Composition for the treatment of Parkinson's disease

Abstract

The present invention provides a novel water-soluble composition comprising a sesquiterpene coumarin concentrate containing sesquiterpene coumarins from species of the genus Ferula in various proportions, for the treatment of Parkinson's disease by inhibition of MAO enzymes (MAO-A and MAO-B). The present invention also provides a method for preparing a sesquiterpene coumarin concentrate from an ethyl acetate extract and asafoetida (a oleogum resin of Ferula asafoetida) using silica gel column chromatography, wherein the mobile phase consists of various compositions of hexane, toluene, chloroform, ethyl acetate, and methanol. Furthermore, the present invention provides a method for preparing a formulation using a multi-step process, which includes preparing a water-soluble formulation after preparing the sesquiterpene coumarin concentrate. The final composition exhibits improved water solubility, long-term stability, MAO inhibitory activity, and compliance with pharmaceutical standards.

Description

[Technical Field]

[0001] The present invention relates to a method for preparing a water-soluble composition containing a sesquiterpene coumarin concentrate fraction containing sesquiterpene coumarins derived from Ferula species in various ratios. The present invention further provides a method for preparing the water-soluble composition using a method comprising 1) extraction, fractionation and preparation of a sesquiterpene coumarin concentrate fraction containing sesquiterpene coumarins in various ratios, and 2) preparation of the water-soluble composition. The water-soluble composition improves the solubility, dispersibility, stability and compliance with pharmaceutical standards of the hydrophobic sesquiterpene coumarins. More specifically, the present invention relates to the use of the above-mentioned water-soluble composition containing sesquiterpene coumarins that inhibit monoamine oxidases (MAO, i.e., MAO-A and MAO-B) and impart beneficial effects to the treatment and management of Parkinson's disease. [Background technology]

[0002] Parkinson's disease (PD) is caused by the progressive loss of dopaminergic neurons in the substantia nigra (SN). It is the second most common progressive neurodegenerative disease, particularly prevalent in the elderly, and is characterized by a decrease in dopamine levels in the striatal region. This decrease in dopamine leads to the characteristic main symptoms of Parkinson's disease: tremor, bradykinesia, and rigidity (Dauer & Przedborski, 2003; Lee & Yankee, 2021; Maiti et al., 2017; Mhyre et al., 2012; Rizek et al., 2016). The rapid increase in the population affected by this disease is placing a tremendous medical and economic burden on society. Because the precise causes and site specificity of Parkinson's disease are not well understood, current medications can only alleviate patients' symptoms by one of the following: a) increasing dopamine levels, b) using dopamine receptor agonists, or c) reducing dopamine breakdown by inhibiting monoamine oxidase (Cho et al., 2021; Finberg, 2018; Muller, 2012; Riederer & Laux, 2011). For early-stage Parkinson's disease, strategic inhibition of MAO (MAO-A and MAO-B) is used to maintain control of L-dopa dosage (Cho et al., 2021; Finberg, 2018; Krishna et al., 2014; Muller, 2012; Nagatsua & Sawadab, 2009; Riederer & Laux, 2011). While some chemically synthesized MAO inhibitors are used clinically, they are not without adverse events such as hypertension, nocturnal awakenings, orthostatic hypotension, arrhythmias, psychiatric symptoms, hallucinations, extrapyramidal symptoms, dyskinesia, and serotonin syndrome. Furthermore, discontinuation of these drugs carries a risk of withdrawal syndrome, similar to that associated with antidepressants and antiparkinsonian drugs (Koschel et al., 2021; MAO-B Inhibitors (Rasagiline, Selegiline, Safinamide) | Parkinson's UK; Rabinak & Nirenberg, 2010; Solla et al., 2015, 2017). Therefore, to overcome the side effects associated with chemically synthesized drugs, natural substances derived from medicinal plants are being developed as promising MAO inhibitors.On the other hand, natural compounds have low water solubility, which further hinders their solubility, efficacy, formulation into dosage forms, compliance with pharmaceutical standards, and patient adherence to medication.

[0003] Therefore, in light of the problems with conventional technologies reported to date, there is a recognized need for the rapid development of novel water-soluble compositions based on natural substances for the treatment and management of Parkinson's disease. As a result, the present invention has developed a water-soluble composition containing a sesquiterpene coumarin concentrate fraction containing sesquiterpene coumarins extracted from species of the genus Ferula, particularly Ferula assafoetida, in various proportions.

[0004] Sesquiterpene coumarins are naturally occurring compounds biosynthesized in plants of the genus Ferula (family: Apiaceae). The most commonly used of these plants is Ferula asafoetida, widely known as asafoetida or awei. This plant is used in cooking as a distinctive flavoring. The exudate of Ferula asafoetida, more precisely its oleogum resin, is known to contain volatile organic sulfides, sesquiterpenes, umbelliferone, ferulic acid, and sesquiterpene coumarins (Abd El-Razek et al., 2001; Iranshahy & Iranshahi, 2011; Mohammadhosseini et al., 2015, 2019; Mohammadhosseini & Nekoei, 2014; Yatham et al., 2021). Aqueous methanol extracts of Ferula asafoetida have been reported to inhibit the enzyme acetylcholinesterase (Ali et al., 2013; Jazayeri et al., 2014). Aqueous extracts of Ferula asafoetida have been reported to exhibit anticonvulsant effects (Bagheri et al., 2014). Farnesiferol C, one of the components of Ferula asafoetida, showed moderate neuroprotective effects against oxygen-glucose deficiency in vitro (Chang et al., 2013). Sesquiterpene coumarins isolated from Ferula sinkiangensis, a closely related species, suppressed neuroinflammation (Xing et al., 2017). Aqueous extracts of the gum resin of asafoetida showed neuroprotective effects and improved nerve cell viability (Homayouni Moghadam et al., 2013). Extracts from various parts of Ferula asafoetida showed efficacy in mice against naloxone-induced morphine withdrawal symptoms via gamma-aminobutyric acid (GABA) (Khanavi et al., 2017). Extracts of Ferula asafoetida also showed neuroprotective effects against glutamate-induced neurotoxicity (Tayeboon et al., 2013).The oleogum resin of Ferula asafoetida reduced pyridoxine-induced peripheral neuropathy in mice (Homayouni Moghadam et al., 2014), and Ferula asafoetida resin extracted with ethanol at 80°C inhibited hMAO-B (Zarmouh et al., 2016). [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] One objective of the present invention is to develop pharmaceutical compositions containing sesquiterpene coumarins extracted from species of the genus Ferula, specifically Ferula asafoetida, in various ratios. Another object of the present invention is to develop a sesquiterpene coumarin concentrate that confers excellent MAO (MAO-A and MAO-B) inhibitory activity. Another object of the present invention is to develop a pharmaceutical composition in which sesquiterpene coumarin concentrates containing sesquiterpene coumarins having acyclic, monocyclic, and bicyclic skeletons, in which sesquiterpenes spontaneously bond with coumarin via ether bonds, exhibit synergistic effects. Another objective is to develop a sesquiterpene coumarin concentrate that can be further incorporated into a water-soluble composition that has improved water solubility and dispersibility, long-term stability, and pharmaceutical parameters. [Means for solving the problem]

[0006] Therefore, the present invention relates to a general chemical structure -

[0007] [ka]

[0008] The present invention provides a water-soluble composition for the treatment of Parkinson's disease by inhibition of MAO (MAO-A and MAO-B), comprising sesquiterpene coumarins having [specific properties].

[0009] The sesquiterpene coumarin concentrated fraction in the water-soluble composition shown above contains sesquiterpene coumarins selected from the following in various ratios -

[0010] Ferucrin (1), Assafoetidanol A (2), Ligpersein A (3), Kamolonol (4), 10'-Acetoxy-11'-hydroxyumbelliprenin (5), Epi-conferdione (6), Karatabicinol (7), Kellenin (8), Ferocaulidin (9), Farnesiferol A (10), Galbanic acid (11), Szowitsia coumarin B (12), Farnesiferol B (13), Gummosin (14), Farnesiferol C (15), Szowitsia coumarin A (16), Methyl galbanate (17).

[0011]

Chemical formula

[0012] The water-soluble composition inhibits MAO enzymes (MAO-A and MAO-B) and improves the solubility, dispersibility, stability and pharmaceutical parameters of the sesquiterpene coumarin concentrated fraction.

[0013] This method is essentially different from the known prior art in the following steps.

[0014] Step 1: Preparation of the sesquiterpene coumarin concentrated fraction A method for preparing a sesquiterpene coumarin concentrated fraction, comprising the following steps: (a) extracting oleo-gum resin of the genus Ferula (asafetida, i.e., asafoetida) multiple times with an organic solvent having a polarity index of 2 to 6, preferably ethyl acetate (polarity index 4.4), collecting the extract, and concentrating it until a residue free of ethyl acetate is obtained; (b) separating the ethyl acetate-free residue obtained in step (a) using gradient elution with hexane-chloroform (100:0 to 0:100 v / v) and chloroform-methanol (100:0 to 70:30 v / v) on normal-phase silica gel to obtain a sesquiterpene coumarin concentrated fraction containing sesquiterpene coumarin having umbelliferone and ferulic acid in various ratios; (c) further separating the fraction obtained in step (b) using gradient elution with acetonitrile-water on a reversed-phase (C18) HPLC column bonded with octadecylsilane to obtain pure sesquiterpene coumarin; (d) the sesquiterpene coumarin concentrated fraction in step (b) contains ferulicin (0.05 to 42.31%), asafetidanol A (0.013 to 1.17%), ligpersin A (0.31 to 36.29%), camonol (1.50 to 46.67%), 10'-acetoxy-11'-hydroxyumbelliprenin (1.52 to 2.86%), epi-conferdione (0.24 to 12.24%), karatabicinol (0.07 to 6.13%), kellerin (1.43 to 17.63%), ferocaudine (0.19 to 2.23%), farnesiferol A (0.13 to 14.31%), galbanic acid (0.05 to 52.56%), szowitsia coumarin B (5.18 to 16.18%), farnesiferol B (0.81 to 10.08%), gummosin (2.58 to 4.03%), farnesiferol C (0.01 to 40.98%), szowitsia coumarin A (0.28 to 7.01%), and methyl galbanate (0.70 to 10.77%).

[0015] Step 2: Preparation of the water-soluble composition A sesquiterpene coumarin concentrate containing sesquiterpene coumarin in various proportions is added in a 1:1 to 1:10 (w / w) ratio to an oil phase containing a suitable fatty acid or fatty acid ester selected individually or in combination from, but not limited to, caprylic acid, isopropyl myristate, olive oil, coconut oil, sunflower oil, and ethyl oleate, and dissolved at 10°C to 40°C under high or low external energy for 5 minutes to 24 hours to obtain a clear and transparent solution. The solution is further treated with a nonionic, cationic, or anionic emulsifier selected from, but not limited to, Tween20, Tween60, Tween80, Span80, Span20, sodium lauryl sulfate, Brij35, dodecyltrimethylammonium chloride, trigol, and cetyltrimethylammonium bromide, in a ratio of 1:1.1 to 1:12.5 (w / w), with or without a co-surfactant, by stirring at high or low energy at 10°C to 30°C for 1 minute to 24 hours. Furthermore, the resulting mixture is dispersed in water in a ratio of 1:0.5 to 1:20 (w / w), by stirring at high or low energy for 1 minute to 24 hours, to obtain a water-soluble composition. [Effects of the Invention]

[0016] The developed water-soluble composition has several advantages, which are briefly listed below. • Water-soluble compositions containing concentrated sesquiterpene coumarin fractions require no harmful chemicals whatsoever. Water-soluble compositions containing sesquiterpene coumarins are cost-effective. The preparation method is very simple and economically viable. • The water-soluble composition improves the solubility, dispersibility, stability, and compliance with pharmaceutical standards of the hydrophobic sesquiterpene coumarin concentrate. Water-soluble compositions containing sesquiterpene coumarins can be formulated in liquid, solid, gel, and semi-solid dosage forms. • Sesquiterpene coumarin concentrate and water-soluble compositions containing sesquiterpene coumarin concentrate exhibit MAO enzyme (MAO-A and MAO-B) inhibitory activity. • Water-soluble compositions containing sesquiterpene coumarins exhibit stability for at least one month in the pH range of 2.0 to 10.0. • Water-soluble compositions containing sesquiterpene coumarins can be stored at a temperature range of 10-40°C for at least 3 months. [Modes for carrying out the invention]

[0017] The present invention provides a novel water-soluble composition comprising a sesquiterpene coumarin concentrate for the management or treatment of Parkinson's disease by inhibition of MAO enzymes (MAO-A and MAO-B). More specifically, the present invention further relates to a method for preparing an ethyl acetate extract and a sesquiterpene coumarin concentrate from asafoetida (i.e., the oleogum resin of Ferula asafoetida) using silica gel column chromatography with a mobile phase consisting of hexane, chloroform, and methanol (polarity 2-6) of various compositions. The specific ratio of sesquiterpene coumarin present in the fraction is determined by HPLC analysis at 325 nm.

[0018] Specifically, the present invention provides a method for preparing a water-soluble composition containing a concentrated fraction of sesquiterpene coumarin derived from a species of the genus Ferula. The present invention further provides a method for preparing the water-soluble composition using the following method:

[0019] 1) Extraction, fractionation, and preparation of sesquiterpene coumarin concentrated fractions - Asafoetida is extracted multiple times using an organic solvent with a polarity index of 2 to 6, particularly ethyl acetate (polarity index: 4.4), by any known method, and then the solvent is evaporated to obtain a solvent-free residue. Furthermore, this solvent-free residue is fractionated by normal-phase silica gel column chromatography using solvents with a polarity index of 0 to 6, specifically hexane, chloroform, and methanol in various combinations. The fractions are concentrated under reduced pressure to obtain a solvent-free residue, which is then analyzed by HPLC at 325 nm.

[0020] 2) Felculin (0.05~42.31%), Asafoetidanol A (0.013~1.17%), Rigpersin A (0.31~36.29%), Camoronol (1.50~46.67%), 10'-Acetoxy-11'-Hydroxyumbelliprenine (1.52~2.86%), Epi-Conferdione (0.24~12.24%), Calatabisinol (0.07~6.13%), Kelerin (1.43~17.63%), Ferrocauridine (0.19~2.23%), Farnesiferol A (0.13~14.31%), Galvanic Acid (0.05~52.56%), Suzowitziak A sesquiterpene coumarin concentrate, containing a sesquiterpene coumarin composition comprising marine B (5.18-16.18%), farnesiferol B (0.81-10.08%), gunmosine (2.58-4.03%), farnesiferol C (0.01-40.98%), suzowitsiacumarin A (0.28-7.01%), and methyl galvanate (0.70-10.77%), is added to a suitable oil phase in a ratio of 1:1 to 1:10 (w / w). The mixture is then treated at 10°C to 30°C with high-energy or low-energy stirring for 1 minute to 24 hours to obtain a clear and transparent solution. Furthermore, this solution is treated with an emulsifier and a co-surfactant in a ratio of 1:1 to 1:12.5 (w / w), with high or low-energy stirring at 10°C to 30°C for 1 minute to 24 hours. Furthermore, the resulting mixture is dispersed in water at a ratio of 1:0.5 to 1:20 (w / w) by stirring at high or low energy for 1 minute to 24 hours to obtain a water-soluble composition.

[0021] The resulting water-soluble composition improves the solubility, dispersibility, stability, and compliance with pharmaceutical standards of the hydrophobic sesquiterpene coumarin concentrate. More specifically, the present invention relates to the use of the above-mentioned water-soluble composition comprising a sesquiterpene coumarin concentrate that inhibits MAO enzymes (MAO-A and MAO-B) and imparts beneficial effects for the treatment and management of Parkinson's disease. The water-soluble composition can be formulated in liquid, solid, gel, and semi-solid dosage forms.

[0022] The water-soluble composition exhibits the following characteristics: The water-soluble composition comprises a suitable oil phase, a co-surfactant, an emulsifier, and sesquiterpene coumarin concentrates containing sesquiterpene coumarins having bicyclic, monocyclic, and acyclic sesquiterpene skeletons dissolved in water, in various proportions. • The composition has the general molecular formula C 24~26 H 26~36 O 3~6 It also contains sesquiterpene coumarins having molecular weights in the range of 362 to 444. Sesquiterpene coumarins include, but are not limited to, felculin (0.05-42.31%), asafoetidanol A (0.013-1.17%), ligpersin A (0.31-36.29%), chamoronol (1.50-46.67%), 10'-acetoxy-11'-hydroxyumbelliprenine (1.52-2.86%), epi-conferdione (0.24-12.24%), karatabisinol (0.07-6.13%), and kererin (1.43-17.63%). The following are selected: ferrocauridine (0.19-2.23%), farnesiferol A (0.13-14.31%), galvanic acid (0.05-52.56%), suzowitziacmarin B (5.18-16.18%), farnesiferol B (0.81-10.08%), gunmosine (2.58-4.03%), farnesiferol C (0.01-40.98%), suzowitziacmarin A (0.28-7.01%), and methyl galvanate (0.70-10.77%). The fatty acids or fatty acid esters contained in the oil phase are selected individually or in combination from caprylic acid, isopropyl myristate, olive oil, coconut oil, and ethyl oleate. Nonionic, cationic, and anionic emulsifiers are selected from Tween20, Tween60, Tween80, Span80, Span20, sodium lauryl sulfate, Brij35, dodecyltrimethylammonium chloride, trigol, and cetyltrimethylammonium bromide. The co-surfactant is selected from propylene glycol and PEG400. • A water-soluble composition containing a concentrated fraction of sesquiterpene coumarin inhibits MAO enzymes (MAO-A and MAO-B). • The water-soluble composition is miscible with polar solvents such as water, but not with nonpolar solvents. The water-soluble composition exhibits stability for at least one month in the pH range of 2.0 to 10.0. The water-soluble composition can be stored for at least three months at a temperature range of 10 to 40°C. Furthermore, the water-soluble composition can be formulated into various dosage forms such as solids, semi-solids, gels, and liquids. The water-soluble composition may be analyzed by HPLC, NMR, FT-IR, DLS, and TEM.

[0023] Therefore, water-soluble compositions containing concentrated sesquiterpene coumarin fractions may have future applications in the pharmaceutical, nutritional supplement, phytochemical, and healthcare industries in the management and treatment of Parkinson's disease through inhibition of MAO enzymes (MAO-A and MAO-B).

[0024] Inventive process related to the present invention This method differs fundamentally from known prior art in the following steps.

[0025] Step 1: Preparation of sesquiterpene coumarin concentrate A method for preparing a concentrated fraction of sesquiterpene coumarin, comprising the following steps: (a) extracting oleogum resin of the genus Ferula (asafoetida, i.e., Asafoetida) multiple times with an organic solvent with a polarity index of 2 to 6, preferably ethyl acetate (polarity index 4.4), collecting the extract and concentrating it until it becomes an ethyl acetate-free residue; and (b) concentrating the ethyl acetate-free residue obtained in step (a) with hexane-chloroform (100:0 to 0:100 v / v) and chloroform-methanol (100:0 to 70:30 (c) Step (b) is to separate the fraction obtained in step (b) using normal-phase silica gel with gradient elution using v / v to obtain a sesquiterpene coumarin concentrated fraction containing sesquiterpene coumarins having umbelliferone and ferulic acid in various ratios, and (d) step (b) is to further separate the fraction obtained in step (b) using gradient elution with acetonitrile-water and reverse-phase (C18) HPLC with octadecylsilane to obtain pure sesquiterpene coumarin, and (d) step (b) is to separate the sesquiterpene coumarin concentrated fraction from feruclin (0.05~42.31%), asafoetidanol A (0.013~1.17%), ligpersin A (0.31~36.29%), chamoronol (1.50~46.67%), 10'-acetoxy- A preparation method comprising the steps of: 11'-hydroxyumbelliprenine (1.52-2.86%), epi-conferdione (0.24-12.24%), karatabisinol (0.07-6.13%), kerelin (1.43-17.63%), ferrocauridine (0.19-2.23%), farnesiferol A (0.13-14.31%), galvanic acid (0.05-52.56%), suzowitziacmarin B (5.18-16.18%), farnesiferol B (0.81-10.08%), gunmosine (2.58-4.03%), farnesiferol C (0.01-40.98%), suzowitziacmarin A (0.28-7.01%), and methyl galvanate (0.70-10.77%).

[0026] Step 2: Preparation of water-soluble composition A sesquiterpene coumarin concentrate containing sesquiterpene coumarin in various proportions is added in a 1:1 to 1:10 (w / w) ratio to an oil phase containing a suitable fatty acid or fatty acid ester selected individually or in combination from, but not limited to, caprylic acid, isopropyl myristate, olive oil, coconut oil, sunflower oil, and ethyl oleate. A clear and transparent solution is obtained by dissolving the fraction by applying high or low levels of external energy at 10°C to 40°C for 5 minutes to 24 hours. The solution is further treated with a nonionic, cationic, or anionic emulsifier selected from, but not limited to, Tween20, Tween60, Tween80, Span80, Span20, sodium lauryl sulfate, Brij35, dodecyltrimethylammonium chloride, trigol, and cetyltrimethylammonium bromide, in a ratio of 1:1.1 to 1:12.5 (w / w), with or without a co-surfactant, by stirring at high or low energy at 10°C to 30°C for 1 minute to 24 hours. Furthermore, the resulting mixture is dispersed in water in a ratio of 1:0.5 to 1:20 (w / w), by stirring at high or low energy for 1 minute to 24 hours, to obtain a water-soluble composition. [Examples]

[0027] The following examples are for illustrative purposes only and should not be construed as limiting the scope of the present invention.

[0028] (Example 1) 1.75 kg of oleogum resin (asafoetida) of the genus Ferula was added to a clean round-bottom flask and extracted with 3.5 L of ethyl acetate under continuous stirring (500 rpm) for 2 hours at room temperature (25-30°C). The ethyl acetate was filtered, and the residue was re-extracted three more times. The pooled ethyl acetate extract was evaporated under reduced pressure at 45-50°C to obtain a reddish oily residue with a strong sulfurous odor. This oily residue was subjected to chromatography using hexane, chloroform, and chloroform:methanol (8:2 v / v) in a silica gel layer to obtain three fine fractions. Furthermore, the fine fraction eluted with chloroform was subjected to silica gel column chromatography, and eluted in order of increasing polarity using hexane and hexane:chloroform. The fine fraction eluted in hexane:chloroform (75:25 v / v) was dried under reduced pressure to obtain a rubbery residue concentrated with sesquiterpene coumarins.

[0029] To prepare the water-soluble composition, 0.5 g of sesquiterpene coumarin concentrate was treated with 3.35 g of caprylic acid with the help of high-energy sonication at 30°C for 30 minutes to obtain a clear and transparent solution. The resulting solution was subjected to high-energy sonication for 1 hour with 6.7 g of Tween20 at 30°C, and then dispersed in 210 g of water after further high-energy sonication for 30 minutes to obtain a translucent, milky white water-soluble composition.

[0030] The obtained sesquiterpene coumarin concentrate and its water-soluble composition were characterized and confirmed to exhibit the following properties.

[0031] The sesquiterpene coumarin concentrate is rubbery and reddish-brown. • Sesquiterpene coumarin concentrates are miscible with nonpolar to moderately polar solvents, but not with water. The sesquiterpene coumarin concentrate contains, as measured by area at 325 nm in HPLC, umbelliferone at 31.09%, galvanic acid at 19.28%, farnesiferol C at 40.98%, suzowitziacumarin A at 2.49%, ferrocauridine at 0.64%, methyl galvanate at 2.13%, and calatabisinol at 1.24%. • The sesquiterpene coumarin concentrate is MAO-A(IC). 50 0.06952 mg / mL) inhibitory activity and MAO-B (IC) 50 It showed inhibitory activity at 0.01319 mg / mL. • The sesquiterpene coumarin concentrate is unstable in the pH range of 2.0 to 10.0 and the temperature range of 30 to 80°C. • Water-soluble compositions containing concentrated sesquiterpene coumarin fractions are white in color. • Water-soluble compositions containing concentrated sesquiterpene coumarin fractions are miscible with water but not with nonpolar solvents. • Water-soluble compositions containing concentrated sesquiterpene coumarin fractions are stable in the pH range of 2.0 to 10.0. • A water-soluble composition containing a concentrated fraction of sesquiterpene coumarin inhibits MAO enzymes. • Water-soluble compositions containing sesquiterpene coumarin concentrates exhibit characteristic peaks in HPLC and NMR.

[0032] (Example 2) 1.75 kg of ferula oleogum resin (asafoetida) was added to a clean round-bottom flask and extracted with 3.5 L of ethyl acetate under continuous stirring (500 rpm) for 2 hours at room temperature (25-30°C). The ethyl acetate was filtered, and the residue was re-extracted three more times. The pooled ethyl acetate extract was evaporated under reduced pressure at 45-50°C to obtain a reddish oily residue with a strong sulfurous odor. This oily residue was subjected to chromatography using hexane, chloroform, and chloroform:methanol (8:2 v / v) in a silica gel layer to obtain three fine fractions. Furthermore, the fraction eluted with chloroform was subjected to silica gel column chromatography, and eluted in order of increasing polarity using hexane and hexane:chloroform. The fine fraction eluted in hexane:chloroform (75:25 v / v) was dried under reduced pressure to obtain a rubbery residue concentrated with sesquiterpene coumarins.

[0033] To prepare the water-soluble composition, 1 g of sesquiterpene coumarin concentrated fraction was treated with 1.75 g of caprylic acid with the help of high-energy sonication at 40°C for 10 minutes to obtain a clear and transparent solution. The resulting mixture was treated with 34.25 g of Tween60 / PG (1:1 w / w) at 25°C with low-energy stirring for 24 hours, and then dispersed in 110 g of water with high-energy sonication for 1 minute to obtain a translucent water-soluble composition.

[0034] The obtained sesquiterpene coumarin concentrate and its water-soluble composition were characterized and confirmed to exhibit the following properties.

[0035] The sesquiterpene coumarin concentrate is a rubbery, semi-solid, reddish-brown substance. • Sesquiterpene coumarin concentrates are miscible with nonpolar to moderately polar solvents, but not with water. The sesquiterpene coumarin concentrate contains, as measured by area at 325 nm in HPLC, ferulic acid at 0.67%, umbelliferone at 17.76%, galvanic acid at 44.56%, farnesiferol B at 0.81%, calatabisinol at 0.62%, ferrocauridine at 0.19%, farnesiferol C at 19.14%, szowitziacumarin A at 1.43%, and methyl galvanate at 1.97%. • The sesquiterpene coumarin concentrate is MAO-A(IC). 50 0.0403 mg / mL) inhibitory activity and MAO-B (IC) 50 It showed inhibitory activity at 0.0040 mg / mL. • The sesquiterpene coumarin concentrate is unstable in the pH range of 2.0 to 10.0 and the temperature range of 30 to 80°C. • The water-soluble composition containing the concentrated fraction of sesquiterpene coumarin is pale yellow. • Water-soluble compositions containing concentrated sesquiterpene coumarin fractions are miscible with water but not with nonpolar solvents. • Water-soluble compositions containing concentrated sesquiterpene coumarin fractions are stable in the pH range of 2.0 to 10.0. • A water-soluble composition containing a concentrated fraction of sesquiterpene coumarin inhibits MAO enzymes. • Water-soluble compositions containing sesquiterpene coumarin concentrates exhibit characteristic peaks in HPLC and NMR.

[0036] (Example 3) 1.75 kg of oleogum resin of the genus Ferula was added to a clean round-bottom flask and extracted with 3.5 L of ethyl acetate under continuous stirring (500 rpm) for 2 hours at room temperature (25-30°C). The ethyl acetate was filtered, and the residue was re-extracted three more times. The pooled ethyl acetate extract was evaporated under reduced pressure at 45-50°C to obtain a reddish oily residue with a strong sulfurous odor. This oily residue was subjected to chromatography using hexane, chloroform, and chloroform:methanol (8:2 v / v) in a silica gel layer to obtain three fine fractions. Furthermore, the fraction eluted with chloroform was subjected to silica gel column chromatography, and eluted in order of increasing polarity using hexane and hexane:chloroform. The fine fraction eluted in hexane:chloroform (75:25 v / v) was dried under reduced pressure to obtain a rubbery residue concentrated with sesquiterpene coumarins.

[0037] To prepare the water-soluble composition, 0.5 g of sesquiterpene coumarin concentrate was treated with 2.5 g of caprylic acid and olive oil (1:1 w / w) with the help of stirring at 30°C for 2 hours to obtain a clear and transparent solution. The resulting mixture was treated with 12.5 g of Tween 20 and sodium lauryl sulfate (8:2 w / w) with the help of low-energy stirring at 20°C for 180 minutes, and then dispersed in 45 g of water with stirring for 3 hours to obtain a milky white water-soluble composition.

[0038] The obtained sesquiterpene coumarin concentrate and its water-soluble composition were characterized and confirmed to exhibit the following properties.

[0039] The sesquiterpene coumarin concentrate is a rubbery, brownish solid. • Sesquiterpene coumarin concentrates are miscible with nonpolar to moderately polar solvents, but not with water. · In the sesquiterpene coumarin concentrated fraction, as the area at 325 nm in HPLC, ferulic acid is 0.79%, umbelliferone is 19.83%, galbanic acid is 52.56%, farnesiferol B is 10.08%, farnesiferol C is 7.66%, szowiakumarin A is 0.72%, and methyl galbanate is 1.36%. · The sesquiterpene coumarin concentrated fraction has MAO-A (IC 50 0.0643 mg / mL) inhibitory activity and MAO-B (IC 50 0.0016 mg / mL) inhibitory activity. · The sesquiterpene coumarin concentrated fraction becomes unstable in the pH range of 2.0 to 10.0 and the temperature range of 30 to 80 °C. · The water-soluble composition containing the sesquiterpene coumarin concentrated fraction is white. · The water-soluble composition containing the sesquiterpene coumarin concentrated fraction is miscible with water but not with nonpolar solvents. · The water-soluble composition containing the sesquiterpene coumarin concentrated fraction is stable in the pH range of 2.0 to 10.0. · The water-soluble composition containing the sesquiterpene coumarin concentrated fraction inhibits MAO enzymes. · The water-soluble composition containing the sesquiterpene coumarin concentrated fraction shows characteristic peaks in HPLC and NMR.

[0040] (Example 4) 1.75 kg of oleogum resin (asafoetida) of the genus Ferula was added to a clean round-bottom flask and extracted with 3.5 L of ethyl acetate under continuous stirring (500 rpm) for 2 hours at room temperature (25-30°C). The ethyl acetate was filtered, and the residue was re-extracted three more times. The pooled ethyl acetate extract was evaporated under reduced pressure at 45-50°C to obtain a reddish oily residue with a strong sulfurous odor. This oily residue was subjected to chromatography using hexane, chloroform, and chloroform:methanol (8:2 v / v) in a silica gel layer to obtain three fine fractions. Furthermore, the fine fraction eluted with chloroform was subjected to silica gel column chromatography, and eluted in order of increasing polarity using hexane and hexane:chloroform. The fine fraction eluted in hexane:chloroform (75:25 v / v) was dried under reduced pressure to obtain a rubbery residue concentrated with sesquiterpene coumarins.

[0041] To prepare the water-soluble composition, 0.5 g of sesquiterpene coumarin concentrate was treated with 1.5 g of olive oil with the help of low-energy stirring at 20°C for 6 hours to obtain a clear and transparent solution. The resulting mixture was subjected to high-energy sonication at 15°C for 15 minutes and then treated with 12 g of Brij 35, propylene glycol, and sodium lauryl sulfate (2:2:1 w / w). Finally, it was dispersed in 24 g of water with the help of low-energy stirring for 45 minutes to obtain a milky white water-soluble composition.

[0042] The obtained sesquiterpene coumarin concentrate and its water-soluble composition were characterized and confirmed to exhibit the following properties.

[0043] The sesquiterpene coumarin concentrate is oily and reddish-brown. • Sesquiterpene coumarin concentrates are miscible with nonpolar to moderately polar solvents, but not with water. The sesquiterpene coumarin concentrated fraction contains 0.58% ferulic acid, 30.96% umbelliferone, 43.67% galvanic acid, 5.94% farnesiferol B, 0.39% szowitziacumarin A, 1.49% farnesiferol C, and 2.37% methyl galvanate, as measured by area at 325 nm in HPLC. • The sesquiterpene coumarin concentrate is MAO-A(IC). 50 0.0631 mg / mL) inhibitory activity and MAO-B (IC) 50 It showed inhibitory activity at 0.0024 mg / mL. • The sesquiterpene coumarin concentrate is unstable in the pH range of 2.0 to 10.0 and the temperature range of 30 to 80°C. • The water-soluble composition containing the concentrated fraction of sesquiterpene coumarin is white. • Water-soluble compositions containing concentrated sesquiterpene coumarin fractions are miscible with water but not with nonpolar solvents. • Water-soluble compositions containing concentrated sesquiterpene coumarin fractions are stable in the pH range of 2.0 to 10.0. • A water-soluble composition containing a concentrated fraction of sesquiterpene coumarin inhibits MAO enzymes. • Water-soluble compositions containing sesquiterpene coumarin concentrates exhibit characteristic peaks in HPLC and NMR.

[0044] (Example 5) 1.75 kg of oleogum resin (asafoetida) of the genus Ferula was added to a clean round-bottom flask and extracted with 3.5 L of ethyl acetate under continuous stirring (500 rpm) for 2 hours at room temperature (25-30°C). The ethyl acetate was filtered, and the residue was re-extracted three more times. The pooled ethyl acetate extract was evaporated under reduced pressure at 45-50°C to obtain a reddish oily residue with a strong sulfurous odor. This oily residue was subjected to chromatography using hexane, chloroform, and chloroform:methanol (8:2 v / v) in a silica gel layer to obtain three fine fractions. Furthermore, the fine fraction eluted with chloroform was subjected to silica gel column chromatography, and eluted in order of increasing polarity using hexane and hexane:chloroform. The fine fraction eluted in hexane:chloroform (50:50 v / v) was dried under reduced pressure to obtain a rubbery residue concentrated with sesquiterpene coumarins.

[0045] To prepare the water-soluble composition, 2.5 g of sesquiterpene coumarin concentrate was treated with 15 g of olive oil and caprylic acid (0.1:1 w / w) with the help of low-energy stirring at 25°C for 12 hours to obtain a clear and transparent solution. The resulting mixture was then treated with 35 g of Span 80 and dodecyltrimethylammonium chloride (8:2 w / w) with the help of high-energy sonication at 30°C for 45 minutes, and further dispersed in 525 g of water with low-energy stirring for 24 hours to obtain an opaque, milky white water-soluble composition.

[0046] The obtained sesquiterpene coumarin concentrate and its water-soluble composition were characterized and confirmed to exhibit the following properties.

[0047] The sesquiterpene coumarin concentrate is oily and reddish-brown. • Sesquiterpene coumarin concentrates are miscible with nonpolar to moderately polar solvents, but not with water. The sesquiterpene coumarin concentrate contains, as measured by area at 325 nm in HPLC, 16.25% umbelliferone, 49.42% galvanic acid, 1.63% farnesiferol B, 5.61% farnesiferol C, 2.28% epi-conferdione, 1.16% ferrocauridine, 3.79% farnesiferol A, and 10.77% methyl galvanate. • The sesquiterpene coumarin concentrate is MAO-A(IC). 50 0.0075 mg / mL) inhibitory activity and MAO-B (IC) 50 It showed inhibitory activity at 0.0047 mg / mL. • The sesquiterpene coumarin concentrate is unstable in the pH range of 2.0 to 10.0 and the temperature range of 30 to 80°C. • Water-soluble compositions containing concentrated sesquiterpene coumarin fractions are white in color. • Water-soluble compositions containing concentrated sesquiterpene coumarin fractions are miscible with water but not with nonpolar solvents. • Water-soluble compositions containing concentrated sesquiterpene coumarin fractions are stable in the pH range of 2.0 to 10.0. • A water-soluble composition containing a concentrated fraction of sesquiterpene coumarin inhibits MAO enzymes. • Water-soluble compositions containing sesquiterpene coumarin concentrates exhibit characteristic peaks in HPLC and NMR analysis.

[0048] (Example 6) 1.75 kg of oleogum resin (asafoetida) of the genus Ferula was added to a clean round-bottom flask and extracted with 3.5 L of ethyl acetate under continuous stirring (500 rpm) for 2 hours at room temperature (25-30°C). The ethyl acetate was filtered, and the residue was re-extracted three more times. The pooled ethyl acetate extract was evaporated under reduced pressure at 45-50°C to obtain a reddish oily residue with a strong sulfurous odor. This oily residue was subjected to chromatography using hexane, chloroform, and chloroform:methanol (8:2 v / v) in a silica gel layer to obtain three fine fractions. Furthermore, the fine fraction eluted with chloroform was subjected to silica gel column chromatography, and eluted in order of increasing polarity using hexane and hexane:chloroform. The fine fraction eluted in hexane:chloroform (50:50 v / v) was dried under reduced pressure to obtain a rubbery residue concentrated with sesquiterpene coumarins.

[0049] To prepare the water-soluble composition, 2.5 g of sesquiterpene coumarin concentrate was treated with 2.5 g of isopropyl myristate with the help of high-energy sonication at 15°C for 8 hours to obtain a clear and transparent solution. The resulting mixture was then treated with 22.5 g of Tween 60 and trigol (1:1 w / w) with the help of high-energy sonication at 30°C for 120 minutes, and further dispersed in 16.5 g of water with low-energy stirring for 10 hours to obtain a translucent brownish water-soluble composition.

[0050] The obtained sesquiterpene coumarin concentrate and its water-soluble composition were characterized and confirmed to exhibit the following properties.

[0051] The sesquiterpene coumarin concentrate is oily and reddish-brown. • Sesquiterpene coumarin concentrates are miscible with nonpolar to moderately polar solvents, but not with water. The sesquiterpene coumarin concentrate contains, as measured by area at 325 nm in HPLC, ferulic acid at 1.16%, umbelliferone at 7.26%, chamoronol at 2.30%, ferrocauridine at 1.03%, epiconferdione at 12.24%, querelin at 17.63%, karatabisinol at 0.42%, farnesiferol A at 14.31%, galvanic acid at 28.46%, farnesiferol C at 1.10%, and methyl galvanate at 4.96%. • The sesquiterpene coumarin concentrate is MAO-A(IC). 50 0.0101 mg / mL) inhibitory activity and MAO-B (IC) 50 It showed inhibitory activity at 0.0012 mg / mL. • The sesquiterpene coumarin concentrate is unstable in the pH range of 2.0 to 10.0 and the temperature range of 30 to 80°C. • The water-soluble composition containing the sesquiterpene coumarin concentrate is yellowish-white. • Water-soluble compositions containing concentrated sesquiterpene coumarin fractions are miscible with water but not with nonpolar solvents. • Water-soluble compositions containing concentrated sesquiterpene coumarin fractions are stable in the pH range of 2.0 to 10.0. • A water-soluble composition containing a concentrated fraction of sesquiterpene coumarin inhibits MAO enzymes. • Water-soluble compositions containing sesquiterpene coumarin concentrates exhibit characteristic peaks in HPLC and NMR.

[0052] (Example 7) 1.75 kg of oleogum resin (asafoetida) of the genus Ferula was added to a clean round-bottom flask and extracted with 3.5 L of ethyl acetate under continuous stirring (500 rpm) for 2 hours at room temperature (25-30°C). The ethyl acetate was filtered, and the residue was re-extracted three more times. The pooled ethyl acetate extract was evaporated under reduced pressure at 45-50°C to obtain a reddish oily residue with a strong sulfurous odor. This oily residue was subjected to chromatography using hexane, chloroform, and chloroform:methanol (8:2 v / v) in a silica gel layer to obtain three fine fractions. Furthermore, the fine fraction eluted with chloroform was subjected to silica gel column chromatography, and eluted in order of increasing polarity using hexane and hexane:chloroform. The fine fraction eluted in hexane:chloroform (50:50 v / v) was dried under reduced pressure to obtain a rubbery residue concentrated with sesquiterpene coumarins.

[0053] To prepare the water-soluble composition, 0.5 g of sesquiterpene coumarin concentrate was treated with 5 g of caprylic acid and isopropyl myristate (1:1 w / w) with the help of low-energy stirring at 20°C for 20 hours to obtain a clear and transparent solution. The resulting solution was then treated with 44 g of Span20, PEG400 and sodium lauryl sulfate (2:6:2 w / w) with the help of high-energy sonication at 15°C for 90 minutes, and further dispersed in 198 g of water with low-energy stirring for 10 minutes to obtain a clear and transparent water-soluble composition.

[0054] The obtained sesquiterpene coumarin concentrate and its water-soluble composition were characterized and confirmed to exhibit the following properties.

[0055] The sesquiterpene coumarin concentrate is oily and reddish-brown. • Sesquiterpene coumarin concentrates are miscible with nonpolar to moderately polar solvents, but not with water. The sesquiterpene coumarin concentrate contains, as measured by area at 325 nm in HPLC, ferulic acid at 1.05%, umbelliferone at 11.10%, chamoronol at 18.42%, epi-conferdione at 5.87%, kerelin at 12.70%, karatabisinol at 2.34%, farnesiferol A at 9.68%, suzowitziaquamarine B at 16.18%, farnesiferol C at 1.0%, suzowitziaquamarine A at 0.61%, and methyl galvanate at 9.08%. • The sesquiterpene coumarin concentrate is MAO-A(IC). 50 0.0095 mg / mL) inhibitory activity and MAO-B (IC) 50 It showed inhibitory activity at 0.0075 mg / mL. • The sesquiterpene coumarin concentrate is unstable in the pH range of 2.0 to 10.0 and the temperature range of 30 to 80°C. • The water-soluble composition containing the concentrated fraction of sesquiterpene coumarin is pale yellow. • Water-soluble compositions containing concentrated sesquiterpene coumarin fractions are miscible with water but not with nonpolar solvents. • Water-soluble compositions containing concentrated sesquiterpene coumarin fractions are stable in the pH range of 2.0 to 10.0. • A water-soluble composition containing a concentrated fraction of sesquiterpene coumarin inhibits MAO enzymes. • Water-soluble compositions containing sesquiterpene coumarin concentrates exhibit characteristic peaks in HPLC and NMR.

[0056] (Example 8) 1.75 kg of oleogum resin (asafoetida) of the genus Ferula was added to a clean round-bottom flask and extracted with 3.5 L of ethyl acetate under continuous stirring (500 rpm) for 2 hours at room temperature (25-30°C). The ethyl acetate was filtered, and the residue was re-extracted three more times. The pooled ethyl acetate extract was evaporated under reduced pressure at 45-50°C to obtain a reddish oily residue with a strong sulfurous odor. This oily residue was subjected to chromatography using hexane, chloroform, and chloroform:methanol (8:2 v / v) in a silica gel layer to obtain three fine fractions. Furthermore, the fine fraction eluted with chloroform was subjected to silica gel column chromatography, and eluted in order of increasing polarity using hexane and hexane:chloroform. The fine fraction eluted in hexane:chloroform (50:50 v / v) was dried under reduced pressure to obtain a rubbery residue concentrated with sesquiterpene coumarins.

[0057] To prepare the water-soluble composition, 0.5 g of sesquiterpene coumarin concentrate was treated with 4.5 g of isopropyl myristate at 10°C for 24 hours with the help of low-energy stirring to obtain a clear and transparent solution. The resulting solution was then treated with 50 g of Tween 80 with the help of high-energy sonication at 30°C for 1 minute, and further dispersed in 110 g of water with low-energy stirring for 30 minutes to obtain a clear and transparent water-soluble composition.

[0058] The obtained sesquiterpene coumarin concentrate and its water-soluble composition were characterized and confirmed to exhibit the following properties.

[0059] The sesquiterpene coumarin concentrate is oily and reddish-brown. • Sesquiterpene coumarin concentrates are miscible with nonpolar to moderately polar solvents, but not with water. The sesquiterpene coumarin concentrate contains, as measured by area at 325 nm in HPLC, ferulic acid at 0.99%, umbelliferone at 13.08%, chamoronol at 46.67%, epi-conferdione at 0.24%, kerelin at 1.43%, karatabisinol at 1.92%, acetoxyumbelliprenine at 1.52%, gunmosine at 4.03%, suzowitsiaquamarine B at 5.18%, suzowitsiaquamarine A at 7.01%, and methyl galvanate at 1.13%. • The sesquiterpene coumarin concentrate is MAO-A(IC). 50 0.0901 mg / mL) inhibitory activity and MAO-B (IC) 50 It showed inhibitory activity at 0.0373 mg / mL. • The sesquiterpene coumarin concentrate is unstable in the pH range of 2.0 to 10.0 and the temperature range of 30 to 80°C. • Water-soluble compositions containing concentrated sesquiterpene coumarin fractions are yellowish in color. • Water-soluble compositions containing concentrated sesquiterpene coumarin fractions are miscible with water but not with nonpolar solvents. • Water-soluble compositions containing concentrated sesquiterpene coumarin fractions are stable in the pH range of 2.0 to 10.0. • A water-soluble composition containing a concentrated fraction of sesquiterpene coumarin inhibits MAO enzymes. • Water-soluble compositions containing sesquiterpene coumarin concentrates exhibit characteristic peaks in HPLC and NMR.

[0060] (Example 9) 1.75 kg of oleogum resin (asafoetida) of the genus Ferula was added to a clean round-bottom flask and extracted with 3.5 L of ethyl acetate under continuous stirring (500 rpm) for 2 hours at room temperature (25-30°C). The ethyl acetate was filtered, and the residue was re-extracted three more times. The pooled ethyl acetate extract was evaporated under reduced pressure at 45-50°C to obtain a reddish oily residue with a strong sulfurous odor. This oily residue was subjected to chromatography using hexane, chloroform, and chloroform:methanol (8:2 v / v) in a silica gel layer to obtain three fine fractions. Furthermore, the fine fraction eluted with chloroform was subjected to silica gel column chromatography, and eluted in order of increasing polarity using hexane and hexane:chloroform. The fine fraction eluted in hexane:chloroform (50:50 v / v) was dried under reduced pressure to obtain a rubbery residue concentrated with sesquiterpene coumarins.

[0061] To prepare the water-soluble composition, 0.5 g of sesquiterpene coumarin concentrate was treated with 4.3 g of coconut oil and caprylic acid (1:1 w / w) with the help of low-energy stirring at 35°C for 3.5 hours to obtain a clear and transparent solution. The resulting solution was then treated with 15.75 g of Tween 80, propylene glycol, and dodecyltrimethylammonium chloride (6:2:2 w / w) with the help of high-energy sonication at 20°C for 30 minutes, and further dispersed in 98 g of water with low-energy stirring for 12 hours to obtain a clear and transparent water-soluble composition.

[0062] The obtained sesquiterpene coumarin concentrate and its water-soluble composition were characterized and confirmed to exhibit the following properties.

[0063] The sesquiterpene coumarin concentrate is oily and reddish-brown. • Sesquiterpene coumarin concentrates are miscible with nonpolar to moderately polar solvents, but not with water. The sesquiterpene coumarin concentrate contains, as measured by area at 325 nm in HPLC, ferulic acid at 1.74%, umbelliferone at 19.55%, acetoxyumbelliprenine at 2.86%, chamoronol at 43.28%, karatabisinol at 6.13%, gunmosine at 2.58%, suzowitsiaquamarine B at 5.94%, suzowitsiaquamarine A at 4.72%, and farnesiferol A at 0.13%. • The sesquiterpene coumarin concentrate is MAO-A(IC). 50 0.0300 mg / mL) inhibitory activity and MAO-B (IC) 50 It showed inhibitory activity at 0.0038 mg / mL. • The sesquiterpene coumarin concentrate is unstable in the pH range of 2.0 to 10.0 and the temperature range of 30 to 80°C. • The water-soluble composition containing the concentrated fraction of sesquiterpene coumarin is light brown in color. • Water-soluble compositions containing concentrated sesquiterpene coumarin fractions are miscible with water but not with nonpolar solvents. • Water-soluble compositions containing concentrated sesquiterpene coumarin fractions are stable in the pH range of 2.0 to 10.0. • A water-soluble composition containing a concentrated fraction of sesquiterpene coumarin inhibits MAO enzymes. • Water-soluble compositions containing sesquiterpene coumarin concentrates exhibit characteristic peaks in HPLC and NMR.

[0064] (Example 10) 1.75 kg of oleogum resin (asafoetida) of the genus Ferula was added to a clean round-bottom flask and extracted with 3.5 L of ethyl acetate under continuous stirring (500 rpm) for 2 hours at room temperature (25-30°C). The ethyl acetate was filtered, and the residue was re-extracted three more times. The pooled ethyl acetate extract was evaporated under reduced pressure at 45-50°C to obtain a reddish oily residue with a strong sulfurous odor. This oily residue was subjected to chromatography using hexane, chloroform, and chloroform:methanol (8:2 v / v) in a silica gel layer to obtain three fine fractions. Furthermore, the fine fraction eluted with chloroform was subjected to silica gel column chromatography, and eluted in order of increasing polarity using hexane and hexane:chloroform. The fine fraction eluted in chloroform was dried under reduced pressure to obtain a rubbery residue concentrated with sesquiterpene coumarins.

[0065] To prepare the water-soluble composition, a clear and transparent solution was obtained by treating 0.5 g of sesquiterpene coumarin concentrate with 2 g of coconut oil with the help of high-energy sonication at 30°C for 4 hours. The resulting solution was then treated with 10 g of Tween60 and PEG400 (1:1 w / w) with the help of high-energy sonication at 20°C for 30 minutes, and further dispersed in 37.5 g of water by low-energy stirring for 5 hours to obtain a clear and transparent water-soluble composition.

[0066] The obtained sesquiterpene coumarin concentrate and its water-soluble composition were characterized and confirmed to exhibit the following properties.

[0067] The sesquiterpene coumarin concentrate is oily and reddish-brown. • Sesquiterpene coumarin concentrates are miscible with nonpolar to moderately polar solvents, but not with water. The sesquiterpene coumarin concentrate contains, as measured by area at 325 nm in HPLC, ferulic acid at 39.10%, umbelliferone at 8.97%, felculin at 6.89%, chamoronol at 1.50%, ligpersin A at 36.29%, karatabisinol at 0.75%, farnesiferol A at 1.37%, farnesiferol C at 0.23%, szowitziacumarin A at 0.48%, and methyl galvanate at 0.70%. • The sesquiterpene coumarin concentrate is MAO-A(IC). 50 0.0699 mg / mL) inhibitory activity and MAO-B (IC) 50 It showed inhibitory activity at 0.0068 mg / mL. • The sesquiterpene coumarin concentrate is unstable in the pH range of 2.0 to 10.0 and the temperature range of 30 to 80°C. • The water-soluble composition containing the concentrated fraction of sesquiterpene coumarin is light brown in color. • Water-soluble compositions containing concentrated sesquiterpene coumarin fractions are miscible with water but not with nonpolar solvents. • Water-soluble compositions containing concentrated sesquiterpene coumarin fractions are stable in the pH range of 2.0 to 10.0. • A water-soluble composition containing a concentrated fraction of sesquiterpene coumarin inhibits MAO enzymes. • Water-soluble compositions containing sesquiterpene coumarin concentrates exhibit characteristic peaks in HPLC and NMR.

[0068] (Example 11) 1.75 kg of oleogum resin (asafoetida) of the genus Ferula was added to a clean round-bottom flask and extracted with 3.5 L of ethyl acetate under continuous stirring (500 rpm) for 2 hours at room temperature (25-30°C). The ethyl acetate was filtered, and the residue was re-extracted three more times. The pooled ethyl acetate extract was evaporated under reduced pressure at 45-50°C to obtain a reddish oily residue with a strong sulfurous odor. This oily residue was subjected to chromatography using hexane, chloroform, and chloroform:methanol (8:2 v / v) in a silica gel layer to obtain three fine fractions. Furthermore, the fine fraction eluted with chloroform was subjected to silica gel column chromatography, and eluted in order of increasing polarity using hexane and hexane:chloroform. The fine fraction eluted in chloroform was dried under reduced pressure to obtain a rubbery residue concentrated with sesquiterpene coumarins.

[0069] To prepare the water-soluble composition, 0.5 g of sesquiterpene coumarin concentrate was treated with 4 g of ethyl oleate with the help of low-energy stirring at 40°C for 15 hours to obtain a clear and transparent solution. The resulting solution was then treated with 27 g of Brij35 and PEG400 (1:1 w / w) with the help of high-energy sonication at 10°C for 60 minutes, and further dispersed in 126 g of water with low-energy stirring for 2 hours to obtain a translucent, milky white water-soluble composition.

[0070] The obtained sesquiterpene coumarin concentrate and its water-soluble composition were characterized and confirmed to exhibit the following properties.

[0071] The sesquiterpene coumarin concentrate is a sticky solid that is reddish-brown in color. • Sesquiterpene coumarin concentrates are miscible with nonpolar to moderately polar solvents, but not with water. The sesquiterpene coumarin concentrate contains 85.20% ferulic acid, 2.09% umbelliferone, 7.37% felculin, 1.17% asafoetidanol A, 1.48% ligpersin A, 0.52% calatabisinol, 0.27% farnesiferol A, and 0.17% galvanic acid, as measured by area at 325 nm in HPLC. • The sesquiterpene coumarin concentrate is MAO-A(IC). 50 0.0568 mg / mL) inhibitory activity and MAO-B (IC) 50 It showed inhibitory activity at 0.0141 mg / mL. • The sesquiterpene coumarin concentrate is unstable in the pH range of 2.0 to 10.0 and the temperature range of 30 to 80°C. • The water-soluble composition containing the concentrated fraction of sesquiterpene coumarin is white. • Water-soluble compositions containing concentrated sesquiterpene coumarin fractions are miscible with water but not with nonpolar solvents. • Water-soluble compositions containing concentrated sesquiterpene coumarin fractions are stable in the pH range of 2.0 to 10.0. • A water-soluble composition containing a concentrated fraction of sesquiterpene coumarin inhibits MAO enzymes. • Water-soluble compositions containing sesquiterpene coumarin concentrates exhibit characteristic peaks in HPLC and NMR.

[0072] (Example 12) 1.75 kg of oleogum resin (asafoetida) of the genus Ferula was added to a clean round-bottom flask and extracted with 3.5 L of ethyl acetate under continuous stirring (500 rpm) for 2 hours at room temperature (25-30°C). The ethyl acetate was filtered, and the residue was re-extracted three more times. The pooled ethyl acetate extract was evaporated under reduced pressure at 45-50°C to obtain a reddish oily residue with a strong sulfurous odor. This oily residue was subjected to chromatography using hexane, chloroform, and chloroform:methanol (8:2 v / v) in a silica gel layer to obtain three fine fractions. Furthermore, the fine fraction eluted with chloroform:methanol (8:2 v / v) was subjected to silica gel column chromatography, and eluted using hexane and hexane:chloroform in order of increasing polarity. The fine fraction eluted in chloroform was dried under reduced pressure to obtain a rubbery residue concentrated with sesquiterpene coumarins.

[0073] To prepare the water-soluble composition, 0.5 g of sesquiterpene coumarin concentrate was treated with 3.5 g of caprylic acid with the help of low-energy stirring at 10°C for 8 hours to obtain a clear and transparent solution. The resulting solution was then treated with 4 g of sodium lauryl sulfate at 25°C with low-energy stirring for 1 hour, and further dispersed in 27.5 g of water after high-energy sonication for 30 minutes to obtain a translucent white water-soluble composition.

[0074] The obtained sesquiterpene coumarin concentrate and its water-soluble composition were characterized and confirmed to exhibit the following properties.

[0075] The sesquiterpene coumarin concentrate is oily and reddish-brown. • Sesquiterpene coumarin concentrates are miscible with nonpolar to moderately polar solvents, but not with water. The sesquiterpene coumarin concentrate contains 98.61% ferulic acid, 0.05% felculin, 0.13% asafoetidanol A, 0.31% ligpersin A, 0.07% calatabisinol, 0.01% farnesiferol C, and 0.05% galvanic acid, as measured by area at 325 nm in HPLC. • The sesquiterpene coumarin concentrate is MAO-A(IC). 50 0.0526 mg / mL) inhibitory activity and MAO-B (IC) 50 It showed inhibitory activity at 0.0198 mg / mL. • The sesquiterpene coumarin concentrate is unstable in the pH range of 2.0 to 10.0 and the temperature range of 30 to 80°C. • The water-soluble composition containing the concentrated fraction of sesquiterpene coumarin is yellowish-brown in color. • Water-soluble compositions containing concentrated sesquiterpene coumarin fractions are miscible with water but not with nonpolar solvents. • Water-soluble compositions containing concentrated sesquiterpene coumarin fractions are stable in the pH range of 2.0 to 10.0. • A water-soluble composition containing a concentrated fraction of sesquiterpene coumarin inhibits MAO enzymes. • Water-soluble compositions containing sesquiterpene coumarin concentrates exhibit characteristic peaks in HPLC and NMR.

[0076] (Example 13) 1.75 kg of oleogum resin (asafoetida) of the genus Ferula was added to a clean round-bottom flask and extracted with 3.5 L of ethyl acetate under continuous stirring (500 rpm) for 2 hours at room temperature (25-30°C). The ethyl acetate was filtered, and the residue was re-extracted three more times. The pooled ethyl acetate extract was evaporated under reduced pressure at 45-50°C to obtain a reddish oily residue with a strong sulfurous odor. This oily residue was subjected to chromatography using hexane, chloroform, and chloroform:methanol (8:2 v / v) in a silica gel layer to obtain three fine fractions. Furthermore, the fine fraction eluted with chloroform was subjected to silica gel column chromatography, and eluted in order of increasing polarity using hexane and hexane:chloroform. The fine fraction eluted in chloroform was dried under reduced pressure to obtain a rubbery residue concentrated with sesquiterpene coumarins.

[0077] To prepare the water-soluble composition, 0.5 g of sesquiterpene coumarin concentrate was treated with 2 g of caprylic acid and ethyl oleate (1:1 w / w) with the help of high-energy sonication at 35°C for 5 minutes to obtain a clear and transparent solution. The resulting solution was then treated with 5 g of dodecyltrimethylammonium chloride at 30°C after low-energy stirring for 20 hours, and further dispersed in 60 g of water after high-energy sonication for 60 minutes to obtain a clear and transparent water-soluble composition.

[0078] The obtained sesquiterpene coumarin concentrate and its water-soluble composition were characterized and confirmed to exhibit the following properties.

[0079] The sesquiterpene coumarin concentrate is oily and reddish-brown. • Sesquiterpene coumarin concentrates are miscible with nonpolar to moderately polar solvents, but not with water. The sesquiterpene coumarin concentrate contains 45.88% ferulic acid, 0.76% umbelliferone, 3.52% ligpersin A, 0.59% karatabisinol, 1.54% ferrocauridine, 42.31% ferucrin, and 3.14% galvanic acid, as measured by area at 325 nm in HPLC. • The sesquiterpene coumarin concentrate is MAO-A(IC). 50 0.1014 mg / mL) inhibitory activity and MAO-B (IC) 50 It showed inhibitory activity at 0.0135 mg / mL. • The sesquiterpene coumarin concentrate is unstable in the pH range of 2.0 to 10.0 and the temperature range of 30 to 80°C. • The water-soluble composition containing the concentrated fraction of sesquiterpene coumarin is light brown in color. • Water-soluble compositions containing concentrated sesquiterpene coumarin fractions are miscible with water but not with nonpolar solvents. • Water-soluble compositions containing concentrated sesquiterpene coumarin fractions are stable in the pH range of 2.0 to 10.0. • A water-soluble composition containing a concentrated fraction of sesquiterpene coumarin inhibits MAO enzymes. • Water-soluble compositions containing sesquiterpene coumarin concentrates exhibit characteristic peaks in HPLC and NMR.

[0080] Thus, the compositions developed in this invention contain sesquiterpene coumarins derived from species of the genus Ferula, particularly Ferula asafoetida, which have MAO (MAO-A and MAO-B) inhibitory activity. Sesquiterpene coumarins include sesquiterpenes having acyclic, monocyclic, and bicyclic skeletons that spontaneously bind to coumarin via ether bonds. Sesquiterpene coumarins are nonpolar compounds with low water solubility, which can hinder their absorption and compliance with pharmaceutical standards. Furthermore, sesquiterpene coumarins contain ether bonds that are easily hydrolyzed in the acidic environment of the stomach. Therefore, the present invention aims to impart improved solubility, dispersibility, stability, and compliance with pharmaceutical standards by incorporating the sesquiterpene coumarin concentrate fraction into a water-soluble composition.

[0081] Abbreviation MAO: Monoamine oxidase HPLC: High-Performance Liquid Chromatography nm: nanometer FT-IR: Fourier Transform Infrared Spectroscopy NMR: Nuclear magnetic resonance method I C 50 : Half-number inhibitory concentration gm: grams w / w:mass ratio

[0082] (References) TIFF2026518486000003.tif201150TIFF2026518486000004.tif208149TIFF2026518486000005.tif208150TIFF2026518486000006.tif176150

Claims

1. A composition comprising a concentrated sesquiterpene coumarin fraction in an oil phase containing a fatty acid or fatty acid ester, together with an emulsifier, a co-surfactant, and water.

2. Sesquiterpene coumarin has the general molecular formula C 24～26 H 26～36 O 3～6 The composition according to claim 1, having a molecular weight in the range of 362 to 444.

3. Sesquiterpene coumarins include felculin (0.05–42.31%), asafoetidanol A (0.013–1.17%), ligpersin A (0.31–36.29%), chamoronol (1.50–46.67%), 10'-acetoxy-11'-hydroxyumbelliprenine (1.52–2.86%), epi-conferdione (0.24–12.24%), karatabisinol (0.07–6.13%), kerelin (1.43–17.63%), and ferrocauridine (0. The composition according to claim 1, selected from 19-2.23%, farnesiferol A (0.13-14.31%), galvanic acid (0.05-52.56%), suzowitziaquamarin B (5.18-16.18%), farnesiferol B (0.81-10.08%), gunmosine (2.58-4.03%), farnesiferol C (0.01-40.98%), suzowitziaquamarin A (0.28-7.01%), and methyl galvanate (0.70-10.77%).

4. The composition according to claim 1, wherein the oil phase containing a fatty acid or fatty acid ester is selected, but is not limited to, caprylic acid, isopropyl myristate, olive oil, coconut oil, and ethyl oleate, either alone or in combination.

5. The composition according to claim 1, wherein the emulsifier is selected from the group consisting of nonionic, cationic, and anionic emulsifiers.

6. The composition according to claim 5, wherein the nonionic emulsifier is Tween20, Tween60, Tween80, Span80, Span20, and Brij35.

7. The composition according to claim 5, wherein the cationic emulsifier is dodecyltrimethylammonium chloride or cetyltrimethylammonium bromide.

8. The composition according to claim 5, wherein the anionic emulsifier is sodium lauryl sulfate or trigol.

9. The composition according to claim 1, wherein the cosurfactant is selected from propylene glycol and PEG400.

10. The composition according to claim 1 for use in the treatment of Parkinson's disease.

11. A method for preparing the composition described in claim 1, a. A step of obtaining a clear and transparent solution by treating a concentrated sesquiterpene coumarin fraction with an oil phase containing fatty acids or fatty acid esters in a ratio of 1:1 to 1:10 (w / w) at 10°C to 40°C for 5 minutes to 24 hours with high or low external energy input, b. A step to obtain a mixture by treating the solution obtained in step (a) with a nonionic, cationic, or anionic emulsifier in a ratio of 1:1.1 to 1:12.5 (w / w), regardless of the presence or absence of a co-surfactant, and stirring at high or low energy at 10°C to 30°C for 1 minute to 24 hours. c. A step to obtain a water-soluble composition by dispersing the mixture obtained in step (b) with water in a ratio of 1:0.5 to 1:20 (w / w) by stirring for 1 minute to 24 hours. A method that includes this.

12. A method for preparing a sesquiterpene coumarin concentrate according to claim 11, (a) A step of extracting oleogum resin of the genus Ferula multiple times with an organic solvent with a polarity index of 2 to 6 to obtain an extract, (b) A step of collecting the extract from step (a) and concentrating it until it becomes a residue that does not contain ethyl acetate, (c) A step to obtain a sesquiterpene coumarin-enriched fraction by separating the ethyl acetate-free residue from step (b) with silica gel and then eluting it using a hexane-chloroform and chloroform-methanol gradient. A method that includes this.

13. A method for treating Parkinson's disease in a subject, comprising administering to the subject a therapeutically effective amount of the water-soluble composition according to claim 1.

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