International Journal of Ayurveda a Review on Gyrinops Walla Gaertn

ISSN:2456-6640 International Journal of Ayurveda (A Monthly Scientific Journal of Kiban Research Publications) www.kibanresearchpublications.com

RESEARCH ARTICLE

A Review on Gyrinops walla Gaertn. (Walla) and In Vitro Study on Anthelmintic Effect against Larvae of Haemonchus contort us and Toxocara canis

Ediriweera ERHSS1*, Rajapakshe RPVJ2, Ratnasooriya WD3

1. Department of Nidana Chikithsa, Institute of Indigenous Medicine, University of Colombo, Rajagiriya, Sri Lanka. 2. Department of Pathobiology, Faculty of Veterinary and Animal Science, University of Peradeniya, Peradeniya, Sri Lanka. 3. General Sir John Kotalawella Defence University, Ratmalana, Sri Lanka.

*Corresponding Author: E-mail: [email protected] Abstract: Gyrinops walla Gaertn. (Family: Thymelaeaceae; Sinhala name: Walla; Walla Patta) is a common tree growing in moist regions of Sri Lanka. Sri Lankans use Walla stems for making handles for hoes and bark in lieu of ropes. This is an attempt to collect scattered medicinal knowledge and to observe anthelmintic effects of G. walla in-vitro. Information was assimilated from books on traditional medicine and traditional physicians. G. walla is administered internally as juice of fresh leaves, herbal gruel and boluses for keeping inside the mouth. It is used externally as ‘Mellum’ and ‘Aalepa’. Bark is used in ‘Patta bandeema’ (binding splints in fractures). When treating cobra and viper bites, it is administered through the nose (Nasya Karma). G. walla is used to treat worm infections, tonsillitis and hemorrhoids inhumans and also to deworm calves. Hence, in vitro larval migratory inhibition assay was carried out with Toxocara canis and Haemonchus contortus using water extract of G. walla. This study revealed 80.25% and 42.22% larval migration inhibition respectively. Chloroform extract of twigs and leaves displayed activity against Eagle’s carcinoma in the nasopharynx in cell culture. Some villagers in Sri Lanka believe that brushing with G. walla charcoal may cause teeth to fall out. Agarwood-type resin, used in manufacturing fragrances and perfumes, is also obtained from G. walla. Gyrinops walla is a plant with many medicinal, commercial and domestic usages.

Keywords: Gyrinops walla, Walla Patta, Patta Walla, Toxocara canis, Haemonchus contort us, Anthelmintic properties. Article received: 05 July 2018 Revised: 13 July 2018 Published: 20 July 2018

Introduction Gyrinops walla (Family: Thymelaeaceae; which leads to severe anaemia and death. Sinhala name: Walla Patta; Walla) is a The present study was conducted to gather medium-tall tree which grows up to 15 m in medicinal uses of G. walla and to evaluate its height with straight, slender trunk and anthelmintic properties against larvae of small, rounded crown [1]. G. walla is the only Haemonchus contortus and Toxocara canis agarwood producing tree growing in Sri through in vitro studies. Lanka which is believed to be endemic [2]. G. Material and Method walla is used in Sri Lankan traditional medicine in treatment of various diseases; Medicinal uses of Gyrinops walla were especially worm infections [3]. collected from Ayurveda and Sri Lankan traditional medical books, interviewing Toxocariasis is an infection caused by the physicians, research journals and internet. ingestion of larvae of the dog round worm Anthelmintic properties against larvae of Toxocara canis, or the cat round worm, Toxocara canis and Haemonchus contort us Toxocara cati. Haemonchosis in goats is an were evaluated through in vitro studies. It infection caused by Haemonchus contortus was conducted in three steps.

 Preparation of medicine Eggs reach infective stage within 30-40 days. Thereafter eggs were washed twice by  Assessment of anthelmintic properties centrifugation at 150g for 15 minutes with against larvae of Toxocara canis 0.15 M Phosphate Buffered saline (PBS) (pH  Assessment of anthelmintic properties 7.2) to remove H2SO4 and the other organic against larvae of Haemonchus matter and the eggs were recounted at 1:100 dilutions by the McMaster technique.  Contort us Viability of the T. canis embryonated eggs Preparation of Medicine was assessed by the light stimulation method 120 g of fresh leaves of Gyrinops walla before use as described by O ’Lorcain [5]. (Walla) were taken and mixed with 1920 ml The Storage and Maintenance of Larval of water and boiled down to 240 ml to Cultures prepare the decoction. The storage and maintenance of larval Anthelmintic Properties Against Larvae cultures of Toxocara larvae to be used for of Toxocara canis experimental purposes often have to be Collection of Toxocara canis Eggs stored, and this was done satisfactorily in a shallow layer of water. Eggs of Toxocara canis were obtained from the faeces of young puppies and were Forty millilitres of a suspension containing embryonated for larval preparations as not more than 3000 larvae per ml were described by Rajapakse [4]. All positive placed in a tissue culture flask and kept in an samples were mixed in one litre of water incubator maintained at 10 0C. As the containing 0.05% Teepol (Lankem Ceylon storage at low temperatures would induce Ltd., Colombo) in a measuring cylinder and inhibition of some population, care was taken washed five times by sedimentation method not to use larvae while they were being to remove all fat and other fine materials. conditioned. This means that the usage before 4 weeks of storage (larvae had been Thereafter, the sediment was re-suspended stored for 2 weeks to ensure a normal in 500ml of saturated salt solution and the establishment rate) or after 16 weeks, was suspension was centrifuged (at 1000g for 10 avoided. minutes) in order to separate the eggs. The surface layer of the supernatant solution In Vitro Larval Migration Inhibition containing the T. canis eggs was collected Assay using a Pasture pipette and washed with The larvae migration inhibition (LMI) water through a filter of 100µm pore size in bioassay developed by Wag land [6] and order to remove the coarse fibrous matter. modified by Rabel [7]. Was used to determine The filtrate was then poured through a filter the effectiveness of the plant extract against of pore size 50µm where T. canis eggs infective larvae. One millilitre of the remained on the filter. decoction of Gyrinops walla (Walla) was Development of Infective Eggs of taken and diluted by adding 9 ml of Toxocara canis phosphate buffered saline (PBS) so as to obtain a transparent solution. Then the pH Freshly harvested eggs were stored in 0.1 N value (6.5) and density was measured in this H2SO4 at a depth of 0.5 cm in Petri dishes (10 solution (1.03g /ml). As the positive control, cm x 1.5 cm) in an incubator (Lindberg and levamisole 200 g/ml was used and May Pvt. Ltd., Australia) at 14.5 0C. At this phosphate buffered saline (PBS) was used as temperature the development of eggs was the negative control. arrested without any substantial reduction of their viability. Then 200 l of larval suspensions were added to wells containing 800 l of either controls The eggs could be stored in this manner for (positive and negative) or plant extract and 60 days. Whenever infective eggs were were incubated at 37 oC in the wells of required, Petri dishes containing the tissue culture plates. Three wells (replicate required number of eggs were placed at room samples) were run for each concentration of temperature (220C - 240C). In the course of each decoction and for the controls. All the this second incubation, the culture was incubations were carried out in 24 well tissue rocked gently once a day to ensure aeration.

culture plates overnight (18 hours), at 37 0C Faecal cultures were prepared using faeces and pH 7.2. Following day, solutions were collected from infected goats. The faeces were transferred to sieves (20 m mesh at one end) broken up finely, using a large pestle and and left for 24 hours at room temperature for mortar, mixed with sterile dung or sawdust in active larvae to migrate through the sieves. 1:1 ratio, and dampened with distilled water On the next day, sieves were removed, until the mixture was moist and crumbly. Lugol’s iodine (0.1 ml) was added to the well Then the mixture was kept in wide-mouthed and the number of larvae which had glass jars or enamel trays and incubated at migrated was counted under the microscope. room temperature for 10-14 days. The Viability and Activity of the Post The cultures were maintained by aerating the Migratory Larvae with Different Plant lower layers every day and, to prevent drying, Remedies were Observed and Recorded by adding a few drops of water in order to as follows maintain moisture. After 14 days, cultures were baermannized using wide-mouthed glass  Grade 0 = Dead; No recovery after prolonged jars. The larvae were counted and assessed for immersion in saline viability and identification was carried out  Grade 1 = Inactive but occasional movement before being stored at 10 0C. can be observed; The Storage and Maintenance of Larval  Grade 2 = Inactive but intermittent Cultures movement can be observed clearly; The storage and maintenance of larval  Grade 3 = Slow moving; cultures was carried out as described under Toxocara canis.  Grade 4 = Active. In Vitro Larval Migration Inhibition Anthelmintic properties against larvae of Assay Haemonchus contort us (In Vitro) In vitro larval migration inhibition assay was Collection of the Eggs of Haemonchus carried out as described under Toxocara canis contortus but infective larvae in unsheathed forms were Fecal samples were collected directly from the used. Sheathes were removed by incubating rectum of goats in Kekirawa veterinary range, the larvae in sodium hypochlorite solution Sri Lanka. Fecal egg count was determined (0.025% available chlorine) for 10 minutes at using the modified McMaster technique [8]. room temperature, washing several times and Faeces of high eggs per gram (EPG) of >5000 concentrating to approximately 2500 from each animal were collected for this larvae/ml PBS. study. All positive samples were then Result subjected to fecal culture for collection of infective larvae. Review on Gyrinops Walla (Walla) Fecal Culture and Isolation of Scientific classification of Gyrinops Haemonchus contortus Larvae walla

Kingdom Plantae Phylum Tracheophta Class Magnoliopsida Order Malvales Family Thymelaeaceae Genus Gyrinops Species walla

Synonyms of Gyrinops walla A small tree with a slender erect trunk and a small rounded head, branchlets numerous, Sinhala Name: Walla, Patta walla, Walla slender, erect, bark thin, smooth, reddish patta brown, buds silky; leaves simple, alternate, English Name: Sri Lankan Agar wood exstipulate, 3.7—10 cm long, oblong- Botanical Name: Gyrinops walla, Cameraria lanceolate, acute at base, shortly acuminate, zeylanica Moon obtuse or subacute, entire with a very narrow Description of Gyrinops walla hyaline edge, quite glabrous, slightly shining, lateral veins very fine and numerous, midrib

©2016-2018, IJA. All Rights Reserved 3 Ediriweera ERHSS et. al.| International Journal of Ayurveda | August 2018 | Vol. 03| Issue 08 |01-08 prominent beneath, petioles very short; anthers sessile; ovary superior, on a rather flowers regular, bisexual, small, yellowish long stalk, 2-locular with a pendulous ovule white on slender pubescent pedicels, 3—5 in in each loculus, style long, stigma capitate; shortly stalked umbels from axils of present fruit capsule 1.8 cm long, obovate, or fallen leaves, bracts small, hairy, compressed, pointed, coriaceous, reddish caducous; perianth tubular, slender with a brown, dehiscing loculicidally into 2 valves; ring of short hairs and scales above stamens, seeds covered with brown hair, pointed, pubescent, segments S, oval, subacute, produced into a long tail-like process at base. spreading; stamens 5 in one row, inserted in Flowering in December [9]. mouth of perianth-tube opposite segments,

Image of Gyrinops walla (Walla)

Distribution of Gyrinops walla (Walla) Gyrinops walla occurs in India and Ceylon. It is rather common in the moist regions up to 4,000 feet altitude in Ceylon [9].

Figure 2: Distribution of Gyrinops walla

belongs to the same family (Thymelaeaceae) Ayurveda pharmacodyanamic are widely used in Ayurveda to treat various properties of Gyrinops walla (Walla) diseases. Authors are of the opinion that Ayurveda pharmacodyanamic properties of Gyrinops walla may possess same properties Gyrinops walla are not described in as in Aquilaria malaccensis and those Ayurveda texts. Aquilaria malaccensis properties are given in table 1 [10].

Table 1: Ayurveda pharmacodyanamic properties of Gyrinops walla and Aquilaria malaccensis Ayurveda pharmacodyanamic Gyrinops walla (Walla) Aquilaria malaccensis properties (Thymelaeaceae) (Thymelaeaceae) Rasa Not Known Katu, Tikta Guna Not Known Laghu, Ruksha, Theekshna Veerya Not Known Ushna Vipaka Not Known Katu, Dosha Karma Not Known Kaphavatanashaka

Tender leaves, leaves, stem bark Part use in Medicine

Table 1: Medicinal uses of Gyrinops walla (Walla) Indication Prescription Worm infections In worm infections, fresh juice extracted from tender leaves of Gyrinops walla (Walla) is mixed with bee’s honey and orally consumed to expel the worms [11]. Tender leaves of G. walla and Hydrolea zeylanica (Family: Hydrophyllaceae, Sinhala name: Diyakirilla ) are pounded well, wrapped in a banana leaf and cooked by keeping in the hearth under charcoal embers. Then the juice is extracted and given with Ayurveda preparations of Krimiripu Kalka or Krimiraja Kalka to treat worms[3]. Haemorrhoids Tender leaves of G. walla are spread over haemorrhoids to stop bleeding [12]. Fistula in ano Tender leaves of G. walla and fresh rhizome of Curcuma domestica (Family: Zingiberaceae; Sinhala name: Kaha) are pounded and applied on fistula in ano. Cobra poison Tender leaves of G. walla and Hedyotis fruticosa (Family: Rubiaceae, Sinhala name: Weraniya) are cut into small pieces and mixed well. The mixture is heated on a pan and kept on the site of cobra bite and bandaged in treatment of cobra poisoning [13]. Viper poison In viper poisoning, Nasya Karma (Errhine therapy) is performed using juice extracted from tender leaves of G. walla [14]. Leaves and stem bark of G. walla are pounded and juice is extracted. Rice is cooked using seeds of Setaria italica (Family: Poaceae, Sinhala name: Thana Hal) and extracted juice of G. walla is added to this rice to prepare Chyme [15]. Common Krait poison and Sri In Common Krait poison and Sri Lankan Krait poisoning, Nasya Karma Lankan Krait (Errhine therapy ) is performed using juice extracted from tender leaves of G. walla [14]. Boils Leaves of G. walla, Lagerstroemia speciosa (Family: Lythraceae, Sinhala name: Muruta), Momordica charantia (Family: Cucurbitaceae, Sinhala name: Batu Karawila) and Fluggea leucopyrus (Family: Phyllanthaceae, Sinhala name: Katu Pila) are pounded together with common salt and turmeric powder. The mixture is heated in a pan and applied on boils [15]. Swelling due to allergies Tender leaves of G. walla, Hibiscus furcatus (Family: Malvaceae, Sinhala name: Napiritta) and Ficus hispida (Kotadimbula), scraped coconut and turmeric powder are pounded together, then heated in a pan and applied on area swollen due to allergy [16]. Infection in the nail bed Tender leaves of G. walla, Callicarpa tomentosa ( Family: Verbenaceae, Sinhala name: Illa) and Psiadia ceylanica (Family: Asteraceae, Sinhala name: Pupula Wel), scraped coconut and jaggery of Saccharum officinarum ( Family: Poaceae, Sinhala name: Uk) are pounded together, heated in a pan and is applied on infected nail [16]. Extraction of aching teeth Very tender leaves of G. walla, piece of fresh rhizome of C. longa and common salt are crushed together, kept on aching tooth overnight. This helps to loosen the tooth [17]. Tender leaves of G. walla are ground and should be kept fifteen minutes on the gum of the moving tooth. This should be done for several days. The tooth will fall off without any pain after a few days [18]. Tonsillitis Tender leaves of G. walla, dried leaves of Piper betle (Family: Piperaceae, Sinhala name: Bulath) and Peperomia pellucida (Family: Piperaceae, Sinhala name: Gas Bulath) are ground, made into small boluses and kept at the back the mouth and resulting saliva is swallowed 12]. Tender leaves of G. walla, are ground, made into boluses. Oil of seeds of Madhuca longifolia (Family: Sapotaceae, Sinhala name: Mee) is applied on the boluses. These boluses are kept at the back the mouth and swallow the saliva [12].

Indications of Gyrinops walla in  Tender leaves of G. walla, Caesalpinia Veterinary Practice bonduc (Family: Fabaceae, Sinhala name: Kumburu Wel), Vitex negundo (Family:

Verbenaceae, Sinhala name: Nika), Eagle’s carcinoma of the nasopharynx in cell Capsicum frutescens (Family: Solanaceae, culture (KB) (3) showing ED505.6 and Sinhala name: Miris) and Citrus 0.75µg/ml for twigs leaves respectively [21]. aurantifolia (Family: Rutaceae, Sinhala  Subasinghe et. al. reported that the name: Dehi) and roots of C. frutescens, compound of G. walla resin is extremely Toddalia asiatica (Family: Rutaceae, similar with that of commercially available Sinhala name: Kudumirissa), P. betle and agarwood resins [22]. Subasinghe et. al. Piper chavya (Family: Piperaceae, Sinhala stated that among the 19 constituents name: Siviya Wel) are ground with lime identified by GCMS in the agarwood resins, juice until it turns to a fine paste. 20gm of free fatty acids and isopropyl naphthalene, this paste is given with medicated water 2-phenylethyl chromone compounds were prepared by boiling pods of C. frutescens in found to be common for most of the G. walla treatment of impaired appetite in calves trees tested. Commonly found sesquiterpene [19]. compounds from the G. walla resin were  Tender leaves of G. walla, Ficus Jinkhol, γ-eudemon, valerenol and valerinal. benghalensis (Family: Moraceae, Sinhala Similar compounds have been reported in name: Nuga) and F. leucopyrus, stems of resin from Aquliaria spp. which is the more leaves of Azadirachta indica (Family: established source of agarwood [2]. Meliaceae, Sinhala name: Kohomba) and Anthelmintic Properties against Larvae Bamboo salt are ground into a fine paste. of Toxocara canis and Haemonchus 20gm of this paste is given with 30ml of contort us (In Vitro) fresh juice extracted from leaves of Abrus precatorius (Family: Fabaceae, Sinhala As shown in the Table 2 and Figure 1, name: Olinda) in treatment of Mun decoction of Gyrinops walla (Walla) was Adappan (Nasal discharge and tremors) in 80.25% effective in inhibiting Toxocara larval cattle [19]. migration and 42.22% effective in inhibiting Haemonchus larval migration. Whereas Chemical Constituents larval migration inhibition of Toxocara canis Tannins, saponins, steroid glycosides, and Haemonchus contort us with Levamisole flavonoids and alkaloids are present in leaf, were 99.7% and 96.6% respectively. stem and bark [20]. A number of inactive constituents have been identified. These The viability of post-migratory larvae of compounds are friedelan-3β-yl acetate, Toxocara canis and Haemonchus contort us friedelin, friedelan-3β-ol, apigenin-7, 4’ - with controls and with decoction of G. walla dimethyl ether, luteolin- 7,3’,4’-trimethyl are presented in Table 3 and figure 2. ether, (+)- syringaresinoll, velutin, pilloin, Maximum number of migrated larvae of genkwanin, sitoindoside 1 and mangiferin. Toxocara and Haemonchus was observed in The cucurbitacin 1 content of CHCl3 extract the negative control PBS. Least number of was 0.0018% for the twigs and 0.015% for the migrated larvae was observed in the positive leaves [21]. control Levamisole where all the larvae were dead after migration. 12.6% of Toxocara and Research 10.4% of Haemonchus larvae migrated in  Yeh Schun and Geoffrey, reported that the decoction of G.walla. All the migrated twigs and leaves of Gyrinops walla were Toxocara larvae were dead or in Grade 1, 2, 3 exhaustively extracted with petroleum and or 4 and all migrated Haemonchus larvae MeOH, and the MeOH extract was were dead or in Grade 1 or 2. partitioned between CHCl3 and H2O. The CHCl3 extract displayed activity against

Table 2: Percentages of in vitro larval migratory inhibition of Toxocara canis and Haemonchus contort us infective larvae with decoction of Gyrinops walla (Walla) Percentage (%) of larval migration inhibition (LMI) Treatment Toxocara canis Haemonchus contortus Levamisole 200 μg / ml in PBS (Positive Conrol) 99.7 96.6 Phosphate buffered saline (Negative control) 0 0 Gyrinops walla (Walla) (density 1.03g /ml) 80.25 42.22

Table 3: Viability of post-migratory larvae of Toxocara canis and Haemonchus contortus larvae with decoction of Gyrinops walla (Walla) Percentage (%) of viability of Toxocara Percentage (%) of viability of

Treatment canis larvae Haemonchus contortus larvae

Total Total

Grade 1 Grade 2 Grade 3 Grade 4 Grade 0 Grade 1 Grade 2 Grade 3 Grade 4 Grade 0 Levamisole 0 0 0 0 0 0 0 0 (Positive control) 55 55 0.1 0.1 Phosphate 0 0 0 0 72.5 72.5 buffered saline (Negative control)) 0 0 0 0.2 61.5 61.7 Gyrinops walla 0.19 0.67 1.91 8.38 1.48 12.63 8,63 1.61 0.2 0 0 10.4. (Walla) (density 4 1.03g /ml) Grade 0 = Dead; No recovery after prolonged immersion in saline; Grade 1 = Inactive but occasional movement can be observed; Grade 2 = Inactive but intermittent movement can be observed clearly; Grade 3 = Slow moving; Grade 4 = Active

Figure 1: Percentages of in vitro larval migratory inhibition of Toxocara canis and Haemonchus contort us infective larvae with decoction of Gyrinops walla (Walla)

Figure 2: Viability of post-migratory larvae of Toxocara canis and Haemonchus contort us larvae with decoction of Gyrinops walla (Walla)

against Eagle’s carcinoma of the Discussion nasopharynx in cell culture of G. walla are Sri Lankan traditional physicians use scientifically proven. Based on Ayurveda Gyrinops walla (Walla) to treat worm principals, it can be assumed that Gyrinops infections, haemorrhoids, fistula in ano, walla (Walla) also possess Tikta, Katu Rasa, snake poisoning, boils, infection in the nail Laghu, Theekshna and Ruksha Guna, Ushna bed, swelling due to allergies, extraction of Veerya and Katu Vipaka as in Aquilaria moving or loose teeth and tonsillitis. Activity malaccensis (Thymelaeaceae) [10]. As per

Ayurveda, these properties reduce Kapha Conclusion Dosha which provides a friendly surrounding It is concluded that Gyrinops walla (Walla) to growth and propagation of worms. Sri Gaertn. Is effective in inhibiting larval Lankan traditional physicians are familiar migration of Toxocara canis and Haemonchus with Krimighna (wormicidal) property of contortus and also has multi-faceted Gyrinops walla and use this plant in medicinal values. treatment of worm infections in humans. The present study also supports their knowledge on wormicidal property of Gyrinops walla. References 1. Subasinghe SMCUP (2013) Agar wood 11. Anonymous (1970) Vaidyaka hasthasaraya production of Walla Patta (Gyrinops walla): The hevath sinhale ath veda potha, Modern Poth tree of future, available from: Mudranalaya, Colombo, Sri Lanka, 209. https://www.researchgate.net/publication/26423 12. Anonymous(1994) Talpathe piliyam 3, 9820_Agarwood_production_of_walla_patta_Gy Department of Ayurveda, Colombo, Sri rinops_walla_The_tree_of_future, accessed on Lanka,48, 216. 6/5/2017. 13. Rev Kngaloka, Sapa N (1968) visha samhara, 2. Subasinghe SMCUP, Hettiarachchi DS(2015) Singha Mudranalaya, Telijjawila, Sri Lanka, Characterisation of agarwood type resin of 41. Gyrinops walla Gaertn growing in selected populations in Sri Lanka, Industrial Crops and 14. Gunsena D (1980) Visha vaidya prakaranaya, Products, 69:76-79. Modern Poth Mudranalaya, Colombo, Sri Lanka, 50-59. 3. Aylapperuma ESDA, Vatiika Prakaranaya, Vidyawardhana Press (1914) Colombo, Sri 15. Anonymous (1993)Talpathe piliyam 11, Lanka,6-7, 35. Department of Ayurveda, Colombo, Sri Lanka,146, 4. Rajapakse RPVJ, Wasanthathilake VWSM, Lloyd S, Fernando ST (1992) Collection of eggs 16. Anonymous (1993) Talpathe Piliyam 4, and Hatching and Culturing Second stage Department of Ayurveda, Colombo, Sri larvae of Toxocara vitulorum in vitro, The Lanka, a, 140, 178. Journal of Parasitology, 78 (6):1090-1092. 17. Rev Knanawimala, Karambagale K (2011) ath 5. O’Lorcain P (1995) the effects of freezing on the veda potha, Ratna publishes, Colombo, Sri viability of Toxocara canis and T. Lanka, 156. catiembryonated eggs, Journal of 18. Rev Knanawimala Deshiya K (2011) vaidya Helminthological, 69(2):169-71. shabda koshaya, Samayawardhana publishes, 6. Wagland BM, Jones WO, Hribar L, Bendixsen Colombo, Sri Lanka, a, 463. T, Emery DL (1992) A new simplified assay for 19. Anonymous (1993) Talpathe piliyam 10, larval migration inhibition, International Department of Ayurveda, Colombo, Sri Journal for Parasitological, 22(8):1183-1185. Lanka, b, 38, 130. 7. Rabel B, McGregor R, Douch PG (1994) 20. Dharmadasa RM, Siriwardana A, Improved bioassay for estimation of inhibitory Samarasinghe K, Adhihetty P (2013) effects of ovine gastrointestinal mucus and Standardization of Gyrinops walla Gaertn anthelmintics on nematode larval migration, (Thymalaeaceae): Newly Discovered, International Journal of Parasitological, Fragnant Industrial Potential, Endemic Plant 24(5):671-676. from Sri Lanka, World Journal of Agricultural 8. Cringoli G, Rinaldi L, Maurelli MP, Research, 1(6):101-103. Morgoglinone ME, Musella V, Utzinger J (2011) 21. Yeh Schun, Geoffrey (1985) A Cordell, Studies Ancylostomacaninum: calibration and in the Thymelaeaceae 111. Constituents’ of comparison of diagnostic accuracy of floatation Gyrinops walla, Journal of natural products, in tube, Mc Master and FLOTAC in faecal 48(4): 684 -685. samples of dogs, Experimental Parasitological, 128(1): 32-37. 22. Subasinghe SMCUP, Hettiarachchi DS, Rathnamalala E(2012) Agar wood-type Resin 9. Jayaweera DMA (1982) Medicinal plants from Gyrinops walla Gaertn: A New (Indigenous and Exotic) Used in Ceylon, Discovery, Journal of Tropical Forestry and National foundation, Sri Lanka. Environment 2(2):43-48. 10. Sharma PV (1991) Dravyaguna Vijnana 2,

Chaukambha Bharati Academy, India, 726 - 728.