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Phytochemical Analysis and in Vitro Anthelmintic Activity of Imperata Cylindrica Underground Parts Pawi Bawitlung Lalthanpuii and Kholhring Lalchhandama*

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Phytochemical Analysis and in Vitro Anthelmintic Activity of Imperata Cylindrica Underground Parts Pawi Bawitlung Lalthanpuii and Kholhring Lalchhandama* Lalthanpuii and Lalchhandama BMC Complementary Medicine and Therapies (2020) 20:332 BMC Complementary https://doi.org/10.1186/s12906-020-03125-w Medicine and Therapies RESEARCH ARTICLE Open Access Phytochemical analysis and in vitro anthelmintic activity of Imperata cylindrica underground parts Pawi Bawitlung Lalthanpuii and Kholhring Lalchhandama* Abstract Background: Imperata cylindrica is considered as an agricultural weed, but it is a valuable plant in the traditional medicines of Southeast Asia. In the Mizo traditional medicine of India and Myanmar, the rhizomes and roots are used as a remedy for bacterial, fungal and intestinal helminth infections. Methods: An extract of the whole underground parts was prepared in Soxhlet apparatus using chloroform as a solvent. After concentrating in a vacuum rotary evaporator, the extract was analysed using gas chromatography- mass spectrometry. Anthelmintic activity was tested in vitro against the tapeworm Raillietina tetragona and the roundworm Ascaridia galli. Scanning electron microscopy was used to examine the structural changes on the helminths after treatment with the plant extract. Results: Twenty-two compounds were identified from the plant extract out of which fatty acids were the predominant compounds. Palmitic acid was the most abundant. Bioactive phytosterols such as campesterol and stigmasterol were also detected. The plant extract was significantly effective on both the helminths and showed dose-dependent anthelmintic activity as that of albendazole. The tapeworm treated with the plant extract showed deformities on the suckers, clumping of the spines, tegumental folds and erosion of microtriches. Extensive damage was also seen on the roundworm including cuticular shrinkage, collapse of the lips, and formation of warty surface throughout the body. Conclusion: I. cylindrica extract effectively killed and caused detrimental effects on parasitic tapeworm and roundworm. The study therefore validates the traditional usage among the Mizo people, and guarantees further investigation on the exact compound(s) and mechanism of action. Keywords: Anthelmintic, Medicinal plant, Roundworm, Scanning electron microscopy, Tapeworm Background infect 1.5 billion people worldwide [3], while schisto- Helminthiasis is one of the most persistent scourges of somes (flukes) account for 220 million cases [4]. the health and welfare of humans, livestock animals and Tapeworms are the least prevalent among them but are wildlife. It remains the major factor of human debility, responsible for the worst outcomes such as neurocysti- poverty, cognitive weakness and sometimes death [1], as cercosis, which cause permanent brain damage and well as of huge economic losses in animal industry [2]. premature fatality. Helminthiasis also intensifies other According to the current estimate, roundworms alone infections such as those of Plasmodium falciparum, human immunodeficiency virus (HIV), and Mycobac- * Correspondence: [email protected] terium tuberculosis [5]. No new drugs have been de- Department of Life Sciences, Pachhunga University College, Aizawl, Mizoram veloped for several decades [6], while anthelmintic 796001, India © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Lalthanpuii and Lalchhandama BMC Complementary Medicine and Therapies (2020) 20:332 Page 2 of 9 resistance is evolving at an accelerated pace in the shade at 21–25 °C. The dried parts were ground to fine most important helminth parasites of livestock ani- powder using electric blender. mals [7] and humans [8]. The plant extracts were prepared in a 5-l capacity The tapeworm Raillietina tetragona Molin, 1858, and Soxhlet apparatus using three solvents such as methanol the roundworm Ascaridia galli Schrank, 1788, are the (polar solvent), chloroform (medium polar solvent) and most prevalent and thereby the most important parasites hexane (non-polar solvent). The extracts were concen- of birds in different parts of the world [9–11]. They trated by removing and recovering the solvents in a vac- cause detrimental health conditions and pathological uum rotary evaporator (Buchi Rotavapor® R-215) [14]. symptoms including anaemia, droopiness, emaciation The final extracts were obtained as semisolid precipitates and diarrhoea [12]. The consequences such as loss of ap- and were stored at 4 °C until use. The chloroform ex- petite, weight loss, reduced egg production and death tract was found to contain the most abundant com- lead to economic losses in poultry farming [13]. In spite pounds and showed the highest biological activity, and of their huge influences in avian health and poultry pro- thus was used for complete chemical analysis and an- duction, there are no prescription anthelmintic drugs. thelmintic assay. Medicinal plants are as a source of many important pharmaceutical drugs, but they have not yet produced a Chemicals and drug single anthelmintic drug. An interesting plant is Imper- All chemicals were standard analytical grades procured ata cylindrica (L.) Raeusch. (family - Poaceae), the from HiMedia Laboratories Private Limited, Mumbai, underground parts (rhizomes and roots) of which are India. Acetonitrile for gas chromatography and tetra- used in Mizo traditional medicine as a treatment for in- methylsilane for electron microscopy were products of testinal helminthiasis, fungal infections, blood urine Merck Life Science Private Limited, Mumbai, India. A (haematuria), blood vomit (haematemesis), and nose- standard anthelmintic, albendazole (ZENTEL®) was a bleed (epistaxis) [14]. Although the plant is regarded as product of GlaxoSmithKline Pharmaceuticals Ltd., a noxious weed [15], it is recognised in Southeast Asian Mumbai, India. cultures as an antibacterial, anticoagulant (styptic), anti- pyretic (febrifuge), water pill (diuretic), skin smoothen- Chemical analysis ing (emollient), salivating (sialagogue), and blood The chemical constituent of the plant extract was ana- soothing (tonic) agent [16]. Its effects on circulatory sys- lysed in a single quadrupole gas chromatography-mass tem such as in vasodilation and blood flow have been re- spectrometry system (Thermo Scientific TRACE™ 1300 ported [17, 18]. ISQ™ LT). Acetonitrile was used to dissolve the extract. Practically no attention has been given to this plant in GC elution was done in a non-polar column TR-5MS terms of chemical analysis and biological activities (260F142P) having a dimension of 30 m × 0.25 mm × against parasites. It is therefore challenging to study its 0.25 μm and film thickness of 0.25 μm. Temperature of effect on intestinal parasites. the injector port was set at 250 °C. The oven A remarkable fact is that while most anthelmintic temperature was initially set at 70 °C for 2 min and in- drugs are helminth specific, i.e. they are effective only crementally increased at 10 °C up to 250 °C. Helium was against a particular group of helminths, I. cylindrica is used a carrier gas and passed at a constant flow rate of acclaimed to be equally effective against both tapeworms 1 ml/min. One microliter of the sample was injected in a and roundworms [14]. Identification of important com- splitting ratio of 1:50. The ionisation electron energy of pounds present in the plant extract and anthelmintic the mass spectrometer was set at 70 eV. Ion source and tests using both tapeworm and roundworm will be use- transfer line temperature were set at 250 °C. The run- ful for further investigations on the pharmacology of this ning duration was 55 min. The final chromatogram was plant. generated with Thermo Scientific™ Xcalibur™ software. Compounds were identified based on their chemical formula, retention time, and molecular weight from the Methods libraries of Wiley Registry™ and National Institute of Plant material and extraction Standards and Technology (NIST) database. I. cylindrica was collected from Ngopa, Mizoram, India, which is located between 23.8861° latitude north and Anthelmintic test 93.2119° longitude east. The plant specimen with vou- Anthelmintic activity was studied in vitro on the survival cher number PUC-I-2018-01 was authenticated at the of tapeworm R. tetragona, and the roundworm A. galli Botanical Survey of India (BSI), Shillong, Meghalaya, [19]. The helminth parasites were recovered from the India. The aerial parts were discarded and the under- intestines of freshly sacrificed local chicken, Gallus ground parts including rhizomes and roots were dried in Gallus domesticus Linnaeus, 1758. Use of chicken was Lalthanpuii and Lalchhandama
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