WO2013020368A1

WO2013020368A1 - Rhodiola rosea extracts and isolated compounds and uses thereof for treating neurodegenerative diseases

Info

Publication number: WO2013020368A1
Application number: PCT/CN2012/001058
Authority: WO
Inventors: Nancy Yuk Yu Ip; Fanny Chui Fun Ip; Guangmiao Fu; Ho Kee KOON; Yu Pong NG
Original assignee: The Hong Kong University Of Science And Technology
Priority date: 2011-08-08
Filing date: 2012-08-08
Publication date: 2013-02-14
Also published as: CN103857400B; CN103857400A

Abstract

This invention relates to extract, fractions and isolated compound of Rhodiola rosea, and uses thereof for treating neuropathological and neurodegenerative diseases. The extracts and compounds of the present invention inhibit the aggregation of alpha-synuclein. In one embodiment, Rhodiola rosea extracts and compounds of the present invention can be used to treat synucleinopathies including PD, dementia with Lewy bodies, pure autonomic failure, multiple system atrophy, and Alzheimer's disease.

Description

DESCRIPTION

RHODIOLA ROSEA EXTRACTS AND ISOLATED COMPOUNDS AND USES THEREOF FOR TREATING NEURODEGENERATIVE DISEASES

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. provisional application Serial No. 61/573,022, filed August 8, 2011, which is herein incorporated by reference in its entirety.

BACKGROUND

Parkinson's disease (PD) is a disabling, progressive neurodegenerative disorder. The clinical manifestations of PD include resting tremors, rigidity, bradykinesia, and postural instability with cognitive and emotional disorders. The primary characteristic pathology of PD is the loss of dopaminergic neurons in the substantia nigra pars compacta and the presence of intracytoplasmic inclusions known as Lewy bodies.

The etiology and the pathogenesis of PD are not completely understood. Both environmental and genetic factors may contribute to the progression of PD (Broussolle & Thobois, 2002). Most PD patients have the sporadic forms of PD, while several susceptive genes have been identified as associated with familial forms of PD. Three missense point mutations (A53T, A30P, and E46K) and genomic duplication or triplication of the α-synuclein gene have been reported as a cause of familial PD (Conway et al., 1998).

The α-synuclein protein is predominately expressed in neurons, especially at synaptic terminals, and plays a role in synaptic function and neural plasticity (Sidhu et al., 2004). The pathological α-synuclein exists as insoluble, filamentous aggregates containing abnormally nitrated, phosphorylated, and ubiquitinated residues in Lewy bodies and Lewy neurites. It has been reported that a-synucleinopathy is an important pathogenic mechanism of neurodegenerative diseases (Vekrellis et al., 201 1).

The α-synuclein protein has high propensity to adopt various conformations, with a strong tendency to self-aggregate into oligomers that further aggregate into fibrils deposited as Lewy bodies and in other diseases. The mutant forms of α-synuclein are more inclined to form aggregates as shown in in vitro and in animal models (Giasson et al., 2002; Lee et al, 2002). Alpha-synuclein has also been identified as a major component of Lewy bodies and Lewy neurties in dementia with Lewy bodies (DLB) (Spillantini et al., 1998), Alzheimer's disease, multiple system atrophy (MSA), and other neurodegenerative disorders (Halliday et al, 2011).

Additionally, a-synuclein protein levels increase with age in the human substantia nigra. Neurodegenerative phenotypes in human patients and animal models indicate that high expression levels of a-synuclein and the abnormal aggregation of this protein play a role in the pathogenesis of PD. A53T α-synuclein transgenic mice (under the control of the mouse prion- related protein promoter) show a marked reduction in motor function, which could ultimately result in fatal motor paralysis with advancing age (Giasson et al., 2002). Their motor neurons of A53T α-synuclein transgenic mice exhibit axonal degeneration near fibrillary a-synuclein inclusions, which resemble part of the structure of the Lewy bodies.

Recent evidence also suggests that the aggregated insoluble oligomer (protofibril) of a- synuclein plays an important role in the pathogenesis of PD. It has been demonstrated that a- synuclein protofibril forms elliptical or circular amyloid pores that can puncture the cell membrane and result in the release of cell contents and cell death (Lashuel et al., 2002).

In addition to the α-synuclein oligomers toxicity, recent studies have been shown that loss of mitochondrial complex I function and the generation of oxidative stress are found in the brains of PD patients (Keeney et al., 2006), and these may be involved in the progression of selective nigral dopaminergic degeneration in PD.

6-hydoxydopamine (6-OHDA) is a chemical that has been widely used to induce parkinsonism in experimental animals (Lane & Dunnett, 2008). 6-OHDA enters the neurons via the dopamine and noradrenaline reuptake transporters; therefore, 6-OHDA is commonly used in conjunction with a selective noradrenaline reuptake inhibitor (such as desipramine) to selectively kill dopaminergic neurons only. 6-OHDA is considered as an endogenous toxin, as oxidation of dopamine can lead to the generation of 6-OHDA in vitro (Jellinger et al., 1995). Substantial evidence has shown that 6-OHDA generates reactive oxygen species and reduces the activities of glutathione and superoxide dismutase (Betarbet et al., 2002). Following the intracerebral injection of 6-OHDA, striatal neurons start to degenerate in 24 hr and striatal dopamine is depleted in 2-3 days later (Asanuma et al, 1998). MPTP (l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine) is considered as an environmental toxin, and could play a role in the pathogenesis of PD. MPTP itself is not toxic; however, its active metabolite generated in the brain, a positively charged chemical toxin (MPP+), interferes with oxidative phosphorylation in mitochondria, and causes depletion of ATP and subsequently cell death. MPP+ is taken up by dopaminergic neurons via the dopamine transporter. MPP+ also inhibits the synthesis of catecholamines, reduces levels of dopamine and norepinephrine, and inactivates tyrosine hydroxylase. In addition, MPP+ has been found to up-regulate the expression and aggregation of a-synuclein in SH-SY5Y cells (Kalivendi et al., 2004). Parkinsonism can be induced in rodents by intracerebroventicular injection of MPP+ (Cavalla et al., 1985). Locomotor activity of these PD mice can be evaluated in the Rotorod Test and Open Field Test.

Currently, there are no treatments that halt or reverse the progression of PD. Commercially available drugs only relieve the symptoms of the disease to improve quality of life in PD patients. Since there are a wide range of symptoms and complications in PD patients, the choice of medications varies considerably between individuals. The most frequently prescribed medication for PD are therapeutics that boost the production of dopamine in the brain. Levodopa, which is modified by a brain enzyme to produce dopamine, is the most common medication for PD. Over the years, a number of dopamine agonists have been developed for treatment of PD; however, the therapeutic effect decreases after a period of treatment. Furthermore, some patients report side effects such as gastrointestinal ailments and psychological cognitive problem {e.g., confusion, hallucinations, psychosis, etc). Thus, improved therapeutics for PD are needed.

Rhodiola rosea (RR), also known as Roseroot or Golden Root, is a species of Rhodiola in the Crassulaceae plant family. Rhodiola rosea grows in mountains and hard rocks at an altitude over 3000 meters. The root of Rhodiola was described two thousand years ago in "Shen Nong Ben Cao Jing" as a top-grade herb due to its low toxicity, as well as its activity in modulating Qi, nourishing the blood and lung systems, supporting kidney function, and its anti-aging effect.

Rhodiola rosea has also been documented outside of China in the Materia Medica of a number of European countries since the 18^th Century, and has been used since as early as Viking times. "Modern Practical Materia Medica" discloses that RR has activities including central inhibition, anti-fatigue, strengthened cardiac effects, anti-inflammatory, reduction of blood sugar level, anti-peroxidation, and anti-microwave radiation activity. Modern biological researches have demonstrated that RR extracts have anti-fatigue, anti-oxidant, cognitive enhancement, anti- depression, anti-stress, anti-virus, anti-bacteria, anti-tumor, and anti-inflammatory activities (Panossian et al., 2010).

Rhodiola rosea contains a variety of chemical constituents, including phenylpropanoids (e.g., rosavin, rosin, rosarin), phenyl ethanol derivatives (e.g., salidroside, tyrosol), flavanoids (e.g., rodiolin), monoterpenes (rosiridol, rosiridin), triterpenes (e.g., daucosterol), and phenolic acids (e.g., gallic acids). It has been reported that salidroside has anti-apoptotic, antiinflammatory, antioxidative, anti-depressant, and neuroprotective effects. Specifically, salidroside was found to protect PC 12 cells against MPP(+)-induced apoptosis by inhibiting the NO pathway. Rosiridin has been reported as a monoamine oxidase inhibitor and perhaps accounts for the anti-depression effect of RR. Rosavin is known to have inhibition effects on the growth of bacteria Neisseria gonorrhoeae and have reactive oxygen species scavenging activity. (Panossian & Wagner, 2005).

BRIEF SUMMARY

The subject invention provides novel and advantageous materials and methods for preventing and/or treating neurological and/or neurodegenerative diseases and disorders. In one embodiment, the subject invention provides Rhodiola rosea extracts, and compounds isolated from Rhodiola rosea, for preventing, treating, or ameliorating synucleinopathies such as Parkinson's disease.

In one specific embodiment, the subject invention pertains to compounds of formula I, having the following structure:

wherein R| - R₃ are, independently, -H, -OH, methoxy, ethoxy, halo, amino, acyl, or thiol; and R4 - R₉ are, independently, hydrogen, alkyl or acyl.

In one embodiment, the subject invention pertains to compounds of formula II, having the following structure:

(II)

wherein Ri - R₄ are, independently, hydrogen, alkyl or acyl; and R₅ - R₇ are, independently, -H, -OH, methoxy or ethoxy, halo, amino, acyl, or thiol.

In one embodiment, the subject invention pertains to compounds of formula III, having the following structure:

wherein Ri - R₃ and R₇-R₉ are, independently, -H, -OH, methoxy or ethoxy, halo, amino, acyl, or thiol; and R₄ - R₆ are, independently, hydrogen, alkyl or acyl.

In one embodiment, the subject invention pertains to compounds of formula IV, having the following structure:

wherein R_\ - R₆ are, independently, hydrogen, alkyl or acyl; and R₇-R₉ are, independently, -H, -OH, methoxy or ethoxy, halo, amino, acyl, or thiol.

In one embodiment, the subject invention pertains to compounds of formula V, having the following structure:

wherein R_\ - R4 are, independently, hydrogen, alkyl or acyl; and R_¾ is hydroxyl, halo, haloalkyl, amino, alkylamino, alkoxy, thiol, cyano, or -COOH.

In one embodiment, the subject invention pertains to compounds of formula VI, having the following structure:

wherein R] - R are, independently, -H, -OH, methoxy or ethoxy, halo, amino, acyl, or thiol; and R₄ - Rio are, independently, hydrogen, alkyl or acyl (e.g., acetyl).

In one embodiment, the subject invention pertains to compounds of formula VII, having the following structure:

(VII) wherein Ri - R₄ are, independently, hydrogen, or acyl (e.g., acetyl).

In one embodiment, the subject invention pertains to compounds of formula VIII, having the following structure:

wherein Ri - R₃ and R₇ - R₉ are, independently, -H, -OH, acyl, halo, haloalkyl, amino, alkylamino, alkyl, hydroxylalkyl, alkoxy, thiol; and R₄ - R₆ are, independently, hydrogen, alkyl or acyl (e.g., acetyl).

In one specific embodiment, the subject invention pertains to therapeutic uses of rosavin, 6-O-galloyl rosin, rosarin, icariside D2, mongrhoside, gallic acid, 6-O-galloyl arbutin, and rhodiocyanoside A.

In one embodiment, the subject invention provides for therapeutic or pharmaceutical compositions comprising a therapeutically effective amount of the Rhodiola Rosea extract of the present invention, and, optionally, a pharmaceutically acceptable carrier. In another embodiment, the subject invention provides pharmaceutical or therapeutic compositions, comprising an isolated or substantially pure compound selected from formula I (such as rosavin) to formula VIII, or a salt thereof, and optionally, a pharmaceutically acceptable carrier.

In one embodiment, the subject invention provides a method for preventing, treating, or ameliorating a disease or condition where inhibiting the aggregation of a-synuclein protein would be beneficial. In one embodiment, the method comprises administering, to a subject in need of such treatment, an effective amount of a composition comprising the Rhodiola rosea extract of the subject invention, a biologically-active substituent isolated from Rhodiola rosea (e.g., rosavin), and/or a compound of formula I through formula VIII.

In certain embodiments, the subject invention prevents, treats or ameliorates neurodegenerative diseases including, but not limited to, Parkinson's disease, synucleinopathies, amyotrophic lateral sclerosis, Alzheimer's disease, dementia with Lewy bodies (DLB), pure autonomic failure (PAF), multiple system atrophy (MSA), and Huntington's disease.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows that Rhodiola rosea extract inhibited alpha-synuclein aggregation in vitro. Filter trap assay was performed by incubating total extract of Rhodiola rosea (RRTE, 0.001-100 μg/ml) with alpha-synuclein (0.07 μg) recombinant protein for 7 days. Western blot analysis was performed to detect alpha-synuclein aggregates. Assay was performed in duplicate lanes and repeated at least 2 times. Congo red at 5 μΜ was used as positive control. Concentration of DMSO in RR samples was at 0.2%.

Figure 2 shows that extracts and subfractions of Rhodiola rosea inhibited alpha- synuclein aggregation in vitro. Filter trap assay was performed by incubating total extract or the subfractions of RR (0.1 to 10 μg/ml) with alpha-synuclein (0.07 μg) recombinant protein for 7 days. Western blot analysis was performed to detect alpha-synculein aggregates. Assay was performed in duplicate and repeated at least 2 times. Congo red at 5 μΜ was used as positive control. Concentration of DMSO in RR samples was at 0.2%.

Figure 3 A shows that the rosavin compound (RR-C36) isolated from Rhodiola rosea inhibited alpha-synuclein aggregation in vitro. Filter trap assay was performed by incubating compound RR-C36 or RR-C20 (salidroside) (0.2, 2, 20 μΜ) with alpha-synuclein (0.07 μg) recombinant protein for 7 days. The amount of alpha-synuclein aggregates increased after 7 day incubation and congo red inhibited this increase (upper panels). RR-C36 from RRWA inhibited the alpha-synuclein aggregation in a dose-dependent manner (lower panels). The assay was performed in duplicate and repeated at least 2 times. Congo red at 5 μΜ was used as positive control. Concentration of DMSO in RR samples was at 0.2%. Figure 3B shows that the RR-C36 compound (rosavin) inhibited aggregation of alpha-synuclein and induced disaggregation of pre- aggregated alpha-synuclein. For the inhibition assay, recombinant alpha-synuclein in TBS was incubated with RR-C36 for 7 days. For the disaggregation assay, RR-C36 was added to the recombinant alpha-synuclein after 5 days of aging (aggregation), followed by 2 days of incubation. The samples were subject to filter trap assay. RR-C36 inhibited alpha-synuclein aggregation and induced disaggregation of alpha-synuclein in a dose-dependent manner. Figure 3C shows that RR-C22 (6-O-galloyl rosin) and RR-C49 (rosarin) inhibited aggregation of alpha- synuclein after co-treatment with recombinant alpha-synuclein for 7 days. The assay was performed in duplicate and repeated at least 2 times. Congo red at 5 μΜ was used as positive control. The concentration of DMSO in RR samples was 0.2%. Figure 3D shows that RR-C31 (icariside D2) and RR-C47 (mongrhoside) inhibited aggregation of alpha-synuclein after co- treatment with recombinant alpha-synuclein for 7 days. The assay was performed in duplicate and repeated at least 2 times. Congo red at 5 μΜ was used as positive control. The concentration of DMSO in RR samples was 0.2%. Figure 3E shows that the RR-C04 (gallic acid) and RR-C34 (6-O-galloyl arbutin) inhibited aggregation of alpha-synuclein after co-treatment with recombinant alpha-synuclein for 7 days. The assay was performed in duplicate and repeated at least 2 times. Congo red at 5 μΜ was used as positive control. The concentration of DMSO in RR samples was 0.2%.

Figure 4A shows that Rhodiola rosea butanol fraction (RRBU), Rhodiola rosea water fraction (RRWA), and rosavin (RR-C36) inhibited aggregation of alpha-synuclein, as determined by atomic force microscopy (AFM). Recombinant alpha-synuclein in TBS was incubated with RRBU, RRWA, or RR-C36 for 7 and 14 days at room temperature with 0.3% DMSO serving as control. Large size oligomers and protofibrils were observed in the samples treated with DMSO. RRBU, RRWA and RR-C36 inhibited the formation of large size oligomers and protofibrils as shown in the graph of height analysis from AFM images. Figure 4B shows that rhodiocyanoside A (RR-C41) inhibited aggregation of alpha-synculein after 7-day incubation as revealed by atomic force microscropy images and height analysis.

Figure 5 shows that various RR fractions exhibited anti-aggregation activity on Αβ1-42 peptide. The total extract, and various fractions of RR, and rosavin (RR-C36) were incubated with recombinant human Αβ1-42 for 3 days at 37°C. After incubation, the samples were subject to the thioflavin T (ThT) binding assay. RR fractions (RRBU and RRWA) showed anti- aggregation activity on Αβ1-42 aggregation.

Figure 6 shows that RRBU and RRWA prevented MPP+ induced caspase-3 cleavage and inhibited MPP+-induced alpha-synuclein aggregation in SH-SY5Y cells. SH-SY5Y cells were pretreated with RRBU (50 μg/ml), RRWA (50 μg ml), or DMSO (0.1%) for 2 hr. MPP+ (1 mM) was then used to treat the cells for 20 hr. Total cell lysates were then collected for Western blotting against cleaved caspase-3 and alpha-synuclein. Probing of GAPDH was served as loading control.

Figure 7 shows that water (WA) fraction of Rhodiola rosea rescued TH loss in 6-OHDA injected mice. (A) Experimental design. (B) Protein expression of tyrosine hydroxylase (TH) was determined by Western blot analysis. Briefly, 3-month-old C57B/6 mice were treated with water fraction of Rhodiola rosea (RRWA) (i.p., 10 or 100 mg/kg) daily 5 days before and 3 days after the stereotaxic injection of 6-OHDA. 6-OHDA (50 μg) was injected into the cerebroventricles of the mice and striata tissues were collected 3 days after surgery. (C) The Rotorod Test was conducted to examine the motor deficits of mice after 6-OHDA injection.

Figure 8 shows that RRBU, RRWA, and RR-C36 improved motor function in human A53T alpha-synuclein transgenic mice. A53T transgenic mice treated with RRBU, RRWA, or RR-C36 showed improvement of motor activity, as the results showed the treated mice had increased travel distance in the Open Field Test.

Figure 9 shows the amount of RR-C36 detected in mouse plasma and brain using LCMS/MS analysis. RR-C36 was detected in mouse plasma and the brain 15 minutes after intraperitoneal injection.

Figures 10A-H show HPLC chromatograms of the extract and fractions from Rhodiola rosea and the detection of RR-C20 (salidroside), RR-C36 (rosavin), RR-C41 (rhodiocyanoside A), RR-C31 (icariside D2), RR-C34 (6-O-galloyl arbutin), and RR-C47 (mongrhoside A). The mobile phase of A-D started with 2% ACN while E-H was 10% ACN. Detection wavelength: 220 nm.

DETAILED DESCRIPTION

The subject invention provides novel and advantageous materials and methods for preventing and/or treating neurological and/or neurodegenerative diseases and disorders. In one embodiment, the present invention relates to Rhodiola rosea (RR) extracts, and fractions thereof, as well as compounds isolated from Rhodiola rosea (RR) that exhibit anti-Parkinson's effects. Advantageously, the Rhodiola rosea (RR) extract, fractions, and isolated compounds of the present invention inhibit the aggregation of alpha-synuclein, reduce the tyrosine hydroxylase loss in 6-OHDA-induced PD animal model, and rescue the SH-SY5Y cells from cell death in the presence of neurotoxins; therefore the Rhodiola rosea (RR) extracts, fractions, and isolated compounds of the present invention can be used for treatment of neurodegenerative diseases including Parkinson's disease and synucleinopathies.

Specifically, the subject invention shows that the total extract of Rhodiola rosea (RRTE), the butanol (BU) and water (WA) fractions of the total extract, as well as a compound isolated from Rhodiola rosea - rosavin - inhibits the oligomerization and protofibril formation of a- synuclein. Rosavin inhibits the a-synuclein aggregation. The butanol (RRBU) and water (RRWA) fractions of Rhodiola rosea extract also inhibit the aggregation of amyloid-beta peptide, inhibit the activation of caspase-3 in the presence of MPP+, reduce the high molecular weight a- synuclein, and increase monomer expression in SH-SY5Y cells. The water (RRWA) fraction of the Rhodiola rosea extract also attenuates motor deficits in mice that received 6-OHDA injection.

Compounds

In one embodiment, the subject invention pertains to compounds of formula I, having the following structure:

(I) wherein Ri - R₃ are, independently, -H, -OH, methoxy or ethoxy, halo, amino, acyl, or thiol; and R₄ - R9 are, independently, hydrogen, alkyl or acyl.

In one embodiment, the subject invention pertains to a compound of formula I - rosavin ((25,3 ?,45,55,6 ?)-2-[(E)-3-phenylprop-2-enoxy]-6-([(25,3R,45,55)-3,4,5-trihydroxyoxan-2- yl]oxymethyl)oxane-3,4,5-triol), having the following structure:

(Π)

wherein Ri - R₄ are, independently, hydrogen, alkyl or acyl; and R₅ - R₇ are,

independently, -H, -OH, methoxy or ethoxy, halo, amino, acyl, or thiol.

In one embodiment, the subject invention pertains to a compound of formula II - salidroside (2-(4-hydroxyphenyl)ethyl β-D-glucopyranoside), having the following structure:

In one embodiment, the subject invention pertains to a compound of formula III - 6-0- galloyl rosin, having the following structure:

In one embodiment, the subject invention pertains to compounds of formula IV, having the following structu

wherein Ri - R₆ are, independently, hydrogen, alkyl or acyl; and R7-R9 are, independently, -H, -OH, methoxy or ethoxy, halo, amino, acyl, or thiol.

In one embodiment, the subject invention pertains to a compound of formula IV - rosarin ((E)-3-Phenyl-2-propenyl]6-0-a-L-arabinofuranosyl-p-D-glucopyranoside;[(E)-3-Phenyl-2- propenylJ6-0-(a-L-arabinofuranosyl)-P-D-glucopyranoside), having the following structure:

(V) wherein R[ - R₄ are, independently, hydrogen, alkyl or acyl; and R₅ is hydroxyl, halo, haloalkyl, amino, alkylamino, alkoxy, thiol, cyano, or -COOH.

In one embodiment, the subject invention pertains to a compound of formula V - icariside D2, having the following structure:

wherein R_\ - R₃ are, independently, -H, -OH, methoxy or ethoxy, halo, amino, acyl, or thiol; and R₄- Rio are, independently, hydrogen, alkyl or acyl (e.g., acetyl).

In one embodiment, the subject invention pertains to a compound of formula VI - mongrhoside, having the following structure:

(VII) wherein Ri - R₄ are, independently, hydrogen, alkyl or acyl (e.g., acetyl).

In one embodiment, the subject invention pertains to a compound of formula VII rhodiocyanoside A, having the following structure:

wherein R_\ - R₃ and R₇ - R₉ are, independently, -H, -OH, acyl, halo, haloalkyl, amino, alkylamino, alkyl, hydroxylalkyl, alkoxy, thiol; and R₄ - R are, independently, hydrogen, alkyl or acyl (e.g., acetyl).

In one embodiment, the subject invention pertains to a compound of formula VIII - 6-0- galloyl arbutin, having the following structure:

In one embodiment, the subject invention pertains to therapeutic uses of the compounds isolated from Rhodiola rosea.

The term "alkyl" means linear saturated monovalent radicals of one to eight carbon atoms or a branched saturated monovalent of three to eight carbon atoms. It may include hydrocarbon radicals of one to four or one to three carbon atoms, which may be linear. Examples include methyl, ethyl, propyl, 2-propyl, isopropyl, n-butyl, iso-butyl, tert-butyl, pentyl, and the like. In certain embodiments, the alkyl group is a linear or branched chain Ci to C₆ alkyl group, Ci to C$ alkyl group, Ci to C₄ alkyl group, Ci to C₃ alkyl group, ethyl, or methyl group.

The term "hydrocarbon" or "hydrocarbyl" refers to organic compounds or radicals consisting exclusively of the elements carbon and hydrogen. Hydrocarbyl includes alkyl, alkenyl, and alkynyl moieties.

The term "acyl" means a radical -C(0)R wherein R is hydrogen, alkyl or cycloalkyl, or heterocycloalkyl. In one embodiment, the R group of the radical -C(0)R is a Ci to C₄ alkyl. Examples of acyl groups include, but are not limited to, formyl, acetyl, and ethylcarbonyl.

The term "halo" means fluoro, chloro, bromo, and iodo.

The term "hydroxy" means the radical -OH.

The term "substituted," as used herein, refers to an embodiment wherein at least one hydrogen atom of a compound or chemical moiety is replaced with a second chemical moiety. Non-limiting examples of substituents are those found in the exemplary compounds and embodiments disclosed herein, as well as halogen; alkyl; alkenyl; alkynyl; hydroxy; alkoxyl; amino; haloalkyl (e.g., trifluoromethyl); and -COOH. All chemical groups disclosed herein can be substituted, unless it is specified otherwise. For example, "substituted" alkyl, alkenyl, or alkynyl moieties described herein are moieties that are substituted with a second chemical moiety such as a hydrocarbyl moiety, halo, alkoxy, and -COOH. Substituted alkyl groups include, but are not limited to, haloalkyl, hydroxyalkyl, carboxylalkyl, and aminoalkyl.

The term "haloalkyl" means alkyl substituted with one or more same or different halo atoms. Representative examples of haloalkyl groups include, but are not limited to, -CH₂C1, - CH₂Br, -CF₃, -CH₂CH₂C1, and -CH₂CC1₃.

The term "amino," as used herein, refers to -NH .

The term "alkylamino" means a radical -NHR or - R₂ where each R is independently an alkyl group. In certain embodiments, the alkyl group of alkylamino is a Ci to C₄ alkyl. Representative examples of alkylamino groups include, but are not limited to, methylamino, (1- methylethyl)amino, dimethylamino, methylethylamino, and di(l-methyethyl)amino.

The term "hydroxyalkyl" means an alkyl radical as defined herein, substituted with one or more, preferably one, two or three hydroxy groups. In certain embodiments, hydroxyalkyl is a C| to C₆ alkyl, or preferably a Q to C₄ alkyl, substituted with one or more hydroxy groups. Representative examples of hydroxyalkyl include, but are not limited to, hydroxymethyl, 2- hydroxyethyl, 2-hydroxypropyl, 3-hydroxypropyl, l-(hydroxymethyl)-2-methylpropyl, 2- hydroxybutyl, 3-hydroxybutyl, 4-hydroxybutyl, 2,3-dihydroxypropyl, 2-hydroxy- l- hydroxymethylethyl, 2,3-dihydroxybutyl, 3,4-dihydroxybutyl and 2-(hydroxymethyl)-3-hydroxy- propyl, preferably 2-hydroxyethyl, 2,3- dihydroxypropyl, and l-(hydroxymethyl)2-hydroxyethyl.

The term "alkoxy," as used herein, refers to the radical -OR_x , wherein R_x is a Q to C₆ alkyl group. In one embodiment, R_x is a Ci to C₄ alkyl group. Exemplary alkoxy groups include, but are not limited to, methoxy, ethoxy, and propoxy.

In certain embodiments, the subject invention pertains to isolated or substantially pure compounds represented by formula I through VIII. The term "substantially pure," as used herein, refers to more than 99% pure.

As used herein, "isolated" refers to extracts or compounds that have been removed from any environment in which they may exist in nature. For example, an isolated compound or extract would not refer to the compound or extract as it exists in plants from which the compound can be isolated. In preferred embodiments, the compounds and extracts of the subject invention are at least 75% pure, preferably at least 90% pure, more preferably are more than 95% pure, and most preferably are more than 99% pure (substantially pure).

The subject invention further embodies stereoisomers of the compounds. The term "stereoisomer" encompasses all enantiomerically/stereomerically pure and enantiomerically/stereomerically enriched compounds disclosed herein.

In one embodiment, the subject invention pertains to enantiomeric forms of the compounds. The enantiomeric forms of the compounds of the invention are substantially free from one another (i.e., in enantiomeric excess). In other words, the "R" forms of the compounds are substantially free from the "S" forms of the compounds and are, thus, in enantiomeric excess of the "S" forms. Conversely, "S" forms of the compounds are substantially free of "R" forms of the compounds and are, thus, in enantiomeric excess of the "R" forms. In one embodiment of the invention, the enantiomeric compounds are in at least about 80% enantiomeric excess. In a preferred embodiment, the compounds are in at least about 90% enantiomeric excess. In a more preferred embodiment, the compounds are in at least about 95% enantiomeric excess. In an even more preferred embodiment, the compounds are in at least about 97.5% enantiomeric excess. In a most preferred embodiment, the compounds are in at least about 99% enantiomeric excess.

The subject invention also encompasses salts, solvates, hydrates, and polymorphs of the compounds of formula I through VIII, and uses thereof.

In one embodiment, the subject invention does not encompass therapeutic use of gallic acid, 6-O-galloyl arbutin, and 6-O-galloyl rosin disclosed in PCT/CN2010/001982.

Rhodiola rosea Extracts

One aspect of the subject invention provides methods for preparing Rhodiola rosea extracts. The subject methods can also be used to isolate biologically- active chemical constituents from Rhodiola rosea. Also provided are Rhodiola rosea extracts prepared in accordance with the subject invention. In one embodiment, the subject invention provides a method for preparing Rhodiola rosea extract and/or for isolating biologically-active chemical constituents from Rhodiola rosea, wherein the method comprises, consists essentially of, or consists of the steps of:

a) providing a sufficient quantity of raw material of Rhodiola rosea;

b) extracting the raw material of Rhodiola rosea with a first solvent comprising an alcohol to yield a Rhodiola rosea alcohol extract;

c) recovering the Rhodiola rosea alcohol extract; and optionally,

d) concentrating the Rhodiola rosea alcohol extract.

Preferably, the raw material of Rhodiola rosea is dried and ground into powder. Preferably, the raw materials are Rhodiola rosea roots.

In certain embodiments, solvents for the preparation of Rhodiola rosea extract can include, but are not limited to, alcohols (e.g., C1 -C4 alcohols, such as methanol, ethanol, propanol); Ci-C₃ ketones (e.g. acetone); acetic acid; acetate, ethyl acetate, and water.

In one embodiment, the first solvent comprises one or more alcohols selected from C 1-C3 alcohols, such as methanol, ethanol, and propanol.

In one embodiment, the first solvent comprises, or is, a water-alcohol mixture. The alcohol-water (e.g., ethanol-water, methanol-water) mixture can comprise about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% alcohol (e.g., ethanol, methanol).

In one embodiment, the extraction method further comprises:

mixing the Rhodiola rosea alcohol extract with water; and

partitioning the water-Rhodiola rosea alcohol extract mixture with one or more organic solvents to yield one or more organic-solvent fractions and a water fraction.

In certain embodiments, the organic solvent can be selected from acetonitrile, THF, chloroform, toluene, ethylene chloride, chlorobenzene, dichlorobenzene, alcohols (e.g., C 1 -C20 alcohols (e.g. methanol, ethanol, propanol, n-propanol, n-butyl alcohol), C]-C₂o ketones (e.g. acetone, methyl ethyl ketone), Ci-C₂o alkyls (e.g., butane, isobutane, pentane, isopentane, hexane, cyclohexane, heptane, octane, isooctane), acetic acid, acetate, petroleum ether, ethyl acetate, methylene chloride, tetrachloromethane, or any combination thereof.

In one embodiment, the extraction method comprises: providing a sufficient quantity of raw material of Rhodiola rosea;

extracting the raw material of Rhodiola rosea with a first solvent comprising an alcohol (such as ethanol) to yield a Rhodiola rosea alcohol extract;

recovering the Rhodiola rosea alcohol extract; and optionally, concentrating the Rhodiola rosea alcohol extract;

mixing the Rhodiola rosea alcohol extract with water; and

partitioning the water-Rhodiola rosea alcohol extract mixture successively with one or more solvents selected from petroleum ether, ethyl acetate, and n-BuOH to yield petroleum ether, ethyl acetate, n-BuOH, and a water fraction.

In one embodiment, the method further comprises isolating rosavin and/or salidroside from the Rhodiola rosea extract of the invention, such as the Rhodiola rosea alcohol (e.g., ethanol, n-BuOH) extract, and the water fraction of the Rhodiola rosea alcohol (e.g., ethanol, n- BuOH) extract of the invention.

In one embodiment, the extraction method is performed at room temperature. In another embodiment, the extraction method is performed at a temperature of 10°C - 100°C, or any temperatures therebetween, including but not limited to, 15°C - 90°C, 20°C - 80°C, and 60°C - 90°C.

In one embodiment, the raw material of Rhodiola rosea is mixed with a solvent for at least about 15 minutes to extract the biologically- active chemical constitutes. Preferably, the extraction time is at least about 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 4 hours, or 5 hours.

The Rhodiola rosea extract can be collected by, for example, filtration to remove the residues. In one embodiment, the Rhodiola rosea extract may be further evaporated to produce solid or semi-solid compositions. In another embodiment, the Rhodiola rosea extract may be concentrated and/or purified.

In a further embodiment, the subject method comprises creating a chemical profile for the Rhodiola rosea extract, by using techniques such as NMR analysis and chromatography, for example, silica gel column chromatography. The subject invention further provides Rhodiola rosea extracts produced by the subject extraction methods. In a specific embodiment, the Rhodiola rosea extract has a chemical profile as shown in Figures 10A-H.

The term "consisting essentially of," as used herein, limits the scope of the invention to the specified steps and those that do not materially affect the basic and novel characteristic(s) of the subject invention, i.e., a method for preparing Rhodiola rosea extract and/or for isolating biologically-active chemical constituents from Rhodiola rosea. For instance, by using "consisting essentially of," the method for preparing Rhodiola rosea extract does not contain any unspecified steps of extracting or contacting Rhodiola rosea, for example, additional step(s) of extracting or contacting Rhodiola rosea with unspecified solvent(s), or extracting Rhodiola rosea under condition(s) (e.g., a different temperature) different from the specified condition. Also, by using the term "consisting essentially of," the process may comprise steps that do not materially affect the extraction of biologically-active chemical constituents from Rhodiola rosea including collecting or recovering the Rhodiola rosea extract; concentrating the Rhodiola rosea extract; combining multiple Rhodiola rosea extracts into a single composition; lyophilizing or drying the Rhodiola rosea extract into a solid or semi-solid composition; formulating the Rhodiola rosea extract into a pharmaceutical composition such as solutions, suspensions, tablets, capsules, granules, powders, decoctions, and tinctures; mixing the Rhodiola rosea extract with pharmaceutically-acceptable carriers, excipients, flavoring agents, buffering agents, and/or emulsifying agents; and packaging the Rhodiola rosea extract.

Treatment of Neurological and Neurodegenerative Diseases

Another aspect of the subject invention provides therapeutic uses of the Rhodiola rosea (RR) extracts and fractions (e.g., water and/or alcohol such as butanol fractions), and/or compounds of formula I (e.g., rosavin) through formula VIII, and salts thereof, as well as therapeutic compositions comprising one or more of the aforementioned ingredients, for treatment of neurodegenerative diseases and neuropathological conditions, including Parkinson's disease and synucleinopathies.

Advantageously, it is discovered that the total extract of RR, and the subsequent butanol and water fractions of the Rhodiola rosea (RR) total extract, have neuroprotective activity, inhibit the aggregation of alpha-synuclein, and reduce the tyrosine hydroxylase protein expression in 6-OHDA induced PD animal model. In addition, rosavin, a compound isolated from Rhodiola rosea, inhibits alpha-synuclein aggregation. The Rhodiola rosea (RR) extracts and isolated compounds of the present invention inhibit aggregate-prone a-synuclein from its oligomerization. Soluble a-synuclein oligomers are toxic and can ultimately result in neuronal cell death in neurodegenerative diseases.

In one embodiment, the subject invention provides a method for preventing, treating, or ameliorating a disease or condition where inhibiting the aggregation of α-synuclein protein, inhibiting the aggregation of amyloid-beta peptide, and/or inhibiting the activation of caspase-3 would be beneficial. In one embodiment, the method comprises administering, to a subject in need of such treatment, an effective amount of a composition comprising the Rhodiola rosea extract of the subject invention, a biologically- active substituent isolated from Rhodiola rosea (e.g., rosavin), and/or a compound of formula I through formula VIII.

In one embodiment, the Rhodiola rosea extract is the water fraction and/or alcohol fraction (the partitioning solvent is an alcohol such as methanol, ethanol, propanol, and butanol, or an alcohol-water mixture) of an alcohol extract (the extraction solvent is an alcohol such as methanol, ethanol, propanol, and butanol, or an alcohol-water mixture).

In certain embodiments, the subject invention prevents, treats, or ameliorates neurodegenerative diseases including, but not limited to, Parkinson's disease, synucleinopathies, amyotrophic lateral sclerosis, Alzheimer's disease, dementia with Lewy bodies (DLB), pure autonomic failure (PAF), multiple system atrophy (MSA), and Huntington's disease. In certain embodiments, the subject invention prevents, treats, or ameliorates acute and chronic disorders of the CNS, including neuropathological conditions such as neuropathic pain, stroke, brain trauma, and epilepsy. In certain embodiments, the subject invention prevents, treats, or ameliorates Lewy body diseases (LBD) including, but not limited to, Parkinson's disease, Diffuse Lewy body disease (DLBD), Lewy body variant of Alzheimer's disease, multiple system atrophy (MSA), and combined PD and Alzheimer's disease.

The synucleinopathies represent a group of neurodegenerative disorders that contain aggregates of insoluble α-synuclein protein in selectively susceptible populations of neurons and glia. The protein α-synuclein is the major component of Lewy bodies found in PD patients, and plays a central role for the pathogenesis of PD. Synucleinopathies include Parkinson's disease (PD), dementia with Lewy bodies (DLB), pure autonomic failure (PAF), and multiple system atrophy (MSA). Clinically, synucleinopathies are characterized by a chronic and progressive decline in motor, cognitive, behavioral, and autonomic functions, depending on the distribution of the lesions. The deposition of aggregates of synuclein in neurons and glia suggests that a common pathogenic mechanism may exist for these disorders. The association between a- synuclein and neurodegenerative phenotypes in human patients indicates increased expression levels and the abnormal aggregation of a-synuclein plays a role in the pathogenesis of PD.

The present invention is the discovery that the total extract of RR, the subsequent butanol and water fractions of the RR total extract, and the compound RR-C36 (rosavin) inhibit the aggregation of a-synuclein and, thus, are useful for treating PD as well as other synucleinopathies.

In one embodiment, the method comprises administering an effective amount of a pharmaceutical composition comprising the Rhodiola rosea extract of the subject invention, a biologically-active substituent isolated from Rhodiola rosea (e.g., rosavin), and/or a compound selected from formula I - VIII as the active ingredient.

In certain embodiments, the pharmaceutical composition comprises at least 75% by weight, or any weight percent higher than 75% (including but not limited to, higher than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%), the Rhodiola rosea extract of the subject invention, a biologically-active substituent isolated from Rhodiola rosea (e.g., rosavin), and/or a compound selected from formula I - VIII as the active ingredient.

The term "subject," as used herein, describes an organism, including mammals such as primates, to which treatment with the compositions according to the present invention can be provided. Mammalian species that can benefit from the disclosed methods of treatment include, but are not limited to, apes, chimpanzees, orangutans, humans, monkeys; domesticated animals such as dogs, cats, horses, cattle, pigs, sheep, goats, chickens; and other animals such as mice, rats, guinea pigs, and hamsters.

In one specific embodiment, the term "treatment" includes (i) ameliorating a symptom associated with PD or PD-related disorders in a patient diagnosed with PD or PD-related disorders; and/or (ii) relieving (such as attenuating the progress of) or remedying PD or PD- related disorders in a patient diagnosed with PD or PD-related disorders.

In one embodiment, the subject in need of treatment in accordance with the present invention has, or is diagnosed with, a neurodegenerative disease, such as Parkinson's disease, synucleinopathies, amyotrophic lateral sclerosis, Alzheimer's disease, dementia with Lewy bodies (DLB), pure autonomic failure (PAF), multiple system atrophy (MSA), Huntington's disease, and Lewy body diseases (LBD).

In one embodiment, the present invention provides a method for inhibiting the aggregation of a-synuclein protein, inhibiting the aggregation of amyloid-beta peptide, and/or inhibiting the activation of caspase-3, wherein the method comprises administering, to a cell in need of such treatment, an effective amount of a composition comprising the Rhodiola rosea extract of the subject invention, a biologically-active substituent isolated from Rhodiola rosea (e.g., rosavin), and/or a compound selected from formula I - VIII. In one embodiment, the cell is a cell of a mammalian subject, preferably, a human subject.

Alpha-synuclein is a protein that, in humans, is encoded by the SNCA gene. An alpha- synuclein fragment, known as the non-Abeta component (NAC) of Alzheimer's disease amyloid, originally found in an amyloid-enriched fraction, is shown to be a fragment of its precursor protein, NACP, by cloning of the full-length cDNA. It was later determined that NACP was the human homologue of Torpedo synuclein. Therefore, NACP is now referred to as human alpha- synuclein. The amino acid sequences of the alpha-synuclein proteins of various species are publically available and can be readily obtained by a person skilled in the art via databases such as GenBank. In one embodiment, the alpha-synuclein protein is of human origin.

Amyloid beta (Αβ) is a peptide of 36-43 amino acids that is processed from the amyloid precursor protein. Αβ is the main component of deposits found in the brains of patients with Alzheimer's disease. The amino acid sequences of the amyloid beta proteins of various species are publically available and can be readily obtained by a person skilled in the art via databases such as GenBank. In one embodiment, the amyloid beta protein is of human origin.

Caspase-3, also known as CPP32/Yama/apopain, is encoded by the CASP3 gene, and is a member of the cysteine-aspartic acid protease (caspase) family. Caspase-3 is formed from a 32 kDa zymogen that is cleaved into 17 kDa and 12 kDa subunits. When the procaspase is cleaved at a particular residue, the active heterotetramer can then be formed by hydrophobic interactions, causing four anti-parallel beta- sheets from pl7 and two from pl2 to come together to make a heterodimer, which in turn interacts with another heterodimer to form the full 12-stranded beta- sheet structure surrounded by alpha-helices that is unique to caspases. When the heterodimers align head-to-tail with each other, an active site is positioned at each end of the molecule formed by residues from both participating subunits, though the necessary Cys-285 and His-237 residues are found on the pl7 (larger) subunit. Caspase-3 is involved in the cleavage of amyloid-beta 4A precursor protein, which is associated with neuronal death in Alzheimer's disease.

The amino acid sequences of the caspase-3 proteins of various species are publically available and can be readily obtained by a person skilled in the art via databases such as GenBank. In one embodiment, the caspase-3 protein is of human origin.

The term "treatment" or any grammatical variation thereof (e.g. , treat, treating, and treatment etc.), as used herein, includes but is not limited to, ameliorating or alleviating a symptom of a disease or condition, reducing, suppressing, inhibiting, lessening, or affecting the progression, severity, and/or scope of a condition.

The term "prevention" or any grammatical variation thereof (e.g. , prevent, preventing, and prevention etc. ), as used herein, includes but is not limited to, delaying the onset of symptoms, preventing relapse to a disease, increasing latency between symptomatic episodes, or a combination thereof. Prevention, as used herein, does not require the complete absence of symptoms.

The term "effective amount," as used herein, refers to an amount that is capable of treating or ameliorating a disease or condition or otherwise capable of producing an intended therapeutic effect. In certain embodiments, the effective amount enables at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the aggregation of a-synuclein protein.

Therapeutic Compositions and Formulations

In another aspect, the present invention provides a pharmaceutical composition for treating a neurodegenerative disease or neuropathological condition in a subject. In one embodiment, the composition comprises an effective amount of the Rhodiola rosea extract or isolated compounds of Rhodiola rosea and a pharmaceutically acceptable carrier or excipient.

The subject invention provides for therapeutic or pharmaceutical compositions comprising a therapeutically effective amount of the Rhodiola rosea extract of the present invention, and, optionally, a pharmaceutically acceptable carrier. The subject invention also provides therapeutic or pharmaceutical compositions comprising compounds isolated from Rhodiola rosea (such as rosavin) in accordance with the subject invention. The present invention also embodies dietary supplements and health food or drink formulations comprising the Rhodiola rosea extract of the invention.

In one embodiment, the therapeutic or pharmaceutical compositions comprise a therapeutically effective amount of the water fraction, and/or an alcohol (e.g., the solvent contains an alcohol such as methanol, ethanol, propanol, and butanol, and optionally, water) fraction of the Rhodiola Rosea extract of the present invention, and, optionally, a pharmaceutically acceptable carrier.

In one embodiment, the subject invention also provides therapeutic compositions, comprising an isolated or substantially pure compound selected from formula I through formula VIII, or a salt thereof, and optionally, a pharmaceutically acceptable carrier.

The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum oil such as mineral oil; vegetable oil such as peanut oil, soybean oil, and sesame oil; animal oil; or oil of synthetic origin. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.

Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The therapeutic composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, capsules, granules, powders, sustained-release formulations and the like. The composition can be formulated with traditional binders and carriers such as triglycerides. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. Such compositions contain a therapeutically effective amount of the therapeutic composition, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

The therapeutic or pharmaceutical compositions of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include, but are not limited to, salts formed with hydrochloric, phosphoric, acetic, oxalic, tartaric acids, sodium, potassium, ammonium, calcium, ferric hydroxides, etc.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients, e.g., compound, carrier, of the pharmaceutical compositions of the invention.

The compositions of the subject invention can also be formulated consistent with traditional Chinese medicine practices. The composition and dosage of the formulations that are effective in the treatment of a particular disease, condition, or disorder will depend on the nature of the disease, condition, or disorder by standard clinical techniques.

The traditional Chinese medicine in prescription amounts can be readily made into any form of drug suitable for administering to humans or animals. Suitable forms include, for example, tinctures, decoctions, and dry extracts. These can be taken orally, applied through venous injection or mucous membranes. The active ingredient can also be formulated into capsules, powder, pallets, pastille, suppositories, oral solutions, pasteurized gastroenteric suspension injections, small or large amounts of injection, frozen powder injections, pasteurized powder injections, and the like. All of the above-mentioned methods are known to people skilled in the art, described in books and commonly used by practitioners of herbal medicine.

A tincture is prepared by suspending raw medicinal materials (e.g. herbs and fungus) in a solution of alcohol, such as, for example, wine or liquor. After a period of suspension, the liquid (the alcohol solution) may be administered, for example, two or three times a day, one teaspoon each time.

An extract is a concentrated preparation of the essential constituents of a medicinal raw material. Typically, the essential constituents are extracted from the raw medicinal materials (e.g. herbs and fungus) by suspending the raw medicinal materials in an appropriate choice of solvent, typically, water, ethanol/water mixture, methanol, butanol, iso-butanol, acetone, hexane, petroleum ether, or other organic solvents. The extracting process may be further facilitated by means of maceration, percolation, repercolation, counter-current extraction, turbo-extraction, or by carbon-dioxide hypercritical (temperature/pressure) extraction. After filtration to rid of herb debris, the extracting solution may be further evaporated and thus concentrated to yield a soft extract (extractum spissum) and/or eventually a dried extract, extractum siccum, by means of spray drying, vacuum oven drying, fluid-bed drying, or freeze-drying. The soft extract or dried extract may be further dissolved in a suitable liquid to a desired concentration for administering or processed into a form such as pills, capsules, injections, etc.

Routes of Administration

The compounds and compositions of the subject invention can be administered to the subject being treated by standard routes, including oral, inhalation, or parenteral administration including intravenous, subcutaneous, topical, transdermal, intradermal, transmucosal, intraperitoneal, intramuscular, intracapsular, intraorbital, intracardiac, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection, infusion, and electroporation, as well as co-administration as a component of any medical device or object to be inserted (temporarily or permanently) into a subject. In preferred embodiments, the compounds and compositions of the subject invention are administered to a subject by oral administration.

The amount of the therapeutic or pharmaceutical composition of the invention which is effective in the treatment of a particular disease, condition or disorder will depend on the route of administration, and the seriousness of the disease, condition or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. In general, the dosage ranges from about 0.001 mg/kg to about 3 g/kg.

For instance, suitable unit dosages may be between about 0.01 to about 500 mg, about 0.01 to about 400 mg, about 0.01 to about 300 mg, about 0.01 to about 200 mg, about 0.01 to about 100 mg, about 0.01 to about 50 mg, about 0.01 to about 30 mg, about 0.01 to about 20 mg, about 0.01 to about 10 mg, about 0.01 to about 5 mg, about 0.01 to about 3 mg about, 0.01 to about 1 mg, or about 0.01 to about 0.5 mg. Such a unit dose may be administered more than once a day, e.g. two or three times a day.

The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary, depending on the type of the condition and the subject to be treated. In general, a therapeutic composition contains from about 5% to about 95% active ingredient (w/w). More specifically, a therapeutic composition contains from about 20% (w/w) to about 80% or about 30% to about 70% active ingredient (w/w).

Once improvement of the patient's condition has occurred, a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, may be reduced as a function of the symptoms to a level at which the improved condition is retained. When the symptoms have been alleviated to the desired level, treatment should cease. Patients may however require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.

In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease, condition or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

Materials and Methods

Preparation of Rhodiola rosea total extract

Air-dried roots of Rhodiola rosea harvested at Yili, Xin-Jiang, China were purchased from Chengdu pharmaceutical company, Chengdu, Sichuan province, China. The air-dried roots of R. rosea (300 g) were immersed in 1.5 L 70% ethanol (material to solvent ratio at 1 to 5) for 30 min. The herb-solvent mixture was then refluxed 3 times for 2 hr each (1.5 L, 1.5 L, 1.5 L, 2 hr/time). The extract was filtered and the filtrate was evaporated to dryness in vacuum to yield 44.0 g of the total extract (TE).

Preparation ofRRBU and RRWA fractions The Rhodiola rosea total extract (TE) (50.0 g) was suspended in 300 ml water and then partitioned successively with 300 ml petroleum ether (60-90°C), 300 ml ethyl acetate, and 300 ml n-BuOH. Each extraction was performed 3 times and the extracts were filtered and combined. Evaporation of these fractions resulted in a total of 4.8 g petroleum ether extract (PE), 13.8 g ethyl acetate (EA), 19.2 g n-BuOH extract (BU), and 10.6 g water extract (WA).

Filter trap assay for the detection of a-synuclein aggregation

Recombinant a-synuclein was purchased from Genway (# 10-663-45667). The protein was diluted to a concentration of 1 μg μΐ and was stored in aliquots at -80°C before use.

An aliquot was thawed on ice and spun at 12000xg to bring down the pre- formedaggregates. The protein (0.07 μg) was then incubated with Rhodiola rosea samples or DMSO (0.2%) or congo red (5 μΜ) in lx Tris-Buffered Saline (lx TBS: 20 raM Tris, pH 7.5; 500 mM NaCl, pH 7.5 with HC1) at room temperature (RT) for 7 days. After incubation, the samples were loaded onto the Bio-Dot SF Microfiltration Apparatus (Bio Rad) according to the manufacturer's instructions. After vacuum filtration, the α-synuclein aggregates were trapped on the 0.45 μιη nitrocellulose membrane (S&S). The amount of trapped protein was determined by Western Blot analysis using the anti-a-synuclein antibody (BD transduction laboratories, # 610787), 1 :5000 (1 hr RT) and horseradish peroxidase (HRP)-conjugated horse anti-mouse IgG secondary antibody (Cell Signaling Technology) 1 :5000 (1 hr RT). Protein expression was then visualized via an enhanced chemiluminescence detection kit (ECL; Amersham).

6-OHDA injected mouse PD model and the detection of tyrosine hydroxylase expression

6-hydroxydopamine (6-OHDA, 50 μg, Sigma) or the same volume of the vehicle (L- ascorbic acid, 0.02%; Sigma) was injected into the cerebral ventricles of male C57BL/6 mice (10 - 12-week-old) provided by the animal care facility of HKUST. Desipramine (25 mg/kg) was intraperitoneally administered at 1 hour before the injection of 6-OHDA to block noradrenaline reuptake in order to protect neurons other than dopaminergic neurons.

Mice were anaesthetized using chloralhydrate (400 mg/kg, i.p.) and were placed into a stereotaxic frame adaptor for mice (Koft). 6-OHDA was dissolved at a concentration of 10 μg/μl in saline containing 0.02% ascorbic acid and 5 μΐ was injected at a rate of 0.5 μΐ/min. The needle (Hamilton) was left in place for 7 min after the injection before retraction.

The injection was performed using the following co-ordinates: 0.5 mm anteroposterior and 1.0 mm mediolateral from bregma, 2.0 mm dorsoventral from skull. 3 days after stereotaxic injection, striatal tissues were dissected, weighed and homogenized with 1: 10 ratio of lysis buffer (20 mM Tris [pH 7.6], 150 mM NaCl, 1 mM EDTA, 1 mM NaF, 0.5% NP-40 and protease inhibitors). The homogenates were incubated at 4°C for 20 min, followed by centrifugation at 20,000 x g at 4°C for 10 min. The supernatants were collected and the protein concentration was determined by DC protein assay kit (Bio-rad).

The protein samples were mixed with SDS sample buffer and boiled at 100°C for 5 min. Equal amounts of the proteins were separated on 10% SDS-PAGE gel and transferred to a nitrocellulose membrane. The membrane was incubated with antibodies to tyrosine hydroxylase (TH, 1 :4000, Millipore) and β-actin (1:2000, Sigma). Blots were then incubated with HRP- conjugated secondary antibody (1 :4000, Cell Signaling Technology), followed by chemiluminescent detection (ECL; Amsersham).

Determination of compound composition infraction RRBU and RRWA

HPLC conditions:

The HPLC-DAD method has been developed for the analysis and the quality control of RR-TE, CF, BU, WA and standard compounds. A Waters HPLC system consisting of a 600 pump, a 717 auto-sampler and a UV/VIS Photodiode Array 2996 Detector was used for all analyses.

Chromatographic separations were carried out on a SunFire CI 8 column (Particle size 5 μπι, 4.6 mm x 150.0 mm) with acetonitrile (as Solvent A) and water (as Solvent B) in the mobile phase at a flow rate of 1.0 ml/min at room temperature. A gradient elution was applied from 2% to 70% of solvent A starting from 0 to 45 min (0-33 min 2%~50% ACN; 33-40 min, 50%~70% ACN; 40-43 min, 70% ACN; 43-45 min, 70%~2% ACN for Fig. 10A-D, and from 0-40 min, 10% to 85% ACN; 40-42 min, 85% to 10% CAN for Fig. 11 A-D). The wavelength used to detect the compounds was 220 nm. Samples were dissolved in MeOH and water with a ratio of 2: 1, and filtered through a 0.45 μηι Millipore syringe filter unit. Twenty microliter samples were injected for HPLC analysis.

Determination ofRR-C36 level in mouse brain and plasma after intraperitoneal injection

The RR-C36 compound (rosavin) was dissolved in saline and intraperitoneally injected into an 8-week-old C57B1/6 mouse at 300 mg/kg (10 ml/kg). At 15, 30 and 60 min after administration, the mouse was anesthetized and blood was drawn from the eye socket into an EDTA blood collection tube. The mouse was then perfused with saline for 30 min and the brain was dissected. The brain was then homogenized in 300 μΐ water. Plasma was collected after centrifugation. 1 ml of trifluoroacetate was then added to the plasma and brain homogenate and vortexed to mix thoroughly. Centrifugation was then applied to bring down the debris. The supernatant was transferred and evaporated under vacuum. 0.1 ml acetonitrile (ACN) was then added to each tube, vortexed and sonicated for 5 min. Any insolubles were spun down by centrifuging at 13800 rpm for 5 min. 75 μΐ supernatant was carefully transferred into an insert for LC-MS/MS analysis. The injection volume was 10 μΐ.

HPLC conditions

An Agilent 1200 series HPLC apparatus was used, equipped with a G- 1312 binary pump, G-1313 auto-sampler, and G-1316 column oven. This system is operated by an HP Chemstation software (Agilent Technologies, Palo Alto, CA, USA). Chromatography was performed on an Agilent Zorbax C18 column (50 x 3.0 mm, 1.8 μπι) at a column temperature of 40°C. The mobile phase consisted of (A) 0.1% aqueous formic acid and (B) methanol. Ten microliters of each sample solution was injected and eluted by the following program at the flow rate of 0.5 mL/min: 0-5 min, 30-100% B; 5-6 min, 100% B; 6-6.1 min, 100-30% B; 6.1-10 min, 30% B.

MS/MS detection: API 4000+ MS

The MS analysis was carried out on an AB SCIEX 4000+ triple quadrupole system equipped with a Turbo V source with Electrospray ionization probe. ESI in multiple-reaction- monitoring (MRM) mode was performed in the positive ion mode. In the MRM mode, quadrupole 1 is fixed at a set parent ion, quadrupole 2 is used as a collision chamber to induce fragmentation, and quadrupole 3 is fixed at a set daughter ion. The ESI conditions were as follows: declustering potential 60V, entrance potential 10V, collision cell exit potential 12V, collision energies 60 V curtain gas 20 (arbitrary units), collision gas 5 (arbitrary units), ion spray voltage 5500 kV, source temperature 400 °C, ion source gas 1 : 40 (arbitrary units), ion source gas 2: 20 (arbitrary units). The parent/daughter ion pairs used are as 451>117.

Locomotor activity test for PD mouse

Rotorod Test

The Rotorod Test assesses the motor coordination, balance, and equilibrium of rodents, and is a sensitive indicator of subtle defects in rodent motor coordination that may be caused by a test compound. In the test, a rodent is placed on a rotorod (rotating cylinder) powered by a small motor, and the manner in which the animal walks on the drum is noted. The locomotor activity of the mice was tested on an accelerating rotorod (Panlab). Mice were allowed to adapt to the machine one day before training. The mice were placed on a horizontal plastic rod rotating at an initial speed of 4 rpm, and the rotational velocity of the rod was linearly increased from 4 to 40 rpm within 10 min. The latency of the time that each mouse maintained its balance while walking on the top of the rod was measured. Test subjects were pre-trained 3 times the day before treatment or surgery. After surgery, the latency for the subjects to fall was recorded.

Open Field Test

The Open Field Test measures the effect of a test compound on the locomotor activity of the subjects. Locomotor activity was measured by placing each mouse in an open field (a square base (50x 50 cm) surrounded by a 40 cm high darkened wall) for 30 min. Before each trial, the field was cleaned with 70% ethanol and then wiped with wet cotton to prevent possible bias due to odor clues left by previous mice. Each mouse was placed individually in the center of the field and its activity in the field was recorded by a videocamera mounted 2 m above. Scoring of each mouse was performed using Noldus EthoVision XT software. Mice that exhibited a difference of over 2 SD from the mean on the time spent at the corner or peripheral or central region of the field and total distance travelled were excluded.

Atomic force microscopy

Recombinant a-synuclein was purchased from GenWay Biotech, Inc. (San Diego CA) and stored at -20°C. Protein solutions were defrosted and centrifuged for 10 min at 20,000 x g to remove any preformed aggregates or contaminating particles.

Aggregation experiments were performed by adding 2 μΐ of TCM drugs or DMSO dissolved in TBS buffer (pH 7.5) to 40 μΐ aliquots of a-synuclein (1 mg/mL). 10 μΐ of the drug and α-synuclein mixture were further aliquoted into siliconized microcentrifuge tubes (Sigma- Aldrich, St. Louis, MO) for time points of day 0, 3, 7, and 14. Day 0 samples were immediately fixed to freshly cleaved muscovite mica substrate, as described below. Day 3, 7, and 14 samples were incubated in a temperature controlled shaker (C25 Incubator Shaker, New Brunswick Scientific, Edison, NJ) at 37°C with shaking at 250 rpm. At each time point, 2.1 μΐ of each aliquot was applied to a freshly cleaved muscovite mica substrate (3 mm discs, SPI Supplies, West Chester, PA) and incubated for 20 minutes. The mica surface was rinsed with filtered water (7 x 100 μΐ) to remove salts and loosely bound protein. Samples were air dried overnight for AFM imaging the following day. Samples were imaged using a Veeco MultiMode Scanning Probe Microscope (Santa Barbara, CA) with Bruker Scanasyst-Air probes (Camarillo, CA).

All measurements were carried out in the tapping mode under ambient conditions, using ScanAsist in air configuration. Imaging was carried out at a scan rate of 0.977 Hz with 512 data points per line. 2 μπι images were captured at four predefined locations to demonstrate that structures were consistent over the whole face of each mica chip. Images were analyzed using Nanoscope Analysis 1.4 (Bruker).

Detection of a-synuclein and cell death in SH-SY5Y cells

Human neuroblastoma cell line, SH-SY5Y, was maintained in MEM (Invitrogen, Carlsbad, CA) supplemented with 10% FBS, 100 U/ml penicillin and 1 mg/ml streptomycin, at 37°C in 5% C0₂. For Western blot analysis of cleaved caspase-3 and alpha-synuclein aggregation, 1 x 10⁵ cells per ml of SH-SY5Y cells were seeded in a 100-mm tissue plate. After 24-hr incubation, cells were pretreated with RRBU and RRWA in MEM with 2% FBS for 2 hr. Cells were then treated with 1 mM MPP+ (Sigma-Aldrich, St. Louis, MO) for 20 hr. Cells were lysed in modified RIPA lysis buffer (150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, 0.5% deoxycholic acid, 2 μg/ml aprotinin, 1 mM PMSF, 5 mM benzamidine, 1 mM sodium orthovanadate and 10 μg/ml soybean trypsin inhibitor in 50 mM Tris buffer, pH 7.4).

Protein quantification reagent was purchased from BioRad laboratories (Hercules, CA). Following separation by SDS-PAGE gel electrophoresis, the proteins were transferred onto a nitrocellulose membrane. After blocking with 0.1% Tween-20 and 5% non-fat dry milk in Tris- buffered saline at room temperature for 1 hr, the membrane was then incubated with primary antibody (1: 1000) at 4°C overnight, and with horseradish peroxidase (HRP)-conjugated secondary antibody (1 :2000) for 1 hr. Antibodies to cleaved caspase-3 and secondary antibodies (HRP-conjugated goat anti-mouse, anti-rabbit antibodies) were purchased from Cell Signaling Technology (Beverly, MA). Antibody to a-synuclein is from BD Transduction Laboratories (San Jose, CA). Antibody to GAPDH was obtained from Ambion (Invitrogen).

Detection was performed using Enhanced Chemiluminescence (ECL) Western Blot System (GE Healthcare, Buckinghamshire, UK). Quantification of the Western blots was performed using ImageJ.

Human A53T transgenic mouse PD model

Transgenic mice overexpressing human A53T a-synuclein under the control of mouse prion promoter (also referred to herein as human A53T transgenic mice) were used for the behavioral and biochemical studies (Giasson, 2002, Neuron 34:521-233). Genotyping was performed as described by Jackson Laboratory. Briefly, genomic DNA was extracted from mouse ears by proteinase K digestion. Homozygous transgenic mice (A53T) and age-matched non-transgenic mice (wild-type, WT) were identified by quantitative PCR of DNA samples. Both A53T (n= 4-6) and WT (n= 2-3) mice were randomly allocated into different experimental groups. Detection of a-synuclein in transgenic mouse PD models

Expression of a-synuclein in transgenic mice was detected by biochemical fractionation as described (Ihara, 2007, Neuron 53:519-33). Spinal cord from each mouse was dissected, weighed and homogenized by sonication in 3 ml/g of Triton X-100 soluble buffer (10 mM Tris- HC1 [pH 7.6], 150 mM NaCl, 1% Triton X-100 and protease inhibitors) and then incubated at 4°C for 20 minutes. The homogenate was centrifuged at 15,000 x g at 4°C for 30 minutes. After centrifugation, the supernatant was preserved as "Triton-soluble fraction" and the pellet was further sonicated and extracted with 1 ml/g of 0.1% SDS buffer (10 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS and protease inhibitors). After centrifugation at 15,000 x g at 4°C for 30 minutes, the supernatant was preserved as "SDS- soluble fraction". The remaining pellet was dissolved and sonicated in 1 ml/g of 3% SDS buffer (3% SDS and 5% β-mercaptoethanol). The lysate was boiled for 5 minutes and kept as "SDS- insoluble fraction". The samples were mixed with SDS sample buffer and boiled at 100°C for 5 min right before loading and protein separation in 15% SDS-PAGE gel. The proteins were then transferred to nitrocellulose membrane and the membrane was incubated with antibodies to a- synuclein (1 : 1000, BD Transduction Laboratories), LB509 (1:500, Invitorgen) and GAPDH (1 : 10,000, Ambion). Blots were then incubated with HRP-conjugated secondary antibody (1 :4000, Cell Signaling Technology), followed by chemiluminescent detection (ECL; GE Healthcare).

Detection of Αβ(1-42) aggregates by thioflavin T assay

The relative degree of amyloids-aggregation is determined by thioflavin T (ThT) that specifically binds to fibrillar structures. Αβ(1-42) peptide was purchased from rPeptide (Bogart, GA). Preparation of Αβ(1-42) aggregates was undertaken according to the manufacturer's protocol.

In brief, Αβ(1-42) peptide was first dissolved in water at 6 mg/ml and then diluted in PBS to 1 mg/ml. Thirty μΐ of Αβ(1-42) solution was incubated with RR fractions or isolated compounds at 37°C for 3 days. ThT (Sigma- Aldrich) was then added to each sample to a final concentration of 20 μΜ. Each sample was measured in terms of fluorescence intensity using the FLEX Station (Molecular Devices, Sunnyvale, CA). The fluorescence arbitrary units were measured with an excitation wavelength of 430 nm and an emission wavelength at 485 nm with a cutoff at 455 nm.

EXAMPLES

Following are examples that illustrate embodiments for practicing the invention. These examples should not be construed as limiting. All solvent mixture proportions are by volume unless otherwise noted.

EXAMPLE 1 - RHODIOLA ROSEA EXTRACT INHIBITS THE AGGREGATION OF ALPHA-SYNUCLEIN PROTEIN

Total extract (TE) of Rhodiola rosea (RR) was prepared. Recombinant-alpha-synuclein in TBS was incubated with the extract (0.07 g) for 7 days at 37°C. After incubation, the samples were filtered with a Bio-Dot SF microfiltration apparatus (Bio Rad) according to the manufacturer's instructions. After filtration, the amount of trapped alpha-synuclein was determined by Western blot analysis.

As shown in Figure 1, in non-treated control samples, the amount of the aggregated form of alpha-synuclein protein increased significantly on day 7, when compared to day 0. Similar amount of the aggregated form of alpha-synuclein protein was found in day-7 samples treated with DMSO, when compared to non-treated control. Samples treated with congo red, a known inhibitor against alpha-synuclein aggregation, were used as positive control. Incubation of the alpha-synuclein protein with TE of RR (RRTE) at 1 μg to 100 μg inhibited alpha-synuclein aggregation, and such inhibition effect was comparable to that of congo red (Fig. 1).

EXAMPLE 2 - WATER AND ETHANOL FRACTIONS OF RHODIOLA ROSEA EXTRACT INHIBIT THE AGGREGATION OF ALPHA-SYNUCLEIN PROTEIN

Total extract (TE) and fractions of Rhodiola rosea (RR) were prepared. Recombinant- alpha-synuclein in TBS was then incubated with the extract ^g/ml) for 7 days at 37°C. After incubation, the samples were filtered with a Bio-Dot SF microfiltration apparatus (Bio Rad) according to the manufacturer's instructions. After filtration, the amount of trapped alpha- synuclein was determined by Western blot analysis. Recombinant alpha-synuclein was incubated with RR total extract (RRTE) and RR fractions (RRPE (Rhodiola rosea petroleum ether), RREA (Rhodiola rosea ethylacetate), RRBU (Rhodiola rosea butanol) and RRWA (Rhodiola rosea water)) for 7 days. The filter trap assay was performed as described in Example 1. The non-treated samples were negative control, and the congo red-treated samples were positive control.

The results show that RRTE, RREA, RRBU and RRWA inhibited aggregation of alpha synuclein at a concentration as low as 1 μg/ml, while RRPE did not inhibit alpha synuclein aggregation even at 10 μg/ml. RRTE at 3 μg/ml inhibited the alpha-synuclein aggregation, and the inhibition effect was comparable to that of congo red (positive control). Butanol (BU), EA (ethylacetate), and water (WA) fractions were prepared from the total extract. The results show that the BU, EA, and WA fractions (at 1 g ml) potently inhibited the alpha-synuclein aggregation, and such inhibition effects were greater than that of TE (Fig. 2).

EXAMPLE 3 - INHIBITION OF THE AGGREGATION OF ALPHA-SYNUCLEIN PROTEIN

The RR-C20 compound (salidroside) and the RR-C36 compound (rosavin) were isolated from RRWA. Recombinant alpha-synuclein in TBS was then incubated with the RR-C20 compound and the RR-36 compound, respectively, for 7 days at 37°C. After incubation, the samples were filtered with a Bio-Dot SF microfiltration apparatus (Bio Rad) according to the manufacturer's instructions. After filtration, the amount of trapped alpha-synuclein was determined by Western blot analysis.

As shown in Figure 3A, a-synuclein aggregates increased after 7-day incubation and congo red inhibited the increase in α-synuclein aggregation. RR-C36 (rosavin) isolated from RRBU or RRWA inhibited the alpha-synuclein aggregation in a dose-dependent manner.

Figure 3B shows that RR-C36 (rosavin) inhibited the aggregation of alpha-synuclein in vitro in a dose-dependent manner. The IC₅₀ of RR-C36 anti-aggregation effect was -0.36 μΜ, as determined by PRISM GraphPad (version 5.03).

The disaggregation effect of RR-C36 was examined by incubating pre- aggregated alpha- synuclein (incubating alpha-synuclein for 5-days) with RR-C36 for 2 days. RR-C36 at 10 μΜ induced disaggregation of pre-aggregated alpha-synuclein. Figure 3C shows that a-synuclein aggregates increased after 7-day incubation, and congo red inhibited the increase in a-synuclein aggregation. RR-C22 (6-O-galloyl rosin) and RR-C49 (rosarin) isolated from RR inhibited the alpha-synuclein aggregation in a dose- dependent manner.

Figure 3D shows that α-synuclein aggregates increased after 7-day incubation, and congo red inhibited the increase in α-synuclein aggregation. RR-C31 (icariside D2) and RR-C47 (mongrhoside) isolated from RR inhibited the alpha-synuclein aggregation in a dose-dependent manner.

Figure 3E shows that α-synuclein aggregates increased after 7-day incubation, and congo red inhibited the increase in α-synuclein aggregation. RR-C04 (gallic acid) and RR-C34 (6-O- galloyl arbutin) isolated from RR inhibited the alpha-synuclein aggregation in a dose-dependent manner.

EXAMPLE 4 - RHODIOLA ROSEA FRACTIONS, RR-C36, AND RR-C41 INHIBIT OLIGOMERS FORMATION OF ALPHA-SYNUCLEIN

Alpha-synuclein formed distinct morphologies in vitro as observed in atomic force microscopy (Aperti et al, 2006). Smaller, larger spheroidal oligomers and fibrillar species were detected based on the height differences. Non-fibrillar oligomers such as spheres are 2-6 nm in height, filaments are 5 nm in height and fibrils are 8-10 nm in height. Fractions of Rhodiola rosea (RR), RR-C36 and RR-C41 were incubated with recombinant alpha-synuclein in TBS for 7 and 14 days at 37°C. After incubation, the samples were spotted on mica and were analyzed using atomic force microscopy. As shown in Figure 4 A & 4B, oligomers or short fibrils of alpha-synuclein were observed in DMSO-treated samples; in contrast, fractions RRBU, RRWA, compound RR-C36 (rosavin) and RR-C41 (rhodiocyanoside A) inhibited the formation of larger alpha-synuclein aggregates.

EXAMPLE 5 - RR FRACTIONS EXHIBIT ANTI-AGGREGATION ACTIVITY OF Αβ1-42

To examine whether the total extract of RR, various RR fractions, and rosavin ( RR-C36) inhibit the aggregation of amyloid-beta peptide (Αβ1-42), recombinant human Αβ1-42 were incubated with RRTE, various RR fractions and rosavin, respectively, for 3 days at 37°C. After incubation, the samples were subjected to the thioflavin T (ThT) binding assay. Fractions RRBU and RRWA showed anti-aggregation activity on Αβ1-42 aggregation, as revealed by the reduction in ThT fluorescence units. RR-C36 did not reduce the amount of Αβ1-42 aggregates.

EXAMPLE 6 - RRBU AND RRWA PREVENT MPP+ INDUCED NEURONAL CELL DEATH AND RRWA INHIBITS MPP+ INDUCED ALPHA-SYNUCLEIN AGGREGATION

MPP+ treatment of SH-SY5Y cells induces cell apoptosis, the aggregation of alpha- synuclein, as well as the formation of high-molecular weight aggregates (Kalivendi et al., Alpha- synuclein up-regulation and aggregation during MPP+-induced apoptosis in neuroblastoma cells: intermediacy of transferrin receptor iron and hydrogen peroxide. J. Biol. Chem. (2004) 279: 15240-15247).

In this Example, SH-SY5Y cells were first pretreated with RRBU (50 RRWA (50 μg/ml), or DMSO (0.1%) for 2 hr, and then incubated with MPP+ (1 mM) for 20 hr. Total cell lysates were collected for Western blotting against cleaved caspase-3 and alpha synuclein.

As shown in Figure 6, RRBU and RRWA reduced the expression level of cleaved or activated forms of caspase-3 in the presence of MPP+. Probing with GAPDH served as the loading control. In addition, both RRBU and RRWA reduced formation of high molecular weight alpha-synuclein, and increased the amount of monomers in the cell lysate.

EXAMPLE 7 - WA FRACTION OF RHODIOLA ROSEA REDUCES THE LOSS OF TYROSINE HYDROXYLASE IN 6-OHDA INJECTED MICE

6-hydroxydopamine (6-OHDA), a natural dopaminergic toxin, was injected, via the intracerebroventricular route, into the mice pretreated with RRWA for 5 days. The mice were then treated with RRWA daily for an additional 3 days after the stereological surgery. Striatum of the mice were dissected and proteins were extracted. The expression of tyrosine hydroxylase (TH) in the striatum was analyzed using Western blot. Fig. 7A illustrates the experimental design.

Tyrosine hydroxylase is an enzyme that catalyzes the conversion of the amino acid L- tyrosine to dihydroxyphenylalanine - the precursor of dopamine. Decreased expression of tyrosine hydroxylase indicates the loss of dopaminergic neurons. As shown in Fig. 7B, 6-OHDA injected mice showed a drastic reduction in the protein expression of tyrosine hydroxylase in the striata, when compared to ascorbic acid-injected mice (served as sham control). Treatment of water fraction of Rhodiola rosea (RRWA) at 10 mg/kg or 100 mg/kg restored the TH level in 6-OHDA-treated mice as revealed by Western blot analysis (Fig. 7B).

The locomotor activity of the 6-OHDA injected mice was evaluated in the Rotorod Test. The test was performed 1 day before (pre-test) and 3 days after the stereological surgery, using a standard rotorod apparatus at an accelerating speed of 4 rpm to 40 rpm over 600 s. The mean fall latency of each mouse for the three trials was measured. Three days after 6-OHDA injection, the fall latency dropped about 67%. Mice treated with RRWA showed improvement in motor behavior after 6-OHDA lesion. As shown in Figure 7C, the locomotor deficit was attenuated in RRWA-treated mice. The results show that RRWA reduces TH loss induced by 6-OHDA, and attenuates motor deficit after 6-OHDA lesion.

EXAMPLE 8 - RRWA, RRBU AND ROSAVIN AMELIORATE MOTOR DEFICITS IN HUMAN A53T ALPHA-SYNUCLEIN TRANSGENIC MICE

The effects of RRBU, RRWA and RR-C36 were examined in human A53T alpha- synuclein transgenic (A53T) mice. Five-to-six-month old A53T mice showed impaired motor activity, as compared to non-transgenic (WT) mice, as determined by the Open Field Test (Fig. 8, left panel). A53T mice were randomly grouped and treated orally with RRBU (150 mg/kg), RRWA (150 mg/kg) or RR-C36 (rosavin) (20 mg/kg) daily for 4 weeks. Motor function of the treated mice was examined in the Open Field Test after the 4-week treatment with RR-C36, RRWA, or RRBU. RR-C36, RRWA and RRBU improved the motor function of the A53T mice, as the results show that the treated mice traveled greater distance in an open field (Fig. 8, right panel).

EXAMPLE 9 - DETECTION OF ROSAVIN IN MOUSE PLASMA AND BRAIN AFTER INTRAPERITONEAL INJECTION

Rosavin (RR-C36) was administered to a mouse via intraperitoneal injection. 15, 30 and 60 min after intraperitoneal injection, RR-C36 was detected in mouse brain and plasma using LCMS/MS detection. The level of RR-C36 was semi-quantified using known concentrations of RR-C36 mixed with brain homogenate and plasma. The actual concentrations of RR-C36 in brain and plasma were interpolated from the standards.

As shown in Fig. 9, RR-C36 can be detected in the brain and plasma of the mouse 15 minutes after administration.

EXAMPLE 10 - EXTRACTION, ISOLATION AND STRUCTURAL ELUCIDATION OF COMPOUNDS ISOLATED FROM RHODIOLA ROSEA

Air-dried roots of Rhodiola rosea harvested at Yili, Xin-Jiang, China were purchased from Chengdu pharmaceutical company, Chengdu, Sichuan province, China. The air-dried roots of R. rosea (7.5 kg) were re-fluxed three times with 70% aqueous EtOH (35 L, 35 L, 35 L, 2 hr each). The 70% ethanol extract was concentrated in vacuum to yield a residue (900 g). The residue was suspended in 1.5L H₂0 and then partitioned successively with petroleum ether (60- 90°C, 1.5Lx3), ethyl acetate (1.5Lx3), and n-BuOH (1.5Lx3). Evaporation of these fractions resulted in a total of 90 g petroleum ether extract, 250 g EtOAc extract, 360 g «-BuOH extract, and 190 g water extract.

A portion of n-BuOH extract (200 g) was subject to silica gel column chromatography, eluting with EtOAc EtOH/H₂0 with a ratio of 20:2: 1, 16:2: 1, 10:2: 1, 6:2: 1, 4:2: 1, resulting in 66 fractions. Based on the TLC behavior, these fractions were combined and afforded 10 sub- fractions (Fr.A-Fr.J). Compounds RR-C04 (gallic acid, 15 mg) from Fr.A, RR-C20 (salidroside, 1.8 g) from Fr.B, RR-C47 (mongrhoside, 5 mg ) from Fr.C, RR-C31 (icariside D2, 12 mg), RR- C34 (6-O-galloyl arbutin, 16 mg), RR-C36 (rosavin, 800 mg) and RR-C49 (rosarin, 8 mg) from Fr.D, RR-C22 (6-O-galloyl rosin, 6 mg) and RR-C41 (rhodiocyanoside A, 360 mg) from Fr.F, were isolated using repetitive column chromatography (silica gel column, eluted with CHC1₃ and methanol with a ratio of 50: 1-5:1 ; or Sephadex LH-20 column, eluted with methanol).

The structures of compounds RR-C4, RR-C20, RR-C22, RR-C31, RR-C34, RR-C36, RR-C41, RR-C47 and RR-C49 were elucidated based on Ή NMR and ^{l 3}C NMR, analysis. NMR data of these compounds are shown in Table 1, Table 2 and Table 3. Table 1. The Ή and ¹³C NMR Data of Compounds RR-C20, RR-C36 and RR-C41 (CD₃OD,

400 MHz)

RR-C20 RR-C36 RR-C41

Position 'HNMR ¹³C NMR 'HNMR ¹³C 'H MR ^,3C

NMR NMR

1 131.0 138.1 1.97 (3H, d, J=1.2Hz) 20.4

2 7.06 (IH, d, J= 8.0Hz) 131.2 7.45 (IH, d, J=7.2 Hz) 127.5 112.6

3 6.70 (IH, d, J= 8.0Hz) 116.4 7.35 (IH, t, J=7.2Hz) 129.6 6.48 (IH, m) 145.0

4 157.1 7.26 (IH, t, J=7.2 Hz) 128.8 4.42 (IH, m) 68.4

4.53 (IH, m)

5 6.70 (IH, d, J= 8.0Hz) 116.4 7.35 (IH, t, J=7.2 Hz) 129.6 118.3

6 7.06 (IH, d, J= 8.0Hz) 131.2 7.45 (IH, d, J=7.2 Hz) 127.5

7 2.82 (2h. t, J= 7.2Hz) 36.6 6.73 (IH, d, J=16.0Hz) 133.7

8 3.84 (IH, m) 72.3 6.40 (IH, dt, J=16.0, 126.7

4.04 (IH, m) 6.0Hz)

9 4.55 (IH, dt, J=12.8, 66.9

5.2Hz)

4.34 (IH, dt, J=12.8,

1.60Hz)

Glc-1 4.28 (JH, d, J=8.0 Hz) 104.6 4.46 (IH, d, J=7.6Hz) 103.4 4.32 (IH, d, J=7.6 Hz) 103.7

Glc-2 3.20 (IH, m) 75.4 3.35 (IH, m) 74.9 3.20 (IH, m) 74.8

Glc-3 3.35 (IH, m) 78.3 3.53 (IH, m) 76.7 3.36 (IH, m) 77.8

Glc-4 3.28 (IH, m) 71.9 3.51 (IH, m) 74.1 3.27 (IH, m) 71.2

Glc-5 3.37 (IH, m) 78.2 3.89 (IH, m) 77.8 3.37 (IH, m) 77.7

Glc-6 3.73 (IH, m) 63.0 3.92 (IH, m) 69.5 3.86(lH,dd, J=12.0, 1.6 62.4

3.64 (IH, m) 3.74 (IH, m) Hz)

3.69(lH,dd, J=12.0, 5.2 Hz)

Ara-1 4.39 (IH, d, J=6.8Hz) 105.1

Ara-2 3.30 (IH, m) 71.4

Ara-3 3.58 (IH, m) 72.4

Ara-4 3.62 (IH, m) 71.0

Ara-5 4.18 (IH, d, J=l 1.2 Hz) 66.9

3.82 (IH, m)

Table 2. The Ή and ^,3C NMR Data of Compounds RR-C4, RR-C22 and RR-C34 (CD₃OD, 400

MHz)

RR-C4 RR-C22 RR-C34

Position 'HNMR ¹³C NMR 'HNMR ^,3C 'HNMR ^,3C

NMR NMR

1 121.8 138.3 154.0

2 7.05 (IH, s) 110.2 7.34 (IH, d, J=7.6 Hz) 127.7 6.90 (IH, d, J=8.8 Hz) 116.9

3 146.2 7.24 (IH, t, J=7.6Hz) 129.7 6.60 (IH, d, J=8.8Hz) 119.6

4 139.4 7.18 (IH, brd, J=7.6 Hz) 128.9 152.6

5 146.2 7.24 (IH, t, J=7.6Hz) 129.7 6.60 (IH, d, J=8.8 Hz) 119.6

6 7.05 (IH, s) 110.2 7.34 (IH, d, J=7.6 Hz) 127.7 6.90 (IH, d, J=8.8 Hz) 116.9

7 170.2 657 (1H, d, ]=16.0 Hz) 134.4

8 6.33 (IH, ddd, J=16.0, 126.6 5.6, 1.2 Hz)

9 4.42 (1H, m) 72.0

4.27 (1H, m)

Glc-1 4.40 (1H, d, J=8.0 Hz) 103.4 4.70 (1H, d, J=6.8 Hz) 104.0

Glc-2 3.26 (1H, m) 75.8 3.38 (1H, m) 75.7

Glc-3 3.33 (1H, m) 78.7 3.44 (1H, m) 78.1

Glc-4 3.43 (1H, m) 71.0 3.46 (1H, m) 72.0

Glc-5 3.53 (1H, m) 75.3 3.68 (1H, m) 75.1

Glc-6 4.55 (1H, dd, 12.0, 2.0) 65.0 4.57 (1H, brd, J=11.6 65.1

4.44 (1H, dd, 12.0,5.6) Hz)

4.41 (1H, dd, J=11.6,

6.8 Hz)

Γ 121.7 121.6

2' 7.11 (1H, s) 110.4 7.11 (1H, s) 110.5

3' 146.8 146.7

4' 140.0 140.1

5' 146.8 146.7

6' 7.11 (1H, s) 110.4 7.11 (1H, s) 110.4

7' 168.5 168.4

Table 3. The Ή and ¹³C NMR Data of Compounds RR-C31, RR-C47 and RR-C49 (CD₃OD,

400 MHz)

RR-C31 RR-C47 RR-C49

Position Ή NMR ¹³C NMR 'HNMR ¹³C Ή NMR ^l3C

NMR NMR

1 134.5 129.6 138.3

2 7.13 (1H, d, J= 131.1 7.15 (1H, d, J=8.4Hz) 131.2 7.42 (1H, d, J=7.2Hz) 127.5

8.4Hz)

3 7.02 (lH,d,J= 118.0 6.81 (1H, d, J= 8.4Hz) 115.0 7.32 (1H, t, J=7.2 Hz) 129.5

8.4Hz)

4 157.8 159.8 7.24 (1H, t,J=7.2 Hz) 18.5

5 7.02 (lH,d,J= 118.0 6.81 (1H, d, J= 8.4Hz) 115.0 7.32 (1H, t,J=7.2 Hz) 129.5

8.4Hz)

6 7.13 (1H, d, J= 131.1 7.15 (1H, d, J=8.4Hz) 131.2 7.42 (lH,d, J=7.2 Hz) 127.5

8.4Hz)

7 2.75 (2H, t, J=6.8Hz) 39.6 2.84 (2h, t, 1= 7.2Hz) 36.5 6.70 (1H, d, J= 16.0Hz) 133.5

8 3.70 (2H. m) 64.6 3.83 (1H, m) 72.1 6.40 (1H, m) 126.7

4.05 (1H, m)

9or- 3.73 (3H,s) 4.52 (1H, dt, J=12.8, 70.6

OCH3 5.2Hz)

4.30 (1H, dt, J=12.8,

160Hz)

Glc-1 4.28 (1H, d, J=8.0Hz) 102.7 4.30 (1H, d, J=7.6 Hz) 104.5 4.38 (1H, d, J=7.6 Hz) 103.4

Glc-2 3.25 (1H, m) 75.1 3.19 (1H, m) 75.2 3.22 (1H, m) 74.9

Glc-3 3.37 (1H, m) 78.3 3.34 (1H, m) 78.3 3.37 (1H, m) 76.7

Glc-4 3.29 (1H, m) 71.6 3.28 (lH,m) 71.7 3.31 (1H, m) 72.0

Glc-5 3.45 (1H, m) 78.2 3.35 (1H, m) 78.2 3.46 (1H, m) 77.8

Glc-6 3.71 (1H, m) 62.7 3.96 (1H, m) 69.6 4.02 (1H, m) 69.5

3.67 (1H, m) 3.78 (1H, m) 3.72(1 H, m)

Ara or 4.31 (1H, d, J=7.6Hz) 105.3 4.99 (1H, d, J=1.6 Hz) 110.2

Ara(f -1 Ara-2 3.32 (lH. m) 71.8 3.98 (1 H, m) 83.4 Ara-3 3.61 (1 H, m) 72.5 3.82 (1 H, m) 79.1 Ara-4 3.65 (1H, m) 69.7 3.93 (1H, m) 86.0 Ara-5 4.10 (1H, m) 66.9 3.72 (1H, d, J=1 1.2 Hz) 63.0

3.81 (1H, m) 3.62 (1H, m)

All references, including publications, patent applications and patents, cited herein are hereby incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.

The terms "a" and "an" and "the" and similar referents as used in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. Unless otherwise stated, all exact values provided herein are representative of corresponding approximate values (e.g. , all exact exemplary values provided with respect to a particular factor or measurement can be considered to also provide a corresponding approximate measurement, modified by "about," where appropriate).

The use of any and all examples, or exemplary language (e.g. , "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise indicated. No language in the specification should be construed as indicating any element is essential to the practice of the invention unless as much is explicitly stated.

The description herein of any aspect or embodiment of the invention using terms such as "comprising", "having", "including" or "containing" with reference to an element or elements is intended to provide support for a similar aspect or embodiment of the invention that "consists of, "consists essentially of, or "substantially comprises" that particular element or elements, unless otherwise stated or clearly contradicted by context (e.g. , a composition described herein as comprising a particular element should be understood as also describing a composition consisting of that element, unless otherwise stated or clearly contradicted by context). It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application.

REFERENCES

Aperti et al., (2006) Secondarystructure of a-synuclein oligomers: charaterization by Raman and atomic force microscopy. J. Mol. Biol. 355:63-71.

Asanuma et al, (1998) Attenuation of 6-hydroxydopamine-induced dopaminergic nigrostriatal lesions in superoxide dismutase transgenic mice. Neuroscience. 85:907-17.

Betarbet R, Sherer TB, Greenamyre JT. (2002) Animal models of Parkinson's disease. Bioessays. 24:308-18.

Broussolle E, Thobois S. (2002) Genetic and environmental factors of Parkinson's disease. Rev Neurol (Paris) 158: 11-23.

Cavalla et al., Intracerebroventricular administration of l-methyl-4-phenyl- 1,2,3,6- tetrahydropyridine (MPTP) and its metabolite l-methyl-4-phenylpyridinium ion (MPP+) decrease dopamine and increase acetylcholine in the mouse neostriatum. Neuropharmacology. (1985) 24:585-6.

Conway KA, Harper JD, Lansbury PT. (1998) Accelerated in vitro fibril formation by a mutant a-synuclein linked to early-onset Parkinson disease. Nat Med 4: 1318-1320.

Giasson BI, Duda JE, Quinn SM, Zhang B, Trojanowski JQ, Lee VM. (2002) Neuronal a- synucleinopathy with severe movement disorder in mice expressing A53T human a-synuclein. Neuron 34: 521-233.

Halliday et al., Neuropathology underlying clinical variability in patients with synucleinopathies. Acta Neuropathol. (2011) 122: 187-204.

Jellinger K, Linert L, Kienzl E, Herlinger E, Youdim MB. (1995) Chemical evidence for 6- hydroxydopamine to be an endogenous toxic factor in the pathogenesis of Parkinson's disease. J Neural Transm Suppl. 46:297-314.

Kalivendi et al., Alpha- synuclein up-regulation and aggregation during MPP+- induced apoptosis in neuroblastoma cells: intermediacy of transferrin receptor iron and hydrogen peroxide. J. Biol. Chem. (2004) 279: 15240-15247

Keeney PM, Xie J, Capaldi RA, Bennett JP Jr. (2006) Parkinson's disease brain mitochondrial complex I has oxidatively damaged subunits and is functionally impaired and misassembled. J Neurosci 26:5256-64.

Lane E, Dunnett S. (2008) Animal models of Parkinson's disease and L-dopa induced dyskinesia: how close are we to the clinic? Psychopharmacology (Berl). 199:303-12. Lashuel HA, Hartley D, Petre BM, Walz T, Lansbury PT Jr. (2002) Amyloid pores from pathogenic mutations. Nature 418:291.

Lee MK, Stirling W, Xu Y, Xu X, Qui D, Mandir AS, Dawson TM, Copeland NG, Jenkins NA, Price DL. (2002) Human a-synuclein-harboring familial Parkinson's disease-linked Ala-53— > Thr mutation causes neurodegenerative disease with a-synuclein aggregation in transgenic mice. Proc Natl Acad Sci USA 99:8968-73.

Panossian A, Wagner H. (2005) Stimulating effect of adaptogens: an overview with particular reference to their efficacy following single dose administration. Phytother Res. 19:819-38.

Panossian A, Wikman G, Sarris J. (2010) Rosenroot (Rhodiola rosea): traditional use, chemical composition, pharmacology and clinical efficacy. Phytomedicine. 17:481-93.

Sidhu A, Wersinger C, Moussa CE, Vernier P. (2004) The role of α-synuclein in both neuroprotection and neurodegeneration. Ann N Y Acad Sci 1035:250-270.

Spillantini MG, Crowther RA, Jakes R, Hasegawa M, Goedert M. (1998) alpha-Synuclein in filamentous inclusions of Lewy bodies from Parkinson's disease and dementia with lewy bodies. Proc Natl Acad Sci U S A. 95:6469-73.

Vekrellis et al, (2011) Pathological roles of α-synuclein in neurological disorders. Lancet Neurol. 10:1015-25.

Claims

CLAIMS What is claimed is:

1. A method of inhibiting the aggregation of alpha-synuclein, wherein the method comprises administering, to an aggregate of alpha-synuclein, an effective amount of an isolated or substantially pure compound of formula I, or formula III through formula VIII, or a salt thereof, wherein the compound of formula I has the following structure:

wherein R₍ - R₃ of formula I are, independently, -H, -OH, methoxy or ethoxy, halo, amino, acyl, or thiol; and R₄ - R9 of formula I are, independently, hydrogen, alkyl or acyl;

wherein the compound of formula III has the following structure:

wherein R_\ - R₃ and R₇-R₉ of formula III are, independently, -H, -OH, methoxy or ethoxy, halo, amino, acyl, or thiol; and R₄ - R of formula III are, independently, hydrogen, alkyl or acyl; wherein the compound of formula IV has the following structure:

wherein R_\ - R₆ of formula IV are, independently, hydrogen, alkyl or acyl; and R₇-R of formula IV are, independently, -H, -OH, methoxy or ethoxy, halo, amino, acyl, or thiol;

wherein the compound of formula V has the following structure:

wherein Ri - R₄ of formula V are, independently, hydrogen, alkyl or acyl; and R₅ formula V is hydroxyl, halo, haloalkyl, amino, alkylamino, alkoxy, thiol, cyano, or -COOH; wherein the compound of formula VI has the following structure:

wherein Ri - R₃ of formula VI are, independently, -H, -OH, methoxy or ethoxy, halo, , acyl, or thiol; and R₄- Rio of formula VI are, independently, hydrogen, alkyl or acyl; wherein the compound of formula VII has the following structure:

wherein Ri - R4 of formula VII are, independently, hydrogen, alkyl or acyl;

wherein the compound of formula VIII has the following structure:

wherein Ri - R₃ and R₇ - R₉ of formula VIII are, independently, -H, -OH, acyl, halo, haloalkyl, amino, alkylamino, alkyl, hydroxylalkyl, alkoxy, thiol; and R₄- R₆ of formula VIII are, independently, hydrogen, alkyl or acyl.

2. The method according to claim 1, wherein the compound is selected from rosavin, 6-O-galloyl rosin, rosarin, icariside D2, mongrhoside, 6-O-galloyl arbutin, or rhodiocyanoside A.

3. The method according to claim 1 , wherein the aggregate of alpha-synuclein is in a subject, and the compound or a salt thereof is administered to the subject.

4. The method according to claim 3, wherein the subject has a neurodegenerative disease.

5. The method according to claim 4, wherein the neurodegenerative disease is selected from Parkinson's disease, synucleinopathy, amyotrophic lateral sclerosis, Alzheimer's disease, dementia with Lewy bodies, pure autonomic failure (PAF), multiple system atrophy (MSA), Huntington's disease, or Lewy body disease.

6. A method of treating a neurodegenerative disease or a neuropathological condition, wherein the method comprises administering, to a subject in need of such treatment, an effective amount of an isolated or substantially pure compound of formula I, or formula IV through formula VII, or a salt thereof,

wherein the compound of formula I has the following structure:

wherein Ri - R₃ of formula I are, independently, -H, -OH, methoxy or ethoxy, halo, amino, acyl, or thiol; and R₄ - R₉ of formula I are, independently, hydrogen, alkyl or acyl;

wherein the compound of formula IV has the following structure:

wherein R| - R of formula IV are, independently, hydrogen, alkyl or acyl; and R-7-R9 of formula IV are, independently, -H, -OH, methoxy or ethoxy, halo, amino, acyl, or thiol;

wherein the compound of formula V has the following structure:

wherein Ri - R₄ of formula V are, independently, hydrogen, alkyl or acyl; and R5 formula V is hydroxyl, halo, haloalkyl, amino, alkylamino, alkoxy, thiol, cyano, or -COOH; wherein the compound of formula VI has the following structure:

wherein Ri - R₃ of formula VI are, independently, -H, -OH, methoxy or ethoxy, halo, , acyl, or thiol; and R₄ - Ri₀ of formula VI are, independently, hydrogen, alkyl or acyl; and wherein the compound of formula VII has the following structure:

wherein Ri - R₄ of formula VII are, independently, hydrogen, alkyl or acyl.

7. The method according to claim 6, wherein the compound is selected from rosavin, rosarin, icariside D2, mongrhoside, or rhodiocyanoside A.

8. The method according to claim 6, wherein the neurodegenerative disease is selected from Parkinson's disease, synucleinopathy, amyotrophic lateral sclerosis, Alzheimer's disease, dementia with Lewy bodies, pure autonomic failure (PAF), multiple system atrophy (MSA), Huntington's disease, or Lewy body disease.

9. The method according to claim 6, wherein the neurodegenerative disease is synucleinopathy.

10. The method according to claim 6, wherein the neurodegenerative disease is Parkinson's disease.

11. The method according to claim 6, wherein the neuropathological condition is selected from neuropathic pain, stroke, brain trauma, or epilepsy.

12. A method of reducing aggregation of alpha-synuclein, wherein the method comprises administering, to an aggregate of alpha-synuclein, an effective amount of a composition comprising an isolated Rhodiola rosea extract, and, optionally, a pharmaceutically-acceptable carrier.

13. The method according to claim 12, wherein the aggregate of alpha-synuclein is in a subject, and the composition is administered to the subject.

14. The method of claim 12, wherein the composition comprises at least 90% of the Rhodiola rosea extract by weight.

15. The method of claim 12, wherein the Rhodiola rosea extract is a water or alcohol extract.

16. The method of claim 12, wherein the Rhodiola rosea extract is a water, ethanol, or butanol extract.

17. The method according to claim 12, wherein the Rhodiola rosea extract is prepared using a method comprising:

providing a sufficient quantity of raw material of Rhodiola rosea;

extracting the raw material of Rhodiola rosea with a first solvent comprising an alcohol to yield a Rhodiola rosea alcohol extract;

recovering the Rhodiola rosea alcohol extract;

mixing the Rhodiola rosea alcohol extract with water; and

partitioning the wat r-Rhodiola rosea alcohol extract mixture with one or more organic solvents to yield one or more organic- solvent fractions and a water fraction,

wherein the first solvent comprises an alcohol selected from methanol, ethanol, or propanol, and

wherein the organic solvent is selected from petroleum ether, ethyl acetate, methanol, ethanol, propanol, or butanol.

18. The method according to claim 12, wherein the raw material is Rhodiola rosea root.

19. A method of reducing aggregation of amyloid beta peptide, wherein the method comprises administering, to an aggregate of amyloid beta peptide, an effective amount of a composition comprising an isolated Rhodiola rosea extract, and, optionally, a pharmaceutically-acceptable carrier.

20. The method according to claim 19, wherein the aggregate of amyloid beta peptide is in a subject, and the composition is administered to the subject.