INTRODUCTION:

Neurodegenerative disease is a term applied to a variety of conditions which result from a chronic breakdown and deterioration of neurons, particularly, those of central nervous system (CNS)¹. Alzheimer's disease (AD), Parkinson’s disease, multiple sclerosis, amyotrophic lateral sclerosis and spongiform encephalopathy are some of the examples of neurodegenerative diseases^2-4. One of the major pathological features of AD is the abundant presence of amyloid plaques in the brain tissues of affected individuals⁵. These plaques are made up of amyloid-β proteins (Aβ), which are neurotoxic and are actively involved in the neuronal degeneration that occurs in AD6. As a result, modulation of its toxicity has been identified to be an important therapeutic approach to control the onset of AD^6,7,8. In the in vitro model of AD, Aβ has been used to initiate neurotoxicity in various types of cultured cells^9,10,11,12.

Natural products have provided an alternative strategy for AD therapy as they are usually safer and have fewer adverse effects than chemically synthesised drugs¹⁵. Recent findings have shown that natural products have the potential not only to prevent Aβ toxicity, but also to prevent the production of Aβ¹⁶. For example, resveratrol (derived from red grape), curcumin (derived from spice turmeric) and (˗)- epigallocatechin-3-gallate (derived from green tea) have been reported to reduce the effect of Aβ in the cerebral cortex; curcumin is reported to have the ability to bind small Aβ peptides to block Aβ aggregation as well as fibril and oligomer Aβ formation^15,16.

The drugs currently available in the market for the treatment of various learning and memory disorders are associated with several side effects indicating need of substitute medication from alternative system of medicine^17-20. A variety of plants has been reported for the treatment of neurodegenerative disorders, such as AD²¹. A report showed that in traditional practices of medicine, numerous plants have been used to treat cognitive disorders, including neurodegenerative diseases, such as AD and other memory related disorders²².

Hydrocotyle javanica (H. javanica) Thunb. is a naturally growing prostrate herb found throughout the Himalayas and Assam Hills, Nilgiri Hills and Western Ghats of India at altitudes of 2 000 to 8 000 ft²³. The plant belongs to the family Apiaceae (formerly known as Umbelliferae) and the subfamily is Hydrocotyloideae²⁴. Traditionally, the fresh plant parts of H. javanica Thunb. are used as crushed and ingested to cure sore throats and lungs. Kurichiya tribes of Kerala (India) apply the juice of the whole plant on the chest to cure asthma and convulsions²⁵. Pharmacologically it had been proved that the methanolic fraction of the plant showed antibacterial activity against some human pathogenic bacteria which had been published in our earlier publication²⁶

Peristrophe bicalyculata (P. bicalyculata), is upto 60-180 cm in height and found almost throughout India, Afghanistan and Africa. It is commonly known as kali aghedi in Hindi and Kakajangha in Sanskrit. The herb is used for its antibacterial property (tuberculostatic), snake poison, in bone fracture and sprain. Leaf extract is used for fever, cold and cough. Mucilage medicines are used for ear and eye treatments^27-33.The chemical composition of the dried aerial parts of P. bicalyculata reveals that is comprised of 14-methyltritriacont-14-en-15-ol and 35-hydroxynonatriacontanal³⁴. The essential oil shows tuberculostatic activity in vitro against the growth of various strains of Mycobacterium tuberculosis^34,36.

MATERIALS AND METHODS:

Collection and identification:

Collection of two medicinal plants Hydrocotyle javanica and Peristrophe bicalyculata was carried out during the year 2017-2018 on the basis of its medicinal information given by various taxonomist in Pondicherry. Targeted collection based on chemotaxonomic relationships and ethno-medical information derived from Traditional Medicines. The identified plants were authenticated by Dr. N. Loganathan Specialist in Medicinal plants, Hereditary Physician, Pondicherry.

Preparation of Extract:

The collected plant materials were washed with distilled water to remove dirt and soil. The whole plants were further shade dried and then coarsely powdered. The coarse powder of two plants (500g) was extracted with 3 litres of different solvents such as Petroleum ether, n-Hexane, Chloroform, Alcohol and water (60 - 80°C) by continuous hot percolation using Soxhlet apparatus. After completion of extraction it was filtered and the solvent was removed by distillation under the reduced pressure. The extract was stored in desiccator. The extract was vacuum-dried and processed further for Pharmacological evaluation^37-39.

Phytochemical screening:

The different extracts of Hydrocotyle javanica and Peristrophe bicalyculata were subjected to various chemical tests for identification of phytochemical constituents (glycosides, phytosterol, saponins, alkaloids, carbohydrates, flavonoids, tannins, protein and amino acid)^40-42.

In-vitro Neuroprotective evaluation using amyloid β_1-⁴²intoxicated SH-SY5Y cell lines:

Principle:

The ability of the cells to survive a toxic insult has been the basis of most cytotoxicity assays. This assay is based on the assumption that dead cells or their products do not reduce tetrazolium. The assay depends both on the number of cells present and on the mitochondrial activity per cell. The cleavage of MTT to a blue formazan derivative by living cells is clearly a very effective principle on which the assay is based. The principle involved is the cleavage of tetrazolium salt MTT (3-(4,5 dimethyl thiazole-2 yl)- 2,5-diphenyl tetrazolium bromide) into a blue coloured product (formazan) by mitochondrial enzyme succinate dehydrogenase. The numbers of cells were found to be proportional to the extent of formazan production by the cells used.

Procedure⁴³:

The cell culture was centrifuged and the cell count was adjusted to 1.0x105 cells/ml using DMEM medium containing 10% FBS. To each well of a 96 well flat bottom micro-titre plate, 100µl of the diluted cell suspension (approximately 10,000 cells/well) was added with 10ng/ml of LPS. After 24 hours, when the cell population was found adequate, the cells were centrifuged and the pellets were suspended with 100µl of different test sample concentrations prepared in maintenance media. The plates were then incubated at 37^oC for 48 hours in 5% CO₂ atmosphere, and microscopic examination was carried out and observations recorded every 24 hours. After 24 hours, 20 µl of MTT (2mg/ml) in MEM-PR (MEM without phenol red) was added. The plates were gently shaken and incubated for 2 hours at 37^oC in 5% CO₂ atmosphere. The 100µl 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 540nm. The percentage cell viability was calculated using the following formula and concentration of drug or test samples needed to inhibit cell growth by 50% values were generated from the dose response curves.

Mean OD of the individual sample

% Cell viability= × 100

Mean OD of Control

RESULTS:

Phytochemical screening:

The preliminary phytochemical screenings of various extracts of H. javanica and P. bicalyculatta mainly revealed the presence of glycosides, phytosterol, saponins, alkaloids, carbohydrates, flavonoids, tannins, protein and amino acid in table 1 and 2 respectively.

Table:1 Phytochemical Screening of different extracts of Hydrocotyle javanica

SI. No.	Phytoconstituents	Petroleum ether	n- Hexane	Chloroform	Alcohol	Aqueous
1.	Alkaloids	-	-	+	+	+
2.	Carbohydrates	-	-	-	+	+
3.	Glycosides	-	+	-	-	-
4.	Phytosterols	+	+	-	+	+
5.	Saponins	-	-	-	-	+
6.	Fixed oils and Fats	-	-	-	-	-
7.	Tannin and Phenolic compounds	-	-	-	-	+
8.	Proteins and Free amino acids	-	-	+	+	+
9.	Gums and Mucilage	-	-	-	-	-
10.	Flavonoids	-	-	+	+	+
11.	Lignin	-	-	-	+	+
12.	Volatile oil	-	-	-	-	-

Table: 2 Phytochemical Screening of different extracts of Peristrophe bicalyculatta

SI. No.	Phytoconstituents	Petroleum ether	n- Hexane	Chloroform	Alcohol	Aqueous
1.	Alkaloids	-	-	+	+	+
2.	Carbohydrates	-	-	-	+	+
3.	Glycosides	-	+	-	-	-
4.	Phytosterols	+	+	-	+	+
5.	Saponins	-	-	-	+	+
6.	Fixed oils and Fats	-	-	-	-	-
7.	Tannin and Phenolic compounds	-	-	-	-	+
8.	Proteins and Free amino acids	-	-	+	+	+
9.	Gums and Mucilage	-	-	-	-	+
10.	Flavonoids	-	-	+	+	+
11.	Lignin	-	-	-	+	+
12.	Volatile oil	-	-	-	-	-

(+) Indicates presence

(-) Indicates absence

In-vitro Neuroprotective evaluation:

In vitro neuroprotective evaluation of different extracts of H. javanica and P. bicalyculatta were carried out using amyloid β_1-42intoxicated SH-SY5Y cell lines using MTT assay. The percentage cell viability at various concentrations of different extracts of H. javanica and P. bicalyculatta were determined. The cytotoxic effect of the two medicinal plant extracts on the viability of the SH-SY5Y cells is presented as IC₅₀ values in table 3.

Table: 3 In vitro neuroprotective evaluation of different extracts of H. javanica and P. bicalyculattain Aβ_1-42intoxicated SH-SY5Y - MTT assay

SI. No.	Sample Description	SHSY5Y IC₅₀ µg/ml
1.	n-Hexane extract of HJ	17.55082
2.	Chloroform extract of HJ	38.34148
3.	Alcoholic extract of HJ	42.82841
4.	Aqueous extract of HJ	97.85628
5.	n-Hexane extract of PB	98.7468
6.	Chloroform extract of PB	81.27094
7.	Alcoholic extract of PB	19.36739
8.	Aqueous extract of PB	26.90896

In order to examine the possible neuroprotective effects of the two medicinal plant extracts, two concentrations that presented low toxicity from the MTT assay, were selected to determine if they could reduce cell death induced by Aβ in SH-SY5Y cells. An improvement in cell viability, relative to the control, was observed with 87.7% and 78.7% of viable cells were observed with the MTT assay, at 10 µg/ml. (Fig 1). n-Hexane extracts of H. javanica and Alcoholic extract of P. bicalyculatta were found to reduce the effect of Aβ is presented as IC₅₀ value of 17.55082µg/ml and 19.36739µg/ml, but were less effective in comparison with the other solvent extracts screened.

Figure 1: Effect of different extracts of Hydrocotyle javanica (HJ) and Peristrophe bicalyculata (PB) in Aβ_1-42 intoxicated SH-SY5Y cell lines. Statistical significance was determined by one way ANOVA followed by Tukey's multiple comparison tests using Graph pad prism Version 6.0. Values are represented as Mean ± SEM, Superscript *** denotes p<0.001 vs control, ### denotes p<0.001; ## denotes p<0.01; # denotes p<0.05 vs Aβ_1-42 respectively. (Aβ: Amyloid beta; nH: n-Hexane, Chl: Chloroform; Alc: Alcohol; Aq: Aqueous)

DISCUSSION:

The viability of cells exposed to Aβ_1-42 was assessed using the MTT assay. This assay are sensitive, quantitative and reliable colorimetric assays for the determination of cell viability. Both medicinal plants were observed to reduce the effects of Aβ induced neuronal cell death, indicating that they may contain compounds which may be relevant in the prevention of AD progression.

The low toxicity of n-Hexane extracts of H. javanica (IC₅₀17.55082µg/ml) and Alcoholic extract of P. bicalyculatta (IC₅₀19.36739µg/ml) are observed in the present study. In addition, the plant also showed a good effect in reducing Aβ induced cell death.

CONCLUSION:

All eight extracts tested were reduced the effects of Aβ induced neuronal cell death in the present study, indicating that they may contain compounds which may be relevant in the prevention of AD progression. All the experiments in the present study are based on crude extract and are considered to be preliminary and more sophisticated research is necessary to reach concrete conclusions about the findings of the present study. Future work will focus on the isolation and the elucidation of neuroprotective compounds which will be subjected to screening for prevention of Aβ_1-42mediated neuronal degeneration.

ACKNOWLEDGMENTS:

We are expressing our heartfelt gratitude to JSS college of Pharmacy, Ooty for their support during the study.

CONFLICT OF INTEREST:

All authors have none to declare.

ABBREVIATIONS:

Aβ- amyloid beta, AD- Alzheimer’s disease, nHHJ - nHexane extract of H. javanica, CHL HJ – chloroform extract of H. javanica, AlcHJ - alcoholic extract of H.javanica, AqHJ – water/aqueous extract of H. javanica, nHPB - nHexane extract of P. bicalyculatta, CHL PB – chloroform extract of P.bicalyculatta, AlcPB- alcoholic extract of P. bicalyculatta, AqPB – water/aqueous extract of P. bicalyculatta

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Received on 20.09.2019 Modified on 18.11.2019

Research J. Pharm. and Tech. 2020; 13(7): 3351-3355.

DOI: 10.5958/0974-360X.2020.00595.8