Neuroprotective capabilities of Vitex negundo in primary hippocampal neurons Sonia Siddiqui1*, Zafar Saeed Saify2, Khawar Saeed Jamali3, Priya Tufail4, Aiman Kanwal4, Aisha Kamal4 and Faisal Khan4 1Department of Biochemistry, Dow University of Health Sciences, Karachi, Pakistan 2HEJ Research Institute of Chemistry, International Center for Chemical and Biological Sciences (ICCBS), Karachi, Pakistan 3Department of Surgery, Dow University of Health Sciences, Karachi, Pakistan 4Department of Neuroscience, Dr. Panjwani Center for Molecular Medicine and Drug Research (PCMD), International Center for Chemical and Biological Sciences (ICCBS), University of Karachi, Karachi, Pakistan Abstract: Vitex negundu (Vn) is a well-known aromatic shrub commonly used as a traditional folk medicine famous for its potential pharmacological and biological activities. Several chemical compounds are extracted and identified from the different parts of the Vn such as leaves, root, seeds and flowers. Number of researches reported the herb as antimicrobial, anti-androgenic, anti-osteoporotic, and anti-tumour, anti-cancer, anti-inflammatory, anti-oxidant, anti-hyperglycemic and hepatoprotective. The effects of Vn on neurite outgrowth have not been identified till now. Therefore present study was designed to investigate the neurite outgrowth effects of Vn extract in hippocampal neurons. Neurons from P0 mice were isolated and cultured in defined medium containing the different concentrations of Vn (20, 30, 40, 50, 100, 150 and 200 µg/ml) for 48 hrs. The presence of the neurites was confirmed by using ßIII-tubulin antibody which specifically labels only the neurites. Morphometric analysis was done by using Optika Pro-Vision software. The data show that Vn at 30 and 40 µg/ml significantly increased the mean average length of the longest neurite whereas at 150 and 200 µg/ml it significantly decreased the mean average length of the 10 longest neurite in hippocampal neurons. Nevertheless Vn did not show any significant effects on the sum of all the neurite lengths at any concentrations tested. Taken together the result shows that methanolic extract of Vn has potential to produce long neurites at 30 and 40 µg/ml and therefore can be act as a neuroprotective agent in the future drug development. Keyword: Neurite outgrowth, Neuronal regeneration, Vitex nigundo, Hippocampal neurons, Immunostaining, morphometery, neuroinflammation. INTRODUCTION approach (Kulkarni et al., 2008). Another study reported the significant anti-inflammatory effects of fresh mature Vitex negundu (Vn) is a large aromatic shrub which is leaf extract of Vn in formaldehyde and carrageenan also known as five leaved chaste tree widely distributed in induced rat paw oedema (Chawla et al.,1992). Several various regions of the world such as south Asia, other chemical compounds have been isolated from the Indonesia, china, Japan, Pakistan, India, east Africa and plant mainly flavanoids, terpenoids and lignans that are South America (Gautam et al., 2010 and Zheng et al., reported to have convincing medicinal properties (Zehng 2015). All parts of the plant, leaves, roots and seeds have et al., 2015). A type of lignan 6-hydroxy-4-(4-hydroxy-3- been largely used as a folk medicine in south and south methoxyphenyl)-3-hydroxyme-thyl-7-methoxy-3,4- East Asia (Padua et al., 1999). Number of volatile dihydro-2-naphthaldehyde from Vn documented as a compounds have been identified from the crude extract of potent analgesic, anti-inflammatory, anti-oxidant, anti- Vn that possess ant-inflammatory, anti-oxidant, anti- tumor and immuno-stimulatory compound (Zehng et al., fungal, anti-bacterial, analgesic, anti-rheumatism (Zheng 2009). Usually compounds that have anti-inflammatory et al., 2009b; Zheng et al., 2014a), antitumor (Zhou et al., effects are good therapeutic agents that can help to cure 2009), insecticidal (Kamaraj et al., 2009; Kamaraj et al., neurodegenerative diseases. Therefore present study was 2010), antimicrobial (Kamruzzaman et al., 2013), anti- designed to investigate Vn effects on the neurite androgenic, anti-osteoporotic, anti-hyperglycemic and outgrowth from hippocampal neurons. The identification heptoprotective properties. Ethanolic and Water extract of of the therapeutically effective dose concentration of Vn fresh mature leaves of Vn has been reported for would lead to the development of a promising anti- significant analgesic activity in rat and mice (Dharmasiri inflammatory agent to treat neurodegenerative disorders et al., 2003). Methanolic extract of Vn (leaves) shows and helps us to recover the altered morphology of potential anti-inflammatory and anti-oxidant effects that neurons. prevent tissue damage by scavenging free radical *Corresponding author: e-mail: [email protected] Pak. J. Pharm. Sci., Vol.31, No.1(Suppl), January 2018, pp.341-344 341 Neuroprotective capabilities of Vitex negundo in primary hippocampal neurons MATERIALS AND METHODS Morphometery After 48 hours of incubation images were captured Animal ethics through a camera attached to the microscope. Length of All experiments in this study were performed according to neurite was measured from the point of emergence at the established ethical guidelines on embryos or postnatal cell body to the tip of axon terminal, bipolar and pups derived from timed pregnant Balb-C mice (Animal multipolar extension were measured and sum up for House Facility, ICCBS). The animals were housed in consideration of a single neurite length. 10-12 different cages in a standard barrier facility and maintained on a 12 fields from each well is focused and captured. h light: 12 h dark cycle. All experiments in this study Quantification of the morphological parameters was were performed according to the established guidelines. carried out using Optika provision analysis software. At All experiments were performed in PCMD, ICCBS, least 150 cells per treatment were analysed in each University of Karachi. experiment, and results were pooled from at least 3 different experiments. Cell culture Hippocampai from brains were dissected out from the Immunostaining neonatal P0-1 mice. Meninges were removed and small Hippocampal neurons were stained with rabbit anti β-III hippocampus tissues were digested with 0.25% trypsin tubulin (Merk Millipore) antibody diluted in Kreb’s EDTA solution (Sigma Aldrich) for 10-12 min at 37⁰C. Ringer buffer/0.01 % BSA. Triton X-100 (0.1%) is added Mechanical digestion was performed by gently tapping to permeablize the membrane. Cells were fixed with 4% and re-pipetting the suspension. Trypsinization was PFA for 20min and were incubated for 1 hour with stopped by adding culture medium. Cells were primary antibody. Counter staining with secondary centrifuged at 1000 rpm for 10 min at 4°C. Supernatant antibody was done by using Alexa flour 588 for 45 min. was discarded and cell pellet was resuspended into the Each step followed washing with Kreb’s Ringer buffer. fresh medium. Cells were seeded into four well plates Cover slips were mounted by using an immumount on which were previously coated with poly-L-lysine (PLL clean glass slide store it in a cool dry place at 4⁰C.The 0.01%, Sigma Aldrich) for 2 hrs at room temperature. stained pictures were captured by using fluorescence Neurons were cultured in a medium composed of microscope. 10%FBS, 1% glutamax, 1% penicillin/streptomycin and 1% sodium pyruvatein Dulbecco’s Modified Eagle STATISTICAL ANALYSIS Medium (DMEM)/F12 (1:1). Cells were incubated for 48 hours at 37⁰C in 5% CO2 humidified atmosphere. Experiments were repeated at least 3 times and the data is represented as Means ± SD. Mann Whitney U-test was Design of the study applied to evaluate the significance level. A p-value of Neonatal mice pups of postnatal day P0-1were selected. <0.05 is considered as statistically significant. The lengths of neurites were measured after 48 hours of neuronal growth with and without herbal extract Vn. Only RESULTS the free growing neurons with short and long axons were measured from the soma of the cell to the tip of neurite. We have studied the role of methanolic extract of Vn on Cells having neurites less than 20 µm were excluded from the hippocampal neurite outgrowth isolated from the P1 the study. Neurons that were too close and meeting their mice and cultured in a defined medium for 48 hours. neurites to soma of other neurons were also excluded Neurons were cultured in the presence of different doses from the study. This study was completed in 2.5 years. of Vn extract (20, 30, 40, 50, 100, 150, 200 µg/ml). Neurite outgrowth was measured after 48 hours. The Dose response curve neurites were identified by using specific antibody ß-III Stock solution of 1mg/ml is prepared by dissolving tubulin which recognizes and labels only neurites from methanolic extract of Vn in de-ionised water. The the neurons. The present study show that Vn at lower solution was filtered and aliquots were stored at -20⁰C. doses i.e. 30 and 40µg/ml significantly (P<0.05, P<0.0001 Hippocampal neurons were seeded in a density of 10,000 respectively) increased the mean average length of the 10 cells per well. Different selected doses of 20µg/ml, longest neurites (fig. 1). However there was a slight non- 30µg/ml, 40 µg/ml, 50 µg/ml, 100µg/ml, 150µg/ml and significant increase in the longest neurite lengths at 50 200 µg/ml were added into each well. Controls received µg/ml dose (fig. 2). Whereas at 100µg/ml there was a only culture medium. All dilutions were prepared in the slight non-significant decrease in the longest neurite culture medium. The effect of different doses of drug on lengths but at higher concentrations i.e. 150 and 200µg/ml hippocampal neurons was observed after 48hrs of Vn significantly (P<0.005, P<0.0001 respectively) inhibited the mean average lengths of the 10 longest incubation at 37⁰Cin 5% CO2 humidified atmosphere. neurites (fig. 2). On the other hand sum of all the neurites show a different effects compared to the 10 longest 342 Pak.