Isolation of the Sapogenin from Defatted Seeds of Camellia oleifera

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Isolation of the Sapogenin from Defatted Seeds of Camellia oleifera and Its Neuroprotective Effects on Dopaminergic Neurons Yong Ye,* Fei Fang, and Yue Li Department of Pharmaceutical Engineering, School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou 510640, People’s Republic of China ABSTRACT: Sasanqua saponin is a major active compound in the defatted seeds of Camellia oleifera but is always discarded without effective utilization. The sapogenin from hydrolysis of sasanqua saponin was purified, and its amination derivative was investigated on its neuroprotective effects, which were evaluated by animal models of Parkinson disease in mice induced by 1methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The results showed that the sapogenin and its derivative increased dopamine content in striatum and tyrosine hydroxylase (TH) positive cells in substantia nigra and relieved inflammation and behavioral disorder, but the effect on movement was reversed by dopamine receptor antagonist haloperidol and was not intervened by adenosine receptor antagonist CGS 15943. Molecular simulation showed the interaction between dopamine receptor and the sapogenin or its derivative. It is proven that the sapogenin can protect dopamine neurons through antineuroinflammation and activation of dopamine receptor rather than adenosine receptor, and its amination improves the effects. This research provides the prospective prodrugs for Parkinson disease and a new medicinal application of sasanqua saponin. KEYWORDS: sasanqua saponin, amination, Camellia oleifera, neurodegeneration, Parkinson disease, dopaminergic neuron

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g/kg and accumulation coefficient 5.3 in rats.9 It has a stable block called sapogenin with a lot of pharmacological activities of antioxidation, anti-inflammation, immunomodulation, and antitumor,10−12 and it may play a role in neuroprotection. Saponin is one of the active ingredients in many herbs, and some have neuroprotective effects. It is found that ginsenoside Rg1 can reverse the changes of neurotransmitters caused by 1methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP),13 and ginsenoside Re has significant inhibition on apoptosis of nigra neurons in mouse caused by MPTP,14 and gypenosides can cause neuroprotective effects through enhancing activity of antioxidative enzymes and inhibiting generation of hydroxyl radicals and lipid peroxide to reduce Glu-mediated oxidative nerve injury.15 Unlike those saponins, sasanqua saponin from seeds of C. oleifera has a different structure of pentacyclic triterpenoids, whose main active block is sapogenin.16 Our previous research proved the anti-inflammatory and analgesic activities of the sapogenin,17 diclosing its central roles. The protective role of the sapogenin and its derivative on dopaminergic neurons is worth investigation.

INTRODUCTION Parkinson disease (PD) is a chronic neurodegenerative disease caused by extrapyramidal dysfunction, which is related to genetic factors, environmental factors, mitochondrial dysfunction, and so forth,1 and is characterized with the loss of dopaminergic neurons in midbrain and the formation of Lewy body with the main component of α-synuclein.2 Its clinical symptoms will appear since almost 50% of dopaminergic neurons located at substantia nigra compact (SNC) are lost and the concentration of dopamine in the striatum decreases to 80%,3 including motor dysfunction of bradykinesia, tremor, rigidity, and gait disorders as well as movement unassociated symptoms such as the smell sense disorder, autonomic nerve dysfunction, depression, cognitive impairment, sleep disorders, and so forth. Currently, there is no perfect treatment for PD, and traditional treatment strategy is L-dopa replacement therapy. Although the clinical symptoms can be controlled, disease progression cannot be prevented. Moreover, long-term L-dopa treatment can lead to efficacy reduction and severe adverse effects, lowering the quality of patients’ lives.4 In recent years, medication therapy of PD has changed from direct dopamine supplement to multilevel therapy, aiming at reliving the symptoms and impeding the progression of the disease at the same time.5 Natural medicines play an important role in therapy of PD. Camellia oleifera, an edible oil plant, is mainly distributed in China, but the defatted seeds are discarded, leading to environmental pollution and waste of resource. Flavonoids, saponins, and polysaccharides are the main compounds in the defatted seeds of C. oleifera,6 but flavonoids and polysaccharides are unstable in vivo and cannot take effect in the central system because it is difficult for them to pass through the blood brain barrier.7,8 Sasanqua saponin is a safe compound with LD50 4.5 © XXXX American Chemical Society

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MATERIALS AND METHODS Materials. The defatted seeds of Camellia oleifera were collected from The Oil Factory in Meizhou of Guangdong province, China. Macroreticular resin AB-8 was purchased from Cangzhou Resin Company (Hebei, China). Reagents for synthesis were triphenyl chloromethane, triphenyl phosphine, and diethyl azodicarboxylate bought from J & K Scientific Ltd.

Received: March 10, 2014 Revised: May 16, 2014 Accepted: June 8, 2014

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dx.doi.org/10.1021/jf501166w | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

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8 h. Then, acetic anhydride (20.8 g) was added into the mixture and was stirred for 15 h. The solution was added to 4.7 mL of formic acid and was heated to 100−110 °C under reflux for 1 h and then was cooled to 20 °C. Five grams of ammonia, 0.5 g of triphenyl phosphine, and 0.5 g of diethyl azodicarboxylate were mixed into the solution, stirring for 9 h. Finally, 28 g of potassium carbonate was added, stirring for 1 h. Pyridine was distilled off under reduced pressure, and the soluble fractions in water and toluene were removed stepwise by 500 mL of water and toluene. The residue was dissolved in methanol to crystallize and was dried under vacuum condition. Twenty-six grams of the product was gained. The synthetic route is illustrated in Figure 2. Structural Analysis. UV spectra analysis was carried out on UV-3010 ultraviolet spectrometer (Hitachi Company, Japan) scanning from 200 to 600 nm. IR spectra were measured on Nicolet 380 FT-IR spectrometer (Nicolet Apparatus Company, U.S.) with KBr tablets from 4000 to 400 cm−1 with resolution 2 cm−1. Mass spectra were recorded on Bruker Esquire Hct Plus Mass spectrometer with electrospray ionization (ESI, Bruker Company, Germany) in m/z of cation model scanning from 150 to 1200 for 60 min. NMR spectra were determined on 400 MHz AM NMR (Bruker Company, Switzerland) in DMSO-d6 operating at 101 MHz for 13C NMR and at 400 MHz for 1H NMR. PD Model Induced by MPTP. The experiment was performed according to ref 18. Eighty mice were randomly divided into 10 groups of 8 mice for each: normal group, MPTP group, four medicated groups, and four antagonist groups. Medicated groups of mice were intragastricly administered once a day with the sapogenin and its derivative at 25 mg/kg (low dose) and 100 mg/kg (high dose), respectively, for 6 days; the antagonist groups received 1 mg/ kg haloperidol or CGS 15 943 (intraperitoneal injection (i.p.)) plus a high dose of the sapogenin or the derivative (i.g.), and the other groups were fed with normal saline instead. Haloperidol, a dopamine receptor antagonist, can decrease dopamine neurotransmission in the striatum and can induce Parkinson disease-like symptoms in humans.19 CGS 15943 is a competitive adenosine receptor antagonist and a behavioral stimulant in animals.20 These drugs were used in this experiment to deduce whether the drugs took effect through the dopamine receptor pathway or through the adenosine receptor pathway. On the seventh day of administration, the mice except the normal group were intraperitoneally injected with 20 mg/kg MPTP once every 2 h for three consecutive times to prepare the PD mice model. The mice were reared for an additional 7 days and were decapitated after behavioral tests; the striatum of half the brain was quickly separated, and remain of the half was used to determine the levels of TNF-α and IL1β with the ELISA method of the kit description. The other half of the brain was kept for immunohistochemical analysis. The striatum was homogenized in 1 mL 0.1 mol/L pH 7.4 PBS-EDTA buffer. The homogenate was centrifuged at 3000g for 10 min, and the content of dopamine (DA) in supernatant was measured by high-performance liquid chromatographyelectron-capture detector (HPLC-ECD, Agilent Company, U.S.).21 The operating conditions of HPLC were column, Diamonsil C18 (150 × 4.6 mm, 5 μm); mobile phase, 110 mM citrate buffer/100 mM EDTA/70 mM 1-octanesulfonate sodium solution and 20% (V/V) methanol; flow rate, 1 mL/ min; temperature, 30 °C; and injection volume, 20 μL.

(Beijing, China). Drugs for animal tests were MPTP and CGS 15943 purchased from Sigma-Aldrich Company (St. Louis, MO, U.S.) and haloperidol from Shanghai Pharmaceutical Co. Ltd. (Shanghai, China). TNF-α, IL-1β, and tyrosine hydroxylase (TH) immunoassay kits were bought from Jiancheng Biotech. Co. (Nanjing, China). Other chemicals were purchased from Qianhui Reagent Company (Guangzhou, China). Animals. The experiments were carried out on male Kunming mice of weight 30 ± 3 g. The animals were housed under conditions of 25 ± 2 °C, 50 ± 10% humidity with a 12 h light/dark cycle. Food and water were accessible ad libitum. The experiments were performed in accordance with the Chinese Guidelines for the use of laboratory animals, which conforms to the European Union Directive for the protection of experimental animals (2011/63/EU), and received approval (Permit No. 13910) from the Animal Experimentation Ethic Committee of South China University of Technology. All efforts were made to minimize animal suffering and to reduce the number of animals used. Isolation of the Sapogenin. The seeds of C. oleifera were defatted with n-hexane in the Soxhlet apparatus for 8 h. One kilogram of the defatted seeds was extracted with 15 L of 70% (V/V) methanol aqueous solution under reflux at 80 °C for 2 h. The filtrate was vaporized to 5 L in reduced pressure, was flowed at 2 mL/min through the column (diameter 5 cm × height 30 cm) filled with macroreticular resin AB-8, and then was eluted by 2 L of 80% (V/V) ethanol aqueous solution. The eluent was added to 52 mL of hydrochloride acid and was refluxed at 80 °C for 5 h. The precipitate was collected and dissolved in 500 mL of 80% ethanol containing 25 g of sodium hydroxide and was refluxed for the second time at 80 °C for 5 h. The precipitate was collected and crystallized with diethyl ether and was dried in vacuum condition. Then, 82 g of the sapogenin was obtained. The scheme of extraction is illustrated in Figure 1. Synthesis of Sapogenin Derivative. Fifty grams of the sapogenin was dissolved in 300 mL of pyridine, was mixed with triphenyl chloromethane (28.4 g), and was stirred at 50 °C for

Figure 1. Scheme of sapogenin isolation from the defatted seeds of Camellia oleifera. B



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Figure 2. Synthetic route of the sapogenin derivative.

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Behavioral Test. The locomotive activities of mice were monitored by a ZZ-6 spontaneous activity apparatus (Chengdu Taimeng Software Co., China). Moving counts were collected by computer with the infrared-sensitive motion-detection system. Each mouse was placed in the testing chamber for 10 min of adaptation, followed by a 10 min recording.22 The rotarod test was used to detect the balance of mice. Mice were trained three times before the test, and the rotating speed was set to 15 r/min.23 Latency to fall was calculated five times within 180 s. In the traction test, a horizontal metal rod with diameter of 1.5 mm was used as a hanging rod at a height of 30 cm from the ground. Latency to fall was recorded to evaluate their motor coordination.24 Assay of TH Expression. The test was carried out on the basis of the protocol.25 The half brain of a mouse was sunk in paraformaldehyde solution (pH 7.4, 40 g/L) containing 300 g/ L sucrose for 48 h and was cryostat sectioned to slices (60 mm thickness). Nigra slices were incubated in TH antibody with dilution of 1:5000, were stained by diaminobenzidine (DAB), and were mounted to slides for photograph and TH positive cell counting. Simulation of Molecular Interaction. Molecular docking was simulated by Discovery Studio V2.5 software (Accelry Inc., CA, U.S.) in the following procedure.26 Structural data of dopamine receptor was downloaded from Brookhaven Library.27 The missing amino acids and hydrogens were supplemented, and excessive protein conformation was removed. Dopamine receptor was defined as the receptor, and the binding sites and coordinates were defined. A new plot window was opened to draw structures of the sapogenin and its derivative, to optimize 3D geometric structures, and to apply CHARMm force field to ensure correct bond length and bond angle in a state of energy stability.28 CDOCKER Protocol was run to obtain binding parameters, which were used to evaluate the interaction. CDOCKER energy and CKOCKER interaction energy were two important parameters, and their absolute values were in accord with the affinity and action force between the receptor and the ligand. Statistical Analysis. Data were expressed as mean ± standard deviation (x̅ ± s) and were analyzed with SPSS13.0 software. Significant tests among the groups were based on one-way ANOVA and Student−Newman−Keuls (SNK) test.

RESULTS AND DISCUSSION Structure of the Sapogenin and Its Derivative. Sasanqua saponin has certain antioxidative, anti-inflammatory, Table 1. Influence of the Sapogenin and Its Derivative on DA Content in Striatum of Mice Treated with MPTP (x̅ ± s, n = 8)a dose (mg/kg)

group normal saline MPTP sapogenin sapogenin derivative derivative DA antagonist/sapogenin DA antagonist/derivative adenosine antagonist/ sapogenin adenosine antagonist/ derivative

DA content (pmol) ± ± ± ± ± ± ± ± ±

8.1b 7.4 9.8 8.5b 8.7 8.4b 6.2b 7.9b 4.6b

25 100 25 100 1/100 1/100 1/100

63.2 23.4 30.4 40.9 33.2 42.8 41.7 42.7 39.1

1/100

41.3 ± 4.4b

TH+ neurons number 39.6 6.6 12.6 35.8 14.4 36.2 35.6 36.4 32.8

± ± ± ± ± ± ± ± ±

8.1b 3.7 5.8 6.9b 8.0 8.6b 5.8b 6.5b 6.6b

34.2 ± 6.1b

a

Mice were administered (i.g.) with sapogenin or derivative or were injected (i.p.) with haloperidol (dopamine antagonist) or CGS 15943 (adenosine antagonist) once a day for 6 days and then were injected (i.p.) with MPTP. Seven days later, striatum was taken for determination of dopamine by HPLC. TH positive neurons in nigra were counted under microscope in five visual fields (4 mm2) for each of eight slices after immunostaining. bp < 0.01, compared with MPTP group.

and analgesic effects, and the sapogenin from hydrolysis of the sasanqua saponin could increase cellular uptake and could promote its roles in vivo.17 The amino group enhances the interaction of drugs with some receptors29 and may be helpful to PD. Thus, the sapogenin was purified and was derived by amination in hydroxyl and aldehyde groups in order to improve its neuroprotective effects. The purified sapogenin was an amorphous powder and was insoluble in water, chloroform, petroleum ether, and dimethyl ether and was soluble in methanol, ethanol, acetone, and ethyl acetate. mp 251.2−251.7 °C. There was an absorption peak at 207 nm in UV spectra. IR spectra showed a smaller peak of hydroxyl groups at 3435 cm−1 and no characteristic absorption peak of α, β-unsaturated ester, and ether. In MS, m/z was 488 (M+). 1H NMR spectra had no Tig moiety signals but showed C



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Figure 3. TH immunohistochemistry of substantia nigra sections in mice with different treatments. Sapogenin, 100 mg/kg, i.g.; derivative, 100 mg/ kg, i.g.; MPTP, 20 mg/kg × 3 i.p.; haloperidol, 1 mg/kg, i.p.; CGS 15943, 1 mg/kg, i.p. Half brains were sectioned for determination of TH expression by immunohistochemistry. The photos were taken by camera in 50 times magnification.

one carbonyl (d 9.97, J = 12.48). 13C NMR spectra had signals of one carbonyl carbon and two olefinic carbons. The signals in 13 C NMR spectra were assigned as follows: δ208.7 (C-25), 144.5 (C-12), 123.3 (C-13), 74.9 (C-2), 70.3 (C-19), 67.7 (C16), 65.6 (C-31), 52.6 (C-3), 50.1 (C-4), 47.5 (10, 22), 45.9 (C-11, 20), 42.1 (C-17, 18), 38.4 (C-6, 9, 15), 36.1 (C-5, 8), 30.7 (C-21, C-28, 29), 27.1 (C-1), 24.9 (C-14, 34), 19.5 (C-7), 16.8 (C-24, 26, 27). Its property and structure are the same with camelliagenin (C30H48O5) in the reference.30 The derivative of sapogenin had similar properties as the sapogenin. The maximum absorption peak was at 215 nm in UV spectra. IR spectra showed imino groups at 1640 cm−1. MS: m/z 486 (M+). There were signals of imino proton (d 9.61) and amino protons (d 2.71) in 1H NMR spectra. 13C NMR spectra also had no signal of carbonyl carbon. Its formula was speculated as C30H50N2O3. The structures of sapogenin and its derivative are shown in Figure 2. Protection on Dopaminergic Neurons Damaged by MPTP. The MPTP model is one of the well-known animal models of PD and is used to appraise the neuroprotective

effects of the sapogenin and its derivative. MPTP can cross the blood-brain barrier, transforms to 1-methyl-4-phenylpyridine radical with the oxidation of monoamine oxidase B, and then enters dopaminergic nerve cells and selectively destructs dopaminergic neurons, causing a significant reduction of dopamine neurotransmitters in neurons and behavioral changes.31 The result of animal tests showed that DA levels in striatum of mice treated by MPTP were significantly (p < 0.01) lower than the normal saline group, indicating that MPTP caused the damage of the dopaminergic neuron. However, mice fed with the sapogenin and its derivative increased the levels of DA in striatum, and especially, the high dose of the sapogenin and its derivative had significant effects (p < 0.01) on the increase of DA content. Mice treated with the dopamine receptor antagonist haloperidol and the adenosine receptor antagonist CGS 15943 also had higher DA levels in striatum (Table 1). This suggests that high doses of the sapogenin and its derivative can prevent dopaminergic neurons from damage induced by D



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Figure 4. Levels of TNF-α and IL-1β in brain of mice in different treatments. The groups are normal (NS), MPTP (20 mg/kg × 3 i.p.), low dose of sapogenin (SL, 25 mg/kg, i.g.), high dose of sapogenin (SH, 100 mg/kg, i.g.), low dose of derivative (DL, 25 mg/kg, i.g.), high dose of derivative (DH, 100 mg/kg, i.g.), dopamine antagonist plus sapogenin (DAS, haloperidol 1 mg/kg i.p. and sapogenin 100 mg/kg i.g.), dopamine antagonist plus derivative (DAD, haloperidol 1 mg/kg i.p. and derivative 100 mg/kg i.g.), adenosine antagonist plus sapogenin (AAS, CGS15943 1 mg/kg i.p. and sapogenin 100 mg/kg i.g.), and adenosine antagonist plus derivative (AAD, CGS15943 1 mg/kg i.p. and derivative 100 mg/kg i.g.). Data are presented as mean ± standard deviation (x̅ ± s, n = 8). **, p < 0.01, compared with MPTP group.

MPTP and that DA or adenosine receptor antagonist has no adverse effect on DA content. Tyrosine hydroxylase (TH) is the key enzyme for dopamine synthesis in dopaminergic neurons, and its expression is lowered in substantia nigra of patients with Parkinson disease.32 Immunostaining intensity of brain slices in immunohistochemistry can reflect the expression of TH. Compared with normal mice, MPTP treated mice had less staining in substantia nigra of brain slices, indicating that MPTP depressed the expression of TH. However, the intensity had no significant difference between normal control and high dose of the sapogenin or its derivative and between dopamine or adenosine receptor antagonist and sapogenin. This further proves that high dose of the sapogenin and its derivative has the protective effects on dopaminergic neurons and that the antagonists did not reduce the effects. The pictures are shown in Figure 3. TH positive neurons were counted in five visual fields (4 mm2) for each of eight slices, and the average cell number is listed in Table 1. Significant difference (p < 0.01) was found between MPTP group and high dose of sapogenin or its derivative but was not found between antagonist and no antagonist group, suggesting that the dopaminergic neurons’ protection of the sapogenin and its derivative was not affected by dopamine or adenosine receptor antagonist. Inhibition on the Inflammation in Brain. MPTP can induce neuroinflammation through an increase in the expression of TNF-α and IL-1β in the brain.33 The antiinflammatory effects of the sapogenin and its derivative on brain tissues were investigated. The results showed that TNF-α and IL-1β in mice brain of MPTP group were significantly (p < 0.01) increased but decreased in the groups treated with the sapogenin and its derivative. There was no significant difference

Figure 5. (a) Locomotive activity, (b) latency to fall by rotarod test, and (c) latency to fall by traction test in mice of different groups. The groups are NS (normal saline), MPTP (20 mg/kg × 3 i.p.), SL (low dose of sapogenin, 25 mg/kg), SH (high dose of sapogenin, 100 mg/ kg), DL (low dose of derivative, 25 mg/kg), DH (high dose of derivative, 100 mg/kg), DAS (dopamine antagonist haloperidol 1 mg/ kg plus sapogenin 100 mg/kg), DAD (dopamine antagonist haloperidol 1 mg/kg plus derivative 100 mg/kg), AAS (adenosine antagonist CGS 15943 1 mg/kg plus sapogenin 100 mg/kg), and AAD (adenosine antagonist CGS 15943 1 mg/kg plus derivative 100 mg/ E



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between the group of derivative in high dose and normal group, suggesting that the derivative had better control of inflammation in brain. The antagonists had no significant effects on neuroinflammation. The results are illustrated in Figure 4. TNF-α and IL-1β had obvious neurotoxicity on dopaminergic neurons.34 High-level expression of TNF-α could induce great loss of dopaminergic neurons, accompanied by the proliferation of glial monocytes and macrophages.35 TNF-α and IL-1β could also cause phosphorylation and degradation of IKK-β kinase, leading to translocation of NF-κB into the nucleus to promote expression of inflammatory genes.36 This experiment shows that MPTP increases the expression of TNFα and IL-1β in the brain, triggering inflammation and leading to neuron loss. The sapogenin and its derivative inhibit the expression of TNF-α and IL-1β in the mouse brain induced by MPTP and protect dopaminergic neurons in the brain from degeneration by antineuroinflammation. Behavioral Improvement of PD Mice. PD patients not only have dopamine loss in striatum and substantia nigra of brain but also have a lot of motor dysfunction, which can be imitated by animal tests. Spontaneous activities of mice were determined by locomotion test, and the balances were measured by the rotarod test and the traction test. The results showed that MPTP significantly (p < 0.01) lessened the spontaneous activity and latency to fall, suggesting that it caused movement disorder and damaged the motion balance and coordination of mice. The locomotion counts and latency to fall were increased significantly (p < 0.01) after the administration of the sapogenin and its derivative but decreased after injection of haloperidol. This indicated that the sapogenin and its derivative improved the behavioral disorders induced by MPTP, but the effects were reversed by DA receptor antagonist. The derivative had better effects than the sapogenin on behavioral improvement. The results are shown in Figure 5. The structures of sapogenin and its derivative are responsible for their effects on protection of dopaminergic neurons and behavior improvement. They do not have phenolic hydroxyl groups, and it is difficult for them to eliminate the MPTP radical. The neuroprotection contributes to their significant anti-inflammatory effects rather than to their antioxidant effect. They can relieve the motor dysfunction, and the effects are counteracted by dopamine receptor antagonist, indicating they might take effect by activating dopamine receptor rather than adenosine receptor, which is related to PD in many reports.37 The amination of sapogenin shows better effects on amelioration of PD symptoms. It may be attributed to its stronger interaction with dopamine receptor. Interaction with Dopamine Receptor. Molecular simulation is a good way to observe the interaction among groups. The protocol of semiflexible docking (CDOCKER) is proper for interaction of large molecule receptor and small molecule ligand. Major factors on binding stability of receptor and ligand are hydrophobicity and bonding force, which are calculated and judged by free energy. Docking simulation showed that both the sapogenin and its derivative could bind to the dopamine receptor. Each molecule successfully docked in five poses; the binding energy is shown in Table 2. The derivative had higher CDOCKER interactive energy and higher absolute value of CDOCKER energy than the sapogenin, suggesting that it is more powerful to bind to and interact with the dopamine receptor. The group linkage of the sapogenin and its derivative with dopamine receptor is shown in Figure 6, illustrating that one receptor can interact with many more derivatives. The

Figure 5. continued kg). Mice were administered (i.g.) with sapogenin or its derivative or were injected (i.p.) with haloperidol or CGS 15943 for 6 days and then were administered (i.p.) with MPTP. Seven days later, locomotive activity and latency to fall were separately determined. Data are presented as mean ± standard deviation (x̅ ± s, n = 8). **, p < 0.01, compared with MPTP group.

Table 2. Binding Energy of the Sapogenin and Its Derivative with Dopamine Receptor compound sapogenin

derivative

a

pose no.

CDOCKER energy (kcal/mol)

CDOCKER interaction energy (kcal/mol)

1 2 3 4 5 x̅ ± s 6 7 8 9 10 x̅ ± s

−83.46 −84.62 −85.11 −85.19 −85.3 −84.74 ± 0.76 −85.8 −86.31 −86.71 −86.86 −87.03 −86.54 ± 0.49a

47.53 41.96 45.76 45.82 41.58 44.53 ± 2.62 48.85 48.1 48.12 48.66 48.63 48.47 ± 0.34a

p < 0.01, compared with sapogenin.

Figure 6. Group linkage of interaction of (a) the sapogenin and (b) its derivative with dopamine receptor. F



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interaction between the sapogenin or the derivative and the dopamine receptor contributes to their effects on behavioral improvement. The sapogenin was isolated from the defatted seeds of C. oleifera and was used to synthesize a derivative by amination. By means of animal PD models induced by MPTP, it is found that the sapogenin and its derivative have significant effects on inhibition of DA loss, relieving motor deficits and protection of dopaminergic neurons. It is owing to their control of neuroinflammation in central tissues and interaction with dopamine receptor. The sapogenin and its derivative may become the prospective prodrugs for PD. Although the MPTP animal model is widely used for PD therapeutic interventions, it has limitations and cannot produce sleep disturbances and cognitive deficits commonly seen in PD patients.38 Because many factors are involved in PD including gene mutation, environmental impact, and so forth, the key mechanism of PD is still unknown. Therefore, other models such as transgenic animal model will be discussed in our next investigation so as to achieve the clinical application of the sapogenin and its derivative on PD.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86-20-87110234. E-mail: [email protected]. Fax: +86-20-22236337. Funding

The project is supported by National Science Funding of China (No. 81173646). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully thank staff in South China University of Technology for data analysis and ethical committee for approval of animal tests.

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ABBREVIATIONS ANOVA, analysis of variance; CGS 15943, 9-Chloro-2- (2furanyl)- [1,2,4] triazolo [1,5-c] quinazolin-5-amine; DA, dopamine; ELISA, enzyme-linked immuno sorbent assay; MPTP, 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine hydrochloride; PD, Parkinson disease; SNK, Student−Newman− Keuls; TH, tyrosine hydroxylase

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Isolation of the Sapogenin from Defatted Seeds of Camellia oleifera

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