Anthelmintic evaluation of three Ayurvedic formulations: a transmission electron microscopy study in Raillietina sp. (Cestoda)

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Anthelmintic evaluation of three Ayurvedic formulations: a transmission electron microscopy study in Raillietina sp. (Cestoda)

https://doi.org/10.21203/rs.3.rs-3149676/v1

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published 24 Oct, 2023

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Ayurveda is one of the ancient traditional medicine systems in India. However, several Ayurvedic medicines lack scientific evidence about their efficacy. This study reports the in vitro anthelmintic effects of three common Ayurvedic formulations, Krimimudgar Ras, Kriminol, and Birangasav on a poultry cestode Raillietina sp., using transmission electron microscopy (TEM). Adult cestodes were exposed to different concentrations of Ayurvedic formulations and the paralysed parasites from the highest concentration (50 mg/ml) of Ayurvedic formulations, the reference anthelmintic praziquantel (PZQ) together with control were picked up and processed for TEM. The TEM studies of control cestode parasites revealed a normal arrangement of microthrix layer, basal lamina, longitudinal muscle layer, and a normal nucleus and mitochondria. Importantly, the cestodes that were exposed to 50 mg/mL concentration of Krimimudgar Ras revealed the most prominent ultrastructural alterations in the body of parasites in the form of a disrupted microthrix layer, basal lamina, muscle layer and mitochondria. The nucleus also appeared dense and irregular in shape with scattered chromatin and disrupted nuclear membrane. Kriminol-treated worms revealed considerably less damage, whereas Birangasav-treated worms revealed destructive effects in microthrix layer, nucleus and mitochondria. Through the findings of the present study, it can be concluded that of the three common Ayurvedic formulations studied, Krimimudgar Ras causes maximum degree of internal alterations in cestode parasites and thus may be considered as a good anthelmintic agent.

Anthelmintic

Ayurveda

Birangasav

Krimimudgar Ras

Kriminol

Raillietina

Transmission electron microscopy

India takes pride in its numerous traditional healthcare systems, like Ayurveda, Yoga and Siddha. Ayurveda is one of the ancient traditional medicine systems in India and is regarded as ‘Science of Life’ (Debnath et al. 2017; Shi et al. 2020). In recent times, Ayurveda has taken a household name in India and is considered an alternative to modern medicine. The common man in India believes that Ayurvedic drugs are devoid of adverse effects and are relatively safe, since they are not only time-tested but prepared from natural products (Sarkar et al. 2013; Paudyal et al. 2019). A wide publicity of Ayurvedic medicines in various platforms has contributed to their increasing demand in India, and therefore the safety and efficacy and quality control studies on Ayurvedic medicines has become a key concern for the public and health authorities (Mahajon et al. 2023). However, it may be said here that by and large, most Ayurvedic medicines suffer from the lack of scientific trials and validations. Unfortunately, most Ayurveda professional practitioners also have a notion that since Ayurveda is in use from ancient times, their scientific validations seem unwarranted but well-planned research can be carried out for the credibility of Ayurvedic products (Chauhan et al. 2015; Sangle 2023). Nonetheless, a distinction between facts and myths is required and scientific validations of Ayurvedic medicines are a prerequisite for their sale and usage in present times. Although backed by the Government of India’s Drugs and Cosmetics Act, 1940, Ayurveda is still lagging behind in many ways which needs to be addressed by proper scientific studies. The public also needs to be informed clearly about the ongoing clinical trials and systematic awareness should be created about these medicines (Sangle 2023). This will not only provide a better future for Ayurveda in India, but will also pave the way for their swift popularization and will change the face of current clinical research strategies (Acharya 2023).

The claims of Ayurveda are mostly undocumented, and if available, they lack laboratory trails, quality control tests and validations thereby leading to a dearth of authenticated data and restricted acceptance in developed countries. In previous studies, Chen and Yadav (2018; 2019) investigated the in vitro anthelmintic effects of these Ayurvedic formulations on intestinal cestode parasite of domestic fowl, Raillietina sp. using scanning electron microscopy. These studies report the anthelmintic efficacy of these common Ayurvedic formulations, Krimimudgar Ras, Kriminol, and Birangasav on Raillietina sp., using transmission electron microscopy (TEM).

The information about the usage patterns of anthelmintic Ayurvedic medicines was collected in Shillong (Meghalaya) and Guwahati (Assam) through surveys (March, 2016 to December, 2016) undertaken in the Ayurvedic pharmacies and manufacturing units, and through interviews with Ayurvedic practitioners. Thereafter, three most commonly prescribed and marketed anthelmintic Ayurvedic formulations in the area were selected for the study, Krimimudgar Ras, Kriminol syrup, and Birangasav (Fig. 1). These Ayurvedic formulations were procured from Ayurvedic pharmacies in Shillong and Guwahati.

Krimimudgar Ras is a classical Ayurvedic formulation and is a product of Dabur India Limited, whereas, Kriminol syrup is a proprietary Ayurvedic formulation and a product of Vyas Pharmaceuticals, Indore. Birangasav, a classical Asava formulation (syrup), is manufactured by the Assam Ayurvedic Products (ASIDC Limited), Guwahati, and its preparation is based on ‘Bhaishajya Ratnavali’, a reputed classical text of Ayurveda.

The adult live worms of Raillietina sp. (Cestoda) were dissected out from the intestines of domestic chicken (Gallus domesticus) procured from local markets and placed in 0.9% phosphate buffered saline (PBS). These worms (n = 6) were maintained in petri-dishes containing each of the formulation at three different concentrations (10, 30, and 50 mg/mL in PBS) for the solid tablet (Krimimudgar Ras) and percent solutions (0.1, 0.3, 0.5 mL in a volume of 10 mL adjusted by 0.9% PBS) for the liquid formulations (Kriminol syrup, Birangasav). Reference modern cestocidal drug, praziquantel (PZQ, 1 mg/mL) was used as positive control (Mohandas et al. 2013; Khan et al. 2015; Chen and Yadav 2018). One set of cestode parasites in 0.9% PBS served as negative control (Chen and Yadav 2018).

After attaining a state of paralysis, the specimens from the highest concentration of each Ayurvedic formulation, reference drug, together with control group were collected, washed in 10% cold neutral buffered formalin and fixed in modified Karnovsky’s fixative. Parasites were transferred to 0.2 M sodium cacodylate buffer for 4 hr, dehydrated in acetone and embedded in araldite. Ultrathin sections of specimens were stained in uranyl acetate, followed by lead citrate and examined under a Transmission Electron Microscope (JEM-2100, 200kV, JEOL) at NEHU, Shillong.

The transmission electron microscopic studies on control worms revealed a normal arrangement of glycocalyx and microthrix layer. The distal cytoplasm, basal lamina and longitudinal muscle layer were intact (Fig. 2a). A neat nucleus with intact double membrane (Fig. 2b) and mitochondria with prominent cristae (Fig. 2c) were observed. The cestode parasites exposed to 50 mg/mL concentration of Krimimudgar Ras revealed extensive damages. The glycocalyx membrane and microthrix layer was disrupted and wavy with ridges indicating stress in these regions. The syncytial layer showed a number of electron-dense materials (Fig. 2d). The nucleus appeared dense and irregular in shape with scattered chromatin and the nuclear membrane was disrupted at various regions (Fig. 2e). A number of vacuoles were observed in the area outer periphery of the nucleus. Mitochondria appeared oblong with indistinguishable membrane and extremely reduced cristae (Fig. 2f). Kriminol-treated parasites revealed comparatively less damages in the microthrix layer, distal cytoplasm, muscle layer and basal lamina (Fig. 2g). The nucleus was observed to be abnormal lacking nucleolus and the chromatin appeared to be condensed (Fig. 2h). In addition, numerous vacuoles were detected. Also, numerous mitochondria with intact cristae were seen to aggregate (Fig. 2i). Birangasav-treated cestodes revealed notable disruptions in the microthrix layer, with absence of glycocalyx membrane. A break in the basal lamina was observed and the syncytial layer showed disruptions. The contents of the tegumental cytons seemed to have emanated beyond the microthrix layer (Fig. 2j). A nucleus with prominent nucleolus and chromatin concentrated in the periphery was observed. The nuclear membrane however was not detectable in several locations (Fig. 2k). Mitochondria were dilated and cristae were indistinguishable (Fig. 2l). However, it should be noted that Birangasav-treated parasites took the longest time for paralysis and mortality to occur during the in vitro anthelmintic assay.

PZQ-treated worms also displayed numerous damages in the body of the parasites in the form of pits and scars. The glycocalyx membrane and microthrix layer was wrinkled with a loss of unidirectionality and infoldings (Fig. 2m). Damaged nucleus with condensed chromatin and thinning of nuclear membrane were also observed (Fig. 2n). Distorted and sausage-shaped mitochondria with clumped cristae can be observed from the micrograph (Fig. 2o).

The tegument of cestodes comprises of the glycocalyx layer, microthrix layer, distal cytoplasm, basal lamina, and cell organelles, each serving functions such as attachment to the host, absorption of nutrients, protection, sensation and osmoregulation (Dasgupta and Roy 2010). This tegumental region of the cestode is highly prone to attack by anthelmintics (Alvarez et al. 2007). The effects of formulations and PZQ were visualised as destructive in comparison to the untreated worms, with Krimimudgar Ras showing the most prominent effects, followed by Birangasav and Kriminol. It should also be noted that earlier studies on the in vitro anthelmintic study reports that, Birangasav took the longest time for paralysis and mortality to occur (Chen and Yadav 2018).

The literature survey reveals that information about effects of anthelmintic Ayurvedic formulations on ultrastructure of cestodes or other helminths using TEM is deficient in comparison to the effects of medicinal plants on parasitic helminths, which have been widely worked upon (Kundu et al. 2017; Beshay 2018). For example, the alcoholic extract of Millettia pachycarpa, a traditional medicinal plant of Mizoram, has been reported to cause extensive damages to the internal ultrastructure of Raillietina echinobothrida, such as stripping-off of the tegument along with the basal lamina, disruption of the nucleus and nuclear membrane and changes in the size of mitochondria (Roy et al. 2008). Likewise, the extracts from Senna alexandrina and Artemisia absinthium also have been found to have effects on the cestodes, Hymenolepis diminuta and H. nana, respectively, in the form of disruption in the microthrix layer, tegument, and subtegument of parasites (Kundu et al. 2017; Beshay 2018). In a study by Dey and Roy (2018), R. echinobothrida when exposed to the ethanolic extract of Lysimachia ramosa and its phytoproducts, showed erosion of microtriches and distal cytoplasm, exposure of basal lamina into the medium, vacuolization of syncytial layer, disruption of nucleus and mitochondria. Another parasitic helminth, Fasciolopsis buski when treated with the crude extract of Alpinia nigra revealed a total alteration in the tegument and sub-tegument in the form of deformed microtriches, vacuolization, and irregularity of mitochondria (Roy et al. 2012a). Eggs of the nematode Cooperia punctata were also processed for TEM after treating them with the acetonic extract of Gliricidia sepium leaves whereby eggshells were fractured at many places (von Son-de Fernex et al. 2017). Schistosoma mansoni and S. hematobium when incubated in 50–500 µM of curcumin powder showed a disruption in the architecture and motility functions (Abou El Dahab et al. 2019).

The changes in the structure of the protective layers and organelles of the parasite can be attributed to the following reasons. The glycocalyx membrane functions to protect the cestode which when disrupted will bring changes in the golgi complex which aids in the synthesis of this membrane (Lumsden 1975). The microthrix layer in cestodes functions as a region for nutrient absorption, protection, and sensation, and any change or damage would lead to starvation of parasite and make them prone to host immune attack (Challam et al. 2012). For smooth ion transfer from the basal lamina towards the distal cytoplasm, the tegument has to be intact and thus any alteration in these regions will interrupt ion transfer (Threadgold and Read 1970). Also, the treated parasites were observed to possess thin basal lamina which serves as a site for mitochondria synthesis and its distortion could mean the formulation possess damaging effect causing a disruption in the synthesis which ultimately leads to energy disruption. Changes in the shape of mitochondria or cristae were evident which is indicative of energy disruption. In addition, there could also be an impairment in the synthesis of glycogen which in combination with mitochondrial dysfunction would damage the tegument (Bach et al. 2003; Roy et al. 2012b). Disruption in the nucleus, chromatin or nuclear membrane indicates an inhibition in protein synthesis (Stitt and Fairweather 1996). PZQ-treated parasites on the other hand showed more prominent effects in the tegument. Also, a reduction in organelles such as the endoplasmic reticulum could inhibit protein synthesis thereby leading to paralysis and mortality (Kundu et al. 2012; Roy et al. 2012b). Contraction and disintegration of the tegument in the form of infoldings is closely linked to the flux of ions across membranes while necrosis of tissue can be attributed to the changes in membrane transport, myelin degeneration, autophagy (Roy et al. 2008). The aforementioned changes in tegument and subtegument of Raillietina sp. were highly evident in parasites treated with Krimimudgar Ras. Thus, this is the first report on the effects of the selected Ayurvedic formulations on cestode parasites monitored using TEM.

The findings of this study infer that Krimimudgar Ras, a product of Dabur India Limited, is capable of causing maximum degree of internal alterations in cestode parasite, followed by Birangasav and Kriminol. Thus, evidence-based studies on Ayurvedic formulations can contribute to their ubiquitous acceptance without controversy about their efficacy.

Acknowledgements

A fellowship awarded to Risa Parkordor Chen from UGC is acknowledged. TEM facilities were provided by the Sophisticated Analytical Instrument Facility, NEHU Shillong.

Ethic declaration

Funding: This study did not receive any funding from any agency or individual.

Conflict of interest: All authors declare that there is no conflict of interests.

Ethics approval: Not applicable

Consent to participate: Not applicable

Consent for publication: Not applicable

Availability of data and material: All data generated during this study are included in this article.

Code availability: Not applicable

Author contributions: This study was designed and supervised by AKY. RPC executed the study and wrote the first draft. ADS analysed the results and participated in editing of draft. All authors read and approved the final manuscript.

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Anthelmintic evaluation of three Ayurvedic formulations: a transmission electron microscopy study in Raillietina sp. (Cestoda)

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