Protective Effects of Rosa damascena and Its Active Constituent on Aβ(25–35)-Induced Neuritic Atrophy

Dementia is a clinical syndrome characterized by multiple cognitive deficits and causes progressive neurodegeneration leading eventually to death. The incidence of dementia is increasing worldwide with the increase in ageing population. However, no effective treatment is available yet. It has been hypothesized that drugs activating neurite outgrowth might induce neuronal reconstruction and help in the recovery of brain function. Working on this hypothesis, we recently observed that the chloroform extract of the Rosa damascena significantly induced the neurite outgrowth activity and inhibited the Aβ(25–35)-induced atrophy and cell death. Further workup led the isolation of a very long polyunsaturated fatty acid having molecular formula C37H64O2 as an active constituent. The structure of this compound was established by extensive analysis of fragmentations observed in EI-MS mode. The isolated compound protected Aβ(25–35)-induced atrophy and displayed strong neurite outgrowth activity. The length of dendrite in the cells treated with this compound were comparable to those of nerve growth factor (NGF) treated cells.


Introduction
Dementia is one of the most burdensome health conditions worldwide. Dementia causes progressive neurodegeneration leading eventually to death. The incidence of dementia is expected to increase with the increase in ageing population. According to Alzheimer's Disease International (ADI) Delphi consensus study, an estimated 4.6 million people suffer from dementia each year (one new case every 7 s), and 81.1 million people in the world are likely to suffer by the year 2040 [1]. Despite this catastrophic increase in dementia patients worldwide, no effective treatment is available yet. Several acetylcholine esterase inhibitors such as donepezil, rivastigmine and galantamine are most commonly used in the treatment of dementia patients [2]. However, these only slow down the progression of dementia, rather than restoring brain function in a real clinical situation [3]. Therefore, alternative therapeutic strategies are urgently needed. Dementia is caused by neuronal degeneration and atrophy. Neurodegeneration in the central nervous system is an irreversible process; however, formation of new synapses might be possible through the activation of remaining healthy neurons [4]. Therefore, reconstructing the synaptic formation in the brain could be a one of the powerful strategies in dementia treatment [5]. Reconstruction of neuronal network and synaptogenesis require neurite outgrowth as well as dendritic and axonal maturation steps. Therefore, drugs activating these steps could possibly initiate a recovery of brain function.
In this study, we have found that the chloroform extract of the R. damascena (Rosaceae family, collected from the Kashan market in Iran) showed strong neurite outgrowth activity under Aβ(25-35)-induced neuritic atrophy condition and identified its active constituent in the extract.

General
Methods. NMR spectra were taken on a JEOL JNM-LA400 spectrometer with tetramethylsilane (TMS) as an internal standard. HR-EI-MS measurements were performed on a JEOL JMS-700T spectrometer using a direct inlet system at the ionization voltage of 70 eV. Medium pressure liquid chromatograph (MPLC) was performed with BÜCHI MPLC system using a normal BW-820MH silica gel (Fuji Silysia, Aichi, Japan) (column size: 4.0 × 15 cm). Analytical and preparative TLC was carried out on precoated silica gel 60F 254 or RP-18F 254 plates (Merck, 0.25 or 0.50 mmthickness). Decosahexaenoic acid (DHA) and Mead acid used in this study were purchased from Sigma Aldrich.

Immunocytochemistry.
Rat cortical neurons were cultured in 8-well chamber slides at a density of 1.45 × 10 5 cells/ cm 2 . To measure the lengths of axons and dendrites, the cells were treated with each extract, compound or vehicle (0.1% DMSO). The cells were fixed with 4% paraformaldehyde and then immunostained with a monoclonal antibody against phosphorylated neurofilament-H (pNF-H) (1 : 500) as an axonal marker or a polyclonal antibody against MAP2 (1 : 500) as a dendritic marker. Alexa Fluor 488conjugated goat anti-mouse IgG (1 : 300) and Alexa Fluor 568-conjugated goat anti-rabbit IgG were used as second antibodies. A monoclonal antibody against pNF-H was purchased from Sternberger Monoclonals (Lutherville, MD, USA). A polyclonal antibody against microtubule-associated protein 2a and 2b (MAP2) was purchased from Chemicon (Temecula, CA, USA). Alexa Fluor 488-conjugated goat antimouse IgG and Alexa Fluor 568-conjugated goat anti-rabbit IgG were purchased from Molecular Probes (Eugene, OR, USA). Fluorescent images were captured by a fluorescent

Effect of Rose Extract and Its Fractions against Aβ
In order to find out the active constituent that induced neurite outgrowth activity in the chloroform extract of Rose extract, it was subjected to column chromatography in silica gel using gradient methanol-chloroform solvent system to obtain nine fractions. Each of these fractions were again tested for Aβ(25-35)-induced atrophy. As shown in Figure 2, the vehicle group showed the decrease in length of dendrites and axons. Of these tested fractions, fraction-4 showed pronounced effect with significant increase in the length of dendrites and axons; therefore, fraction-4 was further purified by repeated preparative TLC in ethyl acetate-hexane, 2% methanol-chloroform and acetone-benzene (1 : 9) to obtain compound 1 (Figure 3).  132.0, 130.3, 130.2, 128.28, 128.26, 128.1, 127.9, 127.8, and 127.1 ppm). This partial information was coupled with the EI-MS data, and subjected to extensive analysis of MS fragmentation study. Natural product derived PUFAs had methylene-interrupted double bonds of cis-geometry and belonged to either n−3 or n−6 family [19]. The classifiation n−3 or n−6 fatty acids are based on the first double bond that occurs either three or six carbon atoms from the methyl terminus of the fatty acid molecule, respectively. With this information in hand, EI-MS fragmentation analysis (Figure 4) was done as follows.

Identification of Compound 1 and Its Neurite Outgrowth
The compound 1 displayed the molecular ion peak at  Compound 1 was tested for its Aβ(25-35)-induced atrophy. As shown in Figure 5, compound 1 showed activity at a dose of 1 μM. The efficacy of compound 1 was compared with nerve growth factor (NGF) and docosahexaenoic acid (DHA). DHA is essential fatty acid praised for the memory enhancement [20]. Interestingly, compound 1 displayed stronger activity than DHA. Furthermore, the length of dendrite in the cells treated with compound 1 was comparable to those of NGF treated cells. This indicated that compound 1 as the one of the active constituent present in RE responsible for the enhancement of neurite outgrowth.

Discussion
Dementia is a clinical syndrome characterized by multiple cognitive deficits including significant impairment in memory and at least one other sphere of mental activity [21]. The two most common etiologies of dementia are Alzheimer's disease and vascular dementia. Estimates suggest that the numbers of people with dementia worldwide in 2007 was about 33-million, with a likely increase in the coming days [1]. Despite this catastrophic increase in dementia patients worldwide, no effective treatment is yet available [22]. The current treatment includes the use of cholinomimetic agents in the form of acetylcholine esterase (AChE) inhibitors such as donepezil, rivastigmine and galantamine; however, the clinical efficacy of these drugs is still controversial [3]. In this regard, alternative medicines draw considerable attraction these days as a source of potential new treatment for dementia [5,23,24]. Natural medicines such as Withania somnifera and ginseng saponins have been found to induce axonal and dendritic extension [4,25]; while in the present study, we found that the chloroform extract of the R. damascena significantly induced the neurite outgrowth and inhibited the Aβ(25-35)-induced atrophy. Furthermore, RE prevents the neuronal cell death induced by Aβ (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35). The oil of R. damascena have been shown to relieve depression and stress in humans [6], and has a relaxant effect on guinea pig tracheal chains [26]; however, this is the first report on the neurite outgrowth of rose extract. Further work up led the isolation of very long chain polyunsaturated fatty acid (VLFA) compound 1 belonging to n−3 series as one of the active constituents present in the extract. VLFAs are normal components of most tissues, particularly brain, retina and male reproductive tissues. VLFAs with 10-12 double bonds and up to 58 carbon atoms have been reported from the bovine retina [27]. Crustacea Bathynella baicalensis has been shown to contain VLFAs with three to six double bonds and up to 40 carbon atoms [28], while they have been observed rarely in the plant kingdom. The present report is the first example about the occurrence of such VLFAs in R. damascena.
Human development and health depend in many respects on the availability of long chain polyunsaturated fatty acids of 20 or 22 carbons in length that contain up to six methylene-flanked cis-double bonds [29]. Nutritionally important VLFAs include the n−6 fatty acids such as arachidonic acid, and the n−3 fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). In particular, n−3 VLFAs have long been investigated for their importance during human fetal development and the formation and function of the central nervous system, brain and retina. The loss of DHA in the brains of patients with Alzheimer's disease is accompanied by a decrease in memory and learning [30]. DHA administration protects against the oxidative stress and loss of avoidance learning ability caused by the infusion of Aβ into the cerebral ventricle [31]. Although it was reported that DHA stimulated neurite outgrowth in normal neurons such as rat primary hippocampal neurons [32] and PC12 cells [33], no one has reported the effect of DHA on Aβinduced neuritic atrophy. In present study, the compound 1 showed more pronounced protection of Aβ(25-35)-induced neuritic atrophy than the DHA, suggesting the beneficial effect against dementia.
Previous studies indicated that saturated long-chain fatty acids had no effect on neurite extension [34,35] and mammalian cells lack the desaturase enzymes that introduce double bonds at the n−3 and n−6 positions [36]. Therefore, diets rich in these polyunsaturated fatty acid may have direct benefit for the human brain function. Although further studies are required, the present study indicates the presence of VLFAs in rose extract that might have possible health benefit for the patients suffering from dementia. Isolation and identification of other active constituents as well as synthesis and semisynthesis of related compounds are essential for the structure activity relationship study and the discovery of potent lead compound.

Funding
Grant-in-Aid for Scientific Research (No. 16406002) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.