INTRODUCTION:

Since the dawn of time, people have exploited a variety of natural resources to treat illnesses rather than accepting their inevitable deaths^1,2.

Due to the existence of several unique entities, the scientific literature on medicinal flora has recently noticed a boom focused on the utilization of them for their diverse health benefits and therapeutic potentials in the pharmaceutical and nutrition sciences. The World Health Organization (WHO) reported that the phytomedicines usage rate has expanded three times faster than that of conventional medicines, and approximately 80% of the developing-world populations rely on medicinal plants for basic healthcare since they are thought to exert fewer adverse effects. Nearly 33% of the medications manufactured today in affluent nations are derived from plants. Previous studies revealed that phytoconstituents (alkaloids, flavonoids, tannin, glycosides, etc.) have a wide range of pharmacological activities and might be used to treat a number of ailments such as cancer, diabetes, inflammatory disorders, cardiovascular complications, obesity etc.^2-5.

Pandanus fascicularis Lam, also known as screw pine, is an evergreen tree or shrub with delicate blossoms that resembles a palm and is native to South Asia (Bangladesh, India, Japan etc.). It is primarily found in subtropical and tropical climates and is particularly common in mangrove swamps. The plant's parts have traditionally been employed to alleviate rheumatoid arthritis, leprosy, edema, tumors, earaches, fever, skin conditions, ulcers, and dyspepsia. Researchers reported the presence of tannins, carbohydrates, flavonoids, steroids, saponins, hematoporphyrin, decanoic acid, proteins, terpenes, phenols, glycosides, glucose, and alkaloids in different parts of P. fasciculari ^6,7.

Despite the numerous traditional applications of this plant, a very few studies have been conducted on the ethnopharmacological applications and phytoconstituents of root of P. fascicularis. There is a large gap of knowledge about the pharmacological activity of this plant part which must be explored. This prompted us to investigate the in vivo (analgesic, CNS depressant, antidiarrheal, hypoglycemic) and in vitro (antiarthritic, thrombolytic, anthelmintic, and insecticidal) properties of methanolic root extract and its various solvent sections of the aforementioned plant. The finding from our study indicates that the pharmacological activities of root of P. fascicularis can be considered as a benchmark for the future isolation of pure bioactive chemicals for the development of new phytomedicines to treat free radical induced neurodegenerative diseases, diabetes, arthritis, etc.

MATERIALS AND METHODS:

Plant sample collection, authentication, and extraction:

The fresh root of the plant P. fascicularis was collected from the Sundarbans, Bangladesh in January, 2023 and was identified by a taxonomist from the Bangladesh National Herbarium in Mirpur, Dhaka (DACB Accession No: 88421). The crude methanolin extract (MFP) and its fractions(chloroform-CFP, and ethyl acetate- EFP ) were prepared according to previous method⁸.The extracts were then preserved properly to evaluate their in vivo and in vitro pharmacological characteristics.

Chemicals:

Diazepam, morphine, metformin, loperamide, streptokinase, and albendazole were generous gifts from Popular Pharmaceuticals Ltd. (a renowned pharmaceutical company in Bangladesh). Ascorbic acid, acetic acid, and methanol (95%) were purchased from Merck (Germany). Analytical grade materials were used for all other reagents in the investigations.

Test Organisms and Experimental Animals:

Here we used Swiss albino mice of both sexes (age 4-5 weeks, weight 25-30gm) for in vivo pharmacological studies (at 3 distinct doses: 100, 200, and 400mg/kg for the experimented extracts). They were purchased from the Animal Resources Division of the International Center for Diarrheal Disease Research, Bangladesh (ICDDR, B). Prior to pharmacological investigations, all rodents were kept in the standard laboratory condition for near about a week to acclimatize to the laboratory environment⁹. The animal ethical committee of the Southeast University (Dhaka, Bangladesh) authorized each animal experiment protocol (SEU/Pharm/CERC/110/2023). Oral gavage was used to provide each agent via the oral route. Indian earthworms (Pheretima posthuma) and Tribolium castaneum (red flour beetle) were collected from Sher-e-Bangla Agricultural University, Bangladesh.

Preliminary phytochemical screening:

Standard techniques were used to check the presence of distinct bioactive components in the tested extracts (MPF, EAPF and CPF)^10,11.

In vivo Studies:

Analgesic activity:

Analgesic effect of the tested samples was studied through writhing (acetic acid induced)^3,12 and formalin induced pain methods¹³. The following equation was used to compute the proportion of the treated group's writhing inhibition:

% Of inhibition of weithing – [(N_control – N_Test) / control] × 100%

Where, N represents the mean number of writhing for each group.

CNS Depressant activityL:

Plant extracts' CNS depressing action was examined using the open field and hole cross methods according to previous methods with minor modification¹⁴.

Hypoglycemic Test (Oral Glucose Tolerance Test-OGTT):

Healthy rodents were subjected to the oral glucose tolerance test (OGTT) according to the method of Tesfaye et al.¹⁵ and used the equation below to calculate the percent drop in blood sugar level caused by P. fascicularis extracts:

% Reduction in blood glucose = [BGL_control – BGL_test) / BGL_control] × 100%

Here, BGL is the mean blood glucose level for each group.

Antidiarrheal Activity:

The plant samples were also subjected to ensure the anti-diarrheal effect in mice by castor oil and magnesium sulphate induced diarrhea models^12,16.The percentage of defecation inhibition was estimated as follows:

% Inhibition of defecation = [A – B) / A] × 100%

Where, A and B represent the mean number of feces induced by castor oil/magnesium sulphate, and drug/ extract respectively.

In vitro studies:

DPPH radical scavenging assay:

The antioxidant potential of the experimented plant extracts was measured with the previously described DPPH scavenging assay^17-19 technique with little modification. The free radical quenching capacity was measured with the following formula:

%Of inhibition = [(A_blank – A_sample) /A_blank] × 100%

Here, A means absorbance for each group. A graph showing the relationship between test material concentration and DPPH scavenging inhibition was then used to determine the IC₅₀ value (50% inhibition) for each plant sample.

Thrombolytic property:

Using a previously reported approach⁸, we assessed the thrombolytic capability of P. fascicularis root extracts. The % clot lysis was determined by applying the equation beneath²⁰:

%Clot lysis = [Weight of clot lysis / Weight of clot before lysis) × 100%]

Antiarthritic property:

The percentage inhibition of protein denaturation was calculated using the following formula by following previous described antiarthritic test²¹:

%Inhibition = [(Absorbance of control –

Absorbance of test samples /

Absorbance of control] ×100%

Anthelmintic test:

With some minor tweaks, previous approaches^22,23 were used to study the anthelmintic activity of P. fascicularis extracts.

Insecticidal activity screening:

The insecticide capability of the investigated materials was evaluated against T. castaneum using the following formula²⁴.

E_r = (E_o – E_c / 100 – E_c) × 100%)

E_r = Corrected percentage of mortality; E_o = Experiential percentage of mortality; E_c = Control percentage of mortality

Statistical analysis:

The findings were shown as mean ± SEM. One-way analysis of variance (ANOVA) was used to evaluate data between groups, followed by Dunnett's test where p value <0.05 regarded statistically significant (SPSS software, version 26.0, IBM Corporation, Armonk, NY, USA).

RESULTS:

Preliminary phytochemical screening:

Table 1 summarizes the findings of the phytochemical screening of P. fascicularis root extracts. The crude methanolic extract and its fractions showed the presence of numerous beneficial secondary metabolites.

Table 1: Phytochemical screening test of P. fascicularis root extracts

Phytocompounds	MPF	EAPF	CPF
Glycoside(Borntrager’s test)	++	+	++
Carbohydrate(Molisch’s Test & Benedict’s test)	++	++	++
Resin Ferric chloride test)	+	+	++
Alkaloid(Mayer's test & Dragendorff's test)	++	+	+++
Saponin(Frothing test)	+	+	+++
Tannin(Catechin test)	++	+	+++
Steroid(Liebermann Burchard test)	+	+	++
Flavonoid(Shinoda test)	++	+	+++
Phenol(Ferric chloride test)	+++	+	+++
Fixed oil(Stain test)	+	-	-
Terpenoids(Salkowski test)	+	-	++

Note: Here, “+” states the presence and “-” shows the absence of any phytochemical group. Bioavailability key: (+++) ve = robust intensity, (++) ve = medium intensity, (+)ve poor intensity,(−) ve = absent.

In vivo studies: Analgesic activity:

In writhing test, all extracts notably (p<0.01; p< 0.05) decreased the number of writhes in mice compared to the negative control (Table 2) at all doses. The MPF extract at the higher dose (400 mg/kg) exhibited maximum percent of writhing inhibition 49.17%. The median length of time that animals spent beating the injected paw in each of the two phases was considerably reduced by all tested extracts in formalin lick test (Table 3). We noticed that CPF extract at higher dose displayed improved pain prevention in both phases (early phase 55.82% and late phase 58.18%).

Table 2: Effect of root extracts of P. fascicularis on acetic acid-induced writhing in mice

Group	Treatment / Dose (mg/kg)	Number of Writhing	% of Inhibition
Control (I)	Tween 80 solution	30.83 ± 0.98	-------
Standard (II)	Morphine/ 2	9.0 ± 0.63^b	70.81
III	MPF/ 100	23.17± 1.18^a	24.85
IV	MPF/ 200	17.83 ± 0.76^a	42.167
V	MPF/ 400	15.67 ± 0.82^b	49.17
VI	EAPF/ 100	26.17± 0.75^a	26.17
VII	EAPF/ 200	20.67 ± 0.82^a	32.95
VIII	EAPF/ 400	16.50 ± 1.05^b	46.48
IX	CPF/ 100	26.83± 1.17^a	12.97
X	CPF/ 200	21.33 ± 1.21^a	30.81
XI	CPF/ 400	19.17 ± 0.75^b	37.82

Note: The data are presented as mean ± STD (n=5); One-Way Analysis of Variance (ANOVA) trailed by Dunnet's test; a=p<0.05, b=p<0.01 significant compared to the control.

CNS Depressant activity:

The outcomes of open field and hole cross tests are given in Table 4 and Table 5. All extracts significantly reduced the locomotor activity in mice (p<0.01; p< 0.05) in both methods. In comparison to MPF and EAPF extracts, the CPF of showed more notable effects. The findings demonstrated that practically all P fascicularis root extracts considerably (p<0.01; p<0.05) reduced the number of holes the mice crossed over time from their initial value.

Hypoglycemic Test:

From 30 minutes on, all extracts in the OGTT significantly reduced the plasma glucose levels (Table 6). CPF exhibited the strongest hypoglycemic activity among all extracts.

Table 3: Anti-nociceptive activity of P. fascicularis root extracts in formalin-induced lick test

Groups	Treatment /Dose (mg/kg)	Early phase (Sec)	Late phase (Sec)	% Protection of early phase	% Protection of late phase
Control (I)	Tween 80 solution	37.17 ± 1.47	47.83 ± 0.75
Standard (II)	Morphine / 2	15.17 ± 0.75^b	17.0± 0.63^b	59.19	64.46
III	MPF / 100	30.83 ± 1.47^b	32.67 ± 1.63^a	17.06	40.77
IV	MPF / 200	23.50± 1.05^b	28.00 ± 1.26^b	36.78	41.46
V	MPF / 400	19.33 ± 1.03	23.0 ± 0.89^b	47.99	50.51
VI	EAPF / 100	32.5± 1.04^b	27.17± 1.47	12.56	43.19
VII	EAPF / 200	26.0± 0.89^b	30.67 ± 0.82^a	30.05	35.88
VIII	EAPF / 400	21.83 ± 1.33	25.67 ± 0.82	41.27	46.33
IX	CPF / 100	33.66±0.81^a	40.67± 1.21^a	9.44	14.97
X	CPF / 200	30.5 ± 1.04^b	35.0 ± 0.89^b	17.94	26.82
XI	CPF / 400	20.33 ± 0.82^b	20.00± 1.26^b	55.82	58.18

Note: The data are presented as mean ±STD (n=5); One-Way Analysis of Variance (ANOVA) trailed by Dunnet's test; a=p<0.05, b=p<0.01 significant compared to the control.

Table 4: P. fascicularis root extracts' effects in the open field test

Groups	Treatment/Dose (mg/kg)	Number of Movements
Groups	Treatment/Dose (mg/kg)	0 min	30 min	60 min	90 min	120 min
Control (I)	Tween 80 solution	128.4± 0.49	123.0± 0.63	121.2± 0.74	118.4± 0.48	116.6± 0.49
Standard (II)	Diazepam/1	122.8 ±0.84^a	65.2 ± 0.83^b	46.2±1.30^b	17.2± 0.83^b	10.2 ± 0.85^b
III	MPF/100	123.0 ± 1.22	96.8 ± 0.84^a	86.0± 1.58^a	76.0± 0.89^b	67.0 ± 1.58^a
IV	MPF/200	120.8± 0.83^a	87.2 ± 1.48^b	76.4 ±.14^b	54.2 ±1.648^a	55.0 ± 1.87^b
V	MPF/ 400	120.6± 0.89^a	77.8± 0.83	70.2 ± 1.30	62.2 ±1.79^b	48.0 ± 0.71^a
VI	EAPF/100	125.0 ±0.71^a	112.4 ± 0.55^b	99.2± 1.30^a	88.8± 1.64^a	77.8 ± 0.85^a
VII	EAPF/ 200	124.6 ± 0.55	97.6 ± 1.67	80.2± 1.30^a	67.2± 1.64^a	57.6 ± 0.54^a
VIII	EAPF/ 400	121.2 ±1.30^a	80.4 ± 1.52	73.2± 0.84^a	65.0± 1.22^b	46.2 ± 1.09^b
IX	CPF/ 100	126.2 ±0.84^a	119.0 ± 0.71^b	97.8± 0.84^a	87.6± 0.55^a	74.6 ± 1.14^a
X	CPF/ 200	125.2 ±0.45^a	110.6 ± 1.52^a	89.6±1.14^b	76.2± 1.30^b	66.2 ± 1.48^b
XI	CPF/ 400	123.2 ±0.54^a	78.4 ± 0.89^b	69.2±0.83^b	59.6± 1.14^b	54.6 ± 1.52^b

Note: The data are presented as mean ± STD (n=5); One-Way Analysis of Variance (ANOVA) trailed by Dunnet's test; a=p<0.05, b=p<0.01 significant compared to the control.

Hole cross method

Table 5: P. fascicularis root extracts' effects in the hole cross test

Groups	Treatment/Dose (mg/kg)	Number of movements
Groups	Treatment/Dose (mg/kg)	0 min	30 min	60 min	90 min	120 min
Control (I)	Tween 80 solution	21.0±0.63	18.4±0.8	15.2±0.75	14.4±0.74	13.8±0.49
Standard (II)	Diazepam/1	14.8±0.75^a	8.0±1.40^a	4.8±0.75^b	3.2±0.40^b	1.6±0.80^b
III	MPF/100	15.4±0.49^a	13.2±0.75^a	11±0.89^a	7±0.75	6.6±0.80^a
IV	MPF/200	14.41±1.02^a	12±0.89^a	8.0±0.89^a	6.4±0.49	4.4±0.80^b
V	MPF/ 400	12.0±1.02^a	9.4±0.48^a	7.4±0.49^a	5.6±0.80^b	4.0±0.63^a
VI	EAPF/100	19.0±0.49	16.0±0.89^a	14.0±0.63^a	12.8±0.40^a	11.6±0.80^b
VII	EAPF/ 200	17.4±0.89^a	14.0±0.63^a	11.8±0.40^a	11.2±0.98^b	9.6±0.49^a
VIII	EAPF/ 400	16.8±0.75^a	14.4±0.48^b	11.0±0.89^a	9.2±0.97^b	7.4±0.80^b
IX	CPF/ 100	20.0±0.63^a	16.4±0.80^a	15.8±0.40	14.0±0.89^a	12.6±0.49^a
X	CPF/ 200	20.0±0.52^a	14.6±0.98^b	10.4±0.48^a	8.4±0.49	7.8±0.75^a
XI	CPF/ 400	18.0±0.63^a	14.0±0.89^a	10.8±0.98^b	8.6±0.48^b	6.2±0.75^b

Note: The data are presented as mean ± STD (n=5); One-Way Analysis of Variance (ANOVA) trailed by Dunnet's test; a=p<0.05, b=p<0.01 significant compared to the control.

Table 6: Effect of P. fascicularis root extracts' on OGTT in healthy rodents

Groups	Treatment/Dose (mg/kg)	Blood Glucose level
Groups	Treatment/Dose (mg/kg)	0 min	30 min	1 hour	2 hours	3 hours
Control (I)	Tween 80 solution	9.53±0.49	23.78±0.48	18.5± 0.71	15.33±0.62	10.25±0.52
Standard (II)	Metformin/10	8.38 ±0.33^b	16.53±.67^b	10.78±0.79^b	6.22±0.59^b	4.12 ± 0.62^b
III	MPF/100	9.18 ± 0.58	20.67±0.69^a	19.28± 0.38^a	16.55± 0.25^a	10.28 ± 0.29^a
IV	MPF/200	9.22± 0.41	18.58±.67^b	15.27 ±0.29^b	13.95±0.16^b	10.37 0.44^b
V	MPF/ 400	8.30± 0.36	17.27± 0.53	13.10± 0.35	10.2 ± 0.38^a	7.05 ± 0.38^a
VI	EAPF/100	8.45± 0.37	21.67±0.84^b	19.28± 0.38^a	16.55± 0.25^a	13.17 ± 0.29^a
VII	EAPF/ 200	9.21± 0.41	18.58 ± 0.67	15.27± 0.28^b	13.95±0.16^b	10.36 ± 0.44^a
VIII	EAPF/ 400	8.30 ± 0.35	17.26 ± 0.53	13.1± 0.35	10.2± 0.38^b	7.05 ± 0.38^a
IX	CPF/ 100	8.55 ± 0.31^a	21.33±0.40^a	19.63± 0.21^a	17.23± 0.34^a	13.18 ± 0.31^a
X	CPF/ 200	9.25 ± 0.36	19.12±0.57^a	15.63± 0.19^b	13.8± 0.25^b	9.62± 0.23^a
XI	CPF/ 400	8.67 ± 0.61^b	17.6 ± 0.48^b	12.52± 0.19^b	9.57± 0.28^b	6.4± 0.14^b

Note: The data are presented as mean ± STD (n=6); One-Way Analysis of Variance (ANOVA) trailed by Dunnet's test; a=p<0.05, b=p<0.01 significant compared to the control.

Diarrheal Tests:

All tested extracts significantly (p< 0.05 and p< 0.01, versus negative control) and dose-dependently reduced the overall number of diarrheal stools in both diarrhea tests (Table 7 and Table 8). CPF displayed the strongest inhibitory action, which varied with dose, among all the extracts.

Table 7: Effect of P. fascicularis root extracts at various dose levels on castor oil-induced diarrhea in mice

Groups	Treatment / Dose (mg/kg)	Total no. of Feces in 4 hours	% of inhibition
Control (I)	Tween 80 solution	21.67±1.03
Standard (II)	Loperamide/ 2	4.83±1.16^b	77.71
III	MPF/100	17.17±0.75^a	20.77
IV	MPF/200	13.16±1.33^a	39.27
V	MPF/ 400	9.33±0.82^a	56.94
VI	EAPF/100	18.16±0.98^a	16.19
VII	EAPF/ 200	13.83±0.75^a	36.18
VIII	EAPF/ 400	11.50±1.04^b	46.91
IX	CPF/ 100	16.50±0.55^a	23.86
X	CPF/ 200	11.67±0.81^a	46.15
XI	CPF/ 400	8.67±0.81^b	59.99

Note: The data are presented as mean ± STD (n=6); One-Way Analysis of Variance (ANOVA) trailed by Dunnet's test; a=p<0.05, b=p<0.01 significant compared to the control.

Table 8: Effect of P. fascicularis root extracts at various dose levels on magnesium sulphate-induced diarrhea in mice

Groups	Treatment / Dose(mg/kg)	Total no. of feces in 4 hours	% of inhibition
Control (I)	Tween 80 solution	19.33±1.21
Standard (II)	Loperamide/ 2	4.17±0.75^b	78.43
III	MPF/100	15.67±1.03^a	18.93
IV	MPF/200	13.5±0.54^a	30.16
V	MPF/ 400	10.00±0.89^b	48.27
VI	EAPF/100	15.33±0.52^a	20.69
VII	EAPF/ 200	13.83±0.75^b	28.45
VIII	EAPF/ 400	11.67±1.03^a	39.63
IX	CPF/ 100	17.33±0.55^a	10.35
X	CPF/ 200	10.33±1.21^a	46.56
XI	CPF/ 400	8.17±0.41^b	57.73

Note: The data are presented as mean±STD (n=6); One-Way Analysis of Variance (ANOVA) trailed by Dunnet's test; a=p<0.05, b= p<0.01 significant compared to the control.

In vitro studies:

DPPH radical scavenging assay:

The studied materials revealed concentration-dependent quenching properties against the DPPH radical in the antioxidant experiment (Figure 1). The CPF was noticed to be more potent DPPH scavenger with an IC₅₀ value of 39.89 in comparison to the standard ascorbic acid (IC₅₀35.67).

Figure 1: IC₅₀ values of the experimented extracts and Standard.

Thrombolytic test:

The investigated extracts exhibited noteworthy thrombolytic efficacy (Figure 2). CPF exhibited the greatest degree of clot lysis among the studied samples (31.35±2.05%).

Figure 2: Thrombolytic activity of P. fascicularis root extracts. Results are reported as the mean standard deviation of triplicate measurements (mean ± SEM; n=3); and analyzed by One-Way Analysis of Variance (ANOVA) trailed by Dunnet's test. *p<0.05, **p<0.01 significant compared to the control.

Antiarthritic Activity:

Table 9 presents the comprehensive findings of the atiarthritic test. CPF showed the highest percentage of inhibition (87.26±1.02% at 500µg/ml), while MPF showed the lowest percentage of inhibition (60.48±1.89% at 100µg/ml).

Anthelmintic Test:

Table 10 lists the results of the anthelmintic activity of roof extracts of P. in comparison to albendazole .

Table 10: Anthelmintic activity of P. fascicularis root extracts at different concentrations

Groups	Conc. Used (mg/ml)	Time taken for paralysis (min) X±SD	Time taken for death (min) X±SD
Control (I)	-----	-----	-----
Albendazole (II)	20	33.67±1.53	42.00±2.00
MPF (III)	25	77.00±1.00^a	82.00±4.36^a
	50	64.33±1.53^a	69.66±2.52^a
	75	59.00±1.00^b	64.66±4.16
EAPF (IV)	25	68.67±0.50^a	68.33±3.21^a
	50	53.67±2.08^b	61.67±2.51^a
	75	50.33±1.53^b	56.33±1.52^b
CPF (V)	25	56.00±1.00^a	64.66±4.16^a
	50	49.05±0.58^a	53.66±2.08^b
	75	39.66±2.08^b	44.33±1.52^b

Note: The data are presented as mean ± STD (n=6); One-Way Analysis of Variance (ANOVA) trailed by Dunnet's test.; a=p<0.05, b=p<0.01 significant compared to the control.

Insecticidal test

Figure 3 shows the impact of contact poisoning of root extracts of P. fascicularis on adult T. castaneum mortality.

Figure 3: Insecticidal effects of root extracts of P. fascicularis

DISCUSSION:

Plant bioactive constituent assure the medicinal potential and their therapeutic actions ^1-4,25,26. To isolate new active and scarce compounds, phytochemical investigation is crucial. In recent study we extracted the root of P. fascicularis with methanol and fractionated in ethyl acetate and chloroform solvents. The experimented extracts exhibited the presence of various secondary metabolites (Table1 ). The previous literature also supported the presence of 2-phenyl ethyl methyl ether, terpene-4-ol, α–terpeniol, benzyl benzoate, flavanoids, glycosides, alkaloids, steroids, terpenoids, saponins and tannins in different parts of P. fascicularis^,. Traditionally the examined plant parts are used to alleviate rheumatoid arthritis, leprosy, edema, tumors, earaches, fever, skin conditions, ulcers, and dyspepsia^6,7.

Writhing test (acetic acid-induced) and Licking (formalin-induced pain) test were used to assess the analgesic effectiveness of P. fascicularis root extracts. After receiving acetic acid intraperitoneally, the body releases histamine, prostaglandins (PGs), serotonin, Cyclooxygenase (COX), bradykinin, and cytokines which cause pain by contraction of the abdominal muscles^3,4. Our extracts decreased writhing in the current investigation (p<0.01, p<0.05; Table 2). We conducted the Licking test on the plant extracts under study to distinguish between their central and peripheral antinociceptive activities. In our investigation we noticed that all tested samples significantly (p<0.01, p<0.05) reduced the mean time of beating the formalin injected paw in both phases (Table 3). The CPF was found to be most potent in all phases (early phase 55.82% protection and in late phase 58.18% protection). Therefore, based on the outcomes of our study, all extracts also have peripheral and central antinociceptive effects in along with additional anti-inflammatory activity. Bioactive phytoconstituents such as flavonoids, alkaloids, and terpenoids, which have been linked to considerable analgesic and anti-inflammatory effects in previous pharmacological studies^3,4,10,19, were also found in our tested extracts.

The CNS depressive impact of P. fascicularis root extracts was investigated utilizing two neuropharmacological approaches, namely open field, and hole cross, by examining the naturalistic locomotor behavior of mice. These paradigms are well-known traditional methods for examining neuropharmacological action^10,19. Here, CPF extract produced highly significant effects than other extracts at higher dose (400 mg/kg) (Table 4 and Table 5). A decrease in locomotor activity is a sign of CNS depressing activity and is seen as an indication of awareness^19,27. When the voltage-triggered Ca²⁺ channel is hindered, it can either enhance chloride conductivity or increase GABA-induced chloride conduction. As a result, the extracts are likely to either directly activate GABA receptors or amplify GABAergic suppression in the CNS via membrane hyperpolarization, resulting in a decrease in the firing rate of crucial brain neurons^1,3,4. The secondary metabolites (flavonoids, terpenoids, saponin, etc.) of the plant that may have synergistic effects at one or more target sites connected to a physiological function are responsible for the extracts' depressant activity^3,4.

A medicine that effectively treats diabetes will be able to regulate the rise in blood sugar using a variety of mechanisms, and the potential of extracts to avoid hyperglycemia might be assessed with OGTT¹⁵. Because it temporarily boosts the animal's blood glucose level without causing injury to the pancreas, this approach is known as the physiological induction of diabetes mellitus^12,15. In comparison to the reference metformin, the crude methanolic root extract (MPF) and it fractions (EAPF and CPF) demonstrated a remarkable hypoglycemic activity (p<0.05, p<0.01; Table 6) in rodents during the glucose tolerance test that persisted for up to 3 hours which might be due to the presence of, various significant bioactive phytoconstituents like falvonoids, tannin etc.^25,26.

The antidiarrheal effectiveness of the investigated plant extracts was assessed using models of diarrhea induced by castor oil and magnesium sulphate. Castor oil consumption increases the intestinal absorption of ricinoleic acid released by intestinal lumen lipase. By influencing gut motility and ultimately causing diarrhoea, ricinoleic acid exerts a significant laxative effect on mice through the prostaglandin receptor EP2¹². All experimental extracts significantly (p < 0.05, p<0.01) decreased diarrhea by dropping the volume of intraluminal fluid accumulation in a dose-dependent manner in the castor oil-induced diarrheal test (Table 7). Magnesium sulphate, on the other hand, also causes diarrhea by preventing salt chloride and water reabsorption and increasing the volume of intestinal content. Cholecystokinin is released from the duodenal mucosa when this salt is consumed, which causes diarrhea¹⁶. Significant anti-diarrheal efficacy was shown by P. fascicularis root extracts (MPF, EAPF, and CPF) against magnesium sulphate-induced diarrhea (Table 8). At all trial doses compared to the standard, extracts significantly (p<0.05, p<0.01; Table 7 and Table 8) enhanced the percentage of inhibition and also noticeably decreased the number of diarrheal feces. The presence of flavonoids and tannin may be the cause of the extracts capacity to reduce intestinal output^12,16.

Antioxidants with the ability to quench free radicals are crucial in the treatment of different pathological state. Because of this, there is a growing interest in creating natural antioxidants derived from plants that are capable of safeguarding the body from damage caused by oxidative stress brought on by free radicals. The most used technique for assessing the antioxidant capacity of plant materials is the DPPH scavenging assay. The tested plant exhibited moderate antioxidant potential against DPPH free radicals, as shown in Figure 1. Numerous bioactive phytochemical components, particularly phenolic compounds, are crucial for both the antioxidant and free radical scavenging effects of plants which were also found in our extracts.

Recent studies by epidemiologists employing plant and natural products have demonstrated that natural thrombolytic/fibrinolytic medications lower the risk of thrombosis in contrast to synthetic products^12,20,21. In our investigation, crude root extract of P. fascicularis and its fractions revealed notable thrombolytic activity in comparison to reference standard streptokinase. Among the extracts examined, CPF exhibited the highest percentage of clot lysis (31.35±2.05%) followed by EAPF (25.41±1.48%) and MPF (20.83±1.19%) (Figure 2). Numerous investigations have demonstrated that saponins, alkaloids, and tannins are triggering clot lysis activity^8,20,21 which were also confirmed in our extracts.

Tissue protein denaturation, a primary contributor to arthritic diseases, can create autoantigens in numerous situations. Protein denaturation can be caused by several physical and chemical factors, including heat, light, pressure, acids, alcohol, alkali, acetone, heavy metal salts, and dyes, as well as alterations in the disulfide, hydrogen, hydrophobic, and electrostatic interactions in the protein^8,21. All the extracts in this investigation show dose-dependent denaturation of bovine serum albumin. The chloroform fraction of the study (Table 9) was found to be most active at 500μg/ml dose (87.26±1.02%). This study revealed the presence of flavonoids and phenolic substances in plant extracts, which were claimed to have anti-arthritic properties in previous studies^8,21.

Natural resources like plants can also yield novel physiologically active compounds that are compatible with human physiology and have no negative effects or less than those of synthetic anthelmintic compounds^8,21-23. In the anthelmintic study, we distinguish between plant extracts and regular albendazole in terms of the length of earthworm paralysis and death. In this case, we discovered a statistically significant association between extract graded concentrations, exposure periods, and adult parasite mortality. The chloroform fraction demonstrated (Table 10) possible reductions in paralysis (39.66±2.08min) and death (42.0±2.0min) times compared to standard (33.67±1.53min and 44.33±1.52 min, respectively) among all examined extracts at higher dose (75mg/ml). Copious investigations suggested that tannins, flavonoids, alkaloids, and phenolic chemicals are responsible for the anthelmintic activity^8,21-23 which were also confirmed by our study.

Herbal products have recently gained popularity due to their significant potential as insecticidal chemicals^2,24. In our present research we observed that the examined extracts exhibited little insecticidal activity against adult T. castaneum. Previous research reported the presence of bioactive components in plant extracts such as polysaccharides, phenol, flavonoids, phytosterol, saponins and tannins can have repellant, antifeedant, and poisonous properties^27-30.

CONCLUSION:

The root of P. fascicularis was found to contain a large number of secondary bioactive metabolites, such as glycosides, alkaloids, tannin, saponin, flavonoids, terpenoids, and so forth. These metabolites had a variety of effects, including analgesic, CNS depressant, hypoglycemic, antidiarrheal, thrombolytic, antiarthritic, anthelmintic, and insecticidal qualities. At every experimental dosage examined, the impact is immediate, persistent, and statistically significant. Our research leads us to the conclusion that P. fascicularis root may provide pharmaceutical companies with new, safe, effective, and less toxic candidate treatments, hence lowering the cost of treating diseases. However, more investigation is required to identify the bioactive molecule or compounds and comprehend the exact molecular pathways in order to determine a safe and effective dosage and validate the potential for use in treatment and prevention.

CONFLICT OF INTEREST:

The authors state unequivocally that they have no competing interests.

FUNDING INFORMATION:

For this study, the authors received no specific grant or financial assistance from any public, commercial, or non-profit funding bodies.

DATA AVAILABILITY STATEMENT:

The corresponding authors can provide the datasets created and/or examined during this study upon reasonable request.

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Received on 27.12.2023 Revised on 29.06.2024

Accepted on 09.11.2024 Published on 28.01.2025

Available online from February 27, 2025

Research J. Pharmacy and Technology. 2025;18(2):647-655.

DOI: 10.52711/0974-360X.2025.00096

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Sample	% of Inhibition at Concentration (μg/ml)
Sample	100	200	300	400	500
Diclofenac sodium	72.48±1.78	78.75± 0.55	84.46±0.89	90.12±3.08	92.77±0.96
MPF	60.48±1.89^a	62.34±1.36^b	68.34±0.91^a	72.26±0.94^b	74.29±1.05^a
EAPF	63.23±1.89	66.41±1.41^b	71.66±0.59^b	77.19±2.25^a	80.33±0.86^b
CPF	66.55±0.95^b	70.44±1.14^b	75.31±1.12^b	83.26±1.02^b	87.26±1.02^b