Phytochemical analysis and in vitro anthelmintic activity of Imperata cylindrica underground parts

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.

resistance is evolving at an accelerated pace in the most important helminth parasites of livestock animals [7] and humans [8].
The tapeworm Raillietina tetragona Molin, 1858, and the roundworm Ascaridia galli Schrank, 1788, are the most prevalent and thereby the most important parasites of birds in different parts of the world [9][10][11]. They cause detrimental health conditions and pathological symptoms including anaemia, droopiness, emaciation and diarrhoea [12]. The consequences such as loss of appetite, weight loss, reduced egg production and death lead to economic losses in poultry farming [13]. In spite of their huge influences in avian health and poultry production, there are no prescription anthelmintic drugs.
Medicinal plants are as a source of many important pharmaceutical drugs, but they have not yet produced a single anthelmintic drug. An interesting plant is Imperata cylindrica (L.) Raeusch. (family -Poaceae), the underground parts (rhizomes and roots) of which are used in Mizo traditional medicine as a treatment for intestinal helminthiasis, fungal infections, blood urine (haematuria), blood vomit (haematemesis), and nosebleed (epistaxis) [14]. Although the plant is regarded as a noxious weed [15], it is recognised in Southeast Asian cultures as an antibacterial, anticoagulant (styptic), antipyretic (febrifuge), water pill (diuretic), skin smoothening (emollient), salivating (sialagogue), and blood soothing (tonic) agent [16]. Its effects on circulatory system such as in vasodilation and blood flow have been reported [17,18].
Practically no attention has been given to this plant in terms of chemical analysis and biological activities against parasites. It is therefore challenging to study its effect on intestinal parasites.
A remarkable fact is that while most anthelmintic drugs are helminth specific, i.e. they are effective only against a particular group of helminths, I. cylindrica is acclaimed to be equally effective against both tapeworms and roundworms [14]. Identification of important compounds present in the plant extract and anthelmintic tests using both tapeworm and roundworm will be useful for further investigations on the pharmacology of this plant.

Plant material and extraction
I. cylindrica was collected from Ngopa, Mizoram, India, which is located between 23.8861°latitude north and 93.2119°longitude east. The plant specimen with voucher number PUC-I-2018-01 was authenticated at the Botanical Survey of India (BSI), Shillong, Meghalaya, India. The aerial parts were discarded and the underground parts including rhizomes and roots were dried in shade at 21-25°C. The dried parts were ground to fine powder using electric blender.
The plant extracts were prepared in a 5-l capacity Soxhlet apparatus using three solvents such as methanol (polar solvent), chloroform (medium polar solvent) and hexane (non-polar solvent). The extracts were concentrated by removing and recovering the solvents in a vacuum rotary evaporator (Buchi Rotavapor® R-215) [14]. The final extracts were obtained as semisolid precipitates and were stored at 4°C until use. The chloroform extract was found to contain the most abundant compounds and showed the highest biological activity, and thus was used for complete chemical analysis and anthelmintic assay.

Chemicals and drug
All chemicals were standard analytical grades procured from HiMedia Laboratories Private Limited, Mumbai, India. Acetonitrile for gas chromatography and tetramethylsilane for electron microscopy were products of Merck Life Science Private Limited, Mumbai, India. A standard anthelmintic, albendazole (ZENTEL®) was a product of GlaxoSmithKline Pharmaceuticals Ltd., Mumbai, India.

Chemical analysis
The chemical constituent of the plant extract was analysed in a single quadrupole gas chromatography-mass spectrometry system (Thermo Scientific TRACE™ 1300 ISQ™ LT). Acetonitrile was used to dissolve the extract. GC elution was done in a non-polar column TR-5MS (260F142P) having a dimension of 30 m × 0.25 mm × 0.25 μm and film thickness of 0.25 μm. Temperature of the injector port was set at 250°C. The oven temperature was initially set at 70°C for 2 min and incrementally increased at 10°C up to 250°C. Helium was used a carrier gas and passed at a constant flow rate of 1 ml/min. One microliter of the sample was injected in a splitting ratio of 1:50. The ionisation electron energy of the mass spectrometer was set at 70 eV. Ion source and transfer line temperature were set at 250°C. The running duration was 55 min. The final chromatogram was generated with Thermo Scientific™ Xcalibur™ software. Compounds were identified based on their chemical formula, retention time, and molecular weight from the libraries of Wiley Registry™ and National Institute of Standards and Technology (NIST) database.

Anthelmintic test
Anthelmintic activity was studied in vitro on the survival of tapeworm R. tetragona, and the roundworm A. galli [19]. The helminth parasites were recovered from the intestines of freshly sacrificed local chicken, Gallus Gallus domesticus Linnaeus, 1758. Use of chicken was permitted by the Institutional Ethics Committee of Pachhunga University College (PUC-IEC-2016-Z2 of 10/ 08/2016). The worms were collected and washed in Petri dishes containing 0.9% neutral phosphate-buffered saline (PBS) maintained at 37 ± 1°C in a glass-panelled microbiological incubator. I. cylindrica chloroform extract was prepared in exponential concentrations of 1.25, 2.5, 5, 10 and 20 mg/ml by dissolving in PBS supplemented with 1% dimethylsulfoxide (DMSO). A broad-spectrum anthelmintic, albendazole (with a standard dosage of 20 mg/ml) was prepared similarly as a positive control. Negative control consisted of worms kept in Petri dishes containing only PBS with 1% DMSO. All the media were maintained at 37 ± 1°C. A set of five worms were introduced into each media, and each test was performed in triplicates.
Survival was defined as a total loss of motor activity after stimulation by dipping the parasites in lukewarm PBS (45°C). Data were generated as means ± standard deviation and presented in normalised values against the control. Student's t-test was used to determine the statistical significance and the level of significance was considered when p value was less than 0.05.

Scanning electron microscopy
Helminths in control experiment and those treated with 20 mg/ml of the plant extract were processed for scanning electron microscopy to compare the structural appearances based on a standardized method for helminths [20]. They were first fixed in 10% formaldehyde (buffered with 0.1 M sodium cacodylate) at 4°C for 4 h. Then a secondary fixation was done with 1% osmium tetroxide at 4°C for 1 h. They were dehydrated through a series of acetone in increasing concentrations. After treating with tetramethylsilane for 15 min they were dried in an air-drying chamber at 25°C. They were mounted on metal stubs and sputter coated with gold in JFC-1100 (JEOL Ltd., Tokyo, Japan) ion-sputtering chamber. Finally, they were observed under a JSM-6360 scanning electron microscope (JEOL Ltd., Tokyo, Japan) at an electron accelerating voltage of 20 kV.

Anthelmintic activity
The in vitro anthelmintic activity of the chloroform extract of I. cylindrica against the tapeworm, R. tetragona is presented in Anthelmintic activity of the chloroform extract of I. cylindrica on A. galli is shown in Table 3. Roundworms were more resilient that tapeworms and survived up to 216.29 h in control media. Albendazole was again highly

Scanning electron microscopy
Scanning electron microscopy of an untreated R. tetragona is shown in Fig. 2. The tapeworm has an anterior bulb-like scolex which bears the attachment organs called suckers (Fig. 2a). Each sucker is oval shaped and  is lined with rows of pointed projections called the spines (Fig. 2b). The main body consists of a series of body segments or proglottids. The proglottids appear smooth and silky as the tegument (body surface) is made up of microscopic hairs called microtriches (Fig. 2c).
R. tetragona treated with 20 mg/ml of the chloroform extract of I. cylindrica revealed extensive changes on the tegument throughout the body. The scolex as shown in Fig. 3a indicates tegumental erosion and distortion of the suckers. Irregular masses on the general tegument and around the suckers are visible. In each sucker, the spines clumped and a central pit is formed which indicates tegumental erosion (Fig. 3b). Folding of the tegument and degeneration of microtriches are evident on all the proglottids (Fig. 3c).
Scanning electron microscopy of an untreated A. galli is presented in Fig. 4. The terminal end of the anterior part of the body consists of three blob-like structures called the lips. The lips surround the mouth. Sensory organs called papillae are seen as protrusions on the lips (Fig. 4a). The general body surface, called the cuticle, is rigid and smooth. There are straight and parallel transverse rings called annulations throughout the body (Fig. 4b). The tail end bears anal opening or cloaca, precloacal sucker and several sensory protrusions called amphids (Fig. 4c).
The anthelmintic effects are more pronounced on A. galli treated with 20 mg/ml of the chloroform extract of I. cylindrica. The deleterious effects are particularly pronounced on the head region as shown in Fig. 5a. Three lips fully collapsed and the surrounding cuticle is massively shrunken. The main body is extensively shrunk with the formation of irregular warts all over the cuticle. The annulations are completely distorted (Fig. 5b). The deformity extends to the tail region as shown in Fig. 5c. Cuticular shrinkage extends to the tip of the tail. The amphids are degenerated and there is formation of scar in the precloacal sucker.
In the present study, the rhizomes and roots appear to contain a number of important bioactive compounds. For instance, palmitic acid has antibacterial activity against different types of pathogenic bacteria [29,30]. It also shows selective cytotoxicity upon human leukemic cells [31]. 2-Methoxy-4-vinylphenol isolated from pine (Pinus species) exhibits anticancer activity in vitro against different cancer cell lines [32]. It was also shown to exhibit immunomodulatory activity by enhancing antiinflammatory response suppression of NF-κB and MAPK activation, and acetylation of histone H3 [33]. 2, 4-Di-tert-butylphenol from Persea americana is a potent antimicrobial compound against pathogenic microbes such as Aspergillus sp. and Phytophthora cinnamomi [34]. 6-Methylenebicyclo [3.2.0] hept-3-en-2-one is reported from Allium tuberosum and was shown to play an important role in immunity against parasitic infection such as root-knot nematode, Meloidogyne species [35]. Phytosterols are known for their blood-lipid lowering and anticancer activities. Campesterol inhibits fibroblast growth factor and tube formation of human umbilical vein endothelial cells, thereby indicating its role in the prevention of blood cancers [36]. Increased β-sitosterol and campesterol in the blood circulation inhibits proliferation of cancer cells in mice [37]. Campesterol, βsitosterol, and stigmasterol are reported to decrease the  risk of gastric cancer [38] and lung cancer [39]. But campesterol and stigmasterol have no beneficial effect in colon cancer [40]. These compounds are therefore likely cancer-specific and are useful for people with  cardiovascular diseases and cancers [41]. Stigmasterol is further attributed to prevention of osteoarthritis as it inhibits proteins of chondrocytes [42]. It also exhibits hypoglycaemic activity by reducing serum thyroid hormones, triiodothyronine (T 3 ) and thyroxin (T 4 ), as well the activity of hepatic glucose-6-phophatase [43]. Substantiating the therapeutic usage of I. cylindrica as in the Mizo traditional medicine, the findings show that the plant extract does exert an appreciable anthelmintic activity against both tapeworm and roundworm. Damaging effects were seen on the tegument and suckers of the tapeworm R. tetragona, and on the cuticle and lips of the roundworm A. galli. These distinctive effects are to be expected because tapeworms and roundworms belong to two completely distinct classes (phyla) and have major differences in structural and physiological properties [44,45]. In tapeworms, absorption of nutrients or drugs is directly through the body surface, the tegument, and this nature makes drugs act faster [46]. But roundworms are covered with a hard cuticle so that nutrients or drugs are absorbed poorly and slowly; thereby delaying the course of drug action [47]. A prolonged activity is therefore expected for plant extracts such as in the present study that would contain only small quantity of the bioactive compound(s). Tegumental shrinkage, disintegration of microtriches and deformity of the suckers as seen in the present study are the major effects of anthelmintic drugs on tapeworms, albeit with varying degree of damages for different drugs [48,49]. In roundworms, the signature anthelmintic effects include shrinkage and folding of the cuticle, deflation of the lips and damages on the papillae [50,51]. Thus, I. cylindrica extract produced the most notable anthelmintic effects on both tapeworm and roundworm of poultry, and thus has a potential for application in veterinary animals. However, the present in vitro test does not warrant similar efficacy for in vivo condition, therefore, poses the need for in vivo test and identification of the main anthelmintic compound.

Conclusion
The study revealed that I. cylindrica rhizomes and roots contain important bioactive compounds, which exert anthelmintic activity on both tapeworm and roundworm. GC-MS analysis indicated that the chloroform extract of the underground parts is rich in saturated fatty acids. In concurrence with the usage of the plant in the Mizo traditional medicine as an anthelmintic agent, the plant extract was shown to be effective against R. tetragona and A. galli. Tegumental damages on the tapeworm and cuticular distortion on the roundworm revealed by scanning electron microscopy are clear validations of the broad-spectrum anthelmintic activity of the plant. These findings indicate that the plant contains interesting bioactive compound(s) that can serve as lead molecule in anthelmintic development. Authors' contributions KL conceived and designed the study, analysed and interpreted the data, and wrote the first draft of the manuscript. PBL executed the experiments and generated the data. Both authors have read and approved the final manuscript.

Funding
Financial support in the form of Extra Mural Research Funding was granted to KL by the Science and Engineering Research Board (SERB), Government of India (EMR/2016/004053). The funding agency played no role in the concept, execution and outcome of the study.

Availability of data and materials
The datasets during and/or analysed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate
The study was approved by the Institutional Ethics Committee of Pachhunga University College (PUC-IEC-2016-Z2 of 10/08/2016). There are no ethical issues arising from the use of parasites from poultry, and thus, consent is not applicable.

Consent for publication
Not applicable.