INTRODUCTION:

Neurodegenerative disease (NDD), including Alzheimer’s disease (AD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS), are progressive disorders with abnormal protein deposition and selective neuronal loss, leading to motor, and/or cognitive impairments. NDD has afflicted millions of people worldwide and is one of the main reasons for the high incidence of mortality in elderly people.

The World Health Organization (WHO) predicts that NDD will become the second leading cause of death after cardiovascular disease by 2040¹. Despite huge basic and clinical research efforts, no disease-modifying interventions are available to patients and therapeutic strategies are limited to symptomatic treatments at present. The main challenges in the study of NDD drug development are the diversity of etiology and clinical heterogeneity among individuals². Due to lack of biomarkers makes it difficult to detect disease in the early stages. There is an increasing need for more animal models that can outline the complexity of NND pathogenesis.

Herbal therapy of various disease and disorder is as old as mankind. The use of traditional medicine has reported to be efficacious and safe. The need to use medicinal plants as alternatives to classical allopathic medicines in the provision of primary health care cannot be over-looked. Moreover, herbal medicines are considered as time tested and has relatively safer for both human use and environment friendly and hence are used as sources of many lead compounds. They are also economic, easily available and affordable. Therefore, there is need to look inwards to search for herbal medicinal plants with the aim of validating the ethno medicinal use and subsequently an isolation and characterization of compounds which will be added to the potential lists of drugs³. Herbal remedies are plant parts that are used to cure a number of disorders and improve overall health.

Tridax procumbens commonly known as Dagdi Pala in Marathi and coat buttons, tridax daisy in English, is a weed commonly found in India and Nigeria. The plant is native of tropical America and naturalized in tropical Africa, Asia, and Australia. Tridax procumbens has been widely known for its anti-microbial and wound healing properties by ethnomedicinal practitioners. Apart from this, it has also been known for its number of pharmacological activities like neuroprotection, anti-inflammatory, immune-modulating property, anti-diabetic activity, hypotensive effect, bronchial catarrh, dysentery, diarrhoea. All parts of the plant have been reported to possess diverse scale of phytoconstituents⁴. The photochemistry of Tridax procumbens includes major secondary metabolites such as flavonoids, carotenoids, alkaloids, saponins, tannins and terpenes. Various alkaloids present in the plant have proven to exhibit anti-microbial activity against various micro-organisms such as Proteus mirabilis, Escherichia coli, Trychophyton mentagophytes and Candida albicans. Procumbenetin and other flavonoids present in the plant is reported to exhibit hypoglycaemic activity, decrease calcium deposition in kidneys, protect against oxidative distress and lowers VLDL cholesterol level. Leuteolin isolated from the plant is known for its anti-carcinogenic and anti-inflammatory activity. Whereas quercetin is reported to show anti-ulcerogenic activity. Beta-carotene and lutein exhibit protection against photo oxidative damage. It was reported that lignin and flavonoids exhibit antioxidant, anti-inflammatory and neuroprotective activity. The anti-Parkinson’s study was conducted for the crude hexane and chloroform extract of Tridax procumbens (HETP) and (CETP and the extract was successfully proved to possess the said activity. Thus, depending upon these reported data, two fractions of crude chloroform extract of Tridax procumbens (CETPF1) and (CETPF2) was opted for the studying the anti-Parkinson’s activity.

MATERIALS AND METHODS:

Collection and authentication of plant material:

The Tridax procumbens leaves were collected from the Kumbhargaon, Bhigwan, Maharashtra in the month of December 2021. Fresh leaves collected were washed and shade dried. The dried samples were ground to coarse powder with a mechanical grinder (Blender) and powdered samples were kept in clean airtight container. The fresh leaves were identified and authenticated by the Alarsin organization, Andheri (E), Mumbai.

Preparation of plant extract:

For the plant extraction, Successive extraction technique at 50°C with soxhlet extraction method was used. The hexane was used as solvent. Extraction was done until the solvent in siphoning tube becomes colourless. The obtained filtrate was concentrated using rotavac evaporator. The extract was stored in a desiccator at room temperature⁵. The percentage yield obtained of Hexane Extract of Tridax procumbence (HETP) was 2.6% w/w. After extraction with hexane solvent, course powder of leaves was dried, and powder was extracted with chloroform. After completion of the extraction process, the filtrate was concentrated by using rotavac evaporator to get 3.8% w/w yield of Chloroform Extract of Tridax procumbence (CETP)⁶.

Animals:

Zebrafish were procured from Department of Biological Sciences, Chennai. Adult wild type AB strains of zebrafish (3‑5cm) of both the sexes (4‑6‑months old) were used. The fishes were habituated to the laboratory conditions for at least 14 days and housed in a 50‑L tank filled with un‑chlorinated aquarium water at temperature of 28±2°C with constant filtration and aeration. Density of five fishes per litre was maintained⁷. Fishes were kept on 14:10h light/dark cycle and were fed twice a day with aquarium food. The fishes were housed in the animal house of Bharati Vidyapeeth’s College of Pharmacy, Navi Mumbai at 28±2°C and 50-65% humidity under a 14:10hr light and dark cycle. The prior approval was granted by the institutional animal ethics committee (Protocol no. BVCP/IAEC/02/2020), and all the animal experiments were carried out according to standard CCSEA guidelines.

Drosophila melanogaster wild type, Canton special strain was obtained from the National centre for biological sciences, Bangalore, India. The standard culture media was used to grow the flies at 23±1°C and 60% relative humidity under 12 h light/dark cycle⁸.

EXPERIMENTAL DESIGN:

Phytochemical screening of HETP and CETP :

The HFTP and CFTP were dissolved in its respective solvent and used for preliminary phytochemical screening. Different chemical tests were carried out to detect presence of flavonoids, tannins, glycosides, steroids, alkaloids, phenolic compounds, proteins and carbohydrates.

Pharmacological activity of HETP and CETP:

Acute toxicity study in zebra fish:

Acute toxicity was performed on test compound (HETP) and (CETP) extract according to OECD guideline number 203 in 7 zebrafish.

Limit test: The limit test was performed for 96 hrs at 100 mg/L. The fishes were exposed to the (HETP) and (CETP) extract at a maximum dose of 100mg/L for a period of 96 hrs. Mortalities were recorded at 24, 48, 72 and 96 hrs post exposure. Parameters evaluated were swimming at the bottom of tank, motions like to and fro, up and down, skin becomes faint and dull. The complete mortality was observed at the limit test hence the limit test was failed. Hence the main toxicity study was performed⁹.

Main study: According to OECD guideline, starting with 100mg dose divided with 1.6 factor 5 different concentrations were selected for the study i.e., 100mg, 62.5mg/l, 40mg/l, 24.3mg/l, 15mg/l was given to the fish. The fishes were exposed to the (HETP) and (CETP) extract at the dose of 62.5mg/L of extract for 96 hrs. Out of 7,6 fish were died in the 62.5mg/L dose hence this dose was neglected. The next dose i.e., 40 mg dose was found to be safe and hence was selected as maximum tolerable dose.

Rotenone induced catalepsy in zebra fish:

The study included six groups with 10 fish in each group viz., vehicle control, rotenone control (rotenone with vehicle), chloroform (11 and 40mg/L), hexane (11 and 40mg/L). Behavioural testing was done between 10:00 am to 5:00pm. The fish were exposed to respective treatments, followed by rotenone for 30 mins each with 15 min interval between both exposure¹⁰. For behavioral study fish were transferred to examination tank with premarked vertical lines 5cm apart to calculate speed and a horizontal line dividing into two equal halves to determine time spent in upper or lower halves of the fish tank. The parameters were determined at 0, 15, 30, 45 and 60 min for 5 min at each time point¹¹. The erratic swim pattern was also noted during the observations.

On the day 29^th, Adult zebra fish were euthanised by hypothermia for 5min at 0-4⁰C followed by decapitation. Whole brains were dissected to prepare a homogenate for biochemical analysis. The phosphate buffer saline solution was utilized for preparation of homogenates and centrifuged for 5min in centrifuge at 800g. The supernatants were stored at -20ºC for further biochemical analysis such as Superoxide dismutase¹²,

glutathione peroxidase⁸, malondialdehyde¹².

Acute toxicity study in Fruit Fly:

A 7-day acute toxicity study (LC₅₀) was carried out following the method described by Etuh et al. This was to determine the concentration of the extract that kills 50 % of the flies in seven days. Twenty flies (both sexes) were subjected to a series of prepared concentrations of each compound mixed in the diet. The flies are exposed to different solvent extraction of different concentration of. The concentration used for Chloroform extract of Tridax procumbens were 2, 2.5, 5, 7.5mg/ml. The mortality rate was scored every 24-hr interval for seven days^13,14.

Rotenone induced locomotor deficits in fruit fly:

Flies were divided into six groups (n =8), viz., vehicle control (vehicle treated without challenge), rotenone was given through diet and dose given to the flies were HETP (0.05 and 0.02% w/v), CETP (0.04 and 0.02 % w/v).

Negative geotaxis assay in fruit fly:

The flies were subjected to both inducing agent rotenone and treatments with test drug for 7 days. Followed by that the motor function activity was determined using a negative geotaxis assay. For the study, flies were transferred into a graduated flat bottom glass tube with 12cm x 2cm dimension and allowed to attune for at least 5min. The tube was initially tapped so as to confirm all the flies were at the bottom followed by observing the climbing activity. The percentage of flies which escaped the 10cm distance in the next 60 s was recorded.

Column chromatography:¹⁵

The column chromatography was used to separate the fractions of CETP. The mobile phase used was hexane: ethyl acetate (80:20). The Silica gel 60-120 and hexane was used for packing the column. The CETP was mixed with silica and added to the column. The flow rate was adjusted to 0.5ml/min and 5ml aliquots were collected. The TLC was performed of all the aliquots and plate was observed under UV visible spectra chamber. Thus, the two different fractions were obtained by column chromatography from chloroform extract of Tridax procumbens and were labelled as CETP extract fraction 1 (CETPF1) and CETP extract fraction 2 (CETPF2)

Pharmacological activity of bioactive CETPF1 and CETPF2:

Acute toxicity study in zebra fish:

Acute toxicity was performed on test compound CETPF1 and CETPF2 according to OECD guideline number 203 in 7 zebrafish. The detailed procedure was followed as mentioned above⁹. The maximum tolerable dose was thus found to be 24.3mg/l

Rotenone induced catalepsy in zebra fish:

Fish were divided into ten groups (n = 10), viz., vehicle control (vehicle treated without challenge), rotenone control (rotenone with vehicle), CETPF1 and CETPF2 (24.3 and 6mg/L), (24.3 and 6mg/L) respectively. The behavioural and biochemical estimations were done as mentioned above¹⁰.

Acute toxicity study in Fruit Fly:

Twenty flies (both sexes) were subjected to a series of prepared concentrations as 2, 2.5, 5, 7.5mg/ml of each compound i.e., CETPF1 and CETPF2. The study was conducted as explained above^13,14.

Rotenone induced locomotor deficits in fruit fly:

Flies were divided into six groups (n =8), viz., vehicle control (vehicle treated without challenge), rotenone was given through diet and dose given to the flies were CETPF1 (0.04 and 0.02% w/v), CETPF2 (0.04 and 0.02 % w/v). Negative geotaxis assay in fruit fly was done using above procedure.

STATISTICAL ANALYSIS:

One-way ANOVA followed by Dunnett’s multiple comparisons test using Prism Graph Pad version 8.0 (Graph Pad software, Inc., CA, and USA) was used.

RESULT:

Phytochemical screening of HETP and CETP:

Table 1: Result of Phytochemical screening of HETP and CETP⁶

Name of test	Name of test	HETP	CETP
Test for flavonoids	Shinoda test	Positive	Positive
Test for glycosides	Killer killani test	Positive	Positive
Test for steroids	Salkowski test	Positive	Positive
Test for alkaloids	Wagner’s test	Positive	Positive
	Dragendroff’s test	Positive	Negative
	Hager’s test	Positive	Positive
Test for phenolic compounds	Ferric chloride test	Positive	Negative
	Lead acetate test	Positive	Negative
	Acetic acid test	Negative	Positive
Test for proteins	Millions test	Positive	Negative
Test for carbohydrates	Molisch test	Positive	Positive
Test for carbohydrates	Barford test	Positive	Positive

Pharmacological activity of HETP and CETP:

Acute toxicity study in Zebra fish:

At the 65.53mg/L dose of HETP and CETP extract fishes were showing abrupt movements line up and down, to and fro motion also fainting of skin, swimming at the bottom of tank. Hence this dose was neglected. The next dose selected for toxicity study was 40mg/L dose of HETP and CETP extract. At 40mg/L dose of HETP and CETP, no abnormal swim pattern was observed, and mortality observed was 10% mortality. Hence 40 mg/L dose of HETP and CETP extract was found to be maximum tolerable dose. Hence dose obtained from acute toxicity study was 40mg/L of HETP and CETP extract, and low dose obtained was one fourth of the high dose i.e. 10mg/L HETP and CETP extract.

Rotenone induced catalepsy in zebra fish:

Estimation of behavioural parameter:

Latency to travel from one fixed point to another

The performance of Latency to travel from one point to another of fishes treated with hexane and chloroform extract in rotenone induced zebra fishes was shown in table no 2. The rotenone induced catalepsy in fishes pretreated with HFTP, CFTP extract for 28 days showed a significant improvement (p< 0.05) in Latency to travel from one point to another on 7^th, 14^th, 21^stand 28^th day when compared to disease control group. Avarage latency shown by fishes on 7^th, 14^th, 21^stand 28^thday is mentioned in table- 2.

Time spent near the bottom of the tank:

The performance of time spent near the bottom of tank of fishes treated with hexane and chloroform extract in rotenone induced zebra fishes was studied as shown in table no.03. The rotenone induced catalepsy in fishes pretreated with HFTP, CFTP extract for 28 days showed a significant improvement (p< 0.05) in time spent near the bottom of tank when compared to disease control group.

Table 3: Average Time spent near the bottom of the tank on 7^th, 14^th, 21^stand 28^thday of treatment at various time interval after rotenone challenge¹²

GROUPS	Time spent near the bottom of the tank (Sec)
GROUPS	15 Min	30 Min	45 Min	60 Min
Group I (Disease control)	260.06±9.12	272.06±13.10	278.04±6.30	277.07±5.10
Group II (Vehicle control)	40.03±8.62*	35.12±3.13*	29.19±5.23*	21.21±2.67*
Group III(High dose of HETP)	109.20±15.74*^#	106.06±10.1*^#	103.02±9.0*^#	140.01±6.30*^#
Group IV (Low dose of HETP)	115.04±4.2*^#	112.05±17.0*^#	101.05±7.29*^#	156.03±7.87*^#
Group V (High dose of CHTP)	66.09±19.00*^#	60.06±3.50*^#	44.04±9.00*^#	52.01±4.22*^#
Group VI (Low dose of CHTP)	69.09±19.00*^#	63.06±3.50*^#	68.04±9.00*^#	62.01±4.22*^#

Values are expressed as mean ± SD; n=10, where n is the number of fishes per group. *Indicates there is significant difference with p< 0.05 when compared with disease control group and ^#indicates there is significant difference with p< 0.05 when compared with vehicle group by One-way ANOVA followed by Dunnett’s multiple comparisons test. Time in minutes indicates recording of activity after rotenone challenge.

Estimation of biochemical parameters:

Table 4: Effect of HETP And CETP extracts on the Levels of Lipid Peroxidation (MDA), Superoxide Dismutase Assay (SOD) And Reduced Glutathione (GSH) in the Brain of Rotenone Challenged Fishes⁸

GROUPS	MDA (nM/mg of protein)	SOD (U/mg Protein)	GSH (nM/mg of protein)
Group I (Disease control)	1.269±0.02	1.131±0.03	0.140±0.03
Group II (Vehicle control)	0.217±0.01*	3.148±0.03*	1.356±0.03*
Group III (High dose of HETP)	0.350±0.04*^#	1.823±0.02*^#	0.390±0.02*^#
Group IV (Low dose of HETP)	0.378±0.03*^#	1.802±0.01*^#	0.235±0.01*^#
Group V (High dose of CHTP)	0.310±0.03*^#	1.937±0.03*^#	0.438±0.03*^#
Group VI (Low dose of CHTP)	0.357±0.06**	1.809±0.01**	0.234±0.02**

Values are expressed as mean±SD; n=10, where n is the number of flies per group. *Indicates there is significant difference with p<0.05 when compared with disease control group and ^#indicates there is significant difference with p<0.05 when compared with vehicle group by One-way ANOVA followed by Dunnett’s multiple comparisons test.

Based upon pharmacological activity of HETP and CETP, in zebra fish and fruit fly it was concluded that CETP was giving better pharmacological action as compared to HETP hence it was further selected for fractionation using column chromatography.

Column chromatography:

Table 6: Details of fractions obtained by column chromatography of CETP

Fraction No.	Volume (ml)	TLC	RF
1-4	5 ml	No spot detected	-
5-9	5 ml	1 spot detected	0.49
10-13	5 ml	No spot detected	-
14-19	5 ml	No spot detected	-
20-25	5 ml	2 spots detected	0.52 and 0.50
26-30	5 ml	No spot detected	-
31-36	5 ml	No spot detected	-
37-41	5 ml	No spot detected	-
42-46	5 ml	No spot detected	-
47-50	5 ml	No spot detected	-

Each fraction was collected separately in a test tube and numbered consecutively for further analysis on thin layer chromatography. Thin layer chromatography (TLC) provides partial separation of both organic and inorganic materials using thin-layered chromatographic plates especially useful for checking the purity of fractions. Fraction 5 to 9 had shown one spot and fraction 20-25 had shown two spots on TLC. The visualized spots of the components in the plate are marked and the Rf value obtained were 0.49 and 0.52, 0.50 respectively.

The two different fractions obtained by column chromatography from chloroform extract of Tridax procumbens was CETP extract fraction 1 (CETPF1) and CETP extract fraction 2 (CETPF2), were used as a treatment for zebra fish and fruit fly to determine its pharmacological activity.

Pharmacological activity of bioactive Fraction1 and Fraction 2 of CETP:

Acute toxicity studies in zebra fish:

At the 65.53mg/L dose of CETPF1 and CETPF2 fishes were showing abrupt movements line up and down, to and fro motion also fainting of skin, swimming at the bottom of tank. Hence this dose was neglected. The next dose selected for toxicity study was 40mg/L dose of CETPF1 and CETPF2. The 40mg dose was given to next 7 fishes. Out of 7 fishes 5 were died hence this dose was neglected. The 24.3 dose was given to the fishes, and it was found to be safe. Hence dose obtained from acute toxicity study was 24.6mg/L of CETPF1 and CETPF2 and low dose obtained was one fourth of the high dose i.e., 6mg/L CETPF1 and CETPF2 Rotenone induced catalepsy in zebra fish

Rotenone induced catalepsy in zebra fish:

Estimation of behavioural parameter:

Latency to travel from one fixed point to another

The performance of Latency to travel from one point to another of fishes treated with hexane and chloroform extract in rotenone induced zebra fishes was shown in table below. The rotenone induced catalepsy in fishes pretreated with CETPF1 and CETPF2 for 28 days showed a significant improvement (p<0.05) in Latency to travel from one point to another 7^th, 14^th, 21^stand 28^thday when compared to disease control group.

Time spent near the bottom of the tank:

The performance of time spent near the bottom of tank of fishes treated with hexane and chloroform extract in rotenone induced zebra fishes was studied as shown in table below. The rotenone induced catalepsy in fishes pretreated with CETPF1 and CETPF2 for 28 days showed a significant improvement (p< 0.05) in time spent near the bottom of tank when compared to disease control group.

Table 7: Latency to travel from one point to another and Time spent near the bottom of the tank on 7^thday of treatment at various time interval after rotenone challenge

GROUPS	Latency to travel from one point to another (cm)
GROUPS	15 Min	30 Min	45 Min	60 Min
Group I (Disease control)	134.00 ±8.62	114.30±3.13	111.00±5.23	107.00±2.67
Group II (Vehicle control)	454.10±9.12*	459.20±13.10*	465.12±6.30*	469.23±5.10*
Group III(High dose of CETPF1)	336.23±15.78*^#	345.31±10.20*^#	365.04±9.00*^#	380.03±6.37*^#
Group IV(Low dose of CETPF1)	303.13±4.30*^#	305.26±17.40*^#	308.17±7.22*^#	317.23±7.80*^#
Group V(High dose of CETPF2)	345.21±16.20*^#	348.23±12.34*^#	356.19±4.89*^#	385.43±5.32*^#
Group VI (Low dose of CETPF2)	246.28±19.20*^#	248.27±3.56*^#	255.14±9.10*^#	285.04±4.22*^#
	Time spent near the bottom of the tank (Sec)
Group I (Disease control)	478.06±9.12	483.06±13.16	489.04±6.36	498.07±5.15
Group II (Vehicle control)	142.05±8.62*	114.19±3.13*	129.16±5.23*	112.12±2.67*
Group III(High dose of CETPF1)	383.04±15.78*^#	345.34±10.02*^#	335.21±9.0*^#	323.05±6.37*^#
Group IV(Low dose of CETPF1)	403.02±4.30*^#	415.34.±17.34*^#	411.32±7.22*^#	423.21±7.84*^#
Group V(High dose of CETPF2)	300.04±16.02*^#	320.21±12.34*^#	226.21±4.89*^#	220.02±5.32*^#
Group VI (Low dose of CETPF2)	350.05±19.12*^#	338.32±3.56*^#	255.05±9.11*^#	228.03±4.22*^#

Values are expressed as mean±SD; n=10, where n is the number of fishes per group. *Indicates there is significant difference with p<0.05 when compared with disease control group and #indicates there is significant difference with p<0.05 when compared with vehicle group by One-way ANOVA followed by Dunnett’s multiple comparisons test. Time in minutes indicates recording of activity after rotenone challenge.

Table 8: Latency to travel from one point to another and Time spent near the bottom of the tank on 14^th of treatment at various time interval after rotenone challenge

GROUPS	Latency to travel from one point to another (cm)
GROUPS	15 Min	30 Min	45 Min	60 Min
Group I (Disease control)	130.00 ±8.62	112.30±3.13	111.00±5.23	107.00±2.67
Group II (Vehicle control)	460.10±9.12*	459.20±13.10*	465.12±6.30*	470.23±5.10*
Group III(High dose of CETPF1)	334.04±15.78*^#	348.05±10.20*^#	369.02±9.00*^#	383.05±6.37*^#
Group IV(Low dose of CETPF1)	309.04±4.30*^#	309.00±17.40*^#	311.09±7.22*^#	319.04±7.8*^#
Group V(High dose of CETPF2)	339.03±16.20*^#	348.00±12.34*^#	358.04±4.89*^#	388.05±5.32*^#
Group VI (Low dose of CETPF2)	245.01±19.20*^#	240.01±3.56*^#	257.04±9.10*^#	280.03±4.22*^#
	Time spent near the bottom of the tank (Sec)
Group I (Disease control)	481.06±9.12	488.06±13.12	489.04±6.31	498.07±5.10
Group II (Vehicle control)	141.05±8.62*	116.19±3.13*	128.16±5.23*	112.12±2.67*
Group III(High dose of CETPF1)	384.04±15.78*^#	343.34±10.22*^#	335.21±9.00*^#	322.05±6.37*^#
Group IV(Low dose of CETPF1)	402.02±4.03*^#	390.34±17.41*^#	401.32±7.22*^#	388.21±7.18*^#
Group V(High dose of CETPF2)	304.04±16.22*^#	302.21±12.34*^#	219.21±4.89*^#	209.02±5.32*^#
Group VI (Low dose of CETPF2)	345.05±19.20*^#	328.32±3.56*^#	255.05±9.01*^#	220.03±4.22*^#

Values are expressed as mean±SD; n=10, where n is the number of fishes per group. *Indicates there is significant difference with p<0.05 when compared with disease control group and ^#indicates there is significant difference with p<0.05 when compared with vehicle group by One-way ANOVA followed by Dunnett’s multiple comparisons test. Time in minutes indicates recording of activity after rotenone challenge.

Table 9: Latency to travel from one point to another and Time spent near the bottom of the tank on 21^stday of treatment at various time interval after rotenone challenge

Groups	Latency to travel from one point to another (cm)
	15 Min	30 Min	45 Min	60 Min
Group I (Disease control)	123.00 ±8.62	110.30±3.13	111.00±5.23	108.00±2.67
Group II (Vehicle control)	451.10±9.12*	458.20±13.10*	465.12±6.30*	467.23±5.10*
Group III(High dose of CETPF1)	330.09±15.78*^#	345.04±10.20*^#	365.03±9.00*^#	383.01±6.37*^#
Group IV(Low dose of CETPF1)	303.03±4.30*^#	305.04±17.40*^#	308.02±7.22*^#	317.00±7.80*^#
Group V(High dose of CETPF2)	334.05±16.20*^#	344.08±12.34*^#	356.04±4.89*^#	385.00±5.32*^#
Group VI (Low dose of CETPF2)	246.03±19.20*^#	248.04±3.56*^#	255.05±9.10*^#	285.00±4.22*^#
	Time spent near the bottom of the tank (Sec)
	140.05±8.62*	115.19±3.13*	111.16±5.23*	98.12±2.67*
Group I (Disease control)	485.06±9.12	488.06±13.21	489.04±6.33	490.07±5.13
Group II (Vehicle control)	140.05±8.62*	115.19±3.13*	111.16±5.23*	98.12±2.67*
Group III(High dose of CETPF1)	381.04±15.78*^#	345.34±10.22*^#	335.21±9.02*^#	323.05±6.37*^#
Group IV(Low dose of CETPF1)	403.02±4.03*^#	415.34±17.41*^#	411.32±7.22*^#	423.21±7.80*^#
Group V(High dose of CETPF2)	352.04±16.22*^#	344.21±12.34*^#	316.21±4.89*^#	321.02±5.32*^#
Group VI (Low dose of CETPF2)	365.05±19.20*^#	358.32±3.56*^#	334.05±9.01*^#	328.03±4.22*^#

Table 10: Latency to travel from one point to another and Time spent near the bottom of the tank on 28^thday of treatment at various time interval after rotenone challenge

Groups	Latency to travel from one point to another (cm)
Groups	15 Min	30 Min	45 Min	60 Min
Group I (Disease control)	112.00 ±8.62	114.30±3.13	101.00±5.23	107.00±2.67
Group II (Vehicle control)	464.10±9.12*	459.20±13.10*	465.12±6.30*	469.23±5.10*
Group III(High dose of CETPF1)	345.28±15.78*^#	347.06±10.20*^#	363.08±9.00*^#	382.21±6.37*^#
Group IV(Low dose of CETPF1)	306.24±4.30*^#	309.19±17.40*^#	305.23±7.22*^#	319.27±7.80*^#
Group V(High dose of CETPF2)	348.23±16.22*^#	349.21±12.34*^#	352.32±4.89*^#	383.38±5.32*^#
Group VI (Low dose of CETPF2)	248.34±19.12*^#	251.34±3.56*^#	254.00±9.11*^#	284.00±4.22*^#
	Time spent near the bottom of the tank (Sec)
Group I (Disease control)	471.06±9.12	491.06±13.10	489.04±6.30	498.07±5.10
Group II (Vehicle control)	131.05±8.62*	106.19±3.13*	108.16±5.23*	112.12±2.67*
Group III(High dose of CETPF1)	383.04±15.78*^#	345.34±10.20*^#	335.21±9.0*^#	323.05±6.37*^#
Group IV(Low dose of CETPF1)	442.02±4.13*^#	415.34±17.40*^#	411.32±7.22*^#	423.21±7.18*^#
Group V(High dose of CETPF2)	350.04±16.12*^#	344.21±12.34*^#	331.21±4.89*^#	303.02±5.32*^#
Group VI (Low dose of CETPF2)	361.05±19.02*^#	358.32±3.56*^#	325.05±9.10*^#	318.03±4.22*^#

DISCUSSION:

Parkinson's disease, also referred to as PD, is a long-term neurodegenerative illness driven by a lack of dopamine. This can be caused by degeneration of dopamine-producing neurons in the brain's substantia nigra pars compacta region and intraneuronal deposition of Lewy bodies, that are brought on by protein aggregation of α-synuclein. Movement-related symptoms such as tremors in the hands, arms, legs, jaw, or trunk; stiffening or rigidity of the extremities and neck; bradykinesia, or slowness of movement; abnormalities in postural stability, or poor balance and festinating gait, are among the key clinical features of PD¹⁰.

In this study, the Chloroform extract of Tridax procumbens and fraction two of chloroform obtained by column chromatography were studied for their protective effect against rotenone induced catalepsy in zebra fish and locomotor deficits in drosophila flies depicting the PD model. On long-term use of rotenone, it disrupts the dopaminergic neurons functioning and hence causes extrapyramidal syndrome which affects the motor coordination systems. The phytoconstituents that are already reported to be present in Tridax procumbens are ellagic acid, catechin, luteolin, galgravin, kaempferol, silymarin; have neuroprotective, anti-inflammatory and antioxidant activity; suggesting possible role of Tridax procumbens in the treatment of PD⁶^,⁵.

It was evident that in zebra fish model the rotenone induced the catalepsy as it significantly increased the time spent near bottom and decreased in latency of travel from one point to another as compared to vehicle control animals. In the initial part of the study, chloroform extract and hexane extract was given to the zebra fish and fruit fly, in which chloroform extract had shown the effective treatment in rotenone induced catalepsy in zebra fish and fruit fly, whereas in the second part of study chloroform extract of Tridax procumbence was taken for fractionation using column chromatography, two different fractions (CETPF1 and CETPF2) were obtained and given to the animals to check the pharmacological activity in zebra fish and fruit fly. CETPF2 (24.6mg/L) showed the protective effect against catalepsy induced by rotenone. The improvement in the locomotion was indicated in both the models 28 days post treatment with CETPF2 (24.6mg/L)which was comparable to that of vehicle group¹⁶^,¹⁷.

Thus, it was notable that on long term treatment the cumulative levels of CETPF2 are responsible to get accumulated giving significant efficacy post treatment. The rotenone specifically leads to disruption of mitochondrial-1 complex which is crucial in normal physiology and hence contributes to PD pathogenesis. ¹⁸^,¹⁹.

The SOD, GSH and Lipid peroxidation (by MDA as biomarker) were studied as indicator of oxidative stress in the brain. The arachidonic acid which is a unsaturated fatty acid present abundantly in the tissue cell is the major source of generation of oxidative stress chain reaction as a virtue of this the MDA formation is increased in oxidative degradation of the cellular lipids. Thus, the SOD and GSH which are the endogenous enzymes, levels go down while the MDA production go up in disease induced animals indicating oxidative stress. It leads to altered structure of bio membrane and hence imbalance in the physiology of membrane bound enzymes and membrane permeability²⁰^,²¹. The depletion of the endogenous enzymes specifically GSH levels in substantia nigra region is reported in PD indicative of dopaminergic neuronal degeneration. The oxidative stress would be further generated as a virtue of inability to neutralise the hydrogen peroxide and hence free radicals generated causing neurodegeneration²²^,¹³. The result obtained in the brain homogenate of the study rats showed a significant reduction in the free radical load and hence proved to be deterrent for oxidation of lipids. Post treatment with CETP (40 and 10mg/L) and CETPF2 (24.6 and 6mg/L) the peroxidation of lipid was significantly decreased in comparison to disease group animals. The SOD and GSH levels in CETP (40 and 10 mg/L) and CETPF2 (24.6 and 6 mg/L) treated animals were found significantly high when compared with the disease control animals. Thus, the rotenone-induced disease control group showed a significant increase in the levels of lipid peroxidation and a decrease in the levels of SOD and GSH in the brain as compared to the vehicle control treated animals¹²^,²³.

All these indicate an increase in the oxidative stress in the brain of animals treated with rotenone. Pre-treatment with CETP (40 and 10mg/L) and CETPF2(24.6 and 6 mg/L) resulted in a decrease in lipid peroxidation and increase in the levels of SOD and GSH, indicating its free radical scavenging effect in the brain of rotenone treated animals. The activity of CETP (40 and 10mg/L) and CETPF2(24.6 and 6mg/L) at both doses is comparable to the vehicle group²⁴^,²⁵ .

Rotenone is frequently used and accepted to induce PD like sign and symptoms in fruit flies. It easily crosses the blood-brain barrier and has tendency to build up in the mitochondria. Once inside the organelle it binds particularly to the dehydrogenase present i.e. NADH complex I dehydrogenase. The inhibition of dehydrogenase is further responsible for neurotoxicity of dopaminergic neurons and locomotor deficits in substantia nigra region. The flies challenged with rotenone exhibited severe locomotor dysfunction and hence were unable to fly²⁶^,²⁷. On the other hand the flies who were exposed to treatment drugs i.e., CETP at doses of (0.05 and 0.025% w/v) and CETPF (0.04 and 0.02% w/v) along with the rotenone showed significant betterment in locomotor activity. Thus, based on both the models wherein the improvement in the locomotion was visible when treated with test samples i.e., CETP and CETPF2; it can be stated that the neuroprotection was exerted typically of dopaminergic neurons²⁸^-29.

CONCLUSION:

The finding of the present study confirmed the neuroprotective and antioxidant properties of a chloroform extract of Tridax procumbence leaves and fraction two of chloroform extract in a zebra fish and fruit fly model of PD that was induced by rotenone and suggested that Tridax procumbence may be a useful supplement for preventing the cell death of dopaminergic neurons in patients with PD. In conclusion, we can say that zebrafish may become effective tool for high through put screening for various diseases. They can be used with ease and effectiveness for initial screening of drugs before subjecting them to rodent testing.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors would like to thank National centre for biological sciences, Bangalore, India and National cell science centre, Pune for providing us flies.

REFERENCE:

1. Wang X. Zhang JB. He KJ. Wang F. Liu CF. Advances of Zebrafish in Neurodegenerative Disease: From Models to Drug Discovery. Frontiers in Pharmacology. 2008; 12: 1-19. https://doi.org/10.3389/fphar.2021.713963

2. Kozioł E. Skalicka-Woźniak K. Michalak A. Kaszubska K. Budzyńska B. Xanthotoxin Reverses Parkinson’s Disease-Like Symptoms in Zebrafish Larvae and Mice Models: A Comparative Study. Pharmacological Reports. 2021; 73: 122-9. https://doi.org/10.1007/s43440-020-00136-9

3. Dhande SR. Patil VR. Protective Effect of Portulaca quadrifida on Rotenone Induced Locomotor Impairment in Drosophila Model. International Journal of Scientific Research and Engineering Development. 2022; 5: 349-51.

4. Dewashish K. Ethnopharmacological and Phytochemical Studies of Tridax Procumbens Linn: A Popular Herb in Ayurveda Medicine. International Journal of Engineering Research. 2020; V9: 758-68. http://dx.doi.org/10.17577/IJERTV9IS090426

5. Sharma B. Kumar P. Extraction and Pharmacological Evaluation of Some Extracts of Tridax Procumbens and Capparis Decidua. International Journal of Applied Research in Natural Products. 2008; 1: 5-12.

6. Anil S. Parvesh G. Phytochemical Studies of the Leaves of Tridax Procumbens. International Journal of Research and Analytical Reviews. 2018; 5: 503-7.

7. Unal I. Ustundag UV. Ates PS. Egilmezer G. Alturfan AA. Yigitbaş T. Alturfan EE. Rotenone impairs oxidant/antioxidant balance both in brain and intestines in zebrafish. International Journal of Neuroscience. 2019; 129: 363-8.

8. Chaudhary P. Dhande S. Evaluation of Anti-Parkinson’s Activity of Ethanolic Extract of Tridax Procumbens (Asteraceae). Indian Journal of Natural Products and Resources. 2020; 11: 9-17. http://nopr.niscpr.res.in/handle/123456789/54489

9. OECD Guidelines for the Testing of Chemicals. Test No. 203: Fish Acute Toxicity Testing, Section 2: Effects on Biotic Systems. June 2019.

10. Makhija DT. Jagtap AG. Studies on Sensitivity of Zebrafish as a Model Organism for Parkinson′s Disease: Comparison with Rat Model. Journal of Pharmacology and Pharmacotherapeutics. 2014; 5: 39-46.

11. Zhang ZJ. Cheang LC. Wang MM. Li GH. Chu IK. Lin ZX. Lee SM. Ethanolic Extract of Fructus Alpinia oxyphylla Protects Against 6-Hydroxydopamine-Induced Damage of PC12 Cells In Vitro and Dopaminergic Neurons in Zebrafish. Cellular and Molecular Neurobiology. 2012; 27-40. 10.1007/s10571-011-9731-0

12. Sarbishegi M. Gorgich A. Khajavi O. Komeili G. Salimi S. The neuroprotective Effects of Hydro-Alcoholic Extract of Olive (Olea Europaea l.) Leaf on Rotenone-Induced Parkinson’s Disease in Rat. Metabolic Brain Disease. 2018; 33: 79-88. https://doi.org/10.1007/s11011-017-0131-0

13. Siima AA. Stephano F. Munissi JE. Nyandoro SS. Ameliorative effects of flavonoids and polyketides on the rotenone induced Drosophila model of Parkinson’s disease. Neurotoxicology. 2020; 81: 209-15. https://doi.org/10.1016/j.neuro.2020.09.004

14. Tochukwu OC. Alexander EM. Salu JM. Toxicity of Azadirachta Indica Hydroethanolic Leaf Extract in Adult Drosophila melanogaster (Harwich Strain). Journal of Advances in Biology and Biotechnology. 2021; 12–23. https://doi.org/10.9734/jabb/2021/v24i430208

15. Shinde V. Garad R. Jain S. Manoj K. Badal B. Column Chromatography. International Journal of Creative Research Thoughts. 2023; 11(5): M591-605.

16. Yun JW. Ahn JB. Kang BC. Modeling Parkinson’s Disease in the Common Marmoset (Callithrix Jacchus): Overview of Models, Methods, and Animal Care. Laboratory Animal Research. 2015; 31 (4): 155- 65. https://doi.org/10.5625%2Flar.2015.31.4.155

17. Santo GD. Veras BO. Rico E. Magro JD. Agostini JF. Vieira LD. Calisto JF. et. al. Hexane extract from SpoSndias mombin L. (Anacardiaceae) Prevents Behavioral and Oxidative Status Changes on Model of Parkinson’s Disease in Zebrafish. Comp. Comparative Biochemistry and Physiology Part C: Toxicology and Pharmacology. 241 (202)108953. https://doi.org/10.1016/j.cbpc.2020.108953

18. Rahman M. Islam B. Biswas M. Alam AK. In Vitro Antioxidant and Free Radical Scavenging Activity of Different Parts of Tabebuia Pallida Growing in Bangladesh. BMC Research Notes. 2015; 8:621. https://doi.org/10.1186/s13104-015-1618-6 available on https://bmcresnotes.biomedcentral.com/articles/10.1186/s13104-015-1618-6

19. Saleem S. Kannan RR. Zebrafish: An Emerging Real-Time Model System to Study Alzheimer’s Disease and Neurospecific Drug Discovery. Cell Death Discovery. 2018; 4: 45. https://doi.org/10.1038/s41420-018-0109-7 available on https://www.nature.com/articles/s41420-018-0109-7

20. McGrath P. Li CQ. Zebrafish: A Predictive Model For Assessing Drug-Induced Toxicity. Drug Discovery Today. 2008; 13: 394–401. https://doi.org/10.1016/j.drudis.2008.03.002

21. Robea MA. Balmus IM. Ciobica A. Satguru S. Plavan G. Gorgan LD. Savuca A. Nicoara M. et al. Parkinson’s Disease-Induced Zebrafish Models: Focussing on Oxidative Stress Implications and Sleep Processes. Oxidative Medicine and Cellular Longevity. 2020; 1370837 https://doi.org/10.1155/2020/1370837 available on https://www.hindawi.com/journals/omcl/2020/1370837/

22. Burgos GE. Vacas IU. Sanchez M. Gomez-Serranillos MP. Nutritional Value of Moringa Oleifera Lam. Leaf Powder Extracts and Their Neuroprotective Effects via Antioxidative and Mitochondrial Regulation. Nutrients. 2021; 13(7): 2203. https://doi.org/10.3390/nu13072203 available on https://www.mdpi.com/2072-6643/13/7/2203

23. Rajalakshmi S. Vijayakumar S. Praseetha PK. Neuroprotective Behaviour of Phyllanthus Emblica (L) on Human Neural Cell Lineage (PC12) against Glutamate-Induced Cytotoxicity. Gene Reports. 2019; 17: 100545. https://doi.org/10.1016/j.genrep.2019.100545 available on https://www.sciencedirect.com/science/article/pii/S2452014419301876

24. Vaz RL. Outeiro TF. Ferreira JJ. Zebrafish as an Animal Model for Drug Discovery in Parkinson’s Disease and Other Movement Disorders: A Systematic Review. Frontiers in Neurology. 2018; 9. 10.3389/fneur.2018.00347 available on https://www.frontiersin.org/journals/neurology/articles/10.3389/fneur.2018.00347

25. Khatri D. Juvekar A. Abrogation of Locomotor Impairment in a Rotenone-Induced Drosophila Melanogaster and Zebrafish Model of Parkinson′s Disease by Ellagic Acid and Curcumin. International Journal of Nutrition, Pharmacology, Neurological Diseases. 2016; 6(2): 90–6. 10.4103/2231-0738.179969

26. Peterson EK. Long HE. Experimental Protocol for Using Drosophila as an Invertebrate Model System for Toxicity Testing in the Laboratory. Journal of Visualized Experiments. 2018; 137: e57450. https://doi.org/10.3791%2F57450 available on https://www.jove.com/t/57450/experimental-protocol-for-using-drosophila-as-an-invertebrate-model

27. Rahul NF. Jyoti S. Siddique YH. Effect of Kaempferol on the Transgenic Drosophila model of Parkinson’s Disease. Scientific Report. 2020; 10: 1–14. https://doi.org/10.1038/s41598-020-70236-2 available on https://www.nature.com/articles/s41598-020-70236-2

28. Makowicz E. Jasicka-Misiak I. Teper D. Kafarski P. HPTLC Fingerprinting—Rapid Method for the Differentiation of Honeys of Different Botanical Origin Based on the Composition of the Lipophilic Fractions. Molecules. 2018; 23(7). https://doi.org/10.3390/molecules23071811

29. Aryal B. Lee Y. Disease Model Organism for Parkinson Disease: Drosophila Melanogaster. BMB Reports. 2019; 8: 250–8. https://doi.org/10.5483%2FBMBRep.2019.52.4.204

Received on 16.07.2022 Modified on 11.08.2023

Research J. Pharm. and Tech 2024; 17(7):3141-3150.

DOI: 10.52711/0974-360X.2024.00491

GROUPS	MDA (nM/mg of protein)	SOD (U/mg Protein)	GSH (nM/mg of protein)
Group I (Vehicle control)	0.217±0.01*	3.148±0.03*	1.356±0.03*
Group II (Disease control)	1.269±0.02	1.131±0.03	0.140±0.03
Group III(High dose of CETPF1)	0.225±0.08*^#	1.981±0.02*^#	0.347±0.02*^#
Group IV(Low dose of CETPF1)	0.226±0.06*^#	1.705±0.04*^#	0.236±0.01*^#
Group V(High dose of CETPF2)	0.344±0.04*^#	2.139±0.03*^#	0.358±0.04*^#
Group VI (Low dose of CETPF2)	0.363±0.02*^#	1.944±0.02*^#	0.315±0.03*^#

Collection and authentication of plant material:

Preparation of plant extract:

Animals:

EXPERIMENTAL DESIGN:

Phytochemical screening of HETP and CETP :

Pharmacological activity of HETP and CETP:

Acute toxicity study in zebra fish:

Rotenone induced catalepsy in zebra fish:

Acute toxicity study in Fruit Fly:

Rotenone induced locomotor deficits in fruit fly:

Negative geotaxis assay in fruit fly:

Column chromatography:15

Pharmacological activity of bioactive CETPF1 and CETPF2:

Acute toxicity study in zebra fish:

Rotenone induced catalepsy in zebra fish:

Acute toxicity study in Fruit Fly:

Rotenone induced locomotor deficits in fruit fly:

STATISTICAL ANALYSIS:

RESULT:

Phytochemical screening of HETP and CETP:

Pharmacological activity of HETP and CETP:

Acute toxicity study in Zebra fish:

Rotenone induced catalepsy in zebra fish:

Estimation of behavioural parameter:

Latency to travel from one fixed point to another

Time spent near the bottom of the tank:

Estimation of biochemical parameters:

Acute toxicity studies in fruit fly:

Rotenone induced locomotor deficits in fruit fly:

Negative geotaxis assay in fruit fly

Column chromatography:

Pharmacological activity of bioactive Fraction1 and Fraction 2 of CETP:

Acute toxicity studies in zebra fish:

Rotenone induced catalepsy in zebra fish:

Estimation of behavioural parameter:

Latency to travel from one fixed point to another

Time spent near the bottom of the tank:

Estimation of Biochemical Parameters:

Acute toxicity studies in fruit fly:

Rotenone induced locomotor deficits in fruit fly:

Negative geotaxis assay in fruit fly:

DISCUSSION:

ACKNOWLEDGMENTS:

REFERENCE:

Column chromatography:¹⁵