ORIGINAL ARTICLE Bali Medical Journal (Bali Med J) 2017, Volume 6, Number 3: 17-22 P-ISSN.2089-1180, E-ISSN.2302-2914 Wudani leaf extract (Quisqualis indica linn) as ORIGINAL ARTICLE traditional medicine to control the incidence of worm CrossMark Doi: http://dx.doi.org/10.15562/bmj.v6i1.371 Published by DiscoverSys Ida Bagus Komang Ardana,1* Made Suma Anthara,2 Anak Agung Gede Oka Dharmayudha,2 Anak Agung Ngurah Subawa,3 D. K. Harya Putra4 Volume No.: 6 ABSTRACT Issue: 3 Background: The present research work was aimed to study whether of 1 ml/40 and 2 ml/40 ml was very high, that was 88.8% and application of 10% extract of wudani leaf (Quisqualis indica Linn) 88.5%, respectively. Furthermore, for eggs of Paramphistomum sp., may decrease the potential of becoming embryos of eggs of Fasciola assessment at day-10 showed that the ovicidal ability of the dose of gigantica and Paramphistomum sp. worms under in-vitro evaluation. 2 ml/40 ml was the highest (23.3%) and significantly higher (P < First page No.: 17 Methods: It was used three doses of the extract; those were 0.05) than that of the dose of 1 ml/40 ml (21.6%), 0.5 ml/40 ml 0.5 ml/40 ml of physiological NaCl, 1.0 ml/40 ml NaCl and 2.0 ml/40 ml (11.6%) and of the control (9.4%). On the other hand, there were no of NaCl and with 5 repetitions for each dose. Assessments of the ovicidal significant difference were noted between the dose of 1 ml/40 ml, effect of extract that is reducing the potential of eggs to become embryo 2 ml/ 40 ml and the control (P > 0.05). Likewise, at day-30 of P-ISSN.2089-1180 were conducted at day-10 and day-30 at the Parasitology Laboratory of assessment, the ovicidal ability of 1 ml/40 ml (51.9%) and 2 ml/40 ml The Faculty of Veterinary Medicine, Udayana University, Bali. (53.4%) was significantly higher (P < 0.01) than that of control. The Results: Results showed that assessment at day-10, the ovicidal high ovicidal ability of 10% extract of wudani leaf at the dose of 1 – E-ISSN.2302-2914 ability of the extract at 0.5 ml/40 ml NaCl for Fasciola gigantica eggs 2 ml/40 ml NaCl may be related to damage of egg shells caused by was 23.8% and was significantly lower (P < 0.05) than that of the active ingredient of the extract. dose of 1 ml/40 ml (33.1%) and of the dose of 2 ml/40 ml (35.9%). Conclusion: Since infection of lungworm predominantly occurs in However, it was significantly higher than the control (10.4%). cattle, the present results may reveal the opportunity of applying the Moreover, the ovicidal ability of dose of 1 ml/40 ml and 2 ml/40 ml wudani leaf extract to control lungworm infection in cattle. Further did not differ significantly (P > 0.05). At Day-30, the ovicidal ability study is definitely needed.

Keywords: eggs of Fasciola sp. and Paramphistomum sp., extract of wudani (Quisqualis indica Linn), ovicidal ability, in-vitro study. Cite This Article: Ardana, I., Anthara, M., Dharmayudha, A., Subawa, A., Harya Putra, D. 2017. Wudani leaf extract (Quisqualis indica linn) as traditional medicine to control the incidence of cattle worm. Bali Medical Journal 6(1): 17-22. DOI:10.15562/bmj.v6i1.371

1Veterinary Clinic Pathology INTRODUCTION 2Veterinary Phamacology, Faculty of Veterinary Medicine, Udayana It has been known that in Indonesia, disease infes- reported that prevalence of Toxocara vitulorum University, Bali-Indonesia tations are still major problems for cattle rearing infection in Bali cattle raised in eastern part of Bali 3 Faculty of Medicine Udayana and production. One of those is Fasciolosis due is 39.4%, whereas there were no records reported University, Bali-Indonesia 2 4Department of Physiology, to infection of Fasciola hepatica, which may cause for Indonesia. However, survey conducted in Faculty of Science Udayana a total loss of 513 million rupiah per year. The 2014 noted that Paramphistomum sp. were found University, Bali-Indonesia disease causes loss of body weight, untreatable liver in almost all cattle slaughtered at Pesanggaran and Veterinary Pathology Clinic, damage and reproductive constraints.1 Moreover, it Darmasaba abattoirs in Bali. Veterinary Pharmacology has been reported that worm infections have widely The high prevalence of worm infection in cattle spread in Indonesia. Generally, the prevalence of may be related to farmer ignorance and their worm infection in Indonesia is very high, particu- lack of knowledge in preventing the diseases, the * Correspondence to: Ida Bagus larly in agricultural landscape, near lakes and areal unaffordable expensive medicines to control the Komang Ardana, Veterinary Clinic Pathology Faculty of Veterinary with high temperature and humidity. In Bali, the diseases, and the improper use of medicines that Medicine, Udayana University, high prevalence of Fasciola infestation was noted lead to disease resistant. Due to particularly prob- Bali-Indonesia. in the Region of Karangasem (18.29%) and it can lems related to availability and the expensive price [email protected] be related with seasonal change, poor management of medicines, alternative solution to control the and sanitation and low level of education of farm- diseases should be found for the benefit of farmers. Received: 06 October 2016 ers. Meanwhile, the prevalence of Fasciola hepatica It has been known that Indonesia has abundance Accepted: 12 December 2016 infection recorded in abattoir in Samarinda in of herbs that has medical properties and, therefore, Published: 02 January 2017. 2009 was 55.56%.1 Furthermore, Agustina et al. research work should be encouraged to explore

Open access: www.balimedicaljournal.org and ojs.unud.ac.id/index.php/bmj 17 ORIGINAL ARTICLE

them. One of those herbs that has been known to and the reaction is considered positive have such medical properties is wudani (Quisqualis when white sediment is formed. indica Linn) that available widely throughout 2. Detection of flavonoid was conducted as Indonesia and has various names i.e dani, udani, follows. bidai, kacekluk, kaceklik, wedani, tikao, rabet- dani, and saradengan. At the level of community, a. With the use of 10% NaOH reactant; one ml wudani belonging to the Combretaceae family of the extract was added with few drops of has been used as traditional medicine to cure reactant and reaction is considered positive various illness such as , headache, rheu- when specific color is formed. matism, immune-modulator, anti-inflammation, b. With the use of Wilstater reactant; one ml antioxidant, and vermicide.3 Condensed tannins of the extract was added with few drops of (CT) extracted from various forage can markedly concentrated HCl and Mg powder. Positive decrease the viability of the larval stages of several reaction occurs when red-orange color is nematodes in and goats. formed. Suma Antara et al reported that application of c. With the use of Smith-Metacalve reactant; 10% wudani extract to piglets at a dose of 5 ml/8 kg one ml of the extract was added with few of body weight each day for 3 days can dimin- drops of concentrated HCl and then is ish infection of Ascaris suum and Trichuris sp.4 warmed. White color formed indicates that However, they did not find out whether its effect is the reaction is positive. through its property as either ovicidal, larvaecidal, 4. Detection of saponin was conducted by adding or vermicidal. Thus, the work reported in this paper hot water to the extract and then agitated. dealt with such research questions. Reaction is considered positive when foam is formed. METHODS 5. Detection of poliphenol was conducted by adding few drops of 1% FeCl3 reactant into one Extraction and Suspension of wudani leaf ml of the extract; Positive reaction occurs when The extract was made by macerating 50 grams of black or dark blue color is formed. fresh wudani leaf, ground, added with 70% etha- 6. Detection of steroid and tripernoid was nol, and following which it was stored in capped performed by adding few drops of unhidrate container away from sunlight for 2 days. After acetate and concentrated H2SO4 into one ml of 2 days of storage, it was then filtered and the residue the extract. When green-blue color is formed, obtained was macerated again with 70% ethanol it indicates the positive reaction towards by following the same procedure. Maceration was steroid and the color formation of red-violet, done until it was obtained clear macerate which brown indicates the positive reaction towards then evaporated in rotating vacuum evaporator at tripernoid. 40°C and then dried in freeze drier. Suspension of extract wudani leaf was made at concentration of Determination of ovicidal ability 10% w/v; 10 grams of the extract was added with Collected eggs of Fasciola gigantica and aquabidest till the total volume was 100 ml. Paramphistomum sp. obtained from the abattoir was initially put to hatch in media of 40 ml of Phytochemical screening of the wudani leaf physiological NaCl and with density of 7-9 eggs/ extract µl. Then they were added with 10% wudani Phytochemical screening was used in order to leaf extract at dose of 0.5 ml/40 ml NaCl (P1), detect the content of available chemical substances 1 ml/40 ml (P2), and 2 ml/40 ml NaCl (P3). The that have biological activities. Method to detect the control group (P0) was without wudani leaf extract. availability of alkaloid, flavonoid, tannin, saponin Six duplications were made for each group. After and steroid are as follows. 24 hours, the wudani extract was taken away and replaced by physiological NaCl and then incu- 1. Detection of alkaloid: bated for as long as 30 days. The doses used in the a. With the use of Wagner reactant; one ml of current work were based on previous results by wudani leaf extract is added with few drops Antara et al.4 They recorded that 10% wudani leaf of reactant; the reaction is considered posi- extract, at a dose of 5 ml/8 kg of body weight or tive when brown sediment is formed. 0.625 ml/kg of body weight was effective in killing b. With the use of Meyer reactant; one ml of Ascaris suum in pigs. Moreover, in a preliminary extract is added with few drops of reactant study on cows, they found an effective dose of

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Table 1 Results on assessment of chemical substances found in wudani leaf extract Phytochemical No. screening Reactant Change of color Note 1 Alkaloid HCl 2N + Wagner reactant + Brown sediment occurred; Chloroform Alkaloid (+) chloroform phase was red in color. 2. Flavonoid Wilstater Brownish green changes into dark brown. Flavonoid (+) Brownish green changes into yellowish green Bate Smith-Metcalfe brownish green changes into blackish green NaOH 10% Brownish green change into green 3. Saponin aquadest, warmed, stirred k, + HCl Stabile foam was formed; Saponin (+) 2M

4. Fenol FeCl3 Brownish green changes into blackish green Fenol (+) 5. Triterpenoid/ Lieberman-Burchard Brownish green changes into blackish green Steroid (+) Steroid H2SO4 Brownish green changes into blackish green

6. Tannin FeCl3 Brownish green changes into blackish green Tannin (+) Gelatin White sediment occurred Note: (+) indicate the availability of chemical substance

Table 2 Ovicidal ability of 10% wudani leaf extract towards eggs of Ovicidal ability of wudani leaf extract Fasciola gigantica following incubation for 30 days towards Fasciola gigantica eggs Table 2 showed the ovicidal ability of 10% wudani Ovicidal ability (%) leaf extract towards eggs of Fasciola gigantica. It has 10 days 30 days been noted that at day 10 of assessment, ovicidal Group Fasciola gigantica Fasciola gigantica ability of the extract towards the eggs was signif- P0 10.346 a 13.098 a icant (P < 0.05) whereas on day 30 it was highly significant (P < 0.01). P1 23.812 b 51.494 b From the current results, it can be noted that P2 33.104 c 86.810 c ovicidal ability of the extract at 0.5 ml/40 ml NaCl P3 35.849 c 88.502 c was 23.8% which is significantly lower (P < 0.05) Note compared to that of dose of 1 ml/40 ml (33.1%) and P0 : Control of those of 2 ml/40 ml NaCl (35.9%). However, it was P1 : Dose of 0.5 ml extract/40 ml physiological NaCl significantly higher than ovicidal ability of the control P2 : Dose of 1 ml extract/40 ml physiological NaCl P3 : Dose of 2 ml extract/40 ml physiological NaCl group (10.4%). On the other hand, the ovicidal ability of dose of 1 ml/40 ml and 2 ml/40 ml was not differ 0.625/50 ml of rumen fluid or 0.0125 ml/ml for significantly (P > 0.05). Moreover, ovicidal ability similar purposes. assessed at day 30 showed a highly significant increase The incubated eggs were aerated every day in (P < 0.01). Ovicidal ability of the extract at dose of order to observe egg development and embryo 1 ml/40 ml and of 2 ml/40 ml was very high, that ability of each egg was noted at day 10 and day 30 is 86.8% and 88.5%, respectively, but did not differ of incubation. Data for embryo ability was then significantly. When compared to dose of 0.5 ml/40 ml collected and examined as follows. The number of (51.5%) and the control group (13.1%), however, a fertile eggs was calculated from 100 eggs from each highly significant difference (P < 0.01) was noted. group and the embryo ability is the percentage of fertile eggs or eggs with embryo in it.5 The ability Ovicidal ability of 10% wudani leaf extract of 10% wudani leaf extract in killing embryo or towards eggs of Paramphistomum sp. its ovicidal ability was calculated by dividing the Table 3 showed the ovicidal ability of 10% wudani number of unhatched eggs with the number of eggs leaf extract towards eggs of Paramphistomum sp. examined times 100%. during in-vitro assessment. It has been recorded that the extract has a signif- RESULTS icant ovicidal ability towards the eggs that were assessed at day 10 and day 30 of incubation. At day 10 Prior to its application, screening of wudani leaf of incubation, the ovicidal ability of the extract at dose extract was made for its contents and the results of 2 ml/40 ml was the highest (23.3%) and signifi- were shown in Table 1. cantly higher (P < 0.05) than that of dose of 1 ml/

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Table 3 Ovicidal ability of 10% wudani leaf extract towards eggs of Paramphistomum sp. incubated for 10 and 30 days Ovicidal ability (%) 10 days 30 days Group Paramphistomum Paramphistomum P0 9.364 a 8.686 a P1 11.626 ab 40.246 b P2 21.630 bc 51.972 c P3 23.342 c 53.466 c Note: P0 : Control Figure 3 Morphology of egg shell of Fasciola P1 :Dose of 0.5 ml extract/40 ml physiological NaCl sp. following soaking with 10% P2 :Dose of 1 ml extract/40 ml physiological NaCl P3 Dose of 2 ml extract/40 ml physiological NaCl of wudani leaf extract at a dose of 1 ml/40 ml physiological NaCl for 24 hours. Changing in surface of egg shell occur and porosity can be seen. Magnification: 500 ×

Figure 1 Morphology of egg shell of Fasciola sp. following soaking with phys- iological NaCl for 24 hours; the shell looks normal. Magnification: 500 × Figure 4 Morphology of egg shell of Fasciola sp. following soaking with 10% wudani leaf extract at a dose of 2 ml/40 ml physi- ological NaCl for 24 hours. Changing in shell surface and porosity can be observed. Magnification: 500 ×

Figure 5 Morphology of egg shell of Paramphistomum sp. following soaking Figure 2 Morphology of egg shell of Fasciola sp. after soaking with 10% wudani with physiological NaCl for 24 hours. leaf extract at a dose of 0.5 ml/40 ml physiological NaCl for 24 hours. The shell looks normal. Magnification: Changing in surface of egg shell occurred. Magnification: 500 × 500 ×

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Figure 8 Morphology of egg shell of Paramphistomum sp. following soak- ing with 10% wudani leaf extract at a dose of 2 ml/40 ml physiological NaCl for 24 hours. Similar damage as shown in the previous pictures occurred. Magnification: 500 × Figure 6 Morphology of egg shell of Paramphistomum sp. following soaking The ability of 10% wudani leaf extract in with 10% wudani leaf extract at a dose of 0.5 ml/40 ml physiological damaging egg shells NaCl for 24 hours. A significant damage in shell can be observed. FASCIOLA GIGANTICA EGGS Magnification: 500 × Assessment using a Scanning Electron Microscopy (SEM) on the effect of soaking eggs with 10% wudani leaf extract at dose of 0 ml (control), 0.5 ml/40 ml NaCl, 1 ml/40 ml NaCl, and 2 ml/40 ml NaCl on morphology of egg shell are presented in Figure 1, Figure 2, Figure 3 and Figure 4, respectively.

Eggs of Paramphistomum sp. Assessment using SEM on the effect of soaking of with 10% wudani leaf extract at dose of 0 ml (control), 0.5 ml/40 ml physiological Na Cl, 1 ml/40 ml physiological NaCl and 2 ml/40 ml physiologi- cal NaCl on morphology of egg shells are presented in Figure 5, Figure 6, Figure 7 and Figure 8, respectively.

DISCUSSION The present research work has confirmed that extract of wudani leaf (Quisqualis indica) posi- Figure 7 Morphology of egg shell of Paramphistomum sp. following soaking tively contains alkaloid, flavonoid, saponin, phenol, with 10% wudani leaf extract at a dose of 1 ml/40 ml physiological tripernoid/steroid and tannin. In addition to those NaCl for 24 hours. As in the previous picture, a significant damage contents, it has been found that it also contains can be observed. Magnification: 500 × vitamin, protein, amino acids, and glycoside. So far, as herbal plant, wudani has been used by people 40 ml (21.6%), of 0.5 ml/40 ml (11.6%), and of control as traditional medicine to cure health problems (9.4%). However, among the latest three doses, there related with worm, pain, diarrhea, head ache, rheu- were no significant different (P > 0.05) was noted. matism, as well as used as an imunomodulator, At day 30 of incubation, there was no differ- anti-inflammation, anti-staphilococcus, and anti- ence (P > 0.05) in ovicidal ability between dose oxidant.3 The extract has property as anti-bacteria of 1 ml/40 ml (52%) and of 2 ml/40 ml (53.5%). against Salmonella typhi in vitro as reported by However, it was significantly higher (P < 0.05) Noorhamdani et al.6 compared to dose of 0.5 ml/40 ml (40.3%) and even The current results showed the ovicidal ability of a highly significantly different (P < 0.01) compared wudani leaf extract towards eggs of Fasciola gigan- to that of control (8.7%) was recorded. tica (88.5%) and Paramphistomum sp. (53.5%).

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The significant difference in ovicidal ability SUGGESTION between the two species of worm indicated that its direct contact to egg shells has resulted in different­ Further study is needed to discover the roles of effect to the egg shells. As mentioned above, extract of wudani leaf and the use of partition, wudani leaf extract contains alkaloid and glycoside fraction in order to know more about function of and has ovicidal property. As reported earlier by the substances or the chemical contents that have Yongabi, alkaloid has the lowest ovicidal ability property as anthelmenthic, so that its practical followed by glycoside and papain has the strongest application can be more effective. ability.7 Moreover, as noted in the current SEM study, following soaking in the extract, structure ACKNOWLEDGEMENT of egg shells underwent changes as shown in Figures 2, 3, and 4 for eggs of Fasciola gigantica We thank the Directorate of Research and and in Figures 6, 7, and 8 for the Paramphistomum Community Service for funding the present sp. eggs. research work through the Directorate General The eggs after being exposed to ethanol content of Higher Education, Department of Education of the extract may undergo pathological changes and Culture, Republic of Indonesia as stated in in albumin layer of egg shell in which some parts Letter of Order of Research Task No. 119/UN14.2/ were thickened and others were thinner. Ardana PNL.01.03.00/2015 Dated March 3, 2015. reported that eggs of Ascaris suum soaked for 24 hours in extract of ripe banana seed also exhibit REFERENCES 8 damage in their shells. The damage in egg shells 1. Jusmaldi, and Juliawan Saputra, 2009. Prevalensi Infeksi of Fasciola gigantica and Paraphistomum sp., as Cacing Hati (Fasciola hepatica) pada Sapi Potong di noted in the current study, may be due to activ- Rumah Pemotongan Hewan Samarinda.Bioprospek, Vol.6(2). ity of alkaloid (carpain and carpasemin), tannin 2. Agustina, K.K., A.A.G.O. Dharmayudha, and IW. Wirata. of ethanol extract that may have proteolytic 2013. Prevalensi Toxocara vitulorum Pada Induk Dan property.9,10 It may cause coagulation of albu- Anak Sapi Bali Di Wilayah Bali Timur. Buletin Veteriner Udayana Volume 5 No. 1 : 1-6. min. Changes in the egg shells may affect ability 3. Jyoti, S., Patel Pushpendra Kumar, and Dubey Balkrishna. of eggs to form embryo. Bariah et al reported 2012. Effects of Methanolic Extracts of Quisqualis Indica that coagulation of albumin of egg shells prevent (aerial parts) on passive smoking induced Hyperlipidemia 11 in Rats. Journal of Medical Pharmaceutical. And Allied development of Ascaris lumbricoides eggs. Thus, Sciences. Available from www.jmpas.com the present results for Ascaris lumbricoides and 4. Anthara, S., I Wayan Wirata, and A.A. Gde Oka Paramphistomum sp. have supported or in accor- Dharmayudha. 2013. Ekstrak Daun Wudani Untuk Pengobatan Infeksi Cacing Ascaris suum pada Babi. dance with their finding. 5. Oksanen, A., Eriksen, L., Roepstorff, R., Ilsoe, B., In conclusion, coagulation of albumin Nansen, P., and Lind, P. 1990. Embrionation and Infectivity layer of egg shells of Fasciola gigantica and of Ascaris suum Eggs. A Comparation of Eggs Collected from Worm Uteri with Eggs Isolated from Pig Faeces. Acta Paramphistomum sp. following soaking in 10% vet. Scand. 31(4) : 393 – 8. extract of wudani leaf may lead to decrase in abil- 6. Noorrhamdani, Djoko Santoso, Mirzia Dwi Rahma. 2013. ity to form embryo or embryo development. In Efek Ekstrak Daun Ceguk (Quisqualis indica Linn) sebagai Antibakteri Salmonella typhi in vitro. Jurnal Penelitian. other word, the extract has an ovicidal property. 7. Yongabi, K.A. 2005. Medicinal Plant Biotechnoloby. It’s This herbal plant has been used as traditional Role and Link in Integrated Biosystems: Part I. FMEny/ medicine to cure diseases caused by Ascaris ZER/ Research Centre, Abubakar. Email: yangabika@ 4 yahoo.com and Taenia spp. have found that application of 8. Ardana, I.B.K. 2007. Peran Ovisidal dan Vermisidal Herbal 10% extract of wudani leaf at dose of 5 ml per Serbuk Biji Pepaya (Carica papaya L) matang pada Babi day for 3 days may overcome infection caused Ascariasis. Desertasi. Pascasarjana, Universitas Udayana. 9. Min B.R., and S.P. Hart. 2003. Taninnin for suppression of by Ascaris suum in pigs suffering from various internal parasits. J Anim Sci.81. 102-109. degrees of infection; it also effective in combating 10. Praveen Kumar Ashok, Kumud Upadhyaya. 2012. Tannin Trichuris sp. infection. are Astringent.j Pharm and Phytochemistry.1 (3) :45-50. 11. Bariah, I., Dachlan, Y.P., and Sarmanu, H. 2001. Efek Anthelmintik Biji Pepaya (Carica papaya) Terhadap CONCLUSION Ultrastruktur Telur Ascaris lumbricoides. (Laporan Penelitian) Surabaya: Centre. Unair. In vitro application of 10% extract of wudani leaf is effectively used to overcome disease caused by worms (used as anthelmenthic). Thus, it may be further developed to be used in the future to over- come such problems in cattle. This work is licensed under a Creative Commons Attribution

22 Published by DiscoverSys | Bali Med J 2017; 6 (3): 17-22 | doi: 10.15562/bmj.v6i1.371