Anthelmintic efficacy of Holarrhena pubescens against Raillietina spp. of domestic fowl through ultrastructural, histochemical, biochemical and GLCM analysis

Rachita Saha; Manjil Gupta; Rima Majumdar; Subrata Saha; Pradip Kumar Kar

doi:10.1371/journal.pone.0282033

. 2023 Sep 14;18(9):e0282033. doi: 10.1371/journal.pone.0282033

Anthelmintic efficacy of Holarrhena pubescens against Raillietina spp. of domestic fowl through ultrastructural, histochemical, biochemical and GLCM analysis

Rachita Saha ¹, Manjil Gupta ¹, Rima Majumdar ¹, Subrata Saha ¹, Pradip Kumar Kar ^1,^*

Editor: Josué de Moraes²

PMCID: PMC10501554 PMID: 37708168

Abstract

Globally, traditional knowledge systems are a powerhouse of information which can revolutionise the world, if decoded accurately and logically. Plant-based ethno-traditional and folklore curatives/medicines has a firm basis in the psyche of the common masses of West Bengal and Holarrhena pubescens is a representative example of it. This article communication on depicting the anthelmintic efficacy of ethanolic extract and Ethyl acetate fraction of the stem bark of Holarrhena pubescens against the cestode Raillietina spp. through efficacy studies, ultra-structural observations, histochemical and biochemical analysis on some tegumental enzymes i.e., Acid Phosphatase (AcPase), Alkaline Phosphatase (AlkPase), Adenosine Triphosphatase (ATPase) and 5ˈ-Nucleotidase (5ˈ-Nu) along with Gray Level Co-occurrence Matrix (GLCM) analysis of histochemical study. Praziquantel was used as the reference drug. Investigations revealed 10mg/ml dosage of crude extract was the most efficacious dose and amongst the fractions the ethyl acetate fraction showed the most anthelmintic property. Ultrastructural studies through Scanning Electron Microscope (SEM) and Transmission Electron Microscope (TEM) clearly depicted the damage in head, sucker, proglottids, proximal and distal cytoplasm (DC), microtriches (MT), basal lamina (BL), nuclear membrane (NM), and, nucleolus (NL) in the treated worms. Histochemical studies revealed decrease in staining intensity for all the tegumental enzymes in the treated worms compared to control. The GLCM analysis strongly supported the result of histochemical studies. Biochemical studies revealed marked reduction in enzyme activity in the treated worms with maximum reduction in the activity of 5ˈ- Nu (77.8%) followed by ATPase (63.17%).

Introduction

Neglected Tropical diseases (NTDs) appear globally in about 149 different nations [1], particularly in the less developed ones where socioeconomic status are severely compromised. NTDs—like helminthiasis—cause a greater global burden of ailments rather than more well-known illnesses like malaria and TB, because they are the most prevalent infectious agents affecting humans and animals in underdeveloped nations [2]. Currently, it is estimated that 33% of people living in the developing countries in Sub-Saharan Africa, Asia, and the Americas are infected with helminths [3].

Domestic poultry is one of the major primary source of dietary protein in the world, providing both meat and eggs [4]. Though the productivity of the poultry industry has steadily increased over the years [5], still, the industry faces serious hurdles in production due to helminth infections caused by nematodes, cestodes and trematodes [6–8]. Globally, 66% of the helminth infections are caused by cestodes with Raillietina spp. being the most ubiquitous genus in majority of the prevalence studies [9]. The genus Raillietina consists of tapeworms belonging to Order Cyclophyllidea and Family Davaineidae. The different species namely R. echinobothrida, R. tetragona and R. cesticillus are identified using light microscopy and scanning electron microscopy by morphometric analysis of the scolex and proglottids position of the genital pore, number of eggs per egg capsule, and rostellar opening surface structures [10]. Their life cycle is completed in two hosts with avian and rodent species being the definitive host and ants and beetles being the intermediate host. The adults live in the duodenum of the host where they get anchored to the intestinal wall, draw nutrition, grow and reproduce [11, 12]. The pathogenicity of Raillietina infections in poultry birds is characterised by disruption of intestinal villi, degeneration and necrosis of intestinal epithelial cells, parasitic granulomas, enteritis, anemia, weight loss and decreased egg production [13, 14]. There are few reports of accidental infection of Raillietina spp. in humans occurring due to accidental ingestion of cysticercoid infected ants or beetles [15–17].

Widespread use of over-the-counter anthelmintics has led to the development of resistance to these drugs in most animals [18]. Anthelmintic resistance is also an emerging problem in poultry warranting the search for plant-based alternatives. Various phytochemicals namely phenols, alkaloids, saponins, tannins, terpenoids and flavonoids derived from different plants have been shown to act as anthelmintics by altering the cell membrane permeability, interfering with mitochondrial function and energy generation, affecting calcium utilisation, inhibiting neurotransmission with subsequent paralysis and affecting egg hatching [19]. In many underdeveloped nations, livestock producers sometimes lack to access basic veterinary service and medications and medicinal plants continue to be utilised in concoction with veterinary pharmaceuticals to cure animal maladies [20, 21]. So, traditional, indigenous or ethnomedicinal knowledge of ethnic/local population can provide us with valuable inputs for the development of curatives from natural products with anthelmintic potential.

Holarrhena pubescens is a medicinal plant used by local Rajbangshi or Koch community in Cooch Behar, West Bengal, India [22, 23]. Holarrhena pubescens Wall. ex G. Don, Syn -Holarrhena. antidysenterica (Roth) Wall. ex A. DC. belongs to family Apocynaceae, (Common Name: Indrajao, Kutraj, Kutaja, Bitter Oleander, Kurchi) and is indigenous to the eastern part of Tropical Africa and Tropical Asia (Indo-China). It is distributed in the subtropical forests of India, Myanmar, Nepal, Bhutan, China and Malaysia [24]. H. pubescens is a huge deciduous tree that grows to 9–18 meters in height and is commonly found in plains and mountains [25]. The leaves are oblong and elliptic in shape. Flowers are fragrant white corymbose cymes. The corolla is lobed and oblong in shape. Fruits are long, having thin follicles with white markings. The seeds are globous and linear-oblong in shape. The flowers bloom from April to July and fruiting occurs from August to October [26]. Astringent, antimalarial, antidysenteric, anthelmintic, stomachic, febrifugal, antibacterial, and antioxidant characteristics have been observed in several portions of the plant (stem, root, leaves, and flower) [27]. The methanolic extract of the stem bark of this plant showed hypoglycaemic action [25]. Previous studies has shown that the plant’s leaf, bark, fruit, and seed all have therapeutic benefit. The leaf of the plant is employed in pest control, and the entire plant is utilized as green manure. Its applications has been documented in Ayurveda, Homoeopathy, and folklore medicine. The flavonoids and phenolic components in Holarrhena pubescens aqueous and methanolic bark extract showed high antioxidant activity [25] H. pubescens alkaloids were shown to be useful in treating acute dysentery, inflammatory bowel illness, diarrhoea, and, worm infections in the gut [28]. It is also reported that H. pubescens, may be helpful to an extent, in treating Amoebiasis [29] and this plant also has anti-diabetic, anti-amnesic and anti-inflammatory property [28].

About 40 alkaloids have been found in the stem bark, root bark, and seeds of Holarrhena pubescens [24]. The barks of stem and root (in total) had the greatest concentration of alkaloids (up to 4.3%), followed by leaves (1.0–1.5%) and seeds (0.6–1.8%). Only 0.4% of the 4.3% alkaloids present in the barks of stem and root are accessible to blooming plants. According to the Ayurvedic Pharmacopoeia of India [30], the bark contains around 2% alkaloids. This plant’s stem bark, seeds, and roots were found to be high in steroidal alkaloid compounds such asisoconessine, kurchine, conessidine, conkurchicine, and holarrhimine, as well as conessine, which possesses antimalarial and antibacterial activities [31, 32].

Whole worm-based assays are commonly used for identification of new anthelmintic compounds. Unlike target based approaches, the main focus of these assays are whether the compounds are able to kill or disable the worms in vitro [33]. Worm motility, morphological changes, ATP production and enzyme activity are some of the features assessed in these assays [34]. The current investigation focuses on the plausible anthelmintic property of the ethanolic extract of stem bark of H. pubescens through ultrastructural, histochemical, GLCM analysis and biochemical studies of the alterations in the activity of the tegumental enzymes namely Acid Phosphatase (AcPase), Alkaline Phosphatase (AlkPase), Adenosine Triphosphatase (ATPase) and 5ˈ-Nucleotidase (5ˈ-Nu) of Raillietina spp in vitro.

Materials and methods

Preparation of extract from Holarrhena pubescens

Fresh bark was collected from the plant H. pubescens from the town of Cooch Behar (26.3169° N, 89.4457° E), West Bengal, India in month of April- June, Since 2018 till date. Dr. Monoranjan Chowdhury identified the plant and the accession number is- 12638. It was kept at herbarium of Department of Botany, University of North Bengal, Darjeeling, West Bengal. The plant materials were finely chopped and air dried for 24–36 hours after being collected. The dried bark components were soaked in ethanol in a glass container (100 grams in 500 ml) for 15–20 days with frequent stirring. The solution was then filtered using Whatman Filter Paper (No. 14), and then dried using a Rotary Vacuum Evaporator (Buchi Rotavapor R-100). After drying, the collected crude extracts of plant material were stored under refrigeration at 4°C until further use. Around 7.0–8.0 gram (g) of plant extract was obtained from 100 g of plant material immersed in 500 millilitre (ml) ethanol. Different fractions of the obtained ethanolic extract were separated using solvents with varying polarities such as Hexane, Chloroform, Ethyl acetate, and n-Butanol using a separating funnel and fractional distillation process [35]. 50 ml aqueous solution of crude extract (2.5 gm in 50 ml distilled water) was combined with 50 ml of Hexane and left to stand for 2 hours. The Hexane fraction was subsequently removed from the upper phase using the liquid liquid separation technique. The method was repeated three times using Chloroform (lower phase), Ethyl acetate, and n-Butanol. The final volume of each fraction (150 ml) was collected and dried using a Rotary Vacuum Evaporator (Buchi Rotavapor R-100). Around 0.25 g, 0.14 g, 0.92 g and 1.1 g of Hexane, Chloroform, Ethyl acetate and n-Butanol fractions were obtained from 2.5 g of crude extract respectively.

Collection of parasites

Fresh intestines of Gallus gallus domesticus were obtained from a local market, and live parasites (Raillietina spp.) were isolated from the gut in 0.9 percent Phosphate-Buffered Saline (PBS: NaCl-8g, KH₂PO₄-0.34g, and KHPO₄-1.21g in 1 litre (l) distilled water, pH 7.4) and kept in an incubator set at 37±1° C.

Experiments

Efficacy testing

The model parasite i.e., Raillietina spp. were incubated in glass petridish at 37°C in PBS without plant extract (control), and with Ethanolic crude extract at 1.0, 2.0, 5.0 and 10.0 mg/ml concentration respectively. Praziquantel was used as the reference drug. Three replicates were used for each concentration. The time required for complete inactiveness or paralysis and death of the parasites were recorded. The time for paralysis was determined by observation of the parasites after treatment. Paralysis was considered when the parasites showed loss of smooth undulating motion and death was considered when there was complete absence of any motion in the parasites. Death was further confirmed if the parasites showed no motion even after transferring to warm PBS. After determining the most efficacious dose of the crude extract, efficacy tests were performed with the same dose of different fractions.

Scanning Electron Microscopy

For Scanning Electron Microscopy (SEM) the parasites were fixed in 10% Neutral Buffered Formalin (NBF) at 4°C for 4 hours followed by repeated washing in double-distilled water, dehydration through acetone grades, critical-point-drying (CPD) using liquid CO₂, metal-coating with gold palladium, and viewed in a JEOL-JSM-35 CF scanning electron microscope [36].

Transmission electron microscopy

Control and treated worms were fixed in Karnovosky’s fixative (4% Paraformaldehyde and 1% Glutaraldehyde) in 0.1M Sodium Phosphate buffer (pH 7.4) and then processed for Transmission Electron Microscope TECNAI G20 HR-TEM as per usual method [37] for ultrastructural studies [36].

Histochemical studies

Following Pearse 1968 [38], histochemical localisation of Acid Phosphatase (AcPase), Alkaline Phosphatase (AlkPase), and Adenosine Triphosphatase (ATPase) in control and phytoproduct exposed worms was carried out [39]. The Wachstein and Meisel 1957 [40] approach, which uses adenosine monophosphate as a substrate, was used to examine 5ˈ- Nucleotidase (5ˈ-Nu). For 30 minutes, sections were incubated at 37°C in a freshly produced medium containing 10 ml of 1.25 percent adenosine-5-phosphate, 5 ml of 0.2 M Tris buffer at pH 7.2, 30 ml of 0.2 percent Pb(NO₃)₂, and 5 ml of 0.1 M MgSO₄. All parasites were cut in 12μm-14μm thickness through Cryostat (Leica- CM 3050S) at -15°C -18°C [41].

GLCM analysis

Gray Level Co-occurrence Matrix (GLCM) analysis for the quantification of the histochemical localization of the enzymes was performed following Dragić et al. 2019 [42]. Mean Gray Value (MGV) and Integrated Density (IG) were calculated for all the images. Five parameters namely Angular Second Moment (ASM), Contrast (CON), Co-relation (COR), Inverse Different Moment (INV) and Entropy (ENT) were calculated for GLCM analysis. Out of these, ASM, COR and INV measures the homogeneity of the image and should be more in deeply stained images whereas CON and ENT measures contrast and lack of spatial organization in the image and should be less in deeply stained images. Images obtained from histochemical study for different enzymes were cropped for region of interest (comprising of the tegument, sub tegument and parenchyma), converted to grayscale and subjected to GLCM analysis through the software Image J [43]. A set of 20 images each from different categories like control, Praziquantel treated, crude extract treated and Ethyl acetate fraction treated was used for GLCM analysis for each enzyme. Receiver Operating Characteristic (ROC) curve analysis was also performed to validate the results obtained from GLCM analysis.

Biochemical studies

The method of Plummer, 1988 [44], was used to estimate the activities of AcPase and AlkPase [36]. ATPase activity was determined by estimating the free phosphate released using the method of Kaplan, 1957 [45], and 5’ -Nu was determined by the Bunitian method following Giri and Roy, 2014 [39]. Protein content was calculated using Lowry’s method [46] for all the enzyme assays. The total enzyme activity was determined for AcPase and AlkPase as the amount in micromole (μM) of p-nitrophenol formed per minute per gram wet tissue. In case of ATPase and 5ˈ- Nu, activity was expressed by liberation of μM inorganic phosphate group per mg protein. The activity of tegumental enzymes was expressed by total and specific enzyme activity.

Results

Efficacy testing

The results indicate that the Ethanolic crude extract of Holarrhena pubescens and the fractions of ethanolic extract showed anthelmintic activity on cestode worm Raillietina spp. The control worms survived in PBS for around 71 hours. In the presence of varying concentrations of the ethanolic extract and different fractions, it was observed that the survival time of the treated worms was longer than that of those treated with Praziquantel (PZ) but lower than the Control (C) (Table 1, Fig 1). The concentration of 10 mg/ml was determined as the most efficacious dose and used for further studies. Out of the different fractions used, the Ethyl acetate fraction (EAF) showed most anthelmintic activity.

Table 1. Results of efficacy testing of the ethanolic crude extract of Holarrhena pubescens and fractions of the ethanolic extract on Raillietina spp.

Incubation Medium	Dose (mg/ml) PBS	Time of paralysis (hours)	Time of death (hours)
Control	-	-	71.0±0.59
Praziquantel	0.05	0.49±0.05	0.98±0.04^*
Ethanolic crude extract	1.0	9.81±0.47	12.84±0.48^*
	2.0	5.09±0.15	8.52±0.16^*
	5.0	2.11±0.23	6.89±0.36^*
	10.0	1.02±0.13	4.44±0.21^*
n-Hexane fraction	10.0	6.16±0.35	20.23±0.31^*
Chloroform fraction	10.0	5.32 ± 0.28	19.44±0.25^*
Ethyl acetate fraction	10.0	0.83 ± 0.21	3.72±0.52^*
n-Butanol fraction	10.0	1.08 ± 0.33	4.66±0.52^*

Open in a new tab

*Data represents mean value ±SD.

n = 450. Values significant at P<0.05

SEM observations

In the control worms, the entire surface of the scolex and proglottids shows a smooth appearance due to being covered with dense microtriches (Fig 2a, 2e and 2i). Suckers are smooth with distinct hooks. In the worms treated with the plant material, distortions are visible in the head region characterised by folding of the tegument, eversion of the suckers and loss of the hooks encircling them. In the proglottids, the tegument shows a folded and cracked appearance (Fig 2c, 2d, 2g, 2h, 2k and 2l). Similar observations were also found in the Praziquantel-treated worms (Fig 2b, 2f and 2j). The damage is more apparent in the worms treated with Praziquantel and Ethyl acetate fraction.

Fig 2 — Scanning electron micrographs of the head (a—d), neck (e—h) and proglottids (i—l) of control and treated *Raillietina* spp. Control (a, e, i), Praziquantel exposed (b, f, j), Ethanolic crude extract exposed (c, g, k), Ethyl acetate fraction exposed (d, h, l) (all scale bars = 20μm).

TEM observations

The control worm’s surface, proximal and distal cytoplasm (DC) with microtriches (MT), basal lamina (BL) (Fig 3a) nucleus with double nuclear membrane (NM), nucleolus (NL), chromatin granules (Fig 4a and 4e), and mitochondrial characteristics were all normal, according to transmission electron microscopic studies. Parasites treated with Praziquantel had severe damage in the microtrix layer and distal cytoplasm (Fig 3b). Swollen nuclei with an anomalous singlet nuclear membrane (Fig 4b) and deformed mitochondria lacking cristae were also observed (Fig 4f). The distal cytoplasm of the worms exposed to the plant extract (crude) was completely eroded, and, the basal lamina of the microtriches was exposed to the outer surface (Fig 3c). The nucleolus disintegrated and a swollen nucleus with an uneven nuclear membrane was observed (Fig 4c). In the parasites treated with the plant extract, the majority of the mitochondria developed deformities (Fig 4g). Raillietina spp., treated with the Ethyl acetate fraction, similarly exhibited fully damaged microtriches and distal cytoplasm (Fig 3d), rupture of the nuclear membrane, condensation of chromatin granules (Fig 4d) and disintegration and vacuolization of the mitochondrial membrane (Fig 4h).

Fig 4 — Transmission electron microscopy images of sections of Control (a, e), Praziquantel treated (b, f), Ethanolic crude extract (c, g) Ethyl acetate fraction (d, h) treated *Raillietina* spp showing nucleus (N), nucleolus (NL) and nuclear membrane (NM) and mitochondria (M); Scale bars = 0.5 μm (a—d) and 0.2 μm (e—h).

Histochemical observation

The tegument of control Raillietina spp. showed intense activity of AcPase, AlkPase, ATPase and 5’ Nu compared to sub-tegument, somatic musculature, and parenchyma (Figs 5a–5h and 6a–6h). ATPase activity was almost negligible in the tegument and sub-tegument of the parasite treated with crude and Ethyl acetate fraction of the plant extract compared to control (Fig 6c and 6d). A reduced activity of AlkPase was observed throughout the treated sections of the parasite (Fig 5g and 5h). In the control sections of histochemical localization of AcPase, pronounced stain intensity was observed in the tegument and sub- tegument regions (Fig 5a–5d). However, there was minimal AcPase activity oberved throughout the section of parasite exposed to crude, Ethyl acetate fraction and Praziquantel. The 5’ -Nu activity was also found to be reduced throughout the tegumental and sub-tegumental region in the phytoproducts exposed worms compared to the control (Fig 6e–6h).

Fig 5 — Histochemical evidence of AcPase (a—d) and AlkPase (e—h) activity in *Raillietina* spp., T-Tegument, ST -Subtegument, SM-Somatic musculature, P-Parenchyma, (a, e). Control, (b, f) Praziquantel, (c, g) Crude extract (d, h) Ethyl acetate fraction treated (All scale bars = 50 μm).

Fig 6 — Histochemical evidence of ATPase (a—d) and 5’-Nu (e—h) activity in *Raillietina* spp., T-Tegument, ST -Subtegument, SM-Somatic musculature, P-Parenchyma, (a, e) Control, (b, f) Praziquantel, (c, g) Crude extract treated, (d & h) Ethyl acetate fraction treated (All scale bars = 50 μm).

GLCM results

The calculated values of different GLCM parameters are depicted in Table 2. For ASM, COR and INV, the values are more in the control worms than in the treated worms and for MGV, ID, CON and ENT the control worms show lower values than the treated worms. The results conform to the expected results. MGV and ID showed the lowest value in the control worms of AcPase due to darker staining section compared to the other enzymes. ASM and CON showed highest and lowest values respectively in the control worms for ATPase. COR shows highest vale in the control worms of AcPase. INV has highest value in the control worms of ATPase. ENT has lowest value in the control worms of ATPase. The ROC curve analysis is shown in Fig 7. The area under the curve (AUC) values is depicted in Table 3. All the values are greater than 50% indicating that the process is considerable.

Table 2. GLCM parameters of images obtained from histochemical studies of different enzymes in Raillietina spp.

		MGV	ID	ASM	CON	COR	INV	ENT
AcPase	C	113.03 ±15.07	7064539.0 ±941926.3	0.000473 ±0.000112	189.95 ±58.36	0.0022 ±0.0006	0.13 ±0.0171	7.99 ±0.26
	PZ	^*168.67 ±16.03	^*10542149.4 ±1002093.42	^*0.000172 ±0.000027	^*558.7 ±66.56	^*0.00081±0.00009	^*0.07 ±0.0054	^*8.95 ±0.12
	ECE	^*136.82 ±31.29	^*8551189.8 ±1955700.6	^*0.000372 ±0.00015	^*331.26 ±218.26	0.00172 ±0.00071	^*0.10 ±0.0285	8.28 ±0.5
	EAF	^*146.41 ±34.33	^*9306178.5 ±2190156.44	^*0.000299 ±0.000153	^*433.75 ±239.09	^*0.00137±0.00063	^*0.09 ±0.0296	^*8.53 ±0.54
AlkPase	C	160.11 ±3.18	15287542.5 ±303448.53	0.000248 ±0.000011	311.32 ±38.36	0.001±0.0	0.09 ±0.0431	8.59 ±0.05
	PZ	^*164.71 ±5.4	^*15726452.2 ±515784.83	0.000234 ±0.00004	^*383.33 ±33.5	0.001±0.0	^*0.08 ±0.0045	8.67 ±0.14
	ECE	^*169.76 ±5.79	^*15930277.4 ±671090.57	0.000242 ±0.000027	^*411.49 ±55.56	0.00099±0.00002	^*0.08 ±0.0073	^*8.67 ±0.1
	EAF	^*167.65 ±8.07	^*15825408.9 ±715618.67	0.000236 ±0.000045	^*445.2 ±56.4	^*0.00099±0.00002	^*0.08±0.01125	^*8.71 ±0.12
ATPase	C	175.75 ±12.06	67556571.1 ±4636486.82	0.00079 ±0.000282	88.9 ±14.54	0.00157 ±0.00053	0.23 ±0.0432	7.59 ±0.43
	PZ	182.64 ±16.28	70208272.0 ±6259795.72	^*0.00053 ±0.000124	^*99.49 ±11.8	0.00128 ±0.00048	^*0.14 ±0.0089	^*7.96 ±0.18
	ECE	184.07 ±9.89	70534709.2 ±3908405.44	^*0.00035 ±0.000071	^*124.57 ±15.59	^*0.00107±0.00032	^*0.19±0.0101	^*8.33 ±0.15
	EAF	183.12 ±7.26	70442003.2 ±2923379.36	^*0.00034 ±0.000066	^*118.6 ±12.49	^*0.001±0.00034	^*0.15 ±0.008	^*8.35 ±0.13
5’- Nu	C	169.78 ±8.13	69543524.8 ±3329793.72	0.00028 ±0.00003	153.14 ±26.47	0.00098 ±0.00007	0.14±0.0127	8.53 ±0.1
	PZ	^*19.37 ±6.13	^*77976486.4 ±2509017.94	^*0.0002 ±0.000031	^*173.83 ±29.83	^*0.00054±0.00008	^*0.12 ± 0.0099	8.62 ±0.13
	ECE	^*178.98 ±7.15	^*73310255.7 ±2928377.66	^*0.00023 ±0.000022	^*190.3 ±24.62	^*0.00083±0.00006	^*0.12 ±0.0142	^*8.68 ±0.08
	EAF	^*178.65 ±6.34	^*73228978.2 ±2717574.88	^*0.00025 ±0.00004	^*176.1 ±27.34	^*0.0009±0.00009	^*0.12±0.0132	^*8.8 ±0.14

Open in a new tab

C- Control, PZ-Praziquantel treated, ECE-Ethanolic Crude Extract treated, EAF- Ethyl acetate fraction treated.

Data represent Mean±SD.

* Values significant at P<0.05

Table 3. Area under curve (AUC) values of GLCM parameters of images obtained from histochemical studies of different enzymes in Raillietina spp.

Enzyme	Area under curve (AUC) values (%)
Enzyme	MGV	ID	ASM	CON	COR	INV	ENT
AcPase	81.00	72.00	81.00	86.00	82.00	88.00	80.00
AlkPase	80.00	74.00	85.00	94.00	62.00	83.00	74.00
ATPase	69.00	68.00	87.00	88.00	74.00	63.00	87.00
5’- Nu	84.00	84.00	65.00	78.00	88.00	88.00	84.00

Open in a new tab

Mean Gray Value–MGV, Integrated Density–IG, Angular Second Moment—ASM, Contrast- CON, Co-relation—COR, Inverse Different Moment—INV and Entropy–ENT.

Biochemical observation

The quantitative analysis of the tegumental enzymes of Raillietina spp. showed significant impact on treatment with the plant materials and the reference drug Praziquantel. As shown in results (Table 4 and Fig 8) AcPase activities decreased by 50.33, 39.70 and 60.56 percent, AlkPase activities decreased by 54.05, 65.44 and 39.74 percent, ATPase activities decreased by 37.16, 41.38 and 63.17 percent, and 5´-Nu activity decreased by 71.63, 65.75, and 77.80 percent, after exposure to the Praziquantel, Ethanolic crude extract, Ethyl acetate fraction of the crude extract respectively. In the worms fed with the Ethyl acetate fraction of the plant, the highest inhibition of activity was observed in the case of 5´- Nu followed by ATPase.

Table 4. Results of biochemical analysis of enzyme activity in Raillietina spp.

	Tegumental enzyme (Total/Specific) activity in μM				Percentage changes after treatment (%)
	AcPase	AlkPase	ATPase	5´-Nu	AcPase	AlkPase	ATPase	5’- Nu
C	21.71± 1.99/9.07 ± 0.87	30.09±1.62/11.74±1.25	244.27±13.96/45.78±4.07	771.15±22.06/159.93±18.5	-	-	-	-
PZ	11.84±1.39/^*4.50±0.65	13.97±1.36/^*5.40±0.73	153.68±13.43/^*28.77±2.15	234.98±33.01/^*45.37±4.7	50.33	54.05	37.16	71.63
ECE	12.98±0.7/^*5.47± 0.76	21.35±1.74/^*8.21±0.99	150.25±11.9/^*26.84±2.83	287.6±30.35/^*54.78±7.85	39.7	65.44	41.38	65.75
EAF	8.33±1.09/^*3.58± 0.52	17.68±1.96 /^*7.08±0.89	89.54±8.97/^*16.86±1.95	202.38±24.84/^*35.49±5.92	60.56	39.74	63.17	77.8

Open in a new tab

C- Control, PZ- Praziquantel treated, ECE- Ethanolic crude extract treated, EAF- Ethyl acetate fraction treated.

Data represents mean value ± SD.

*Values are significant at P ≤ 0.05.

Fig 8 — C- Control, PZ- Praziquantel treated, ECE- Ethanolic crude extract treated, EAF- Ethyl acetate fraction treated.

Discussion

In our present study, the ethanolic crude extract treated worms showed a dose dependent increase in the time of paralysis and death indicating the putative anthelmintic potential of Holarrhena pubescens. The control worms survived for a much longer period than the worms treated with either praziquantel or plant extracts under similar conditions. Amongst the fractions of the ethanolic extract, the worms treated with the Ethyl acetate fraction showed the highest reduction in motility and survivability.

The tegument in cestodes forms a digestive-absorptive-protective surface at the host parasite interface [47, 48]. Ultrastructural studies revealed the presence of polymorphic microtriches in the entire region of the scolex as well as in, immature and mature proglottids, suggesting their role in the absorption of nutrients through the whole body surface [49]. The action of most anthelmintic drugs depend on their passive transfer through this external surface i.e. the tegument [50]. The ultrastructural alteration in the tegument are therefore used as a reliable indicator of anthelmintic activity of a compound [51]. Ultrastructural studies (SEM) showed distortion of the smooth texture of the tegument as well as cracking and folding in the treated worms. This is likely to interfere with the nutrient uptake and energy metabolism of the parasite. Similar type of alterations/damages to the tegument by phytoproduct treated worms have been reported by many authors [41, 52–54]. Albendazole also showed similar types of effects on the tegument [55]. The TEM images further show that the microtrich layer, nuclear membrane, chromatin as well as mitochondria, all suffer damages in the phytoproduct treated worms. This indicates the interference of the phytoproducts with the energy metabolism of the worms as well as functioning of the nucleus. Further, the surface topography and subcellular structures are distorted by the plant extract. The findings are consistent with similar studies with different plant extracts [39, 41, 56, 57].

Tegumental enzymes play key role in maintaining ionic homeostatsis, active transport and metabolic regulation within the parasites [58]. In the present study, the tegumental enzymes namely Acid Phosphatase, Alkaline Phosphatase, Adenosine Triphosphatase and 5’-Nucleotidase show pronounced activity in the tegument, subtegument, somatic musculature and parenchyma of the control worms. However, in the phytoproduct as well as Praziquantel treated worms, there is a marked reduction in the activity of all the four enzymes in the aforementioned regions. Similar results were also evident from the biochemical studies wherein there is a marked reduction in the activity of the tegumental enzymes in the Praziquantel and phytoproduct treated worms. Similar type of results were obtained after treatment with other plant extracts as well [39, 59, 60]. Acid Phosphatase is an important lysosomal enzyme in helminths [58] and the reduction in its activity indicates the disruption of lysosomes in the phytoproduct treated worms [61]. Alkaline Phosphatase is essential for the uptake of glucose through the tegument [62] and reduction in its activity may interfere with the glucose uptake as well as energy metabolism in the worms [36, 41]. Adenosine Triphosphatase in helminths plays a role in the transport of various ions and phospholipids across the tegument [63] and reduction in its activity indicates that the crude plant extract as well as the Ethyl acetate fraction interferes with the transport of different ions as well as the energy metabolism of the worms. Reduction in 5’-Nu activity in the treated worms suggest interference with the uptake of nucleosides [64].

Texture analysis of images is used to quantify spatial differences in the pixels. Gray Level Co-occurrence Matrix (GLCM) is one such method of image texture analysis. It has been used for the enzyme histochemistry in neural tissues [65, 66] and analysis of chromatin architecture [67]. We, for the first time are reporting the use of GLCM for enzyme histochemistry analysis in invertebrates. The MGV represents the brightness of the image and the ID is equivalent to the product of the area and MGV. So, the values of these two parameters are lower in the control images as because the intensity of the staining is higher for all the enzymes in the control images compared to the treated images. ASM and INV measure tissue homogeneity and may increase due to the changes in tissue texture due to increased staining. In our analysis, both ASM and INV are higher in the control images with higher staining intensity than in the phytoproduct treated images. CON measures local differences in the image and ENT measures the lack of spatial organisation. Essentially, CON and ENT will increase with the decrease in image homogeneity. So, the values of CON and ENT are lower in the control images compared to the phytoproduct treated images of the worms. High COR values indicate that the pixels are dependent on one another and vice versa. We obtained slightly higher COR values in control images as compared to treated. From Table 2 it is evident that CON and INV are better indicators of the enzyme histochemistry results.

Conclusion

The results of efficacy and ultrastructural analysis clearly show that the stem bark Ethanolic crude extract and Ethyl acetate fraction of Holarrhena pubescens has anthelmintic potential. Histochemical and biochemical analysis has shown marked reduction in the activity of tegumental enzymes, i.e., AcPase, AlkPase, ATPase and 5´- Nu of the Raillietina spp. The result of various experiments has shown that the Ethyl acetate fraction of crude extract of stem bark of H. pubescens has higher effective anthelmintic property. Our study firmly establishes the anthelmintic potential of H. pubescens. Further studies are required on the identification of the active components of the ethanolic extract as well ethyl acetate fraction of the plant and mechanism of their action, so that, they can be used as candidates for the development of anthelmintic drugs for the control of Raillietinosis in poultry.

Supporting information

S1 Table. Results of efficacy testing of ethanolic crude extract of Holarrhena pubescens and its fractions against Raillietina spp.

(XLSX)

Click here for additional data file.^{(17.6KB, xlsx)}

S2 Table. Data for biochemical analysis of enzyme activity- Acid Phosphatase and Alkaline Phosphatase.

(XLSX)

Click here for additional data file.^{(25.9KB, xlsx)}

S3 Table. Data for biochemical analysis of enzyme activity- 5’ Nucleotidase and ATPase.

(XLSX)

Click here for additional data file.^{(24.8KB, xlsx)}

S4 Table

(ZIP)

Click here for additional data file.^{(60.4KB, zip)}

Acknowledgments

The authors gladly appreciate the constant support and help in conducting the experiments with instrumental and infrastructural facility of the Department of Zoology, Cooch Behar Panchanan Barma University, Cooch Behar. We very much thankful to Department of Botany, University of North Bengal, Darjeeling, West Bengal for the accession number of the plant. The authors are grateful to SAIF, North Eastern Hill University (NEHU), Shillong and Centre for Research in Nanoscience and Nanotechnology (CRNN), Kolkata for their technical support. We also acknowledge the TEM facility of SAIF, All India Institute of Medical Science (AIIMS), New Delhi for their kind cooperation.

Data Availability

All relevant data are within the paper and its Supporting information files.

Funding Statement

The authors received no specific funding for this work.

References

1.Gupta S, Farina JM, Mendoza I, Pérez GE, Martínez-Sellés M, Saldarriaga C, et al. Chapter 2—Malaria & Heart. In: Saldarriaga C, Baranchuk A, editors. Neglected Tropical Diseases and other Infectious Diseases affecting the Heart. Academic Press; 2022. pp. 9–23. [Google Scholar]
2.Hotez PJ, Brindley PJ, Bethony JM, King CH, Pearce EJ, Jacobson J. Helminth infections: The great neglected tropical diseases. J Clin Invest. 2008;118: 1311–1321. doi: 10.1172/JCI34261 [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Hotez PJ, Herricks JR. Helminth Elimination in the Pursuit of Sustainable Development Goals: A “Worm Index” for Human Development. PLoS Negl Trop Dis. 2015;9: 1–6. doi: 10.1371/journal.pntd.0003618 [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Permin A. Impact of Helminth Infections on Production of Chickens. Biomed J Sci Tech Res. 2020;29: 22542–22550. doi: 10.26717/bjstr.2020.29.004819 [DOI] [Google Scholar]
5.Permin A, Hansen JW. Epidemiology, diagnosis and control of poultry parasites. Fao; 1998.
6.Ruff MD. Important parasites in poultry production systems. Vet Parasitol. 1999;84: 337–347. doi: 10.1016/s0304-4017(99)00076-x [DOI] [PubMed] [Google Scholar]
7.Van NTB, Cuong N V, Yen NTP, Nhi NTH, Kiet BT, Hoang N V, et al. Characterisation of gastrointestinal helminths and their impact in commercial small-scale chicken flocks in the Mekong Delta of Vietnam. Trop Anim Health Prod. 2020;52: 53–62. doi: 10.1007/s11250-019-01982-3 [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Yagoob G, Khaki A, Raza SHA, Khan R, Hao L, Hosseini SM, et al. Epidemiological and pathological studies on the helminthic parasites in native chickens of Tabriz city, Iran. Genet Mol Res. 2017;16. doi: 10.4238/gmr16039824 [DOI] [Google Scholar]
9.Shifaw A, Feyera T, Walkden-Brown SW, Sharpe B, Elliott T, Ruhnke I. Global and regional prevalence of helminth infection in chickens over time: a systematic review and meta-analysis. Poult Sci. 2021;100: 101082. doi: 10.1016/j.psj.2021.101082 [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Butboonchoo P, Wongsawad C, Rojanapaibul A, Chai JY. Morphology and molecular phylogeny of raillietina spp. (cestoda: cyclophyllidea: davaineidae) from domestic chickens in Thailand. Korean J Parasitol. 2016;54: 777–786. doi: 10.3347/kjp.2016.54.6.777 [DOI] [PMC free article] [PubMed] [Google Scholar]
11.Reid WM, Ackert JE, Case AA. Studies on the Life History and Biology of the Fowl Tapeworm Raillietina cesticillus (Molin). Trans Am Microsc Soc. 1938;57: 65–76. [Google Scholar]
12.Horsfall MW. Observations on the life history of Raillietina echinobothrida and of R. tetragona (Cestoda). J Parasitol. 1938;24: 409–421. [Google Scholar]
13.Samad MA, Alam MM, Bari ASM. Effect of Raillietina echinobothrida infection on blood values and intestinal tissues of domestic fowls of Bangladesh. Vet Parasitol. 1986;21: 279–284. doi: 10.1016/0304-4017(86)90055-5 [DOI] [PubMed] [Google Scholar]
14.Swayne DE. Diseases of poultry. John Wiley & Sons; 2013. [Google Scholar]
15.Chaudhury A. Raillietina Infection. In: Parija SC, Chaudhury A, editors. Textbook of Parasitic Zoonoses. Singapore: Springer Nature Singapore; 2022. pp. 401–406. [Google Scholar]
16.Baer JG, Sandars DF. The First Record of Raillietina (Raillietina) celebensis (Janicki, 1902), (Cestoda) in Man from Australia, with a Critical Survey of Previous Cases. J Helminthol. 1956;30: 173–182. doi: 10.1017/s0022149x00033137 [DOI] [PubMed] [Google Scholar]
17.Chandler AC, Pradatsundarasar A. Two Cases of Raillietina Infection in Infants in Thailand, with a Discussion of the Taxonomy of the Species of Raillietina (Cestoda) in Man, Rodents and Monkeys. J Parasitol. 1957;43: 81–88. [PubMed] [Google Scholar]
18.Fissiha W, Kinde MZ. Anthelmintic Resistance and Its Mechanism: A Review. Infect Drug Resist. 2021;14: 5403–5410. doi: 10.2147/IDR.S332378 [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Zirintunda G, Biryomumaisho S, Kasozi KI, Batiha GE-S, Kateregga J, Vudriko P, et al. Emerging Anthelmintic Resistance in Poultry: Can Ethnopharmacological Approaches Offer a Solution? Front Pharmacol. 2022;12. doi: 10.3389/fphar.2021.774896 [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Tchetan E, Olounladé PA, Azando EVB, Khaliq HA, Ortiz S, Houngbeme A, et al. Anthelmintic Activity, Cytotoxicity, and Phytochemical Screening of Plants Used to Treat Digestive Parasitosis of Small Ruminants in Benin (West Africa). Animals. 2022;12. doi: 10.3390/ani12192718 [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Romero-Benavides JC, Ruano AL, Silva-Rivas R, Castillo-Veintimilla P, Vivanco-Jaramillo S, Bailon-Moscoso N. Medicinal plants used as anthelmintics: Ethnomedical, pharmacological, and phytochemical studies. Eur J Med Chem. 2017;129: 209–217. doi: 10.1016/j.ejmech.2017.02.005 [DOI] [PubMed] [Google Scholar]
22.Chatterjee D. Forest Rights, Livelihood and Nature Conservation in Buxa Tiger Reserve, India: A Critical Appraisal. Contemp Soc Sci.: 50. [Google Scholar]
23.Mitra S, Mukherjee SK. Ethnomedicinal usages of some wild plants of North Bengal plain for gastro-intestinal problems. 2010.
24.Zahara K, Panda SK, Swain SS, Luyten W. Metabolic diversity and therapeutic potential of holarrhena pubescens: An important ethnomedicinal plant. Biomolecules. 2020;10: 1–28. doi: 10.3390/biom10091341 [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Bhusal A. Evaluation of Antioxidative and Antidiabetic Activity of Bark of Holarrhena Pubescens Wall. J Clin DIAGNOSTIC Res. 2014. doi: 10.7860/JCDR/2014/7803.4863 [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Pradhan BK, Badola HK. Ethnomedicinal plant use by Lepcha tribe of Dzongu valley, bordering Khangchendzonga Biosphere Reserve, in north Sikkim, India. J Ethnobiol Ethnomed. 2008;4: 1–18. [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Sinha S, Sharma A, Reddy PH, Rathi B, Prasad NVSRK, Vashishtha A. Evaluation of phytochemical and pharmacological aspects of Holarrhena antidysenterica (Wall.): A comprehensive review. J Pharm Res. 2013;6: 488–492. doi: 10.1016/j.jopr.2013.04.004 [DOI] [Google Scholar]
28.Bala K, Melkani I, Singh AP, Singh AP, Kaur J. Holarrhena antidysenterica in Inflammatory Bowel Disease: a potential review. J Drug Deliv Ther. 2022;12: 221–226. doi: 10.22270/jddt.v12i4.5458 [DOI] [Google Scholar]
29.Singh KP. Clinical studies on amoebiasis and giardiasis evaluating the efficacy of kutaja (holarrhena antidysenterica) in entamoeba histolytica cyst passers. Anc Sci Life. 1986;5: 228–31. Available: http://www.ncbi.nlm.nih.gov/pubmed/22557529%0Ahttp://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=PMC3331474 [PMC free article] [PubMed] [Google Scholar]
30.Anonymous. The Ayurvedic Pharmacopoeia of India. Part-I, V. New Delhi: Government of India, Ministry of Health and Family Welfare, Department of Ayurveda, Yoga-Naturopathy, Unani, Siddha & Homeopathy.; 2010.
31.Verma G, Dua VK, Agarwal DD, Atul PK. Anti-malarial activity of Holarrhena antidysenterica and Viola canescens, plants traditionally used against malaria in the Garhwal region of north-west Himalaya. Malar J. 2011;10: 20. doi: 10.1186/1475-2875-10-20 [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Siriyong T, Voravuthikunchai SP, Coote PJ. Steroidal alkaloids and conessine from the medicinal plant Holarrhena antidysenterica restore antibiotic efficacy in a Galleria mellonella model of multidrug-resistant Pseudomonas aeruginosa infection 11 Medical and Health Sciences 1108 Medical Microbiolo. BMC Complement Altern Med. 2018;18: 1–10. doi: 10.1186/s12906-018-2348-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Kotze AC. Target-based and whole-worm screening approaches to anthelmintic discovery. Vet Parasitol. 2012;186: 118–123. doi: 10.1016/j.vetpar.2011.11.052 [DOI] [PubMed] [Google Scholar]
34.Herath HMPD, Taki AC, Rostami A, Jabbar A, Keiser J, Geary TG, et al. Whole-organism phenotypic screening methods used in early-phase anthelmintic drug discovery. Biotechnol Adv. 2022;57: 107937. doi: 10.1016/j.biotechadv.2022.107937 [DOI] [PubMed] [Google Scholar]
35.Simon M, Nafanda W, Obeta S. In-vivo evaluation for anthelmintic effect of alkaloids extracted from the stem bark of Afzelia africana in rats. J Adv Sci Res. 2012; 100–104. Available: http://www.sciensage.info/jasr [Google Scholar]
36.Roy B, Giri BR. α-Viniferin-Induced Structural and Functional Alterations in Raillietina echinobothrida, a Poultry Tapeworm. Microsc Microanal. 2015;21: 377–384. doi: 10.1017/S1431927614014603 [DOI] [PubMed] [Google Scholar]
37.Chaffey N. Hayat MA. 2000. Principles and techniques of electron microscopy: biological applications. 4th edn. 543pp. Cambridge: Cambridge University Press. £65 (hardback). Ann Bot. 2001;87: 546–548. doi: 10.1006/anbo.2001.1367 [DOI] [Google Scholar]
38.Pearse A. Histochemistry: Theoretical and applied. 3rd ed. Churchill Livingstone, Edinburgh, London, New York.; 1968. [Google Scholar]
39.Giri BR, Roy B. Resveratrol induced structural and biochemical alterations in the tegument of Raillietina echinobothrida. Parasitol Int. 2014;63: 432–437. doi: 10.1016/j.parint.2013.12.008 [DOI] [PubMed] [Google Scholar]
40.Wachstein M, Meisel E. Histochemistry of hepatic phosphatases of a physiologic pH; with special reference to the demonstration of bile canaliculi. Am J Clin Pathol. 1957;27: 13–23. Available: http://www.ncbi.nlm.nih.gov/pubmed/13410831 doi: 10.1093/ajcp/27.1.13 [DOI] [PubMed] [Google Scholar]
41.Dey P, Roy B. Biochemical and ultrastructural changes in Raillietina echinobothrida in vitro exposed to extract of Lysimachia ramosa. J Parasit Dis. 2018;42: 212–219. doi: 10.1007/s12639-018-0985-z [DOI] [PMC free article] [PubMed] [Google Scholar]
42.Dragić M, Zarić M, Mitrović N, Nedeljković N, Grković I. Application of Gray Level Co-Occurrence Matrix Analysis as a New Method for Enzyme Histochemistry Quantification. Microsc Microanal. 2019;25: 690–698. doi: 10.1017/S1431927618016306 [DOI] [PubMed] [Google Scholar]
43.Abràmoff MD, Magalhães PJ, Ram SJ. Image processing with imageJ. Biophotonics Int. 2004;11: 36–41. doi: 10.1201/9781420005615.ax4 [DOI] [Google Scholar]
44.Plummer D. An introduction to Practical Biochemistry. Tata-McGraw Hill, New Delhi.; 1988. [Google Scholar]
45.Kaplan C. Methods in Enzymology. Academic Press, New York; 1957. [Google Scholar]
46.Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein Measurement With The Folin Phenol Reagent. J Biol Chem. 1951;193. Available: http://devbio.wustl.edu/InfoSource/ISPDFs/Lowry1951.pdf [PubMed] [Google Scholar]
47.Lumsden RD. The Tapeworm Tegument: A Model System for Studies on Membrane Structure and Function in Host-Parasite Relationships. Trans Am Microsc Soc. 1975;94: 501–507. Available: http://www.jstor.org/stable/3225522 [PubMed] [Google Scholar]
48.Thompson DP, Geary TG. 12—The Structure and Function of Helminth Surfaces. In: Marr JJ, Müller M, editors. Biochemistry and Molecular Biology of Parasites. San Diego: Academic Press; 1995. pp. 203–232. [Google Scholar]
49.Radha T, Satyaprema VA, Ramalingam K, Indumathi SP, Venkatesh C. Ultrastructure of polymorphic microtriches in the tegument of Raillietina echinobothrida that infects Gallus domesticus (fowl). J Parasit Dis. 2006;30: 153–162. [Google Scholar]
50.Alvarez LI, Mottier ML, Lanusse CE. Drug transfer into target helminth parasites. Trends Parasitol. 2007;23: 97–104. doi: 10.1016/j.pt.2007.01.003 [DOI] [PubMed] [Google Scholar]
51.Becker B, Mehlhorn H, Andrews P, Thomas H. Ultrastructural investigations on the effect of praziquantel on the tegument of five species of cestodes. Zeitschrift für Parasitenkd. 1981;64: 257–269. doi: 10.1007/BF00927373 [DOI] [PubMed] [Google Scholar]
52.Roy B, Lalchhandama K, Dutta BK. Anticestodal efficacy of Acacia oxyphylla on Raillietin echinobothrida: A light and electron microscopic studis. Pharmacologyonline. 2007;287: 279–287. [Google Scholar]
53.Roy B, Dasgupta S, Tandon V. Ultrastructural observations on tegumental surface of Raillietina echinobothrida and its alterations caused by root-peel extract of Millettia pachycarpa. Microsc Res Tech. 2008;71: 810–815. doi: 10.1002/jemt.20623 [DOI] [PubMed] [Google Scholar]
54.Kar PK, Murmu S, Saha S, Tandon V, Acharya K. Anthelmintic efficacy of gold nanoparticles derived from a phytopathogenic fungus, Nigrospora oryzae. PLoS One. 2014;9: 1–9. doi: 10.1371/journal.pone.0084693 [DOI] [PMC free article] [PubMed] [Google Scholar]
55.Lalchhandama K. In vitro Effects of Albendazole on Raillietina echinobothrida, the Cestode of Chicken, Gallus domesticus. J Young Pharm. 2010;2: 374–378. doi: 10.4103/0975-1483.71630 [DOI] [PMC free article] [PubMed] [Google Scholar]
56.Challam M, Roy B, Tandon V. In vitro anthelmintic efficacy of Carex baccans (Cyperaceae): Ultrastructural, histochemical and biochemical alterations in the cestode, Raillietina echinobothrida. J Parasit Dis. 2012;36: 81–86. doi: 10.1007/s12639-011-0087-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
57.Roy B, Dasgupta S, Manivel V, Parameswaran PS, Giri BR. Surface topographical and ultrastructural alterations of Raillietina echinobothrida and Ascaridia galli induced by a compound isolated from Acacia oxyphylla. Vet Parasitol. 2012;185: 322–326. doi: 10.1016/j.vetpar.2011.09.041 [DOI] [PubMed] [Google Scholar]
58.Smyth JD, McManus DP, others. The physiology and biochemistry of cestodes. Cambridge university press; 1989. [Google Scholar]
59.Roy B, Swargiary A, Syiem D, Tandon V. Potentilla fulgens (Family Rosaceae), a medicinal plant of north-east India: A natural anthelmintic? J Parasit Dis. 2010;34: 83–88. doi: 10.1007/s12639-010-0018-z [DOI] [PMC free article] [PubMed] [Google Scholar]
60.Majumdar R, Kar PK, Giri BR, Roy B. Biosynthesis, characterization and anthelmintic activity of silver nanoparticles of Clerodendrum infortunatum isolate. Sci Rep. 2023;13: 7415. doi: 10.1038/s41598-023-34221-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
61.Tandon V, Pal P, Roy B, Rao HS, Reddy KS. In vitro anthelmintic activity of root-tuber extract of Flemingia vestita, an indigenous plant in Shillong, India. Parasitol Res. 1997;83: 492–8. Available: http://www.ncbi.nlm.nih.gov/pubmed/9197399 doi: 10.1007/s004360050286 [DOI] [PubMed] [Google Scholar]
62.Rogers WP. Histological Distribution of Alkaline Phosphatase in Helminth Parasites. Nature. 1947;159: 374–375. doi: 10.1038/159374b0 [DOI] [PubMed] [Google Scholar]
63.Burenina EA. Adenosine triphosphatases of cestodes Bothriocephalus scorpii. J Evol Biochem Physiol. 2007;43: 287–294. [PubMed] [Google Scholar]
64.Pal P, Tandon V. Anthelmintic efficacy of Flemingia vestita (Leguminoceae): Genistein-induced alterations in the activity of tegumental enzymes in the cestode, Raillietina echinobothrida. Parasitol Int. 1998;47: 233–243. [Google Scholar]
65.Zaletel I, Milutinović K, Bajčetić M, Nowakowski RS. Differentiation of Amyloid Plaques Between Alzheimer’s Disease and Non-Alzheimer’s Disease Individuals Based on Gray-Level Co-occurrence Matrix Texture Analysis. Microsc Microanal. 2021;27: 1146–1153. doi: 10.1017/S1431927621012095 [DOI] [PubMed] [Google Scholar]
66.Chen H, Li W, Zhu Y. Improved window adaptive gray level co-occurrence matrix for extraction and analysis of texture characteristics of pulmonary nodules. Comput Methods Programs Biomed. 2021;208: 106263. doi: 10.1016/j.cmpb.2021.106263 [DOI] [PubMed] [Google Scholar]
67.Paunovic J, Vucevic D, Radosavljevic T, Pantic S, Veskovic M, Pantic I. Gray-level co-occurrence matrix analysis of chromatin architecture in periportal and perivenous hepatocytes. Histochem Cell Biol. 2019;151: 75–83. doi: 10.1007/s00418-018-1714-5 [DOI] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0282033.r001

Decision Letter 0

Josué de Moraes

6 Mar 2023

PONE-D-23-03097Anthelmintic efficacy of Holarrhena pubescens against Raillietina spp. in domestic fowl through ultrastructural, histochemical, biochemical and GLCM analysisPLOS ONE

Dear Dr. Kar,

Please submit your revised manuscript by %DATE_REVISION _DUE%. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Josué de Moraes, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Please note that PLOS ONE has specific guidelines on code sharing for submissions in which author-generated code underpins the findings in the manuscript. In these cases, all author-generated code must be made available without restrictions upon publication of the work.

Please review our guidelines at https://journals.plos.org/plosone/s/materials-and-software-sharing#loc-sharing-code and ensure that your code is shared in a way that follows best practice and facilitates reproducibility and reuse.

3. Please provide the full species name and source for the worms used.

4. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

5. Please amend either the abstract on the online submission form (via Edit Submission) or the abstract in the manuscript so that they are identical.

Additional Editor Comments:

Reviewers have now commented on your paper. You will see that they are advising that you revise your manuscript. If you are prepared to undertake the work required, I would be pleased to reconsider my decision. Moreover, the writing must be seriously improved. In several occasions phrases seems not have connection with the following phrase, besides lack of scientific accuracy in several parts of manuscript and typographic mistakes.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: N/A

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Comments to the authors of the article:

1- It is recommended that in the introduction part of the article, a short explanation about the properties of chemical compounds and active ingredients of Holarrhena pubescens plant extract is given.

2- Raillietina worms isolated from the intestines of birds survived for how many hours in the culture media and laboratory conditions, and by what index is the boundary between the paralysis of the worms and the complete death of the worms determined?

3- It is better for the authors to use more recent references (after 2015) in the article. For example, it is recommended that from the article [Garedaghi yagoob, Khaki, A., Raza, S.H.A., (...), Abdelgayed, S.S., Kakar, M.U. Epidemiological and pathological studies on the helminthic parasites in native chickens of Tabriz city, Iran. Genetics and Molecular Research. 2017; 16(4),gmr16039824. DOI http://dx.doi.org/10.4238/gmr16039824. ] to use in them article references.

4- It is recommended that in the materials and methods section of the article, the exact geographical location (for example, the exact latitude and longitude) of the sampling location of the Holarrhena pubescens plant, which is in the region of West Bengal, India, and also the sampling year should be mentioned.

Reviewer #2: The manuscript “Anthelmintic efficacy of Holarrhena pubescens against Raillietina spp. in domestic fowl through ultrastructural, histochemical, biochemical and GLCM analysis” it is interesting and presents good execution in the methodology. However, I would like the authors to consider the following comments:

- In section: Introduction

The introduction is poor. I suggest describing biological, morphological characters, zoonotic and veterinary importance of Raillietin spp. once infections are highly pathogenic in domestic chickens (Gallus gallus domesticus), for example.

In addition, reporting the role of man as an accidental host in Raillietina spp infections. Human infection is rare, but has been reported from Central and South America, Cuba, Iran, Japan, Southeast Asia, the Philippines, French Polynesia, Australia, and the United States (Hawaii).

Please specify the real objective of this study. Why was Raillietina spp chosen as a study model?

-In section: Materials and Methods- Experiments- Efficacy Testing, line 86.

Write more details as parasites were exposed to extracts. Petri dish? Culture plates? medium of culture?

-In section: Results

Fig. 1. The control group cause Mortality of Raillietina spp. ???

Please insert subtitles on the Y axis of the graph (mortality in hours)

-In section: Discussion

The discussion is poor. I suggest discussing the importance of morphological changes in Raillietina spp. presented in SEM and MET, on the influence on the parasite- host relationship.

In addition, emphasize the need to develop new therapeutic alternatives in the veterinary parasitology field, since Raillietina spp. it represents such importance.

Reviewer #3: The reviewed manuscript describes the obtention and study of anthelmintic properties of ethanolic crude extract and their fractions obtained from different solvents from steam bark of Holarrhena pubescens against Raillietina spp. The manuscript affirms the efficacy of extracts base on comparison with Praziquantel (time of paralysis and time of death), besides observation of effects in parasites by Scanning Electron Microscopy, Transmission Electron Microscopy for instance.

The addition of chromatographic profile, dereplication or any other real information about extract and active fractions would improve the manuscript since it does not afford any information about real composition of evaluated extract and fractions.

The manuscript needs considerably improve in terms of scientific accuracy and correction of several typographic mistakes.

The manuscript in the present format is acceptable for publishing in Plos One since the recommendations below should be taken in consideration or answered.

Major corrections:

1. The authors must carefully and judiciously review the writing of manuscript by complete. There are several points that must be rewrite to text become clearer and present scientific accuracy that is needed.

Minor corrections:

1. It would be possible add consistent information of composition of extracts and active fractions of plant species, besides literature data cited?

2. Plant species can vary easily the chemical composition depending on period of year. Add the date of obtention of plant species.

3. Identification of plant species is important to attest its the real identity? Please afford the A the nunber of voucher specimen and Herbarium where it was deposited.

4. (Line17, 18): The authors say that manuscript focuses on ethanolic extract and ethyl acetate fraction, however fractions obtained from different solvents were evaluated in the efficacy test. As result, EtOH and AcOEt presented more efficacy and then this extracts were subject to following procedures. Please could you explain why n-BuOH was not selected for following tests, since the values are quite similar with EtOH extract (Table 1)?

5. (Line 70) Could you explain the observation “(without chlorophyll)”? What does this mean? Any method to remove chlorophyll was used or just to mention that this part of plant is free of chlorophyll?

6. (line 72) “The dried bark was processed…”. What exactly is processed? Please be clear. This phase as others presents in the manuscript lack of scientific accuracy. Probably, processed means “submitted to extraction”. However, the extraction method is described at lines 75, 76, 77 and 78. The authors must rewrite this section aiming a better description of complete process (since obtention of plant material to fraction obtention).

7. (line72) The extraction process took 15-20 days? Could you explain why and describes in detail the complete methodology?

8. (line 248) In discussion section authors bring to manuscript information about phytochemistry aspects of studied plant species base on literature. However, all these points raised by authors necessarily don’t reflect any considerably discussion to paper in this section. These points must be in introduction to present bioactive compounds of studied species. If authors had performed LC-MS/MS analysis of ETOH extract and/or active fractions and dereplication of compounds, a discussion could explored in this section.

9. (line 306) The section conclusion should be rewrite, since points mentioned don’t bring the idea of conclusion of presented work. The ideas should be expanded. I recommend the authors don’t use sentences as “The result of various experiments has shown”.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Yagoob Garedaghi

Department of Parasitology, Faculty of Veterinary Medicine, Tabriz Medical Sciences, Islamic Azad University, Tabriz, Iran.

Email: Yagoob.garedaghi@gmail.com

https://orcid.org/0000-0003-2976-2706

Reviewer #2: No

Reviewer #3: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Attachment

Submitted filename: PONE-D-23-03097_review-er-revised.pdf

Click here for additional data file.^{(1.9MB, pdf)}

PLoS One. 2023 Sep 14;18(9):e0282033. doi: 10.1371/journal.pone.0282033.r002

Author response to Decision Letter 0

12 Jul 2023

Dear Sir,

With respect to the comments made by the reviewers for our submitted article “Anthelmintic efficacy of Holarrhena pubescens against Raillietina spp. of domestic fowl through ultrastructural, histochemical, biochemical and GLCM analysis” I would like to submit our rebuttal as under.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Response:

All the style requirements of PLOS ONE including those for file naming have been met with in accordance to the links provided.

Response:

DOI 10.1101/2023.02.07.527541

3. Please provide the full species name and source for the worms used.

Response:

Two species of Raillietina occur mostly in the study area namely R.echinobothrida and R. tetragona with the prevalence of R.echinobothrida being higher according to our observations. However, since the sample worms obtained were not segregated according to species during the study, we preferred to use Raillietina spp. in denoting the model organism.

The worms were obtained after dissecting the intestines of freshly slaughtered local fowl (Gallus gallus domesticus) procured from the local market of Cooch Behar, West Bengal, India. We have mentioned the same in the Materials and Methods section, line 148-150.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

Response:

All the minimal data set underlying the results have been uploaded as Supplementary Information files [S1_Table, S1_Fig, S4_Table and S8_Fig, S4_Table and S8_Fig(2)] in the submission portal. There are no ethical or legal restrictions to sharing our data.

5. Please amend either the abstract on the online submission form (via Edit Submission) or the abstract in the manuscript so that they are identical.

Response:

Done via the online submission form.

Additional Editor Comments:

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

________________________________________

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: N/A

________________________________________

3. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

________________________________________

4. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

________________________________________

5. Reviewer Comments to the Author

Reviewer #1: Comments to the authors of the article:

Response:

We have included a short account of the chemical compounds and active ingredients of Holarrhena pubescens plant extracts from literature in the introduction section in two paragraphs from line 104-123 in the revised manuscript.

Response:

The control worms survived for about 72 hours in the culture media and laboratory conditions. The boundary between paralysis and death of the worms were determined by the absence of movement in the paralysed worms when transferred to warm PBS. We have included the description in the Materials and methods section line no. 157-161 in the revised manuscript.

3- It is better for the authors to use more recent references (after 2015) in the article. For example, it is recommended that from the article [Garedaghiyagoob, Khaki, A., Raza, S.H.A., (...), Abdelgayed, S.S., Kakar, M.U. Epidemiological and pathological studies on the helminthic parasites in native chickens of Tabriz city, Iran. Genetics and Molecular Research. 2017; 16(4),gmr16039824. DOI http://dx.doi.org/10.4238/gmr16039824. ] to use in them article references.

Response:

As advised, we have included new and recent references in our manuscript. The number of references have increased from 40 to 68 in the revised manuscript.

4- It is recommended that in the materials and methods section of the article, the exact geographical location (for example, the exact latitude and longitude) of the sampling location of the Holarrhenapubescens plant, which is in the region of West Bengal, India, and also the sampling year should be mentioned.

Response:

Fresh bark was collected from the plant H. pubescens from the town of Cooch Behar (26.3169° N, 89.4457° E), West Bengal, India in month of April- June, Since 2018 till date. We have added it in the Materials and method section line no. 134-135 in the revised manuscript.

- In section: Introduction

In addition, reporting the role of man as an accidental host in Raillietinaspp infections. Human infection is rare, but has been reported from Central and South America, Cuba, Iran, Japan, Southeast Asia, the Philippines, French Polynesia, Australia, and the United States (Hawaii).

Please specify the real objective of this study. Why was Raillietina spp. chosen as a study model?

Response:

The introduction section has been restructured considering the comments of the reviewer and all the mentioned points have been included in the revised manuscript.

-In section: Materials and Methods- Experiments- Efficacy Testing, line 86.

Write more details as parasites were exposed to extracts. Petri dish? Culture plates? medium of culture?

Response:

The parasites were cultured in Phosphate Buffer Saline (PBS), pH 7.4 at 370C in petri dishes in an incubator. We have added this in line no. 154 in Materials and Methods section in the revised manuscript.

-In section: Results

Fig. 1. The control group cause Mortality of Raillietina spp. ???

Response:

No, but the worms survived in PBS for a limited period of time i.e.about 71 hours.

Please insert subtitles on the Y axis of the graph (mortality in hours)

Response:

Done as advised.

-In section: Discussion

The discussion is poor. I suggest discussing the importance of morphological changes in Raillietina spp. presented in SEM and MET, on the influence on the parasite- host relationship.

In addition, emphasize the need to develop new therapeutic alternatives in the veterinary parasitology field, since Raillietina spp. it represents such importance.

Response:

The paragraph in the Discussion section line no. 329- 341 has been rewritten to include the points mentioned by the reviewer. Other sections have been rephrased as well as new information has been added to augment the discussion.

The manuscript needs considerably improve in terms of scientific accuracy and correction of several typographic mistakes.

The manuscript in the present format is acceptable for publishing in Plos One since the recommendations below should be taken in consideration or answered.

Major corrections:

Response:

Many parts of the manuscript have been rephrased as well as additional points have been included as suggested to improve the quality of English as well as clarity of scientific expression in the revised manuscript.

Minor corrections:

1. It would be possible add consistent information of composition of extracts and active fractions of plant species, besides literature data cited?

Response:

Information about the composition of the extracts have been added in the introduction section line no. 104-123 in the revised manuscript from literature. However, data on the phytochemical characterisation of our plant extract is still not presentable though we plan to publish the same in the near future.

2. Plant species can vary easily the chemical composition depending on period of year. Add the date of obtention of plant species.

Response:

2018, added in Materials and Methods section line no. 134-135 of the revised manuscript.

3. Identification of plant species is important to attest it’s the real identity? Please afford the A the nunber of voucher specimen and Herbarium where it was deposited.

Respose.

Dr. Monoranjan Chowdhury identified the plant and the accession number is- 12638. It was kept at herbarium of Department of Botany, University of North Bengal, Darjeeling, West Bengal. Mention in Materials and Methods section line no. 135-137 of the revised manuscript

Response:

The ethanolic extract was first used for efficacy testing. Then the same extract was then subjected to fractionation using hexane, chloroform, ethyl acetate and n-butanol. These four fractions obtained from the ethanolic extract were again used for efficacy testing, out of which the ethyl acetate fraction showed the highest efficacy.

Response:

No method of removing chlorophyll was used. However, the outer layer of the obtained bark was peeled to obtain the fresh brownish inner layer. Nevertheless, we have removed the phrase “without chlorophyll” from the revised manuscript.

Response:

We have rephrased the mentioned section in the Materials and Methods line no. 135-145 in the revised manuscript to improve the scientific accuracy.

7. (line72) The extraction process took 15-20 days? Could you explain why and describes in detail the complete methodology?

Response:

The dried bark was kept immersed in ethanol for 15-20 days with repeated stirring to maximize the yield of phytochemicals.

Response:

As advised, we have moved the section dealing with the phytochemistry aspects of the plant to the Introduction section line no. 104-123 in the revised manuscript.

We have not performed any LC-MS/MS analysis of the extract yet.

Response:

The conclusion has been rewritten as advised.

________________________________________

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

We do not wish to make the peer review history public.

Hope this will suffice for now. We are open for discussion for any further comments made by the reviewers.

Thanking you,

Yours sincerely,

Dr. Pradip Kumar Kar

Attachment

Submitted filename: Rebuttal Letter.pdf

Click here for additional data file.^{(500.2KB, pdf)}

PLoS One. doi: 10.1371/journal.pone.0282033.r003

Decision Letter 1

Josué de Moraes

26 Jul 2023

PONE-D-23-03097R1Anthelmintic efficacy of Holarrhena pubescens against Raillietina spp. of domestic fowl through ultrastructural, histochemical, biochemical and GLCM analysisPLOS ONE

Dear Dr. Kar,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. The authors have made significant improvements to the manuscript compared to the first version. However, there are still some typographical errors and sentences that need to be rephrased to enhance the text's conciseness and fluency, which are crucial for a scientific article. I recommend that the authors thoroughly revise the text to enhance its English quality. Utilizing the services of a native English speaker would be beneficial in this regard.

Please submit your revised manuscript by August 15, 2023. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Josué de Moraes, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

I recommend that the authors thoroughly revise the text to enhance its English quality. Utilizing the services of a native English speaker would be beneficial in this regard.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Reviewer #1: it is recommended that from the article [Garedaghi yagoob, Khaki, A., Raza, S.H.A., (...), Abdelgayed, S.S., Kakar, M.U. Epidemiological and pathological studies on the helminthic parasites in native chickens of Tabriz city, Iran. Genetics and Molecular Research. 2017; 16(4),gmr16039824. DOI http://dx.doi.org/10.4238/gmr16039824. ] to use in them article references.

Reviewer #3: The authors improved the manuscript compared to the first version. However, it is necessary several adjustments in the text taking into consideration typographic mistakes and phrases that must be rewritten to let the text concise and fluid which is necessary for a scientific paper. Unfortunately, the section that describes the extraction procedure, as well as the obtention of organic phases, remains without details. Even though the focus of the work is the evaluation of anthelmintic properties of extract and organic phases of H. pubescens, details of the obtention of these materials are required and essential. As a final consideration, it would be helpful to submit the text to a scientific-technical translation company to adjust the manuscript to be published.

Bellow follows specific comments and considerations to the authors.

Minor corrections:

1. Please, maintain a solvent nomenclature standardized. Use the first letter capitalized for all of them or none of them (except at the beginning of a phrase, for instance);

2. Use italics for “n” before “-butanol”.

3. Please, pay attention to spaces between characters, punctuation, and hyphen. Several points must be reviewed.

4. The same aspect is seen for units after numbers. Please follow a pattern and check spaces between numbers and units.

5. Lines 143 and 144 – Unfortunately the procedure described is not clear enough. Please be specific as much as you can.

6. Lines 143 and 144 – It is necessary to inform the right procedure of liquid-liquid extraction as requested previously. Be specific in terms of the used mass of crude extract, how this crude extract was solubilized (what solvent), or partially solubilized, the addition order of solvents, quantities of used solvents the final mass of each phase obtained, for instance.

7. Is not completely clear how and which procedures (liquid-liquid or fractional distillation) were employed or in which stage each of them was used.

8. Line 144 – Could you explain how fractional distillation was employed to obtain the organic phases?.

9. Despite information about the chemistry of plant species was included in the introduction, the text must be reviewed from lines 100 to 122 to become more concise and let the text fluid. The whole idea of this part is impaired by the repetition of ideas and phrases that should appear in a better position to bring consistency to the text.

10. Line 111 – Please, replace “Kutaja” with the scientific name of the plant species.

11. Lines 114 to 117 – Please, include the reference about alkaloids in H. pubescens.

12. From lines 134 to 137 – This part of the text needs to be rewritten to bring fluidity.

13. In many parts of the manuscript, the authors don’t let clear the amount of material or time of procedure used. For example, lines 137, 138, 141, and other parts. Why is not shown the exact amount of material and time used?.

14. Line 141 – As a suggestion replace “stored in a container at” with “stored under refrigeration at”.

15. Line 155 mentions a 20 mg/mL concentration in the row of samples tested for efficacy testing. However, it is not seen this concentration in Table 1. Please add the value in the table or remove the information (line 155).

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Yagoob Garedaghi

1. Department of Parasitology, Faculty of Veterinary Medicine, Tabriz Medical Sciences, Islamic Azad University, Tabriz, Iran.

https://orcid.org/0000-0003-2976-2706

Reviewer #3: No

**********

PLoS One. 2023 Sep 14;18(9):e0282033. doi: 10.1371/journal.pone.0282033.r004

Author response to Decision Letter 1

1 Aug 2023

Dear Sir,

With respect to the comments made by the reviewers for our submitted article “Anthelmintic efficacy of Holarrhena pubescens against Raillietina spp. Of domestic fowl through ultrastructural, histochemical, biochemical and GLCM analysis” I would like to submit our rebuttal as under.

Additional Editor Comments (if provided):

I recommend that the authors thoroughly revise the text to enhance its English quality. Utilizing the services of a native English speaker would be beneficial in this regard.

Response

Response:

The authors revised the manuscript as and where necessary, and, would like to state that the quality of English of this communication conforms to existing standard comparable to articles published in reputed high-impact journals. The author would like to further reiterate that this manuscript has been written as per convention practiced by Parasitologists, world over, for publication of anthelmintic efficacy data. The corresponding author has already published two articles in Plos One which has been appreciated by the scientific community.

1) Pradip Kumar Kar, Sanatan Murmu, Saswati Saha, Veena Tandon, Krishnendu Acharya. Anthelmintic efficacy of gold nanoparticles derived from phytopathogenic fungi Nigrospora oryzae. Plos One (2014) 9(1): e84693, ISSN No. – 1932-6203, January 2014

2) Anirban Ash, Tomáš Scholz, Alain de Chambrier, Jan Brabec, Mikuláš Oros, Pradip Kumar Kar, Shivaji Prabhakar Chavan, Jean Mariaux. Revision of Gangesia (Cestoda: Proteocephalidea) in the Indomalayan Region: morphology, molecules and surface ultrastructure. Plos One (2012) 7(10): 1-28, e46421 ISSN No. – 1932-6203, October 2012

Hence, the authors would be highly obliged if this explanation suffices the quandary on the quality of English of this article, and would like to provide further clarification / justification if so desired by the editorial team.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #3: (No Response)

________________________________________

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #3: Yes

________________________________________

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #3: Yes

________________________________________

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #3: Yes

________________________________________

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #3: Yes

________________________________________

6. Review Comments to the Author

Reviewer #1: it is recommended that from the article [Garedaghiyagoob, Khaki, A., Raza, S.H.A., (...), Abdelgayed, S.S., Kakar, M.U. Epidemiological and pathological studies on the helminthic parasites in native chickens of Tabriz city, Iran. Genetics and Molecular Research. 2017; 16(4),gmr16039824. DOI http://dx.doi.org/10.4238/gmr16039824. ] to use in them article references.

Response:

It has been done. Line number: 72

Bellow follows specific comments and considerations to the authors.

Minor corrections:

1. Please, maintain a solvent nomenclature standardized. Use the first letter capitalized for all of them or none of them (except at the beginning of a phrase, for instance);

Response:

Done as advised.

2. Use italics for “n” before “-butanol”.

Response:

Done as advised.

3. Please, pay attention to spaces between characters, punctuation, and hyphen. Several points must be reviewed.

Response:

Done as advised.

4. The same aspect is seen for units after numbers. Please follow a pattern and check spaces between numbers and units.

Response:

Done as advised.

5. Lines 143 and 144 – Unfortunately the procedure described is not clear enough. Please be specific as much as you can.

Response:

50 ml of aqueous crude extract solution (2.5 grm in 50 ml distilled water) was combined with 50 ml of Hexane and left to stand for 2 hours. The Hexane fraction was subsequently removed from the upper phase using the liquid liquid separation technique. The method was repeated three times using Chloroform (lower phase), Ethyl acetate, and n-Butanol. The final volume of each fraction (150 ml) was collected and dried using a Rotary Vacuum Evaporator (Buchi Rotavapor R-100). Around 0.25 g, 0.14 g, 0.92 g and 1.1 g of Hexane, Chloroform, Ethyl acetate and n-Butanol fractions were obtained from 2.5 g of crude extract respectively. Mentioned in Materials and Methods section line no. 143-148 of the revised manuscript

Response:

Mentioned in Materials and Methods section line no. 143-148 of the revised manuscript

7. Is not completely clear how and which procedures (liquid-liquid or fractional distillation) were employed or in which stage each of them was used.

Response:

Mentioned in Materials and Methods section line no. 143-148 of the revised manuscript

8. Line 144 – Could you explain how fractional distillation was employed to obtain the organic phases?.

Response:

Mentioned in Materials and Methods section line no. 143-148 of the revised manuscript

Response:

Done as advised.

10. Line 111 – Please, replace “Kutaja” with the scientific name of the plant species.

Response:

Done as advised.

11. Lines 114 to 117 – Please, include the reference about alkaloids in H. pubescens.

Response:

Included in line number 115.

12. From lines 134 to 137 – This part of the text needs to be rewritten to bring fluidity.

Response:

Done as advised.

Response:

The plant materials were finely chopped and air dried for 24-36 hours after being collected. The dried bark components were soaked in ethanol in a glass container (100 grams in 500 ml) for 15-20 days with frequent stirring. The solution was then filtered using Whatman Filter Paper (No. 14), and then dried using a Rotary Vacuum Evaporator (Buchi Rotavapor R-100). After drying, the collected crude extracts of plant material were stored under refrigeration at 4°C until further use. Around 7.0-8.0 gram (g) of plant extract was obtained from 100 g of plant material soaked in 500 millilitre (ml) ethanol.

It has been clearly mentioned in the main manuscript earlier, line number 135 -142 that only stem bark is used for crude extract preparation and other subsequent experiments.

14. Line 141 – As a suggestion replace “stored in a container at” with “stored under refrigeration at”.

Response:

Done as advised.

Response:

Removed the information.

________________________________________

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Response:

We do not wish to make the peer review history public.

Hope this will suffice for now. We are open for discussion for any further comments made by the reviewers.

Thanking you,

Yours sincerely,

Dr. Pradip Kumar Kar

Attachment

Submitted filename: Rebuttal Letter.pdf

Click here for additional data file.^{(673KB, pdf)}

PLoS One. doi: 10.1371/journal.pone.0282033.r005

Decision Letter 2

Josué de Moraes

21 Aug 2023

Anthelmintic efficacy of Holarrhena pubescens against Raillietina spp. of domestic fowl through ultrastructural, histochemical, biochemical and GLCM analysis

PONE-D-23-03097R2

Dear Dr. Kumar Kar,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Josué de Moraes, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #3: Yes

**********

6. Review Comments to the Author

Reviewer #3: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #3: No

**********

PLoS One. doi: 10.1371/journal.pone.0282033.r006

Acceptance letter

Josué de Moraes

1 Sep 2023

PONE-D-23-03097R2

Anthelmintic efficacy of Holarrhena pubescens against Raillietina spp. of domestic fowl through ultrastructural, histochemical, biochemical and GLCM analysis

Dear Dr. Kar:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Josué de Moraes

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. Results of efficacy testing of ethanolic crude extract of Holarrhena pubescens and its fractions against Raillietina spp.

(XLSX)

Click here for additional data file.^{(17.6KB, xlsx)}

S2 Table. Data for biochemical analysis of enzyme activity- Acid Phosphatase and Alkaline Phosphatase.

(XLSX)

Click here for additional data file.^{(25.9KB, xlsx)}

S3 Table. Data for biochemical analysis of enzyme activity- 5’ Nucleotidase and ATPase.

(XLSX)

Click here for additional data file.^{(24.8KB, xlsx)}

S4 Table

(ZIP)

Click here for additional data file.^{(60.4KB, zip)}

Attachment

Submitted filename: PONE-D-23-03097_review-er-revised.pdf

Click here for additional data file.^{(1.9MB, pdf)}

Attachment

Submitted filename: Rebuttal Letter.pdf

Click here for additional data file.^{(500.2KB, pdf)}

Attachment

Submitted filename: Rebuttal Letter.pdf

Click here for additional data file.^{(673KB, pdf)}

Data Availability Statement

All relevant data are within the paper and its Supporting information files.

[pone.0282033.ref001] 1.Gupta S, Farina JM, Mendoza I, Pérez GE, Martínez-Sellés M, Saldarriaga C, et al. Chapter 2—Malaria & Heart. In: Saldarriaga C, Baranchuk A, editors. Neglected Tropical Diseases and other Infectious Diseases affecting the Heart. Academic Press; 2022. pp. 9–23. [Google Scholar]

[pone.0282033.ref002] 2.Hotez PJ, Brindley PJ, Bethony JM, King CH, Pearce EJ, Jacobson J. Helminth infections: The great neglected tropical diseases. J Clin Invest. 2008;118: 1311–1321. doi: 10.1172/JCI34261 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0282033.ref003] 3.Hotez PJ, Herricks JR. Helminth Elimination in the Pursuit of Sustainable Development Goals: A “Worm Index” for Human Development. PLoS Negl Trop Dis. 2015;9: 1–6. doi: 10.1371/journal.pntd.0003618 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0282033.ref004] 4.Permin A. Impact of Helminth Infections on Production of Chickens. Biomed J Sci Tech Res. 2020;29: 22542–22550. doi: 10.26717/bjstr.2020.29.004819 [DOI] [Google Scholar]

[pone.0282033.ref005] 5.Permin A, Hansen JW. Epidemiology, diagnosis and control of poultry parasites. Fao; 1998.

[pone.0282033.ref006] 6.Ruff MD. Important parasites in poultry production systems. Vet Parasitol. 1999;84: 337–347. doi: 10.1016/s0304-4017(99)00076-x [DOI] [PubMed] [Google Scholar]

[pone.0282033.ref007] 7.Van NTB, Cuong N V, Yen NTP, Nhi NTH, Kiet BT, Hoang N V, et al. Characterisation of gastrointestinal helminths and their impact in commercial small-scale chicken flocks in the Mekong Delta of Vietnam. Trop Anim Health Prod. 2020;52: 53–62. doi: 10.1007/s11250-019-01982-3 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0282033.ref008] 8.Yagoob G, Khaki A, Raza SHA, Khan R, Hao L, Hosseini SM, et al. Epidemiological and pathological studies on the helminthic parasites in native chickens of Tabriz city, Iran. Genet Mol Res. 2017;16. doi: 10.4238/gmr16039824 [DOI] [Google Scholar]

[pone.0282033.ref009] 9.Shifaw A, Feyera T, Walkden-Brown SW, Sharpe B, Elliott T, Ruhnke I. Global and regional prevalence of helminth infection in chickens over time: a systematic review and meta-analysis. Poult Sci. 2021;100: 101082. doi: 10.1016/j.psj.2021.101082 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0282033.ref010] 10.Butboonchoo P, Wongsawad C, Rojanapaibul A, Chai JY. Morphology and molecular phylogeny of raillietina spp. (cestoda: cyclophyllidea: davaineidae) from domestic chickens in Thailand. Korean J Parasitol. 2016;54: 777–786. doi: 10.3347/kjp.2016.54.6.777 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0282033.ref011] 11.Reid WM, Ackert JE, Case AA. Studies on the Life History and Biology of the Fowl Tapeworm Raillietina cesticillus (Molin). Trans Am Microsc Soc. 1938;57: 65–76. [Google Scholar]

[pone.0282033.ref012] 12.Horsfall MW. Observations on the life history of Raillietina echinobothrida and of R. tetragona (Cestoda). J Parasitol. 1938;24: 409–421. [Google Scholar]

[pone.0282033.ref013] 13.Samad MA, Alam MM, Bari ASM. Effect of Raillietina echinobothrida infection on blood values and intestinal tissues of domestic fowls of Bangladesh. Vet Parasitol. 1986;21: 279–284. doi: 10.1016/0304-4017(86)90055-5 [DOI] [PubMed] [Google Scholar]

[pone.0282033.ref014] 14.Swayne DE. Diseases of poultry. John Wiley & Sons; 2013. [Google Scholar]

[pone.0282033.ref015] 15.Chaudhury A. Raillietina Infection. In: Parija SC, Chaudhury A, editors. Textbook of Parasitic Zoonoses. Singapore: Springer Nature Singapore; 2022. pp. 401–406. [Google Scholar]

[pone.0282033.ref016] 16.Baer JG, Sandars DF. The First Record of Raillietina (Raillietina) celebensis (Janicki, 1902), (Cestoda) in Man from Australia, with a Critical Survey of Previous Cases. J Helminthol. 1956;30: 173–182. doi: 10.1017/s0022149x00033137 [DOI] [PubMed] [Google Scholar]

[pone.0282033.ref017] 17.Chandler AC, Pradatsundarasar A. Two Cases of Raillietina Infection in Infants in Thailand, with a Discussion of the Taxonomy of the Species of Raillietina (Cestoda) in Man, Rodents and Monkeys. J Parasitol. 1957;43: 81–88. [PubMed] [Google Scholar]

[pone.0282033.ref018] 18.Fissiha W, Kinde MZ. Anthelmintic Resistance and Its Mechanism: A Review. Infect Drug Resist. 2021;14: 5403–5410. doi: 10.2147/IDR.S332378 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0282033.ref019] 19.Zirintunda G, Biryomumaisho S, Kasozi KI, Batiha GE-S, Kateregga J, Vudriko P, et al. Emerging Anthelmintic Resistance in Poultry: Can Ethnopharmacological Approaches Offer a Solution? Front Pharmacol. 2022;12. doi: 10.3389/fphar.2021.774896 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0282033.ref020] 20.Tchetan E, Olounladé PA, Azando EVB, Khaliq HA, Ortiz S, Houngbeme A, et al. Anthelmintic Activity, Cytotoxicity, and Phytochemical Screening of Plants Used to Treat Digestive Parasitosis of Small Ruminants in Benin (West Africa). Animals. 2022;12. doi: 10.3390/ani12192718 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0282033.ref021] 21.Romero-Benavides JC, Ruano AL, Silva-Rivas R, Castillo-Veintimilla P, Vivanco-Jaramillo S, Bailon-Moscoso N. Medicinal plants used as anthelmintics: Ethnomedical, pharmacological, and phytochemical studies. Eur J Med Chem. 2017;129: 209–217. doi: 10.1016/j.ejmech.2017.02.005 [DOI] [PubMed] [Google Scholar]

[pone.0282033.ref022] 22.Chatterjee D. Forest Rights, Livelihood and Nature Conservation in Buxa Tiger Reserve, India: A Critical Appraisal. Contemp Soc Sci.: 50. [Google Scholar]

[pone.0282033.ref023] 23.Mitra S, Mukherjee SK. Ethnomedicinal usages of some wild plants of North Bengal plain for gastro-intestinal problems. 2010.

[pone.0282033.ref024] 24.Zahara K, Panda SK, Swain SS, Luyten W. Metabolic diversity and therapeutic potential of holarrhena pubescens: An important ethnomedicinal plant. Biomolecules. 2020;10: 1–28. doi: 10.3390/biom10091341 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0282033.ref025] 25.Bhusal A. Evaluation of Antioxidative and Antidiabetic Activity of Bark of Holarrhena Pubescens Wall. J Clin DIAGNOSTIC Res. 2014. doi: 10.7860/JCDR/2014/7803.4863 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0282033.ref026] 26.Pradhan BK, Badola HK. Ethnomedicinal plant use by Lepcha tribe of Dzongu valley, bordering Khangchendzonga Biosphere Reserve, in north Sikkim, India. J Ethnobiol Ethnomed. 2008;4: 1–18. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0282033.ref027] 27.Sinha S, Sharma A, Reddy PH, Rathi B, Prasad NVSRK, Vashishtha A. Evaluation of phytochemical and pharmacological aspects of Holarrhena antidysenterica (Wall.): A comprehensive review. J Pharm Res. 2013;6: 488–492. doi: 10.1016/j.jopr.2013.04.004 [DOI] [Google Scholar]

[pone.0282033.ref028] 28.Bala K, Melkani I, Singh AP, Singh AP, Kaur J. Holarrhena antidysenterica in Inflammatory Bowel Disease: a potential review. J Drug Deliv Ther. 2022;12: 221–226. doi: 10.22270/jddt.v12i4.5458 [DOI] [Google Scholar]

[pone.0282033.ref029] 29.Singh KP. Clinical studies on amoebiasis and giardiasis evaluating the efficacy of kutaja (holarrhena antidysenterica) in entamoeba histolytica cyst passers. Anc Sci Life. 1986;5: 228–31. Available: http://www.ncbi.nlm.nih.gov/pubmed/22557529%0Ahttp://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=PMC3331474 [PMC free article] [PubMed] [Google Scholar]

[pone.0282033.ref030] 30.Anonymous. The Ayurvedic Pharmacopoeia of India. Part-I, V. New Delhi: Government of India, Ministry of Health and Family Welfare, Department of Ayurveda, Yoga-Naturopathy, Unani, Siddha & Homeopathy.; 2010.

[pone.0282033.ref031] 31.Verma G, Dua VK, Agarwal DD, Atul PK. Anti-malarial activity of Holarrhena antidysenterica and Viola canescens, plants traditionally used against malaria in the Garhwal region of north-west Himalaya. Malar J. 2011;10: 20. doi: 10.1186/1475-2875-10-20 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0282033.ref032] 32.Siriyong T, Voravuthikunchai SP, Coote PJ. Steroidal alkaloids and conessine from the medicinal plant Holarrhena antidysenterica restore antibiotic efficacy in a Galleria mellonella model of multidrug-resistant Pseudomonas aeruginosa infection 11 Medical and Health Sciences 1108 Medical Microbiolo. BMC Complement Altern Med. 2018;18: 1–10. doi: 10.1186/s12906-018-2348-9 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0282033.ref033] 33.Kotze AC. Target-based and whole-worm screening approaches to anthelmintic discovery. Vet Parasitol. 2012;186: 118–123. doi: 10.1016/j.vetpar.2011.11.052 [DOI] [PubMed] [Google Scholar]

[pone.0282033.ref034] 34.Herath HMPD, Taki AC, Rostami A, Jabbar A, Keiser J, Geary TG, et al. Whole-organism phenotypic screening methods used in early-phase anthelmintic drug discovery. Biotechnol Adv. 2022;57: 107937. doi: 10.1016/j.biotechadv.2022.107937 [DOI] [PubMed] [Google Scholar]

[pone.0282033.ref035] 35.Simon M, Nafanda W, Obeta S. In-vivo evaluation for anthelmintic effect of alkaloids extracted from the stem bark of Afzelia africana in rats. J Adv Sci Res. 2012; 100–104. Available: http://www.sciensage.info/jasr [Google Scholar]

[pone.0282033.ref036] 36.Roy B, Giri BR. α-Viniferin-Induced Structural and Functional Alterations in Raillietina echinobothrida, a Poultry Tapeworm. Microsc Microanal. 2015;21: 377–384. doi: 10.1017/S1431927614014603 [DOI] [PubMed] [Google Scholar]

[pone.0282033.ref037] 37.Chaffey N. Hayat MA. 2000. Principles and techniques of electron microscopy: biological applications. 4th edn. 543pp. Cambridge: Cambridge University Press. £65 (hardback). Ann Bot. 2001;87: 546–548. doi: 10.1006/anbo.2001.1367 [DOI] [Google Scholar]

[pone.0282033.ref038] 38.Pearse A. Histochemistry: Theoretical and applied. 3rd ed. Churchill Livingstone, Edinburgh, London, New York.; 1968. [Google Scholar]

[pone.0282033.ref039] 39.Giri BR, Roy B. Resveratrol induced structural and biochemical alterations in the tegument of Raillietina echinobothrida. Parasitol Int. 2014;63: 432–437. doi: 10.1016/j.parint.2013.12.008 [DOI] [PubMed] [Google Scholar]

[pone.0282033.ref040] 40.Wachstein M, Meisel E. Histochemistry of hepatic phosphatases of a physiologic pH; with special reference to the demonstration of bile canaliculi. Am J Clin Pathol. 1957;27: 13–23. Available: http://www.ncbi.nlm.nih.gov/pubmed/13410831 doi: 10.1093/ajcp/27.1.13 [DOI] [PubMed] [Google Scholar]

[pone.0282033.ref041] 41.Dey P, Roy B. Biochemical and ultrastructural changes in Raillietina echinobothrida in vitro exposed to extract of Lysimachia ramosa. J Parasit Dis. 2018;42: 212–219. doi: 10.1007/s12639-018-0985-z [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0282033.ref042] 42.Dragić M, Zarić M, Mitrović N, Nedeljković N, Grković I. Application of Gray Level Co-Occurrence Matrix Analysis as a New Method for Enzyme Histochemistry Quantification. Microsc Microanal. 2019;25: 690–698. doi: 10.1017/S1431927618016306 [DOI] [PubMed] [Google Scholar]

[pone.0282033.ref043] 43.Abràmoff MD, Magalhães PJ, Ram SJ. Image processing with imageJ. Biophotonics Int. 2004;11: 36–41. doi: 10.1201/9781420005615.ax4 [DOI] [Google Scholar]

[pone.0282033.ref044] 44.Plummer D. An introduction to Practical Biochemistry. Tata-McGraw Hill, New Delhi.; 1988. [Google Scholar]

[pone.0282033.ref045] 45.Kaplan C. Methods in Enzymology. Academic Press, New York; 1957. [Google Scholar]

[pone.0282033.ref046] 46.Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein Measurement With The Folin Phenol Reagent. J Biol Chem. 1951;193. Available: http://devbio.wustl.edu/InfoSource/ISPDFs/Lowry1951.pdf [PubMed] [Google Scholar]

[pone.0282033.ref047] 47.Lumsden RD. The Tapeworm Tegument: A Model System for Studies on Membrane Structure and Function in Host-Parasite Relationships. Trans Am Microsc Soc. 1975;94: 501–507. Available: http://www.jstor.org/stable/3225522 [PubMed] [Google Scholar]

[pone.0282033.ref048] 48.Thompson DP, Geary TG. 12—The Structure and Function of Helminth Surfaces. In: Marr JJ, Müller M, editors. Biochemistry and Molecular Biology of Parasites. San Diego: Academic Press; 1995. pp. 203–232. [Google Scholar]

[pone.0282033.ref049] 49.Radha T, Satyaprema VA, Ramalingam K, Indumathi SP, Venkatesh C. Ultrastructure of polymorphic microtriches in the tegument of Raillietina echinobothrida that infects Gallus domesticus (fowl). J Parasit Dis. 2006;30: 153–162. [Google Scholar]

[pone.0282033.ref050] 50.Alvarez LI, Mottier ML, Lanusse CE. Drug transfer into target helminth parasites. Trends Parasitol. 2007;23: 97–104. doi: 10.1016/j.pt.2007.01.003 [DOI] [PubMed] [Google Scholar]

[pone.0282033.ref051] 51.Becker B, Mehlhorn H, Andrews P, Thomas H. Ultrastructural investigations on the effect of praziquantel on the tegument of five species of cestodes. Zeitschrift für Parasitenkd. 1981;64: 257–269. doi: 10.1007/BF00927373 [DOI] [PubMed] [Google Scholar]

[pone.0282033.ref052] 52.Roy B, Lalchhandama K, Dutta BK. Anticestodal efficacy of Acacia oxyphylla on Raillietin echinobothrida: A light and electron microscopic studis. Pharmacologyonline. 2007;287: 279–287. [Google Scholar]

[pone.0282033.ref053] 53.Roy B, Dasgupta S, Tandon V. Ultrastructural observations on tegumental surface of Raillietina echinobothrida and its alterations caused by root-peel extract of Millettia pachycarpa. Microsc Res Tech. 2008;71: 810–815. doi: 10.1002/jemt.20623 [DOI] [PubMed] [Google Scholar]

[pone.0282033.ref054] 54.Kar PK, Murmu S, Saha S, Tandon V, Acharya K. Anthelmintic efficacy of gold nanoparticles derived from a phytopathogenic fungus, Nigrospora oryzae. PLoS One. 2014;9: 1–9. doi: 10.1371/journal.pone.0084693 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0282033.ref055] 55.Lalchhandama K. In vitro Effects of Albendazole on Raillietina echinobothrida, the Cestode of Chicken, Gallus domesticus. J Young Pharm. 2010;2: 374–378. doi: 10.4103/0975-1483.71630 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0282033.ref056] 56.Challam M, Roy B, Tandon V. In vitro anthelmintic efficacy of Carex baccans (Cyperaceae): Ultrastructural, histochemical and biochemical alterations in the cestode, Raillietina echinobothrida. J Parasit Dis. 2012;36: 81–86. doi: 10.1007/s12639-011-0087-7 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0282033.ref057] 57.Roy B, Dasgupta S, Manivel V, Parameswaran PS, Giri BR. Surface topographical and ultrastructural alterations of Raillietina echinobothrida and Ascaridia galli induced by a compound isolated from Acacia oxyphylla. Vet Parasitol. 2012;185: 322–326. doi: 10.1016/j.vetpar.2011.09.041 [DOI] [PubMed] [Google Scholar]

[pone.0282033.ref058] 58.Smyth JD, McManus DP, others. The physiology and biochemistry of cestodes. Cambridge university press; 1989. [Google Scholar]

[pone.0282033.ref059] 59.Roy B, Swargiary A, Syiem D, Tandon V. Potentilla fulgens (Family Rosaceae), a medicinal plant of north-east India: A natural anthelmintic? J Parasit Dis. 2010;34: 83–88. doi: 10.1007/s12639-010-0018-z [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0282033.ref060] 60.Majumdar R, Kar PK, Giri BR, Roy B. Biosynthesis, characterization and anthelmintic activity of silver nanoparticles of Clerodendrum infortunatum isolate. Sci Rep. 2023;13: 7415. doi: 10.1038/s41598-023-34221-9 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0282033.ref061] 61.Tandon V, Pal P, Roy B, Rao HS, Reddy KS. In vitro anthelmintic activity of root-tuber extract of Flemingia vestita, an indigenous plant in Shillong, India. Parasitol Res. 1997;83: 492–8. Available: http://www.ncbi.nlm.nih.gov/pubmed/9197399 doi: 10.1007/s004360050286 [DOI] [PubMed] [Google Scholar]

[pone.0282033.ref062] 62.Rogers WP. Histological Distribution of Alkaline Phosphatase in Helminth Parasites. Nature. 1947;159: 374–375. doi: 10.1038/159374b0 [DOI] [PubMed] [Google Scholar]

[pone.0282033.ref063] 63.Burenina EA. Adenosine triphosphatases of cestodes Bothriocephalus scorpii. J Evol Biochem Physiol. 2007;43: 287–294. [PubMed] [Google Scholar]

[pone.0282033.ref064] 64.Pal P, Tandon V. Anthelmintic efficacy of Flemingia vestita (Leguminoceae): Genistein-induced alterations in the activity of tegumental enzymes in the cestode, Raillietina echinobothrida. Parasitol Int. 1998;47: 233–243. [Google Scholar]

[pone.0282033.ref065] 65.Zaletel I, Milutinović K, Bajčetić M, Nowakowski RS. Differentiation of Amyloid Plaques Between Alzheimer’s Disease and Non-Alzheimer’s Disease Individuals Based on Gray-Level Co-occurrence Matrix Texture Analysis. Microsc Microanal. 2021;27: 1146–1153. doi: 10.1017/S1431927621012095 [DOI] [PubMed] [Google Scholar]

[pone.0282033.ref066] 66.Chen H, Li W, Zhu Y. Improved window adaptive gray level co-occurrence matrix for extraction and analysis of texture characteristics of pulmonary nodules. Comput Methods Programs Biomed. 2021;208: 106263. doi: 10.1016/j.cmpb.2021.106263 [DOI] [PubMed] [Google Scholar]

[pone.0282033.ref067] 67.Paunovic J, Vucevic D, Radosavljevic T, Pantic S, Veskovic M, Pantic I. Gray-level co-occurrence matrix analysis of chromatin architecture in periportal and perivenous hepatocytes. Histochem Cell Biol. 2019;151: 75–83. doi: 10.1007/s00418-018-1714-5 [DOI] [PubMed] [Google Scholar]

PERMALINK

Anthelmintic efficacy of Holarrhena pubescens against Raillietina spp. of domestic fowl through ultrastructural, histochemical, biochemical and GLCM analysis

Rachita Saha

Manjil Gupta

Rima Majumdar

Subrata Saha

Pradip Kumar Kar

Roles

Abstract

Introduction

Materials and methods

Preparation of extract from Holarrhena pubescens

Collection of parasites

Experiments

Efficacy testing

Scanning Electron Microscopy

Transmission electron microscopy

Histochemical studies

GLCM analysis

Biochemical studies

Results

Efficacy testing

Table 1. Results of efficacy testing of the ethanolic crude extract of Holarrhena pubescens and fractions of the ethanolic extract on Raillietina spp.

Fig 1. Graph showing the paralysis time and death time of Raillietina spp treated with different doses and fractions of the ethanolic extract of Holarrhena pubescens.

SEM observations

Fig 2.

TEM observations

Fig 3.

Fig 4.

Histochemical observation

Fig 5.

Fig 6.

GLCM results

Table 2. GLCM parameters of images obtained from histochemical studies of different enzymes in Raillietina spp.

Fig 7. ROC curves for GLCM parameters of images obtained from histochemical studies on different enzymes in Raillietina spp.

Table 3. Area under curve (AUC) values of GLCM parameters of images obtained from histochemical studies of different enzymes in Raillietina spp.

Biochemical observation

Table 4. Results of biochemical analysis of enzyme activity in Raillietina spp.

Fig 8. Graph showing the effect on total and specific activities of different tegumental enzymes of Raillietina spp. treated with crude extract and fractions of Holarrhena pubescens.

Discussion

Conclusion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Josué de Moraes

Roles

Author response to Decision Letter 0

Decision Letter 1

Josué de Moraes

Roles

Author response to Decision Letter 1

Decision Letter 2

Josué de Moraes

Roles

Acceptance letter

Josué de Moraes

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases