In Vitro Neuro-Protective Studies of Phyto-Compounds from Azadirachta indica, Centella asiatica and Gloriosa superba
Received Date: Aug 13, 2018 / Accepted Date: Aug 16, 2018 / Published Date: Aug 23, 2018
The test substances quercitin dehydrate, asiatic acid and colchicine were evaluated for its in vitro neuroprotective activity against Hydrogen peroxide induced toxicity in HEK-293-(Human embryonic kidney cell line). Firstly Test Substances were estimated for cytotoxicity with different concentrations ranging from 100 μg/ml to 1.56 μg/ml, which resulted high toxicity in HEK-293-(Human embryonic kidney) cell line, and hence the nontoxic concentrations were taken for further studies. In Cytoprotective studies Test Substances were showing significant activity in human embryonic kidney cells against hydrogen peroxide toxicant. These compounds are seen to express their activity in AMPA and SNCA expressed cells in HEK-293 cell line.
Keywords: Quercitin dehydrate; Asiatic acid; Colchicines; Neuroprotective; HEK cell line; Hydrogen peroxide toxicant
Since very ancient days various medicinal plants and their phytocompounds form part and parcel of major populations of India, and other South East Asian countries. For the better management of different Central Nervous System (CNS) disorders, chemical and synthetic drugs studied are not seen to be fully effective; hence different phytomedicinal compounds (phyto-chemicals from herbal sources) are successfully utilized with minimum or without side effects even for long term therapy/treatment .
Azadirachta indica (commonly known as neem) have been used in India for over two millennia for its medicinal property. In Ayurveda, Azadirachta indica is reported to have antifungal, antidiabetic, antibacterial, antiviral, contraceptive and sedative properties including neurological property and has been evaluated for a wide spectrum of diseases including cancer, inflammation, ulcer, dementia, immune disorder, hyperlipidemia and liver disease. Phytocompound used in this work is Quercitin dehydrate [2-6].
Centella asiatica (commonly known as thankuni) has been used to treat various disorders and apart from wound healing, the herb is used for the treatment of various skin ailments such as leprosy, lupus, varicose ulcers, eczema, psoriasis, diarrhoea, fever, amenorrhea, diseases of the female genitourinary tract and also for relieving anxiety and improving memory cognition. It is known to rejunivate the brain and nervous system, increase attention span and concentration and also combat aging. Phytocompound used in this work is Asiatic Acid [7-11].
Gloriosa superb (commonly known as agnisikha or ulatchandal), the alkaloid-rich plant is used as a traditional medicine in many cultures in Indian subcontinent . It has been used in the treatment of gout, snakebite, ulcers, neurological , arthritis, cholera, colic, kidney problems, typhus, impotence, nocturnal emission, etc. and is in great demand for medicinal use. Phytocompound used in this work is Colchicine [12-14].
AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) is a subtype of the ionotropic glutamate receptor coupled to ion channels that modulate cell excitability by gating the flow of calcium and sodium ions into the cell usually seen in schizophrenia and parkinsonism and (SNCA) alpha-synuclein which is a major constituent of Lewy bodies, the protein clumps that are seen of Parkinson's disease are the neuroreceptors used in this work.
Outline of the method
The in vitro cytotoxicity was performed for all four test substances on HEK-293 (Human Embryonic kidney cell) to find toxic concentration of Test Substances to evaluate the cytoprotective activity against hydrogen peroxide.
Preparation of test solution
For cytotoxicity studies, 10 mg of Test Substances were separately dissolved and volume was made up with Ham’s F12 supplemented media containing 2% inactivated FBS to obtain a stock solution of 1 mg/ml concentration, Further, sterilized by 0.22 μ syringe filtration. Serial two fold dilutions were prepared from this for carrying out cytotoxic studies. Placebo (Cell culture media with 2% FBS), which demonstrates the suitability of test system to yield a reproducible, appropriate reactive response in the test system .
The monolayer cell culture was trypsinized and the cell count was adjusted to 100,000 cells/ml using Ham’s F12 containing 10% FBS. To each well of the 96 well microtitre plate, 0.1 ml of the diluted cell suspension was added. After 24 h, when a partial monolayer was formed, the supernatant was flicked off, the monolayer once washed with medium and 100 μl of different test concentrations of Test Substances were added on to the partial monolayer in microtitre plates. The plates were then incubated at 37°C for 72 h in 5% CO2 atmosphere, and microscopic examination was carried out and observations were noted every 24 h interval. After 72 h, the drug solutions in the wells were discarded and 50 μl of MTT in PBS was added to each well. The plates were gently shaken and incubated for 3 h at 37°C in 5% CO2 atmosphere. The supernatant was removed and 100 μl of DMSO was added and the plates were gently shaken to solubilize the formed formazan. The absorbance was measured using a microplate reader at a wavelength of 540 nm. The percentage growth inhibition was calculated using the standard formula and concentration of test drug needed to inhibit cell growth by 50% (CTC50) values is generated from the dose-response curves for each cell line.
Cytoprotective study against hydrogen peroxide induced toxicity
The experiment was performed as per the standard protocol, Briefly, exponentially growing HEK-293 (Human embryonic kidney cell line) were trypsinized from the culture flask and 1.5 × 105 cells/ ml were seeded in 96 well microtitre plate. After attaining confluency, culture mediums from the wells were discarded and cells were preincubated with different non-toxic concentrations of Test Substances were along with the Cell control for 1 h in culture medium. After 1 h pre-incubation, 10 μl of stock H2O2 solution was added (stock of 10 mM H2O2) to all the wells except control wells. The plates were incubated for 24 h at 37°C with atmosphere of 5% CO2. After incubation MTT assay was performed to determine the cell viability. From the absorbance values from test and control groups, % protection offered by test substances were against H2O2 induced toxicity was calculated .
The mRNA expression level of SNCA and AMPA were carried out using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Briefly, the HEK-293 cells were cultured in 60 mm petridish and maintained in HAM’S F12 medium for 48 hrs. The DMEM medium was supplemented with FBS and amphotericin. To the dish required concentration of test samples that are phytoconstitute added and incubated for 24 hr. Total cellular RNA was isolated from the untreated (control) and treated cells using Tri Reagent according to manufacturer’s protocol. cDNA was synthesized from total isolated RNA by reverse transcriptase kit according to manufactures instructions (Thermo scientific). Then 50 μl of the reaction mixture was subjected to PCR for amplification of hek-293 cDNAs using specifically designed primers procured from Eurofins India and as an internal control the house keeping gene GAPDH was co-amplified with each reaction.
Amplification conditions for SNCA and AMPA gene
SNCA- 95°C for 5 min followed by 35 cycles of denaturation at 95°C for 30 seconds, annealing Tm for 30 seconds and extension at 72°C for 45 seconds. This was followed by final extension at 72°C for 10 min. Product size: 305 bp. AMPA-94°C for 5 min followed by 35 cycles of denaturation at 94°C for 15 seconds, annealing at Tm for 30 seconds and extension at 72°C for 45 seconds. This was followed by final extension at 72°C for 10 min. Product size: 760 bp.
Results of the current study are presented in Tables 1 and 2 (Figures 1-4).
|S. No||Name of Test Sample||Test Conc. (μg/ml)||%Cytotoxicity||CTC50 (μg/ml)|
|1.||Quercitin dehydrate||100||38.17 ± 0.37||35.60 ± 0.33|
|50||66.06 ± 0.37|
|25||38.17 ± 0.37|
|12.5||26.24 ± 0.23|
|6.2||17.68 ± 0.38|
|3.1||10.49 ± 0.14|
|1.5||5.25 ± 0.19|
|2.||Asiatic Acid||100||66.34 ± 2.14||3.19 ± 0.10|
|50||60.20 ± 0.35|
|25||57.46 ± 0.56|
|12.5||55.04 ± 1.07|
|6.2||53.70 ± 0.14|
|3.1||48.77 ± 1.35|
|1.5||25.80 ± 0.14|
|3.||Colchicine||100||72.25 ± 0.50||3.40 ± 0.15|
|50||61.37 ± 2.28|
|25||56.02 ± 1.33|
|12.5||53.28 ± 1.39|
|6.2||50.40 ± 0.66|
|3.1||47.28 ± 0.72|
|1.5||31.10 ± 4.19|
Table 1: Cytotoxic properties of Test Substances were against HEK-293 cell line.
|S. No||Samples||Concentration Tested (μg/ml)||% Protection|
|65.55 ± 1.0
34.82 ± 0.7
|54.68 ± 1.1
24.76 ± 2.3
|60.65 ± 1.1
37.50 ± 0.7
Table 2: Cytoprotective activity of Test Substances is in Human embryonic kidney Cell line against Hydrogen peroxide induced toxicity.
Firstly Test Substances were estimated for cytotoxicity with different concentrations ranging from 100 μg/ml to 1.5 μg/ml, which resulted to be 35.60 ± 0.33, 3.19 ± 0.10, 3.40 ± 0.15 μg/ml for Quercitin dehydrate, Asiatic Acid and Colchicine, and hence the nontoxic concentrations were taken for further studies. In Cytoprotective assay, Quercitin dehydrate, Asiatic Acid, and Colchicine, showed 35.60 ± 0.33, 3.19 ± 0.10 and 3.40 ± 0.15 percentage protection at higher concentration against hydrogen peroxide induced toxicity in human embryonic kidney cell line respectively.
The test substances (phyto-compounds) quercitin dehydrate and asiatic acid are seen to express their activity in AMPA expressed cells in HEK-293 cell line and colchicine is seen to express their activity in SNCA expressed cells in HEK-293 cell line. This result tally with work of in-silico work of Bagchi et al. which established quercitin dehydrate and asiatic acid as novel drug leads for AMPA receptor published in neuropsychiatry journal  and with the in-silico wCVork of Bagchi et al. which established colchicine as novel drug lead of SNCA receptor published in Information Systems Design and Intelligent Applications, Advances in Intelligent Systems and Computing, by Springer Nature Singapore Pte Ltd .
This work forms a part of SERB-NPDF for Preenon Bagchi, author reference number PDF/2015/000047.
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Citation: Bagchi P, Kar A, Anuradha M (2018) In Vitro Neuro-Protective Studies of Phyto-Compounds from Azadirachta indica, Centella asiatica and Gloriosa superba. Med Chem (Los Angeles) 8: 230-233. DOI: 10.4172/2161-0444.1000517
Copyright: © 2018 Bagchi P, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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