH ₂ (250x
4.6 mm, 5 μm dp); pressure 125 atm, 60 ° C., 2 mL / min; modifier program: 98/2% CO ₂ / MeOH for 3 min, then 90/10 at a rate of 9.4% / min. The results obtained under the condition of increasing to CO ₂ / MeOH are shown.

FIG. 26 SFC separation of kava lactone extract on column NH ₂ (250x
4.6 mm, 5 μmdp); pressure 125 atm, 80 ° C., 2 mL / min; modifier program: 98/2% CO ₂ / MeOH for 3 min, then 90/10 CO at a rate of 0.4% / min. _The results obtained under the condition of increasing to ₂ / MeOH are shown.

FIG. 27 SFC separation of kava lactone extract on column NH ₂ (250x
4.6 mm, 5 μmdp); pressure 125 atm, 60 ° C., 2 mL / min; modifier program: 93/7% CO ₂ / MeOH for 3 minutes, then 90/10 CO at a rate of 0.2% / min. _The results obtained under the condition of increasing to ₂ / MeOH are shown.

FIG. 28: SFC separation of a kava lactone extract was performed using column protein C4 (
250 × 4.6 mm, 5 μmdp); pressure 125 atm, 100 ° C., 2 mL / min; modifier program: 98.5 / 1.5% CO ₂ / MeOH for 2 min, then 0.2.
The results obtained under the condition of increasing the CO ₂ / MeOH to 90/10 at the ratio of% / min are shown.

FIG. 29: SFC separation of a kava lactone extract was performed using column protein C4 (
250 × 4.6 mm, 5 μm dp); pressure 125 atm, 80 ° C., 2.5 mL / min;
Modulator program: 98/2% CO containing 0.1% isopropylamine. ₂ / MeOH for 3 minutes, then 90/10 CO at a rate of 0.4% / min.₂/ MeO
The results obtained under the condition of raising to H are shown.

FIG. 30: SFC separation of kava lactone extract was performed using column diphenyl (25
0x4.6 mm, 5 μmdp); pressure 125 atm for 3 minutes, then 5 atm / min 19
Raise to 5 atm and at 80 ° C., 2 mL / min; Modulator Program: 98/2% C
The results are shown under the condition that O ₂ / MeOH and then the ratio of 0.1% / min to 93/7 of CO ₂ / MeOH were used.

FIG. 31: SFC separation of kava lactone extract using column CN (250 × 4
． 6 mm, 5 μmdp); pressure at 125 atm, 60 ° C., 2 mL / min; modifier program: 98/2% CO ₂ / MeOH for 3 min, then 0.4 / min at 90/90%.
The results obtained under the condition of raising to 10% CO ₂ / MeOH are shown.

Figure 32: Semi-preparative SFC separation of kava lactone extract on column single protein C4 (250 x 4.6 mm, 5 μmdp); pressure 125 atm, 80 ° C, 4 m.
L / min; Modulator Program: 98/2 with 0.1% Isopropylamine
% CO ₂ / MeOH for 3 minutes and then 0.4% / minute to 90/10% CO ₂ / MeOH.

FIG. 33: Semi-preparative SFC separation of kavalactone extract in series with column 2 protein C4 (250 × 4.6 mm, 5 μmdp); pressure 125 atm.
80 ° C., 4 mL / min, modifier program: 98/2% CO ₂ / MeOH containing 0.1% isopropylamine for 3 minutes; 90/10% at a rate of 0.4% / min.
The results are shown under the conditions of increasing to CO ₂ / MeOH; injection volume −50 μL (100 μg / μL).

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 3/10 A61P 3/10 9/10 9/10 19/02 19/02 21/00 21/00 25 / 16 25/16 25/28 25/28 29/00 29/00 101 101 35/00 35/00 35/02 35/02 B01D 11/02 B01D 11/02 A (31) Priority claim number 09/518 , 191 (32) Priority date March 3, 2000 (March 3, 2000) (33) Priority claiming country United States (US) (81) Designated country EP (AT, BE, CH, CY, DE, DK) , ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR , NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS , JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Ashraf-Kuhlassani , Maddie 24060, Virginia, United States, Braxburg , No. 6600 Dee, Hunt Club Road 305 (72) Inventor Taylor, Rally 24060 Virginia, United States Virginia, Braxburg, Walnut Drive North West 2101 F Term (Reference) 4C076 AA36 AA53 BB01 CC01 CC04 CC11 CC11 CC16 CC18 CC21 CC26 CC27 4C088 AB12 AB25 AC05 AC06 AC11 BA08 BA09 BA10 CA03 CA05 CA06 CA09 CA10 CA14 MA35 MA37 MA52 NA14 ZA02 ZA15 ZA16 ZA36 ZA45 ZA66 ZA68 ZA94 ZA96 ZB11 AC24 CA16 CAB AC25 AC12 CA16 AC12 CA25 DA02 DA05

Claims (78)

[Claims] 1. A method for extracting at least one bioactive substance from at least one natural resource, wherein the solvent is chlorinated to extract an amount of at least one bioactive substance from the at least one natural resource. Contacting the non-fluorinated fluorocarbon solvent with at least one natural source; and removing the non-chlorinated fluorocarbon solvent to isolate at least one bioactive agent. 2. At least one natural resource is hippopotamus root, Bilsonima spp., Ascurus californica, Kuratagas mexicana, Simmondsia quinensis, Phaffia spp., Brucella spp., Trunella spp., Hemia salicifolia, Sidium spp., Enterolobium spp. The method of claim 1 selected from the group consisting of :, Chicopetalum oracoides, Liriosma ovata, and Chaunochiton capperelli. 3. A non-chlorinated fluorocarbon solvent is 1,1,1,2.
-The method of claim 2 which is tetrafluoroethane. 4. The method of claim 2 wherein the extraction is performed as a batch, continuous cascade or countercurrent process. 5. The method according to claim 4, wherein the extraction is carried out at a pressure of at least 0-10 bar. 6. The method according to claim 4, wherein the extraction is carried out at a pressure of at least 3.5 to 5.6 bar. 7. The method of claim 2 wherein the extraction is performed with a mixture of non-chlorinated fluorocarbon solvent and at least one other volatile material. 8. The method of claim 7, wherein the at least one other volatile material is selected from the group consisting of butane, propane, carbon dioxide, hexane, ethanol, methanol and combinations thereof. 9. The method of claim 7, wherein the mixture of non-chlorinated fluorocarbon solvent and at least one other volatile material is in a supercritical or near-critical fluid state. 10. The method further comprises treating the at least one natural resource to a powder, paste, dip, or mixture prior to contacting the at least one natural resource with the non-chlorinated fluorocarbon solvent. The method of claim 2. 11. The method of claim 2, wherein at least one natural resource is combined with at least one cosolvent so that the extraction is a liquid-liquid process. 12. The at least one cosolvent comprises an alcohol, a weak acid, a ketone,
The method of claim 11 selected from the group consisting of chlorine derivatives, hydrocarbons, fluorinated hydrocarbons, acetates, ethers and combinations thereof. 13. The method of claim 2, wherein the at least one bioactive agent is a plurality of bioactive agents. 14. The method of claim 13, further comprising the step of separating a plurality of bioactive agents to obtain at least one isolated and purified bioactive agent. 15. The separation is achieved by high pressure liquid chromatography, packed column supercritical fluid chromatography or radial flow chromatography.
Method 4 16. Hippopotamus root, Bilsonima spp., Ascurus californica,
At least one natural resource selected from the group consisting of Kuratagas mexicana, Simmondsia quinensis, Phaffia species, Brucella species, Crunella species, Heimia salicifolia, Sidium species, Enterolobium species, Chicopetalum oracoides, Liriosma ovata and Chaunochiton capperelli. From at least 1
A method of extracting one bioactive substance, the method comprising contacting a non-chlorinated fluorocarbon with at least one natural resource so that the solvent extracts an amount of at least one bioactive substance from the at least one natural resource. , Wherein the extraction is carried out as a batch, continuous cascade or countercurrent process; and the step of removing the unchlorinated fluorocarbon solvent to isolate at least one bioactive substance. 17. A method for separating an analyte contained in an extract, comprising flowing at least one volatile substance through a packed column, wherein at least one volatile substance is flown.
The two volatiles include the steps of being in a near-critical or supercritical fluid state; passing the extract through a packed column; and recovering the separated analyte, where the analyte is at least one bioactive substance. Yes, the method in which the extract is derived from natural resources. 18. The at least one volatile material is ethanol, methanol,
18. The method of claim 17, selected from the group consisting of butane, propane, dichloromethane, tetrafluoroethane, isopropylamine, and combinations thereof. 19. The packed column is a C4 protein column, NH ₂ column, C
18. The method of claim 17, selected from the group consisting of N column, DIOL column, and diphenyl column. 20. Natural resources include hippo root, Bilsonima spp., Ascrus californica, Kuratagas mexicana, Simmondsia quinensis, Phaffia spp., Brucella spp., Crunella spp., Hemia salicifolia, Sidium spp., Enterolobium spp., Chicopetalum spp. 18. The method of claim 17, wherein the method is selected from the group consisting of Oracoides, Liriosma ovata and Chaunochiton capperelli. 21. A continuous cascade extraction method for extracting a plurality of bioactive substances from at least one natural resource, the method comprising placing an amount of at least one natural resource in a plurality of extraction vessels; Passing the volatiles through multiple extraction vessels in a continuous fashion until the desired concentration of the bioactives is achieved; including removing the volatiles to obtain an amount of multiple bioactives Method. 22. At least one natural source is hippo root, Bilsonima spp.,
Ascurus californica, Kuratagas mexicana, Simmondsia quinensis, Phaffia species, Brucella species, Crunella species, Heimia salicifolia,
Sidium species, Enterolobium species, Chicopetalum oracoides, Liriosma
22. The method of claim 21, selected from the group consisting of ovata and chaunochiton capperelli. 23. The method of claim 21, wherein the volatile material is a non-chlorinated fluorocarbon solvent. 24. The non-chlorinated fluorocarbon solvent is 1,1,1,
24. The method of claim 23, which is 2-tetrafluoroethane. 25. The method of claim 21, wherein the extraction is carried out at a pressure of 0-10 bar. 26. The method of claim 21, wherein the extraction is performed with a mixture of non-chlorinated fluorocarbon solvent and at least one other volatile material. 27. At least one other volatile material is butane, propane,
27. The method of claim 26, selected from the group consisting of carbon dioxide, hexane, ethanol, methanol, nitrogen, chloroform, and combinations thereof. 28. At least one non-chlorinated fluorocarbon solvent.
27. The method of claim 26, wherein the mixture of three other volatiles is in a supercritical or near-critical fluid state. 29. The method of claim 21, further comprising the step of treating at least one natural resource into a powder, paste, dip, or mixture prior to placing the at least one natural resource in an extraction vessel. 30. The method of claim 21, wherein at least one natural resource is combined with at least one co-solvent so that the extraction is a liquid-liquid process. 31. The at least one cosolvent comprises an alcohol, a weak acid, a ketone,
31. The method of claim 30, selected from the group consisting of chlorine derivatives, hydrocarbons, fluorinated hydrocarbons, acetates, ethers and combinations thereof. 32. The method of claim 21, further comprising the step of separating a plurality of bioactive agents to obtain at least one isolated and purified bioactive agent. 33. The method of claim 32, wherein the separation is accomplished by radial flow chromatography, high pressure liquid chromatography, or packed column supercritical fluid chromatography. 34. Ingestible for treating neurological and vascular diseases, comprising a therapeutic concentration of at least one bioactive substance extracted from Birsonima sp. By a non-chlorinated fluorocarbon solvent by continuous cascade extraction. Prescription. 35. The formulation of claim 34, further comprising a therapeutic concentration of at least one bioactive agent derived from at least one other natural source. 36. The formulation of claim 35, wherein the at least one natural source is selected from the list consisting of Sidium species, Enterolobium species, and combinations thereof. 37. Bilsonima spp. Is Bilsonima clasifora, Scidium spp. Is Sidium guaiaba, Enterolobium spp. Is enterolobium.
37. The formulation of claim 36 which is cyclocarpam. 38. The formulation of claim 34 in the form of a tablet, capsule, flavoring agent (pastel), or elixir. 39. Containing a therapeutic amount of at least one bioactive agent extracted from Bilsonima sp. And a therapeutic amount of at least one bioactive agent extracted from Csidium sp., Enterolobium sp., Or a combination thereof. Wherein at least one bioactive substance extracted from Bilsonima spp. And at least one bioactive substance extracted from Sidium spp., Enterolobium spp., Or a combination thereof is an organic solvent, water, a mixture of organic solvents / water. An ingestible formulation extracted by, supercritical fluid extraction, dense gas extraction, or a combination thereof. 40. The formulation of claim 39, wherein the organic solvent is methanol, ethanol, ethyl acetate or a combination thereof. 41. Bilsonima spp. Is Bilsonima clasifolia, Scidium spp. Is Sidium guaiaba, Enterolobium spp. Is enterolobium.
40. The formulation of claim 39 which is cyclocarpam. 42. The therapeutically first amount of at least one bioactive substance extracted from Birsonima sp. Is from the group consisting of β-sitosterol, betulin, proline, pipecolic acid, quercetin, catechin and combinations thereof. 42. The formulation of claim 41 selected. 43. An ingestible for use as a cardiovascular tonic containing therapeutic concentrations of multiple bioactive agents extracted from Ascrus and Kuratas species by a non-chlorinated fluorocarbon solvent by continuous cascade extraction. Prescription. 44. The formulation of claim 43, further comprising a therapeutic concentration of at least one bioactive agent derived from at least one other natural source. 45. The formulation of claim 44, wherein the at least one other natural source is Brucella sp. 46. The formulation of claim 45, wherein the Ascrus species is Ascrus californica, the Kuratas species is Kuratas mexicana, and the Brucella species is Brucella microfila. 47. The formulation of claim 43 in the form of tablets, capsules, flavorings, or elixirs. 48. Containing a therapeutic amount of at least one bioactive substance extracted from Ascrus species and a therapeutic amount of at least one bioactive substance extracted from Kuratagas species, wherein the extract is from Ascrus species. At least one bioactive substance and at least one bioactive substance extracted from a clatagas species are organic solvents, water, organic solvent / water mixtures, near-critical or supercritical fluid extractions, high-density gas extractions, or Ingestible prescription extracted with the combination of. 49. The formulation of claim 48, wherein the Ascrus species is Ascrus California and the Kuratagas species is Kuratagas mexicana. 50. The formulation of claim 48, wherein the organic solvent is methanol, ethanol, ethyl acetate, or a combination thereof. 51. A therapeutic amount of at least one bioactive substance extracted from Ascutus spp. Is β-methylalanine, phenylalanine, isohomoleucine, isohomo-6-hydroxyleucine, mino-4-methyl-hex-. Transformer
4-enoic acid, gamma-glutamyl-2-A-hex-4-enoic acid, arbutin,
49. The formulation of claim 48 selected from the group consisting of hydroquinone, epicatechin, coumarin eleuceroside B-1, quebrachitol and combinations thereof. 52. An extract of jojoba for satisfying hunger and weight loss in humans, the extract comprising Simmondsin, wherein Simmondsin is an organic solvent, water, a mixture of organic solvents / water, near. Extracts extracted from jojoba with supercritical or supercritical fluid extractions, high density gas extractions, or non-chlorinated fluorocarbon solvents. 53. Simmondsin is extracted from defatted powder of jojoba.
Extract of 2. 54. The non-chlorinated fluorocarbon solvent comprises 1,1,1
53. The extract of claim 52, which is 2,2-tetrafluoroethane. 55. The extract of claim 52, wherein Simmondsin is extracted from jojoba in a continuous cascade extraction method. 56. The extract of claim 52 which is in the form of tablets, capsules, flavorings or elixirs. 57. A method of satisfying hunger in a human to aid in weight loss, comprising administering to the human an extract of jojoba containing Simmondsin. 58. The method of claim 57, wherein the extract is in the form of tablets, capsules, flavorings or elixirs. 59. The method of claim 57, wherein the extract is obtained from jojoba defatted powder by extraction with a non-chlorinated fluorocarbon solvent. 60. For use as a health tonic comprising a plurality of bioactive agents extracted from Crunella spp. And Phaffia spp. By continuous cascade extraction with a non-chlorinated fluorocarbon solvent, and for supporting sexual function. Ingestible prescription. 61. The prescription of claim 60, wherein the sexual function supported is male, female, or both. 62. The formulation of claim 60, further comprising a therapeutic concentration of at least one bioactive agent derived from at least one other natural source. 63. The formulation of claim 62, wherein the at least one other natural resource is selected from the group consisting of Chicopetalum oracoides, Liriosma ovata, Chaunotitone cappelli, muirapauma and combinations thereof. 64. The formulation of claim 60, wherein the Tsunderella species is Ts. Typhimurium and the Phaffia species is Phaffia paniculata. 65. The formulation of claim 60 in the form of tablets, capsules, flavorings, or elixirs. 66. Containing a therapeutic amount of at least one bioactive substance extracted from Crunella spp. And a therapeutic amount of at least one bioactive substance extracted from Phaffia spp., Wherein the extraction is from Crunella spp. Wherein the at least one bioactive agent and at least one bioactive agent extracted from Phaffia species are organic solvents, water, organic solvent / water mixtures, supercritical fluid extractions, dense gas extractions, or combinations thereof. Ingestible prescription extracted. 67. The formulation of claim 66, wherein the Tsunderella species is Ts. Typhia and the Faffia species is P. paniculata. 68. The formulation of claim 66, wherein the organic solvent is methanol, ethanol, ethyl acetate, or a combination thereof. 69. The therapeutically first amount of at least one bioactive substance extracted from Crunella spp. Is β-sitosterol, arbutin, caffeine, gonzalitocin, hexacosane-1-ol, tetraphyllin B, N. 67. The formulation of claim 66 selected from the group consisting of: triacontane, tricosan-2-one, paracymene, [alpha] -pinene, [beta] -pinene and combinations thereof. 70. The therapeutically first amount of at least one bioactive substance extracted from Phaffia species is allantoin, daucosterol, β-ecdysone, faffinic acid, fafoside A, B, C, D, E and F, polypogin B, β
-Sitosterol, stigmasterol, stigmasterol-3-O-β-D
67. The formulation of claim 66 selected from the group consisting of glucosides and combinations thereof. 71. A plurality extracted from a Hemia species by organic solvent, water, organic solvent / water mixture, near-critical or supercritical fluid extraction, dense gas extraction, or continuous cascade extraction with non-chlorinated fluorocarbon solvent. An ingestible formulation for use as a non-steroidal anti-inflammatory agent containing a bioactive substance of: 72. The formulation of claim 71, further comprising a therapeutic concentration of at least one bioactive agent derived from at least one other natural source. 73. The Hemia spp. Is Hemia salicifolia.
Prescription. 74. The formulation of claim 71 in the form of tablets, capsules, flavorings, or elixirs. 75. A therapeutic amount of at least one bioactive substance extracted from Heimia spp., Wherein the at least one bioactive substance extracted from Heimia spp. Is organic solvent, water, organic solvent / An ingestible formulation extracted with a mixture of water, near-critical fluid extraction, supercritical fluid extraction, dense gas extraction, or a combination thereof. 76. The Hemia spp. Is Hemia salicifora.
Prescription. 77. The formulation of claim 75, wherein the organic solvent is methanol, ethanol, ethyl acetate, or a combination thereof. 78. The therapeutically first amount of at least one biologically active substance extracted from Hemia spp. 76. The formulation of claim 75 selected from the group consisting of -I, demethylrasbin-II, and combinations thereof.