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

Anthelmintic evaluation of three Ayurvedic formulations: a transmission electron microscopy study in Raillietina sp. (Cestoda) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Anthelmintic evaluation of three Ayurvedic formulations: a transmission electron microscopy study in Raillietina sp. (Cestoda) Risa Parkordor Chen, Amar Deep Soren, Arun Kumar Yadav This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3149676/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Oct, 2023 Read the published version in Journal of Parasitic Diseases → Version 1 posted 5 You are reading this latest preprint version Abstract 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 Introduction 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). Materials and Methods 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. Results 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. 2 a). A neat nucleus with intact double membrane (Fig. 2 b) and mitochondria with prominent cristae (Fig. 2 c) 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. 2 d). The nucleus appeared dense and irregular in shape with scattered chromatin and the nuclear membrane was disrupted at various regions (Fig. 2 e). 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. 2 f). Kriminol-treated parasites revealed comparatively less damages in the microthrix layer, distal cytoplasm, muscle layer and basal lamina (Fig. 2 g). The nucleus was observed to be abnormal lacking nucleolus and the chromatin appeared to be condensed (Fig. 2 h). In addition, numerous vacuoles were detected. Also, numerous mitochondria with intact cristae were seen to aggregate (Fig. 2 i). 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. 2 j). A nucleus with prominent nucleolus and chromatin concentrated in the periphery was observed. The nuclear membrane however was not detectable in several locations (Fig. 2 k). Mitochondria were dilated and cristae were indistinguishable (Fig. 2 l). 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. 2 m). Damaged nucleus with condensed chromatin and thinning of nuclear membrane were also observed (Fig. 2 n). Distorted and sausage-shaped mitochondria with clumped cristae can be observed from the micrograph (Fig. 2 o). Discussion 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. Conclusions 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. Declarations 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. References Abou El Dahab M, Shahat SM, Mahmoud SSM, Mahana NA (2019) In vitro effect of curcumin on Schistosoma species viability, tegument structure and egg hatchability. Exp Parasitol 199:1-8. https://doi.org/10.1016/j.exppara.2019.02.010 Acharya R (2023) Emphasizing evidence-based research: a way forward to robust validity of clinical research in Ayurveda. J Res Ayurvedic Sci 7(2):77-79. https://doi.org/10.4103/jras.jras_49_23 Alvarez LI, Mottier ML, Lanusse CE (2007) Drug transfer into target helminth parasites. Trends Parasitol 23(3):97-104. https://doi.org/10.1016/j.pt.2007.01.003 Bach D, Pich S, Soriano FX, Vega N, Baumgartner B, Oriola J, Daugaard JR, Lloberas J, Camps M, Zierath JR, Rabasa-Lhoret R,Wallberg- Henriksson H, Laville M, Palacı´n M, Vidal H, Rivera F, Brand M, Zorzano A (2003) Mitofusin-2 determines mitochondrial network architecture and mitochondrial metabolism. J Biol Chem 278(19):17190-17197. https://doi.org/10.1074/jbc.M212754200 Beshay EVN (2018) Therapeutic efficacy of Artemisia absinthium against Hymenolepis nana : in vitro and in vivo studies in comparison with the anthelmintic praziquantel. J Helminthol 92(3):298-308. https://doi.org/10.1017/S0022149X17000529 Challam M, Roy B, Tandon V (2012) In vitro anthelmintic efficacy of Carex baccans (Cyperaceae): ultrastructural, histochemical and biochemical alterations in the cestode, Raillietina echinobothrida. J Parasit Dis 36(1):81-86. https://doi.org/10.1007/s12639-011-0087-7 Chauhan A, Semwal DK, Mishra SP, Semwal RB (2015) Ayurvedic research and methodology: Present status and future strategies. Ayu 36(4):364-369. https://doi.org/10.4103/0974-8520.190699 Chen RP, Yadav AK (2018) In vitro anthelmintic evaluation of common Indian Ayurvedic anthelmintic drugs: Krimimudgar Ras, Kriminol and Vidangasava against intestinal helminths. Tang Humanitas Med 8(3):15.1-15.5. https://dx.doi.org/10.5667/tang.2018.0017 Chen RP, Yadav AK (2019) Scanning electron microscopic effects of Indian Ayurvedic drugs: Krimimudgar Ras, Kriminol and Vidangasava on a tapeworm, Raillietina sp., Tang Humanitas Med 9(4):9.1-9.4. https://dx.doi.org/10.5667/tang.2019.0024 Dasgupta S, Roy B (2010) Antiparasitic activity of methanolic extract of Acacia oxyphylla (Leguminosae) against Raillietina echinobothrida . J Parasit Dis 34(1):14-19. https://doi.org/10.1007/s12639-010-0001-8 Debnath P, Banerjee S, Adhikari A, Debnath PK (2017) Ayurpharmacoepidemiology enroute to safeguarding safety and efficacy of Ayurvedic drugs in global outlook. J Evid Based Complement Altern Med 22(2):294-298. https://doi.org/10.1177/2156587215624032 Dey P, Roy B (2018) Biochemical and ultrastructural changes in Raillietina echinobothrida in vitro exposed to extract of Lysimachia ramosa. J Parasit Dis 42(2):212-219. https://doi.org/10.1007/s12639-018-0985-z Khan S, Meena AK, Saluja A (2015) In-vitro anthelmintic evaluation of polyherbal formulation Krumighattini tablet. Res J Pharmacol Pharmacodyn 7(4):119-124. https://doi.org/10.5958/2321-5836.2015.00036.1 Kundu S, Roy S, Lyndem, LM (2012) Cassia alata L: potential role as anthelmintic agent against Hymenolepis diminuta . Parasitol Res 111(3):1187-1192. https://doi.org/10.1007/s00436-012-2950-6 Kundu S, Roy S, Nandi S, Ukil B, Lyndem LM (2017) Senna alexandrina Mill. induced ultrastructural changes on Hymenolepis diminuta . J Parasit Dis 41(1):147-154. https://doi.org/10.1007/s12639-016-0768-3 Lumsden RD (1975) Surface ultrastructure and cytochemistry of parasitic helminths. Exp Parasitol 37(2):267–339. https://doi.org/10.1016/0014-4894(75)90078-8 Mahajon B, Chincholikar MB, Tripathi AK (2023) General guidelines for safety/toxicity evaluation of Ayurvedic formulations: Book review. J Drug Res Ayurvedic Sci 8(1):91-93. https://doi.org/10.4103/jdras.jdras_119_22 Mohandas S, Sreekumar TR, Prakash V (2013) Anthelmintic activity of Vidangadi churna. Asian J Pharm Clin Res 6(3):94-95. Paudyal B, Thapa A, Sigdel KR, Adhikari S, Basnyat B (2019) Adverse events with ayurvedic medicines- possible adulteration and some inherent toxicities. Wellcome Open Res 4:23. https://doi.org/10.12688/wellcomeopenres.15096.3 Roy B, Dasgupta S, Giri BR (2012b) Electron microscopic observations on the alterations of tegumental surface of Raillietina echinobothrida treated with root-peel extract of Potentilla fulgens. Microsc Res Tech 75(7):1000-1005. https://doi.org/10.1002/jemt.20972 Roy B, Dasgupta S, Tandon V (2008) Ultrastructural observations on tegumental surface of Raillietina echinobothrida and its alterations caused by the root-peel extract of Millettia pachycarpa . Microsc Res Tech 71(11):810-815. https://doi.org/10.1002/jemt.20623 Roy B, Swargiary A, Giri BR (2012a) Alpinia nigra (Family Zingiberaceae): An anthelmintic medicinal plant of North-East India. Adv Lif Sci 2(3):39-51. https://doi.org/10.5923/j.als.20120203.01 Sangle CS (2023) Public perception of ayurveda-based clinical trials. J Ayurveda Intgr Med 14(1):100678. https://doi.org/10.1016/j.jaim.2022.100678 Sarkar PK, Chaudhari S, Chattopadhyay A (2013) Concept of interactions between consumable substances in Ayurveda with special reference to foods and drugs. Drug Metabol Drug Interact 28(3):147-152. https://doi.org/10.1515/dmdi-2013-0014 Shi Y, Zhang C, Li X (2021) Traditional medicine in India. J Tradit Chin Med Sci 8(1):S51-S55. https://doi.org/10.1016/j.jtcms.2020.06.007 Stitt AW, Fairweather I (1996) Fasciola hepatica : Disruption of the vitelline cells in vitro by the sulphoxide metabolite of triclabendazole. Parasitol Res 82(4):333-339. https://doi.org/10.1007/s004360050122 Threadgold LT, Read CP (1970) Hymenolepis diminuta : Ultrastructure of a unique membrane specialization in tegument. Exp Parasitol 28:246–252. https://doi.org/10.1016/0014-4894(70)90093-7 Von Son-de Fernex E, Alonso-Diaz MA, Valles-de la Mora B, Mendoza-de Gives P, Gonzalez-Cortazar M, Zamilpa A (2017) Anthelmintic effect of 2H-chromen-2-one isolated from Glicidia sepium against Cooperia punctata. Exp Parasitol 178:1-6. https://doi.org/10.1016/j.exppara.2017.04.013 Cite Share Download PDF Status: Published Journal Publication published 24 Oct, 2023 Read the published version in Journal of Parasitic Diseases → Version 1 posted Editorial decision: Major revisions needed 27 Sep, 2023 Reviewers agreed at journal 11 Jul, 2023 Reviewers invited by journal 11 Jul, 2023 Editor assigned by journal 10 Jul, 2023 First submitted to journal 07 Jul, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3149676","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":217678429,"identity":"844f2d59-ec8a-47ce-aa78-41a1fcc5615b","order_by":0,"name":"Risa Parkordor Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBUlEQVRIiWNgGAWjYBACCWYGNiBlwcDADKQS/9kAScbGA0RokYBoecCWBtLSgF8LA0wLSO0DtsNgBl4tku3caQ8+7pFI3N7O/vBDAs95u7Xth4G21NhE49Iizcy73XDGM4nEOYd5jCUSJG4nbzuTCNRyLC23AYcWOWbebdI8ByQSZzDzMEgkGNxONjsA1MLYcBi/lj9gLeyPfyQknEs2O/8QvxZpkBYGsBYGM4mEAwfszG4QsEWyGeiXngMSxkCHmVkkNiQnmN0A2pKAxy8S589ue/DjgI3sDP7jj2/+bLCzNzuf/vDBhxobnFpgwBGmIBHMSCCgHATsMRijYBSMglEwCmAAALW8YDnfY6zsAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-9005-1394","institution":"NEHU: North Eastern Hill University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Risa","middleName":"Parkordor","lastName":"Chen","suffix":""},{"id":217678430,"identity":"90c7675a-2c2a-4bc6-bfec-16c58ed66a46","order_by":1,"name":"Amar Deep Soren","email":"","orcid":"","institution":"B Barooah College","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Amar","middleName":"Deep","lastName":"Soren","suffix":""},{"id":217678431,"identity":"b4f6846d-a9bd-48b2-976e-9b873c64995e","order_by":2,"name":"Arun Kumar Yadav","email":"","orcid":"","institution":"NEHU: North Eastern Hill University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Arun","middleName":"Kumar","lastName":"Yadav","suffix":""}],"badges":[],"createdAt":"2023-07-07 15:05:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3149676/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3149676/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12639-023-01630-6","type":"published","date":"2023-10-24T15:01:26+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":45453770,"identity":"ee46c894-debb-4183-8281-823372840992","added_by":"auto","created_at":"2023-10-30 15:06:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":211099,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3149676/v1/14cdd3b9-8d60-4bcb-8cbd-79cf2623480f.pdf"}],"financialInterests":"","formattedTitle":"Anthelmintic evaluation of three Ayurvedic formulations: a transmission electron microscopy study in Raillietina sp. (Cestoda)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIndia 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 \u0026lsquo;Science of Life\u0026rsquo; (Debnath et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; 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. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Paudyal et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). 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. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). 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. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Sangle \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). 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\u0026rsquo;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 \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). 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 \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe 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 (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) investigated the \u003cem\u003ein vitro\u003c/em\u003e anthelmintic effects of these Ayurvedic formulations on intestinal cestode parasite of domestic fowl, \u003cem\u003eRaillietina\u003c/em\u003e sp. using scanning electron microscopy. These studies report the anthelmintic efficacy of these common Ayurvedic formulations, Krimimudgar Ras, Kriminol, and Birangasav on \u003cem\u003eRaillietina\u003c/em\u003e sp., using transmission electron microscopy (TEM).\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eThe 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.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These Ayurvedic formulations were procured from Ayurvedic pharmacies in Shillong and Guwahati.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eKrimimudgar 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 \u0026lsquo;Bhaishajya Ratnavali\u0026rsquo;, a reputed classical text of Ayurveda.\u003c/p\u003e \u003cp\u003eThe adult live worms of \u003cem\u003eRaillietina\u003c/em\u003e sp. (Cestoda) were dissected out from the intestines of domestic chicken (\u003cem\u003eGallus domesticus\u003c/em\u003e) procured from local markets and placed in 0.9% phosphate buffered saline (PBS). These worms (n\u0026thinsp;=\u0026thinsp;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. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Khan et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Chen and Yadav \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). One set of cestode parasites in 0.9% PBS served as negative control (Chen and Yadav \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAfter 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\u0026rsquo;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.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). A neat nucleus with intact double membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) and mitochondria with prominent cristae (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec) 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). The nucleus appeared dense and irregular in shape with scattered chromatin and the nuclear membrane was disrupted at various regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). Kriminol-treated parasites revealed comparatively less damages in the microthrix layer, distal cytoplasm, muscle layer and basal lamina (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg). The nucleus was observed to be abnormal lacking nucleolus and the chromatin appeared to be condensed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh). In addition, numerous vacuoles were detected. Also, numerous mitochondria with intact cristae were seen to aggregate (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei). 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ej). A nucleus with prominent nucleolus and chromatin concentrated in the periphery was observed. The nuclear membrane however was not detectable in several locations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ek). Mitochondria were dilated and cristae were indistinguishable (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003el). However, it should be noted that Birangasav-treated parasites took the longest time for paralysis and mortality to occur during the \u003cem\u003ein vitro\u003c/em\u003e anthelmintic assay.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePZQ-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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003em). Damaged nucleus with condensed chromatin and thinning of nuclear membrane were also observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003en). Distorted and sausage-shaped mitochondria with clumped cristae can be observed from the micrograph (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eo).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe 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 \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). This tegumental region of the cestode is highly prone to attack by anthelmintics (Alvarez et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). 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 \u003cem\u003ein vitro\u003c/em\u003e anthelmintic study reports that, Birangasav took the longest time for paralysis and mortality to occur (Chen and Yadav \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe 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. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Beshay \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). For example, the alcoholic extract of \u003cem\u003eMillettia pachycarpa\u003c/em\u003e, a traditional medicinal plant of Mizoram, has been reported to cause extensive damages to the internal ultrastructure of \u003cem\u003eRaillietina echinobothrida\u003c/em\u003e, 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. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Likewise, the extracts from \u003cem\u003eSenna alexandrina\u003c/em\u003e and \u003cem\u003eArtemisia absinthium\u003c/em\u003e also have been found to have effects on the cestodes, \u003cem\u003eHymenolepis diminuta\u003c/em\u003e and \u003cem\u003eH. nana\u003c/em\u003e, respectively, in the form of disruption in the microthrix layer, tegument, and subtegument of parasites (Kundu et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Beshay \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In a study by Dey and Roy (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), \u003cem\u003eR. echinobothrida\u003c/em\u003e when exposed to the ethanolic extract of \u003cem\u003eLysimachia ramosa\u003c/em\u003e 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, \u003cem\u003eFasciolopsis buski\u003c/em\u003e when treated with the crude extract of \u003cem\u003eAlpinia nigra\u003c/em\u003e revealed a total alteration in the tegument and sub-tegument in the form of deformed microtriches, vacuolization, and irregularity of mitochondria (Roy et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2012a\u003c/span\u003e). Eggs of the nematode \u003cem\u003eCooperia punctata\u003c/em\u003e were also processed for TEM after treating them with the acetonic extract of \u003cem\u003eGliricidia sepium\u003c/em\u003e leaves whereby eggshells were fractured at many places (von Son-de Fernex et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). \u003cem\u003eSchistosoma mansoni\u003c/em\u003e and \u003cem\u003eS. hematobium\u003c/em\u003e when incubated in 50\u0026ndash;500 \u0026micro;M of curcumin powder showed a disruption in the architecture and motility functions (Abou El Dahab et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe 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 \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1975\u003c/span\u003e). 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. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). 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 \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1970\u003c/span\u003e). 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. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Roy et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2012b\u003c/span\u003e). Disruption in the nucleus, chromatin or nuclear membrane indicates an inhibition in protein synthesis (Stitt and Fairweather \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). 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. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Roy et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2012b\u003c/span\u003e). 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. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The aforementioned changes in tegument and subtegument of \u003cem\u003eRaillietina\u003c/em\u003e 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.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe 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.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA fellowship awarded to Risa Parkordor Chen from UGC is acknowledged. TEM facilities were provided by the Sophisticated Analytical Instrument Facility, NEHU Shillong.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthic declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis study did not receive any funding from any agency or individual.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest:\u0026nbsp;\u003c/strong\u003eAll authors declare that there is no conflict of interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval:\u0026nbsp;\u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate:\u003c/strong\u003e Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material:\u0026nbsp;\u003c/strong\u003eAll data generated during this study are included in this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability:\u0026nbsp;\u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u0026nbsp;\u003c/strong\u003eThis 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.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbou El Dahab M, Shahat SM, Mahmoud SSM, Mahana NA (2019) \u003cem\u003eIn vitro \u003c/em\u003eeffect of curcumin on \u003cem\u003eSchistosoma \u003c/em\u003especies viability, tegument structure and egg hatchability. Exp Parasitol 199:1-8. https://doi.org/10.1016/j.exppara.2019.02.010\u003c/li\u003e\n\u003cli\u003eAcharya R (2023) Emphasizing evidence-based research: a way forward to robust validity of clinical research in Ayurveda. J Res Ayurvedic Sci 7(2):77-79. https://doi.org/10.4103/jras.jras_49_23\u003c/li\u003e\n\u003cli\u003eAlvarez LI, Mottier ML, Lanusse CE (2007) Drug transfer into target helminth parasites. Trends Parasitol 23(3):97-104. https://doi.org/10.1016/j.pt.2007.01.003\u003c/li\u003e\n\u003cli\u003eBach D, Pich S, Soriano FX, Vega N, Baumgartner B, Oriola J, Daugaard JR, Lloberas J, Camps M, Zierath JR, Rabasa-Lhoret R,Wallberg- Henriksson H, Laville M, Palacı\u0026acute;n M, Vidal H, Rivera F, Brand M, Zorzano A (2003) Mitofusin-2 determines mitochondrial network architecture and mitochondrial metabolism. J Biol Chem 278(19):17190-17197. https://doi.org/10.1074/jbc.M212754200\u003c/li\u003e\n\u003cli\u003eBeshay EVN (2018) Therapeutic efficacy of \u003cem\u003eArtemisia absinthium\u003c/em\u003e against \u003cem\u003eHymenolepis nana\u003c/em\u003e: \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e studies in comparison with the anthelmintic praziquantel. J\u003cem\u003e \u003c/em\u003eHelminthol 92(3):298-308. https://doi.org/10.1017/S0022149X17000529\u003c/li\u003e\n\u003cli\u003eChallam M, Roy B, Tandon V (2012) \u003cem\u003eIn vitro \u003c/em\u003eanthelmintic efficacy of \u003cem\u003eCarex baccans\u003c/em\u003e (Cyperaceae): ultrastructural, histochemical and biochemical alterations in the cestode, \u003cem\u003eRaillietina echinobothrida.\u003c/em\u003e J Parasit Dis\u003cem\u003e \u003c/em\u003e36(1):81-86. https://doi.org/10.1007/s12639-011-0087-7\u003c/li\u003e\n\u003cli\u003eChauhan A, Semwal DK, Mishra SP, Semwal RB (2015) Ayurvedic research and methodology: Present status and future strategies. Ayu 36(4):364-369. https://doi.org/10.4103/0974-8520.190699\u003c/li\u003e\n\u003cli\u003eChen RP, Yadav AK (2018) \u003cem\u003eIn vitro\u003c/em\u003e anthelmintic evaluation of common Indian Ayurvedic anthelmintic drugs: Krimimudgar Ras, Kriminol and Vidangasava against intestinal helminths. Tang Humanitas Med 8(3):15.1-15.5. https://dx.doi.org/10.5667/tang.2018.0017\u003c/li\u003e\n\u003cli\u003eChen RP, Yadav AK (2019) Scanning electron microscopic effects of Indian Ayurvedic drugs: Krimimudgar Ras, Kriminol and Vidangasava on a tapeworm, \u003cem\u003eRaillietina\u003c/em\u003e sp., Tang Humanitas Med 9(4):9.1-9.4. https://dx.doi.org/10.5667/tang.2019.0024\u003c/li\u003e\n\u003cli\u003eDasgupta S, Roy B (2010) Antiparasitic activity of methanolic extract of \u003cem\u003eAcacia oxyphylla\u003c/em\u003e (Leguminosae) against \u003cem\u003eRaillietina echinobothrida\u003c/em\u003e. J Parasit Dis 34(1):14-19. https://doi.org/10.1007/s12639-010-0001-8\u003c/li\u003e\n\u003cli\u003eDebnath P, Banerjee S, Adhikari A, Debnath PK (2017) Ayurpharmacoepidemiology enroute to safeguarding safety and efficacy of Ayurvedic drugs in global outlook. J Evid Based Complement Altern Med\u003cem\u003e \u003c/em\u003e22(2):294-298. https://doi.org/10.1177/2156587215624032\u003c/li\u003e\n\u003cli\u003eDey P, Roy B (2018) Biochemical and ultrastructural changes in \u003cem\u003eRaillietina echinobothrida in vitro\u003c/em\u003e exposed to extract of \u003cem\u003eLysimachia ramosa. \u003c/em\u003eJ Parasit Dis\u003cem\u003e \u003c/em\u003e42(2):212-219. https://doi.org/10.1007/s12639-018-0985-z\u003c/li\u003e\n\u003cli\u003eKhan S, Meena AK, Saluja A (2015) \u003cem\u003eIn-vitro\u003c/em\u003e anthelmintic evaluation of polyherbal formulation Krumighattini tablet. Res J Pharmacol Pharmacodyn\u003cem\u003e \u003c/em\u003e7(4):119-124. https://doi.org/10.5958/2321-5836.2015.00036.1 \u003c/li\u003e\n\u003cli\u003eKundu S, Roy S, Lyndem, LM (2012) \u003cem\u003eCassia alata\u003c/em\u003e L: potential role as anthelmintic agent against \u003cem\u003eHymenolepis diminuta\u003c/em\u003e. Parasitol Res\u003cem\u003e \u003c/em\u003e111(3):1187-1192. https://doi.org/10.1007/s00436-012-2950-6\u003c/li\u003e\n\u003cli\u003eKundu S, Roy S, Nandi S, Ukil B, Lyndem LM (2017) \u003cem\u003eSenna alexandrina\u003c/em\u003e Mill. induced ultrastructural changes on \u003cem\u003eHymenolepis diminuta\u003c/em\u003e. J Parasit Dis 41(1):147-154. https://doi.org/10.1007/s12639-016-0768-3\u003c/li\u003e\n\u003cli\u003eLumsden RD (1975) Surface ultrastructure and cytochemistry of parasitic helminths. Exp Parasitol 37(2):267\u0026ndash;339. https://doi.org/10.1016/0014-4894(75)90078-8\u003c/li\u003e\n\u003cli\u003eMahajon B, Chincholikar MB, Tripathi AK (2023) General guidelines for safety/toxicity evaluation of Ayurvedic formulations: Book review. J Drug Res Ayurvedic Sci 8(1):91-93. https://doi.org/10.4103/jdras.jdras_119_22\u003c/li\u003e\n\u003cli\u003eMohandas S, Sreekumar TR, Prakash V (2013) Anthelmintic activity of Vidangadi churna. Asian J Pharm Clin Res\u003cem\u003e \u003c/em\u003e6(3):94-95.\u003c/li\u003e\n\u003cli\u003ePaudyal B, Thapa A, Sigdel KR, Adhikari S, Basnyat B (2019) Adverse events with ayurvedic medicines- possible adulteration and some inherent toxicities. Wellcome Open Res\u003cem\u003e \u003c/em\u003e4:23. https://doi.org/10.12688/wellcomeopenres.15096.3\u003c/li\u003e\n\u003cli\u003eRoy B, Dasgupta S, Giri BR (2012b) Electron microscopic observations on the alterations of tegumental surface of \u003cem\u003eRaillietina\u003c/em\u003e \u003cem\u003eechinobothrida\u003c/em\u003e treated with root-peel extract of \u003cem\u003ePotentilla\u003c/em\u003e \u003cem\u003efulgens. \u003c/em\u003eMicrosc Res Tech 75(7):1000-1005. https://doi.org/10.1002/jemt.20972\u003c/li\u003e\n\u003cli\u003eRoy B, Dasgupta S, Tandon V (2008) Ultrastructural observations on tegumental surface of \u003cem\u003eRaillietina echinobothrida\u003c/em\u003e and its alterations caused by the root-peel extract of \u003cem\u003eMillettia\u003c/em\u003e \u003cem\u003epachycarpa\u003c/em\u003e. Microsc Res Tech\u003cem\u003e \u003c/em\u003e71(11):810-815. https://doi.org/10.1002/jemt.20623\u003c/li\u003e\n\u003cli\u003eRoy B, Swargiary A, Giri BR (2012a) \u003cem\u003eAlpinia nigra\u003c/em\u003e (Family Zingiberaceae): An anthelmintic medicinal plant of North-East India. Adv Lif Sci 2(3):39-51. https://doi.org/10.5923/j.als.20120203.01\u003c/li\u003e\n\u003cli\u003eSangle CS (2023) Public perception of ayurveda-based clinical trials. J Ayurveda Intgr Med 14(1):100678. https://doi.org/10.1016/j.jaim.2022.100678\u003c/li\u003e\n\u003cli\u003eSarkar PK, Chaudhari S, Chattopadhyay A (2013) Concept of interactions between consumable substances in Ayurveda with special reference to foods and drugs. Drug Metabol Drug\u003cem\u003e \u003c/em\u003eInteract\u003cem\u003e \u003c/em\u003e28(3):147-152. https://doi.org/10.1515/dmdi-2013-0014\u003c/li\u003e\n\u003cli\u003eShi Y, Zhang C, Li X (2021) Traditional medicine in India. J Tradit Chin Med Sci 8(1):S51-S55. https://doi.org/10.1016/j.jtcms.2020.06.007\u003c/li\u003e\n\u003cli\u003eStitt AW, Fairweather I (1996) \u003cem\u003eFasciola hepatica\u003c/em\u003e: Disruption of the vitelline cells \u003cem\u003ein vitro\u003c/em\u003e by the sulphoxide metabolite of triclabendazole. Parasitol Res 82(4):333-339. https://doi.org/10.1007/s004360050122\u003c/li\u003e\n\u003cli\u003eThreadgold LT, Read CP (1970) \u003cem\u003eHymenolepis diminuta\u003c/em\u003e: Ultrastructure of a unique membrane specialization in tegument. Exp Parasitol 28:246\u0026ndash;252. https://doi.org/10.1016/0014-4894(70)90093-7\u003c/li\u003e\n\u003cli\u003eVon Son-de Fernex E, Alonso-Diaz MA, Valles-de la Mora B, Mendoza-de Gives P, Gonzalez-Cortazar M, Zamilpa A (2017) Anthelmintic effect of 2H-chromen-2-one isolated from \u003cem\u003eGlicidia sepium \u003c/em\u003eagainst \u003cem\u003eCooperia punctata. \u003c/em\u003eExp Parasitol 178:1-6. https://doi.org/10.1016/j.exppara.2017.04.013\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-parasitic-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jopd","sideBox":"Learn more about [Journal of Parasitic Diseases](https://www.springer.com/journal/12639)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jopd/default.aspx","title":"Journal of Parasitic Diseases","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Anthelmintic, Ayurveda, Birangasav, Krimimudgar Ras, Kriminol, Raillietina, Transmission electron microscopy","lastPublishedDoi":"10.21203/rs.3.rs-3149676/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3149676/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAyurveda is one of the ancient traditional medicine systems in India. However, several Ayurvedic medicines lack scientific evidence about their efficacy. This study reports the \u003cem\u003ein vitro\u003c/em\u003e anthelmintic effects of three common Ayurvedic formulations, Krimimudgar Ras, Kriminol, and Birangasav on a poultry cestode \u003cem\u003eRaillietina\u003c/em\u003e 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.\u003c/p\u003e","manuscriptTitle":"Anthelmintic evaluation of three Ayurvedic formulations: a transmission electron microscopy study in Raillietina sp. (Cestoda)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-07-14 15:20:40","doi":"10.21203/rs.3.rs-3149676/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions needed","date":"2023-09-28T03:19:37+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2023-07-11T16:01:45+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-07-11T13:31:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-07-10T04:31:54+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Parasitic Diseases","date":"2023-07-07T11:05:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-parasitic-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jopd","sideBox":"Learn more about [Journal of Parasitic Diseases](https://www.springer.com/journal/12639)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jopd/default.aspx","title":"Journal of Parasitic Diseases","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"9d991865-2aa4-41d0-96de-2ac66a517262","owner":[],"postedDate":"July 14th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-30T15:03:42+00:00","versionOfRecord":{"articleIdentity":"rs-3149676","link":"https://doi.org/10.1007/s12639-023-01630-6","journal":{"identity":"journal-of-parasitic-diseases","isVorOnly":false,"title":"Journal of Parasitic Diseases"},"publishedOn":"2023-10-24 15:01:26","publishedOnDateReadable":"October 24th, 2023"},"versionCreatedAt":"2023-07-14 15:20:40","video":"","vorDoi":"10.1007/s12639-023-01630-6","vorDoiUrl":"https://doi.org/10.1007/s12639-023-01630-6","workflowStages":[]},"version":"v1","identity":"rs-3149676","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3149676","identity":"rs-3149676","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}