Anthelmintic efficacy of Phlogacanthus thyrsiflorus leaf extract on juvenile and adult worms of Hymenolepis diminuta (Cestoda)

Abstract

The leaves of Phlogacanthus thyrsiflorus are used as an anthelmintic remedy by the tribal communities of upper Assam in India. The present study was carried out to validate the anthelmintic credentials of this plant. Mature and larval Hymenolepis diminuta worms were exposed to varying concentration of methanolic leaf extract of plant and parasites were observed for paralysis and mortality. At the end of the experiment, worms were collected and processed for scanning electron microscopy (SEM) study to observe the effect of extract on tegument of parasite. The in vivo study was carried out in H. diminuta-rat model with 200, 400 and 800 mg/kg concentrations of extract. The in vivo anthelmintic efficacy was assessed by reductions in egg per gram (EPG) and worm counts after necropsy of animals. In vitro studies revealed the earliest mortality of larval worms in 1.05 ± 0.04 h by 30 mg/ml concentration and of adult worms in 2.05 ± 0.08 h. SEM study revealed extensive damage to the suckers, body tegument and microtriches of worms treated with 30 mg/ml concentration of extract. In in-vivo studies, 800 mg/kg dose of extract showed highest efficacy, with 59% and 54.25% reduction in EPG counts and worm count against juvenile worms, and 63.16% and 66.75% reduction in EPG counts and worm counts, respectively against adult worms. Although the effects were comparatively less than the reference drug, nevertheless this study reveals that P. thyrsiflorus possess anthelmintic efficacy and justify its use in traditional medicine against intestinal-worm infections.

Introduction

About a quarter of the world’s population is known to be infected with helminth parasites (Dai et al. 2022). Soil transmitted helminthiasis is usually prevalent in developing countries where there is poor personal hygiene and sanitary conditions (Jeza et al. 2022). In India, rural areas are known to harbour more such helminth infections than urban areas (Kaliappan et al. 2022). Worldwide, several communities are known to use medicinal plants in their traditional medicine to cure several health conditions, including intestinal worm infections (Beasley et al. 2022). In Assam, the Mising, Sonowal Kachari and Deori tribes of upper Assam consume a decoction of Phlogacanthus thyrsiflorus (Acanthaceae) leaves to treat intestinal-worm infections.

P. thyrsiflorus is an evergreen gregarious shrub about 2.5 m in height and found in the sub-tropical Himalayas, Bangladesh and in the North Eastern regions of India (Khare 2007; Komor and Devi 2016). It is also found in Burma, the Malaya peninsula and Indonesia. It grows best in a moist tropical climate at an altitude of 650–1300 m above sea level (Ningombam and Singh 2014). Its leaves and flowers are bitter in taste and consumed as a vegetable (Komor and Devi 2016). Its potent anticancer properties have been recently reported (Chanu et al. 2021). Besides, it has also been reported to possesses hypolipidaemic, antioxidant, analgesic and antimicrobial activities (Mukherjee et al. 2009; Singh and Singh 2010; Tassa et al. 2012). The leaves are also used to treat cough, asthma, jaundice, diarrhoea, dysentery, tuberculosis and malarial fever (Tassa et al. 2012). In addition, the flowers are used to treat pox and skin diseases (Komor and Devi 2016). It is also an integral part of the folk medicine and mythology of the Meeteis in Manipur, India (Ningombam and Singh 2014).

Several bioactive compounds have been isolated from this plant. An isolated diterpene lactone glucoside namely phloganthoside, has been known to possess antimicrobial and anti-hyperglycaemic properties (Chakravarty and Kalita 2012). This plant is known to possess steroids, flavonoids, tannins, and saponins (Kumar et al. 2017). In addition, another study showed the presence of coumarins, polysterols and diterpenes (Saikia et al. 2018). Its antioxidant property has been attributed to 1-naphthalenecarboxaldehyde, terbutylazine-2-hydroxy, sparteine, L-histidine, aritmina, aspidocarpine, hydroquinidine, (-)-bilobalide, and harmine isolated from P. thyrsiflorus (Saikia et al. 2018). P. thyrsiflorus has also been reported to possess anti-cancer properties against HeLa cell lines (Tiwary et al. 2015).

In Assam, the mixture of its leaves and flowers are used to prepare a local recipe known as “akho ata hak” which is believed to protect from various diseases. Since the leaves of P. thyrsiflorus are used by the Mising, Sonowal Kachari and Deori tribes of upper Assam to treat the intestinal-worm infections as a decoction, this study was undertaken to validate the acclaimed efficacy of P. thyrsiflorus leaf methanolic extract (PLE), using in vitro and in vivo studies against H. diminuta, a cestode parasite. Also, this study monitored the effects of extract on the tegument of the parasites using the scanning electron microscopy (SEM).

Materials and methods

Plant material

Fresh leaves of P. thyrsiflorus were collected from their natural habitats in Sadiya, Tinsukia district of upper Assam. Herbariums were submitted to the Department of Botany for identification. The leaves were cleaned, dried in shade, made into powdered form and then extracted in methanol using a Soxhlet apparatus. After extraction, the extract was filtered, and the filtrates were concentrated under reduced pressure in a rotary evaporator (BUCHI Rotavapor™ R-100 Rotary Evaporator). The percentage yield of PLE was 1.16% (w/w).

Experimental animals

Both male and female Wistar rats about 180–200 g in weight, were used in this evaluation. Animals were singly placed in cages with adequate access to food and water. The use of animals was approved by the Institutional Ethics Committee (IEC) (Animal models). All experiments conform to the ARRIVE and IEC directives.

In vitro study

Laboratory maintained infected rats were dissected to recover adult H. diminuta worms, whereas, cysticercoids were collected from the intermediate host, Tribolium confusum. Both larval and adult worms (n = 5) were placed separately in petridishes containing 10, 20 and 30 mg/ml concentrations of PLE in Hanks’ saline and incubated at 37 ± 1 °C. The time taken for paralysis and mortality to occur were noted and compared with the worms placed in reference drug, praziquantel (PZQ).

SEM study

The worms from 30 mg/ml concentration of extract were collected after paralysis, along with control and reference drug-exposed worms. The worms were washed in distilled water, fixed in 3% glutaraldehyde, washed in cacodylate buffer, dehydrated in acetone grades and dried in tetramethylsilane. Worms were then coated in gold and viewed in a Jeol JSM–6360 SEM.

In vivo study

In vivo study was performed using H. diminuta-rat animal model. Wistar rats were infected with cysticercoids dissected out from artificially infected T. confusum beetle. Establishment of the infection in rats was confirmed by the presence of eggs in the faecal matter of the inoculated rats. Infected rats (n = 6) were exposed to three different doses of the methanolic extract, based on their body weights (b.w.) namely, 200 mg/kg (lower dose), 400 mg/kg (recommended dose), and 800 mg/kg (higher dose) b.w. In addition, one group served as the negative control (dosed only with distilled water) and another group served as the positive control and received a single dose of PZQ (10 mg/kg b.w.). For the juvenile stage, animals were dosed for 5 days (day 3−7) post-inoculation (p.i.) of cysticercoids and dissected on day 28 to estimate the reduction in worm counts. For egg per gram (EPG) count, faecal pellets were collected from each cage on day 18, 19 and 20 p.i. following infection with cysticercoids (Deori and Yadav 2016).

Against adult worms, the EPG count was done on day 18−20 p.i. of cysticercoids before treatment with the extract and PZQ (pre-treatment EPG). Animals were dosed with the plant extract and PZQ on days 21−25 p.i. of cysticercoids and a second EPG count was done on days 26−28 (post-treatment EPG). On day 36, all the animals were sacrificed to estimate the reduction in worm count and percentage reduction on EPG and worm counts was calculated (Deori and Yadav 2016).

Statistical analysis

The data from in vitro tests were analyzed by student’s t-test and the p value < 0.05 was considered to be statistically significant between the values from control and treated groups. The data from in vivo anthelmintic tests were analyzed by one way analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test. The p value < 0.05 was considered to be statistically significant. All the statistical calculations were done using GraphPad Prism (version 4.5) and represented as mean ± standard error of the mean (SEM).

Results

In vitro studies revealed the earliest mortality of larval worms in 1.05 ± 0.04 h by 30 mg/ml concentration. PZQ (1 mg/ml) showed paralysis and mortality at 0.55 ± 0.04 and 1.08 ± 0.04 h, respectively. The control group of worms showed survival till 30.33 ± 0.41 h. The mortality time values of juveniles by PLE were noted to be significantly different (p < 0.0001) from the control worms in a dose-dependent manner (Fig. 1a). For the adult worms the earliest mortality was recorded in 2.05 ± 0.08 h. The reference drug PZQ (1 mg/ml) showed paralysis and mortality in 0.50 ± 0.06 and 0.97 ± 0.06 h, respectively. Worms maintained in control medium showed survival up to 22.00 ± 0.50 h. The plant extract exhibited significant decrease (p < 0.0001) in the survival time of parasite when compared to the control in a dose-dependent manner (Fig. 1b).

Fig. 1.

In vitro anthelmintic effects of PLE: a juvenile H. diminuta b adult H. diminuta, *p < 0.0001 compared with control groups, student’s t-test

SEM study of adult worms exposed to PLE at 30 mg/ml concentration revealed damaged scolex with sunken suckers, distorted microtriches and damaged tegumental contour (Fig. 2). Likewise, the larval worms treated with PLE at 30 mg/ml concentration also revealed substantial damages to their surface morphology (Fig. 3). The whole worm was found to be shrunken in size with constricted neck region and damaged suckers. Also, the microtriches were poorly visible due to clumping and disorganization in their structures.

Fig. 2.

Scanning electron micrographs of adult H. diminuta: a–c represents the control worms, showing normal suckers, microtriches and intact tegument; d–f worms exposed to PZQ (1 mg/ml), showing distorted suckers, contracted tegumental architecture, but normal microtriches. g–i exposed to PLE (30 mg/ml) showing partially altered suckers, distorted microtriches and damaged tegument

Fig. 3.

Scanning electron micrographs of artificially excysted juveniles of H. diminuta: a–c represents the control juvenile, showing intact body contour, suckers and microtriches; d–f juveniles exposed to PZQ (1 mg/ml), showing distorted whole-body architecture, sucker and altered microtriches. g–i exposed to PLE (30 mg/ml), showing damaged body structures, suckers and partially distorted microtriches

In in-vivo studies, 800 mg/kg dose of extract showed highest efficacy, with 59% and 54.25% reduction in EPG count and worm count against juvenile worms (Table 1), and 63.16% and 66.75% reduction in EPG count and worm count, respectively against adult worms (Table 2). The efficacy of extract was noted to be slightly higher against the adult than juvenile stages of parasite.

Table 1.

In vivo anthelmintic effect of PLE^* on juvenile stages of H. diminuta infection (n = 6)

Groups	EPG (mean ± S.E.M.) Days 18 − 20	Percentage reduction in EPG counts	Worms recovered/rat (mean ± S.E.M.)	Percentage reduction in worm counts
Control	22,012 ± 450	–	4.00 ± 0.00	0.00
Plant extract
200 mg/kg	11,495 ± 230a	− 47.78	2.33 ± 0.33a	41.75
400 mg/kg	10,813 ± 301a	− 50.88	1.83 ± 0.17a	54.25
800 mg/kg	9024 ± 226a	− 59.00	1.83 ± 0.16a	54.25
Praziquantel
10 mg/kg	2966 ± 175a	− 86.52	0.83 ± 0.31a	79.25

^*Administration of plant extract and praziquantel on days 3−5 post inoculation with four cysticercoids/rat

^ap < 0.001 as compared to control value, one way ANOVA

Table 2.

In vivo anthelmintic effect of PLE^* on adult stages of H. diminuta (n = 6)

Groups	EPG (mean ± S.E.M.)		Percentage difference in EPG (A-B)	Worms recovered/rat (mean ± S.E.M.)	Percentage reduction in worm counts
	Pre-treatment days 18−20 (A)	Post-treatment days 26−28 (B)
Control	22,977 ± 377	235 ± 389d	+ 1.11	3.67 ± 0.21	8.25
Plant extract
200 mg/kg	21,581 ± 274	11,294 ± 302c	− 47.67	2.33 ± 0.21a	41.75
400 mg/kg	21,083 ± 292	9533 ± 178c	− 54.78	1.67 ± 0.21b	58.50
800 mg/kg	20,511 ± 155	7556 ± 149c	− 63.16	1.33 ± 0.21b	66.75
Praziquantel
10 mg/kg	21,944 ± 190	2492 ± 75c	− 88.64	0.67 ± 0.33b	83.25

^*Administration of plant extract and praziquantel on days 21 − 25 post inoculation with four cysticercoids/rat

^ap < 0.01 as compared to control value, one way ANOVA

^bp < 0.001 as compared to control value, one way ANOVA

^cp < 0.001 as compared to pre-treatment value, one way ANOVA

^dp > 0.05 as compared to pre-treatment value, one way ANOVA

Discussion

Helminthiasis is a major concern in India affecting mostly children. To treat helminthiasis, some communities rely on medicinal plants which are known to possess several healing properties (Singh and Kumar 2022). Studies on traditionally used medicinal plants have brought to light a large number of plants possessing anthelmintic properties (Challam et al. 2010; Roy et al. 2012; Deori and Yadav 2016; Soren and Yadav 2021; Mowla et al. 2022). In vitro and in vivo studies are preliminary studies to validate the efficacy of medicinal plants. Workers have used different models to validate the claims of efficacy of medicinal plants. Bazh and El-Bahy (2013) evaluated the anthelmintic potentials of curcumin and ginger against Ascaridia galli and observed a dose-dependent efficacy as also observed in this study. Likewise, Molla and Bandyopadhyay (2016) reported similar findings in the anthelmintic efficacy study of Murraya koenigii against Haemonchus contotrus. In a recent study, Soren et al. (2021) evaluated the anthelmintic properties of Sesbania sesban using the same H. diminuta model and showed a 65.10% reduction in EPG count and 76% reduction in worm counts which was almost similar to the results obtained in this study. The workers also observed a dose-dependent efficacy as recorded in this study as well. They also reported a mortality time of 20.70 h in the adult worms. Similar results were also seen in a study by Gogoi and Yadav (2016) where the authors showed that Caeselpinia bonducella showed 80% reduction in worm count and 84.38% reduction in EPG count which was higher than the results seen in this study. Their in vitro mortality time of adult worms was seen at 32.44 h which shows that P. thyrsiflorus is a much more effective anthelmintic plant.

Several workers have carried out SEM of the body surface of the parasites in context of effects of medicinal plants and found that SEM study supplements in vitro efficacy studies (Xavier et al. 2020; Soren and Yadav 2021). The present study provides insight on the extent of damage caused to the tegument by plant extract. Idris et al. (2022) performed SEM studies on Caenorhabditis elegans to study the effect of Rumex crispus on its tegument. The study revealed extensive damage on the body surface of parasite. Likewise, Soren and Yadav (Soren and Yadav 2021) also carried out SEM to evaluate the effect of plant extract on parasite body surface. However, SEM study on the effects of plant extract on tegument of H. diminuta are very scanty. The present study recorded the effect of plant extract in the form of damages to suckers, tegument and microtriches, which suggests a tegumental mode of action of extract.

Conclusions

The finding of present in vitro and in vivo anthelmintic studies suggests that the leaves of P. thyrsiflorus possesses anthelmintic properties and justify their use in traditional medicine against intestinal-worm infections.

Author contributions

AKY conceptualised and supervised the study. KD performed the experiments. KD, AKY and ADS analysed the data. KD and ADS wrote the first draft. AKY corrected the draft. The final draft was read and approved by all the authors.

Funding

This study did not receive any funding.

Data availability

All generated data have been incorporated in this article.

Code availability

All data, materials and software application support the claims and comply with field standards.

Declarations

Conflict of interest

All the authors declare that they have no competing interests.

Ethical approval

All experiments on animals were approved by the Institutional Animal Ethics Committee (Animal Models) of North-Eastern Hill University, Shillong (Vide, Member Secretary, IEC, NEHU, dated September 1, 2014). Plant was identified at North-Eastern Hill University (Specimen voucher number: NEHU 11920).

Consent to participation

Not applicable.

Consent for publication

Not applicable.