An Endangered Medicinal Plant, Ilex khasiana Exhibits Potent Antiparasitic Activity Against Intestinal Tapeworm

With almost 600 species described so far in the family Aquifoliaceae, Ilex is the only surviving genus. Commonly known as holly, species of Ilex are familiar in traditional Chinese medicine, especially I. kudingcha and I. latifolia, the leaves of which are parts of dietary foods. They are used as therapeutic agents for their analgesic, antidiarrhoeal, antipyretic, astringent, haematostatic, and hepatoprotective properties.1 South American natives use I. paraguariensis leaves as health-benefit drink and are now commercialised as mate tea. The drink is acclaimed for its hepatoprotection, digestive, antirheumatism, and anti-obesity properties, as well as its ability to prevent arthritis, inflammation, hypertension and hypercholesterolemia.2 Bioactive compounds from the plant are experimentally validated to have antiinflammatory,3 anticancer,4 anti-obesity,5 and cholesterol-reducing activities in animal models.6


INTRODUCTION
With almost 600 species described so far in the family Aquifoliaceae, Ilex is the only surviving genus. Commonly known as holly, species of Ilex are familiar in traditional Chinese medicine, especially I. kudingcha and I. latifolia, the leaves of which are parts of dietary foods. They are used as therapeutic agents for their analgesic, antidiarrhoeal, antipyretic, astringent, haematostatic, and hepatoprotective properties. 1 South American natives use I. paraguariensis leaves as health-benefit drink and are now commercialised as mate tea. The drink is acclaimed for its hepatoprotection, digestive, antirheumatism, and anti-obesity properties, as well as its ability to prevent arthritis, inflammation, hypertension and hypercholesterolemia. 2 Bioactive compounds from the plant are experimentally validated to have antiinflammatory, 3 anticancer, 4 anti-obesity, 5 and cholesterol-reducing activities in animal models. 6 I. pubescens is also well known in traditional Chinese medicine for the treatment of coronary heart diseases, and as antiinflammatory and analgesic agents. 1 It is also known to be an effective remedy for hypertension, hyperlipemia, and hepatitis. 7 Compounds from its root are demonstrated as having anticoagulant, 8 antiinflammatory, 9 and neuro-protective activities. 10 I. cornuta is used in the treatment for dizziness and hypertension. 11 Saponins from its roots are validated as possessing antiviral activity against herpes simplex virus type 1, 12 and myocardial (heart tissue) damage inhibition. 13 I. ficoidea and I. centrochinensis were shown to have potent antiinflammatory and antioxidant activities. 14 Out of 410 accepted species recorded in the genus Ilex, I. khasiana Purk. is outstanding in that it is critically endangered according to the IUCN Red List of Threatened Species, 15 and that its cohort of medicinal properties attracts the least interest so far. It is reported as endemic to the Khasi Hills of Meghalaya, northeast India. About 3,000 individual trees are estimated to exist in these areas. 16 But an isolated distribution is also now recorded at Luangmual, Aizawl, Mizoram. Among the Khasi people, a decoction of the bark and the root is used in the treatment of tuberculosis and severe cold. 17 The leaves are known for various medicinal properties including antiparasitic property in Mizo traditional medicine. In fact, the Mizo traditional practitioners use them as a panacea in all sorts of non-specific illnesses. 18 It is therefore important to understand the true pharmacological potentials of this plant as an antiparasitic agent.

Plant specimen and preparation of plant extract
Ilex khasiana is available only at one area in Mizoram, India, as naturally propagated tree at Luangmual, Aizawl (location 23°44.556'N and 92°41.956'E). The plant specimen was authenticated at the Botanical Survey of India, Eastern Circle, Shillong, Meghalaya, as reported earlier. 18 The herbarium is catalogued with an accession number BSI/EC/Tech./2008/577 in the Department of Pharmacy, Regional Institute of Paramedical and Nursing Sciences, Zemabawk, Mizoram, India.
The leaves were washed and dried under the shade at an ambient temperature (23-25°C). The dry leaves were ground to a fine powder. Hot extraction was done using methanol as a solvent in a 5-litre Soxhlet apparatus for 72 hours. The crude extract was concentrated by evaporating and recovering the solvent in a rotary vacuum evaporator (Buchi Rotavapor ® R-215).

In vitro survival test
Antiparasitic activity was tested in vitro on the survival of the parasitic tapeworm Raillietina tetragona Molin, 1858. Live tapeworms were recovered from the intestines of freshly sacrificed local fowls, Gallus gallus Linnaeus, 1758. An hour before the test, the plant extract was prepared in increasing concentrations, i.e. 0.5, 1, 2, 5, 10 and 20 mg/ml in culture plates by dissolving them in 0.9% neutral phosphate-buffered saline (PBS) supplemented with 1% dimethylsulfoxide (DMSO). Corresponding concentrations of albendazole were also prepared as standard references. Albendazole (Zenlee) was a product of UNI-PEX Pharmaceutical Private Limited, New Delhi, India.
Control media consisted only of PBS with DMSO. Batches of two worms were tested for each concentration, and each test was further performed in triplicates. The parasites were incubated at a constant temperature of 37±1°C. Antiparasitic efficacy was assessed in terms of survival in the culture media. Death was defined as complete absence of motor activity even after physical provocation in lukewarm PBS (45°C). The duration of survival was recorded, and data were generated as statistical means ± standard deviation. Significance of the antiparasitic activity was determined using Student's t-test, and a p value less than 0.05 was considered as significantly different.

Scanning electron microscopy
Tapeworms treated with the highest concentration, i.e. 20 mg/ml of the plant extract were chosen for scanning electron microscopy. After confirmation of death, they were immediately fixed in 4% formaldehyde, buffered with sodium cacodylate (0.1 M, pH 7.2), at 4°C for 4 hours. Another fixation was done in 1% osmium tetroxide at 4°C for 1 hour. After 1 hour, the specimens were dehydrated through ascending grades of acetone up to pure acetone. They were then treated with tetramethylsilane for at least 15 minutes. The TMS was dried in an air-drying chamber maintained at 25°C. The dried specimens were mounted on metal stubs, and then sputter coated with gold in an ionsputtering chamber (JFC 1100, JEOL Ltd., Tokyo, Japan). Finally, they were visualised under a scanning electron microscope (JSM-6360, JEOL Ltd., Tokyo, Japan) under an electron accelerating voltage of 20 kV.

RESULTS
The antiparasitic activity of the methanol extract of I. khasiana leaves and albendazole on the tapeworm, Raillietina tetragona, is given in Scanning electron microscopy revealed details of structural changes on the tapeworms treated with 20 mg/ml of I. khasiana leaf extract. Figure  2 is the anterior part of the tapeworm consisting of the bulb-like scolex and the adjoining neck. Large folds were visible at the base of the scolex and on the neck showing tegumental degeneration at the anterior end of the body. A single sucker shown in Figure 3 indicates tegumental shrinkage and disintegration of the spines around the rim. A magnified view of the same sucker (Figure 4) revealed disordered and clumping of the spines and debris-like tegumental erosion.
The main body (strobila) of tapeworms is a series of body segments (proglottids). All the body segments exhibited irregular shrinkage and appeared to undergo severe contractions ( Figure 5). Some portions were sloughed off. A close-up view of one body segment showed that

DISCUSSION
Tapeworms are a group of helminth parasites which are largely ignored and often described as "low priority" infectious agents due to their relative quiescence in most infections. However, they pose serious threats to clinical conditions such as neurocysticersosis in humans, 19 livestock production, 20 and wildlife. 21 They are distinct helminths in having rather modest anatomical organisation; lack of nervous and digestive systems being the unique features. Their external body surface called tegument is the principal body organ as it serves not only as a protective barrier, but also as nutrient-absorptive and sensory site. 22 These functions are carried out through microscopic hairs called microtriches, which are distributed throughout the tegument. Thus, the hallmark effects of antiparasitic drugs are structural damages on the tegument, microtriches and the underlying subtegument tissue. 23,24 The detail structural features of the tegument and its physical and physiological roles in poultry tapeworms are already amply described. 25,26 In our experiment, the methanol extract of I. khasiana leaves caused considerable tegumental damages including shrinkage, distortion of the spines and rostellum, and erosion of the microtriches. The antiparasitic activity was further substantiated by concentrationdependent lethal activity on R. tetragona similar to that of albendazole.
Benzimidazoles are the drugs of choice in all types of helminth infection because of their broad-spectrum activity. The most commonly used the surface is full of tanged fibres, indicating destruction of hair-like microtriches ( Figure 6). The severity of the tegumental shrinkage is more clearly discernible with transverse rows of grooves and ridges.     benzimidazoles, albendazole and flubendazole cause degeneration of the rostellum, destruction of the microtriches, and eruption of aberrant vesicles on the human tapeworm, Echinococcus granulosus. 27 A combination therapy of albendazole and praziquantel resulted in disintegration of the suckers, detachment of the spines, and severe damage of the tegument, associated with removal of the microtriches in E. granulosus and Mesocestoides corti. 28,29 A single treatment of R. echinobothrida with albendazole produced severe tegumental contraction and collapse. In addition, the suckers were destroyed while the rostellum remained intact. 30 Nitazoxanide also induced pore formation on the scolex and distortion of the neck and the strobilar chains of the immature (cysticerci) of Taenia solium. 31 E. granulosus and E. multiclocularis treated with lonidamine and 6-aminonicotinamide showed tegumental darkening, swelling and shortening of the body segments, surface erosion, and severe damages of the rostellum and suckers. 32 It is also apparent that in spite of the common structural damages they induce, each drug exhibit variation in its effect on the intensity and extend of tegumental alterations. We observed that I. khasiana leaf extract indiscriminately acted on the entire body parts of R. tetragona. The clumping of spines and extensive contraction of the body segments appear to be unique.
Our findings indicate that the plant extract has different mode of action and open a new vista for the development of novel antiparasitic drug.

CONCLUSION
We found that I. khasiana leaf extract effectively killed the tapeworm R. tetragona. It caused structural damages on the tegument of the tapeworm, indicating the characteristic antiparasitic effects. Under scanning electron microscopy, distortion of the microtriches, clumping of the spines, erosion of the microtriches and extensive contraction of the tegument were evident. The need for conservation and propagation of this valuable medicinal plant is compelling. Further studies on the chemical components of the medicinal plant, isolation of the active principle, and mode of action of the compound are warranted by our findings.

CONFLICTS OF INTEREST
None declared.

SUMMARY ABOUT AUTHORS
• A species of holly, Ilex khasiana, is a critically endangered medicinal plants that is known to have a variety of uses in traditional medicine. Among its medicinal properties is antiparasitic activity.
• The methanol extract of the leaves was tested in vitro against an intestinal tapeworm, Raillietina tetragona. The plant extract caused dose-dependent antiparasitic activity against the tapeworm as that of albendazole.
• Scanning electron microscopy was used to examine structural changes on the tapeworm. The body surface (tegument) indicated damages which are characteristics of antiparasitic drugs such as shrinkage, erosion, disintegration of the suckers, and removal of microtriches.
• The damaging effects imply that the plant possesses bioactive compounds that can be new lead molecules in drug development. Thus, the findings warrant further investigations on the chemical nature and precise mode of action.

Charles Lalnunfela:
He is a graduate in Genetics from the University of Madras and a PhD scholar at the Department of Pharmacy, Regional Institute of Paramedical and Nursing Sciences, Zemabawk, Mizoram, India.