In vitro cestocidal activity of Persicaria hydropiper (L.) Delarbre, a traditionally used anthelmintic plant in India

Persicaria hydropiper is a medicinal plant used for the treatment of helminth infections among the Naga people of India. To verify the traditional claim of the plant, an in vitro anthelmintic efficacy of the methanol extract of P. hydropiper leaves was investigated based on the paralytic and mortality effects on Raillietina echinobothrida, an intestinal cestode parasite of domestic fowl, following exposure to 10, 20 and 30 mg/ml concentrations of extract. The effects of extract were also studied on the body surface of the parasites by scanning electron microscopy (SEM). The in vitro results showed an efficacy that was dose-dependent. At the highest dose (30 mg/ml), mortality of parasites occurred in 4.79 ± 0.17 h, in comparison to control, where the parasites survived till 45.63 ± 0.18 h. The SEM observations of extract-treated parasites revealed notable impairment in scolex, with distorted suckers and eroded spines. Also, the tegument was observed to be shrunken with impaired microtriches. The results indicate that P. hydropiper leaves possess noteworthy anthelmintic efficacy and justify their use in traditional medicine against intestinal worms.


Introduction
About 1.5 billion people are estimated to be infected with soil-transmitted helminths (WHO 2022), which are considered as one of the most widespread human pathogens. Over the years, the repeated use of anthelmintic drugs has led to concerns about the development of drug resistance in helminths. The evidence from veterinary medicine and reports of reduced efficacy of anthelmintics in human helminths also raise concerns about the risk of resistance in mass drug administration efforts in humans (Nixon et al. 2020). In the quest to find safe and effective anthelmintic drugs, a keen interest in ethnomedicinal studies has emerged in recent years. Owing to the merits that plants or their secondary metabolites may serve as alternative anthelmintics, several medicinal plants have been explored for their anthelmintic properties (Tandon et al. 2011;Soren et al. 2021).
Persicaria hydropiper (L.) Delarbre (Polygonaceae), called locally as "nhachü" by the Angami tribe of Nagaland, is an annual herb of about 40-70 cm in height. It is distributed in temperate and tropical Asia, Northern Africa, Australia and in many regions of Europe (Huq et al. 2014), as well as in the Northeast India (Hazarika and Sarma 2006). In the traditional medicine of Pakistan, P. hydropiper has been used for a long time against inflammation, rheumatoid arthritis, epilepsy, headache, colic pain, fever, chills, joint pain, oedema and infectious diseases (Ayaz et al. 2015). Recent experimental studies on this plant have also established it to possess diverse biological activities, including anticholinesterase, antioxidant and gastroprotective activities (Ayaz et al. 2017). Ayaz et al. (2014) have reported the anthelmintic activity of the whole plant extract of P. hydropiper using adult earthworms (Pheretima posthuma) and roundworms (Ascaridia galli) as test organisms. It can be argued here that the anthelmintic data generated using earthworms as test organisms possess only limited scope, as earthworms are free-living organism, with a different structural and functional organization to those of parasitic roundworms (Tandon et al. 2011).
During our recent ethnomedicinal studies, it was revealed from field surveys that the leaves of nhachü ( Fig. 1) are used by the Angami tribe of Nagaland, India, as a common remedy to treat intestinal worms. The present study was initiated to investigate the anthelmintic properties of P. hydropiper leaves, using Raillietina echinobothrida, the intestinal cestode parasite of domestic fowl. The motility and/or mortality of the worms and alterations in body surface of parasite, studied by scanning electron microscopy, following exposure of parasites to methanol leaves extract of plant, were the parameters of this present study.

Materials and methods
The fresh leaves of P. hydropiper were collected from their natural habitats in Nagaland (India), and identified by Dr. N. Odyuo, a plant taxonomist at Botanical Survey of India, Eastern Regional Centre, Shillong. A voucher specimen (NEHU-12,099) of plant material has been deposited in the Department of Zoology, NEHU, Shillong. The leaves were washed thoroughly in distilled water, dried under shade, ground into fine powder and extracted in methanol. The extract was stored at 4 °C until used for testing.

In vitro anthelmintic assay
Adult live worms of R. echinobothrida were collected from the autopsy of intestines of freshly slaughtered domestic fowls from local markets in Shillong. The worms were washed thoroughly in distilled water to remove debris and transferred to phosphate-buffered saline (PBS) at 37 ± 1 °C to acclimatize for about 30 min. The active worms (n = 6) were then transferred into petri dishes (triplicates) containing 10, 20 and 30 mg/ml concentrations of P. hydropiper leaf extract, with simultaneous maintenance of control in PBS. Praziquantel (PZQ), at 1 mg/ml, was used as reference drug.
The treated and control group of worms were maintained inside an incubator at 37 ± 1 °C. The efficacy of extract/PZQ was assessed on the basis of physical motility of test worms, as evidenced by their paralysis and mortality.

Scanning electron microscopy
With the onset of paralysis, the worms from control medium, and those exposed to 30 mg/ml concentration of extract and PZQ were picked up and fixed in neutral buffered formalin (NBF) at 4 °C. The fixed specimens were dehydrated in ascending grades of acetone, air-dried in tetramethylsilane, gold coated and finally viewed in JEOL JSM-6360 SEM.

Statistical analysis
All data are represented as mean ± standard error mean (S.E.M), assessed through one-way ANOVA, followed by post hoc tests (Tukey's test) using GraphPad Prism. P ≤ 0.001 was considered statistically significant.

In vitro anthelmintic assay
The plant extract showed a dose-dependent anthelmintic efficacy (Fig. 2). At 10 and 20 mg/ml concentrations of extract, the mortalities of worms were recorded at 6.08 ± 0.18 h and 5.13 ± 0.10 h, respectively. However, at 30 mg/ml concentration of extract, the mortality of cestodes was attained quite early, i.e., at 4.79 ± 0.17 h. Worms exposed to reference drug showed mortality at 4.80 ± 0.12 h. On the other hand, the worms maintained in control medium revealed physical activity till 45.63 ± 0.18 h.

Scanning electron microscopy
SEM of control parasites revealed normal contour of the body, with suckers bearing short and pointed spines (Fig. 3a, b) and thickly packed normal architecture of microtriches (Fig. 3c). In contrast, the worms exposed to 30 mg/ml concentration of the extract showed profound impairment on the scolex, with distorted suckers and eroded spines (Fig. 3d). The tegument was noted to be shrunken (Fig. 3e) and microtriches were disrupted, showing an extensive damage (Fig. 3f). PZQ also caused detrimental effects on the scolex of parasites (Fig. 3g), wherein the body surface of parasite showed prominent furrows (Fig. 3h) and microtriches were clustered (Fig. 3i).

Discussion
In the present study, the P. hydropiper leaf extract showed a dose-dependent efficacy on adult R. echinobothrida. A similar dose-dependent efficacy of Lysimachia ramosa leaves extract, with varying concentrations of 5-50 mg/ml, on intestinal helminths, namely, Ascaris suum, Fasciolopsis buski, and R. echinobothrida was reported by Challam et al. (2010). The study showed that at the highest concentration (50 mg/ml) of extract, R. echinobothrida showed mortality in 4.51 ± 0.15 h which was comparable to the mortality time caused by a concentration of 30 mg/ml of extract which is much lesser than that of Challam et al. (2010) as observed in this study. Similarly, Tandon et al. (1997) studied the cestocidal properties of Flemingia vestita tuber peel extract in different concentrations (0.5-50 mg/ml) and reported the mortality of worms at the highest concentration (50 mg/ml) in 6.5 ± 0.4 h, which is higher as compared to mortality of parasites observed by 30 mg/ml concentration of extract in the present study. Challam et al. (2012) and Kundu et al. (2015) also reported the cestocidal activities of Carex baccans and Senna occidentalis and observed mortality of worms in 4.13 ± 0.06 h and 12.82 ± 0.24 h, at the highest dose of extract, which was 50 mg/ml and 80 mg/ml, respectively. The comparison of data from the above-mentioned related studies suggest that worms exposed to 30 mg/ml of P. hydropiper leaf extract attain mortality in a relatively shorter time (4.79 ± 0.17 h), which indicates that P. hydropiper is a comparatively more effective anthelmintic plant, even at lower concentration of extract.
The cestode tegument has been established as the principal target site of different classes of synthetic drugs or natural anthelmintics (Tandon et al. 1997;Lalchhandama 2019;Lalthanpuii and Lalchhandama 2020a, b). In the present study, the treatment of worms with plant extract revealed destruction in the attachment organs as well as the tegument of parasites. The tegument of cestodes is considered as the only interface through which digestion and absorption takes place. Any alteration in the tegument can lead to disruption of the normal functioning of these physiological activities which can ultimately cause paralysis and death of cestodes (Kar et al. 2014). The ultrastructural effects similar to the present study have also been reported on part of the standard cestocidal drug PZQ, which brings out its action by eliminating worms from the host due to paralysis of musculature and tegumental damage (Chan et al. 2017). Several other workers have documented the potency of anthelmintic plants by denoting the topographical and structural alterations in the tegument of cestodes. For example, R. echinobothrida exposed to methanolic extract of Callicarpa arborea bark (20 mg/ml) revealed crumpled scolex, shrunken segments and total loss of microtriches (Lalthanpuii and Lalchhandama 2020a). Also, in another study, the body surface of R. tetragona, on exposure to chloroform extract of Imperata cylindrica (20 mg/ml) revealed distortion of suckers and tegumental erosion (Lalthanpuii and Lalchhandama 2020b). Structural changes on the tegument of Hymenolepis diminuta due to ethanolic extract of Senna alexandrina at 40 mg/ml have also revealed irreversible destruction over the general body surface where the suckers were found to be constricted and the velvety appearance of microtriches was lost (Kundu et al. 2017).

Conclusion
This study indicates that the leaves of P. hydropiper possess significant anthelmintic efficacy and justify their use in traditional medicine against intestinal worms. Further detailed studies are needed to investigate the mechanism of action and toxicity profile of this plant.