Research Article

Acute Toxicity of Extract of Sunan candlenut (Reutealis trisperma (Blanco) Airy Shaw) Seeds Nyi Mekar Saptarini*, Resmi Mustarichie

ABSTRACT

Background: Extract of Sunan candlenut (Reutealis trisperma (Blanco) Airy Shaw) seed, the Euphorbiaceae family, has been shown to have anti-alopecia activity. Extract safety must be determined so that the extract can be developed into herbal preparations. Aim: The purpose of this study was to determine the acute toxicity and toxic symptoms of Sunan candlenut seed extract. Materials and Methods: Acute toxicity assay was conducted on female Swiss Webster mice with various concentrations of Sunan candlenut seed extract (70, 700, 1400, 3500, and 7000 mg/kg BW mice), then observed

the toxic symptoms for 14 days. Results: The LD50 value of Sunan candlenut seed extract was 4954 mg/kg BW mice. This extract has a significant effect on the central nervous system by reducing the motoric activity (P = 2 × 10-4) and retablisment (P = 0.002), and the autonomic nervous system by disturbing ptosis (P = 0.032) and breathing (P = 0.001). Conclusion: The Sunan candlenut seed extract was 6th category, i.e., relatively harmless, based on the Hodge and Sterner toxicity scale.

KEY WORDS: Acute toxicity, LD50 value, Relatively harmless, Toxic symptoms

INTRODUCTION MATERIALS AND METHODS Sunan candlenut (Reutealis trisperma (Blanco) Materials Airy Shaw), the Euphorbiaceae family, is a plant Sunan candlenut seed was collected from 7-year-old from Southeast Asia. This plant grows in lowland tree from the Center for Agricultural Post Harvest [1] to 1000 m asl at 24–30°C. Sunan candlenut seed, Research, Bogor District, West Java Province, empirically, has efficacy as a laxative, anti-lice, Indonesia. The plant was identified by Plant [2,3] anti-dandruff, antiseptic, and treat skin diseases. Taxonomy Laboratory, Faculty of Mathematics and Sunan candlenut seed extract has been proven has Natural Sciences, Universitas Padjadjaran, Indonesia activity of stimulant of hair growth in rabbits using with No.30/HB/07/2019. the Tanaka method at concentrations of 10, 15, 20, and 25%.[4] All chemical reagents with analytical grade were purchased from Merck, i.e., potassium hydroxide, Most species of Euphorbiaceae are poisonous and toxic iron (III) chloride, magnesium powder, vanillin, plants, due to latex that irritates the skin and eyes, and ethanol, amyl alcohol, chloroform, ammonia, [5] chronic exposure causes carcinogenic effects. The hydrochloric acid, sulfuric acid, and ether. Pulvis safety of herbal preparation is related to toxicology Gummi Arabicum (PGA) with pharmaceutical grade [6] and undesirable effects, so an acute toxicity test is was purchased from China. conducted to determine the possible use of this plant as a herbal preparation. The purpose of this study was Female Swiss Webster mice were obtained from to determine the acute toxicity and toxic symptoms of School of Pharmacy, Institute of Technology Sunan candlenut seed extract. Bandung, Indonesia. Mice were nullipara, weight 20–30 g, 5–6 weeks old, and not pregnant. Mice Access this article online were acclimated for 1 week and their body weight was measured every day. Mice were healthy, if body Website: jprsolutions.info ISSN: 0975-7619 weight difference was no more than 20% with normal

Department of Pharmaceutical Analysis and Medicinal Chemistry, Faculty of Pharmacy, Universitas Padjadjaran, West Java, Indonesia

*Corresponding author: Nyi Mekar Saptarini, Department of Pharmaceutical Analysis and Medicinal Chemistry, Faculty of Pharmacy, Universitas Padjadjaran, Jl Raya Bandung Sumedang km 21, West Java 45363 Indonesia. E-mail: [email protected]

Received on: 28-11-2019; Revised on: 18-01-2020; Accepted on: 15-03-2020

Drug Invention Today | Vol 14 • Issue 5 • 2020 753 Nyi Mekar Saptarini and Resmi Mustarichie activity.[7] The acute toxicity assay procedures and metabolites. The extraction efficiency is enhanced protocols were reviewed and approved by the Health by the small particle size,[12] so Sunan candlenut Research Ethics Committee, Faculty of Medicine, seeds were pulverized to increase the surface area. Universitas Padjadjaran with No. 163/UN6.KEP/ The smaller the particle size, the better the yield, EC/2020. due to increased solvent penetration and diffusion of extracted secondary metabolites. The solvent selection Extraction is crucial for solvent extraction, such as maceration.[13] Sunan candlenut seeds were extracted by the Ethanol was chosen as the solvent, due to universal maceration method with 96% ethanol for 3 × 24 h. solvent for phytochemical investigation,[12] safe for Every day, ethanol was replaced by the fresh one. human consumption,[14] and volatile with boiling point All macerate was collected and concentrated by of 78.4°C so easily to concentrated.[15] using a rotary evaporator, then calculated the yield. The gravimetric method was used to determine the A total of 250 g of simplicia produced 62.46 g of moisture content.[8] Farnsworth method based on color concentrated extract with a yield of 24.98 ± 0.12% alteration was used to phytochemical screening.[7] with reddish-brown color, characteristic odor, and oily. The yield related to the amount of chemical Acute toxicity assay compounds contained in the extract.[7] Sunan candlenut Swiss-Webster mice were divided into 6 groups seeds contain alkaloids, saponins, flavonoids, and (n = 5), i.e., five treated groups (Sunan candlenut polyphenols. This result different from Sari’s study seed extract 70, 700, 1400, 3500, and 7000 mg/kg which showed the seeds contains alkaloids, flavonoids, [4] BW) and control (2% PGA suspension). Mice were monoterpenes, and sesquiterpenes. This difference given an oral single dose preparation, then observed was due to the source of seeds, i.e., Bogor District in [4] the toxic symptoms for 14 days. The toxic symptoms this study and Sukabumi District in Sari’s study. The include effects on the autonomic nervous and the extract moisture content was 2.12 ± 0.18% which met [7] central nervous system. The autonomic nervous the requirement, i.e., no more than 10%. The purpose system involves pineal, ptosis, writhing, piloerection, of determination moisture content is to determine the [7] breathing, salivation, lacrimation, defecation, and quality of the extract. urination. The central nervous system involves Female mice were used due to more sensitive than male motoric activity, hanging, retablisment, catalepsy, mice.[9] In this study, the extract concentration was sedatives, tremors, convulsions, Straub, flexion, and determined based on Hodge and Sterner toxicity scale. Hafner. The observation was conducted at before and [10] Pharmacological screening was observed before 0.5, 1, 2, 4, and 24 h after treatment.[9] Observations and at 0.5, 1, 2, 4, and 24 h after treatment. The results of body weight and death are conducted every day showed that all mice did not experience catalepsy, until the 14th day, then the mice were sacrificed. Acute sedation, tremor, convulsions, Straub, writhing, toxicity was evaluated based on Hodge and Sterner piloerection, salivation, lacrimation, defecation, and toxicity scale.[10] urination. All mice still had hanging, flexion, Hafner, Statistical analysis and pineal. This extract has a significant effect on the central nervous system by reducing the motoric Software IBM SPSS statistics base 22.0 was used to activity (P = 2 × 10−4) and retablisment (P = 0.002), data analysis and presented as the mean ± standard deviation. Statistical analysis was determined with one-way ANOVA and considered statistically 30 significant at P < 0.05. Probit analysis was used to [11] 25 determine the LD50 value.

RESULTS AND DISCUSSION 20 )

There was 250 g of Sunan candlenut seeds which ght (g 15 wei obtained by peeling 2 kg of fruit, then washed and control dried. The seed skin of Sunan candlenut was not as body extract 70 mg/g 10 hard as candlenut which is similar to the shell with a extract 700 mg/g slightly slippery outer surface. Seed shells thickness extract 1400 mg/g of about 1–2 mm, brown to blackish. Inside the seeds 5 extract 3500 mg/g were oval-shaped rigid white flesh of seeds. extract 7000 mg/g

0 Maceration was chosen as an extraction method, due 0510 15 to simple equipment and procedure. Extraction is me (days) facilitated by diffusion and solubility of secondary Figure 1: The mice’s body weight during observation (n=5)

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Table 1: Mice mortality during observation Group (n=5) Cumulative mortality (%) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Control 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Extract 70 mg/kg BW 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Extract 700 mg/kg BW 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Extract 1400 mg/kg BW 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Extract 3500 mg/kg BW 40 40 40 40 40 40 40 40 40 40 40 40 40 40 Extract 7000 mg/kg BW 60 60 60 60 60 60 60 60 60 60 60 60 60 60

Table 2: Observation on liver and kidney compare to the control [Table 2]. These organs Group Liver (g) Kidney (g) were observed, due to their role in elimination of the metabolic process of xenobiotics and waste Control (n=5) 1.1511±0.2382 0.2290±0.0212 Extract 70 mg/kg 1.1119±0.1727 0.2577±0.0220 product, so it is vulnerable to the xenobiotics side BW (n=5) effects. Hepatocyte and bile duct cells injuries lead to Extract 700 mg/kg 1.2708±0.3284 0.2596±0.0207 accumulation of bile acid in the liver, which promote BW (n=5) liver damage.[18] Nephrotoxicity can caused by Extract 1400 mg/kg 1.097±0.0122 0.2368±0.0116 poisonous effects of some substances.[19] There was no BW (n=5) significant difference in weight of liver (P = 0.41) and Extract 3500 mg/kg 1.0884±0.1966 0.2165±0.0074 kidney (P = 0.47) compared to the control. BW (n=3) Extract 7000 mg/kg 0.8832±0.2171 0.2679±0.0230 BW (n=2) CONCLUSION The Sunan candlenut seeds extract was relatively and the autonomic nervous system by disturbing ptosis harmless based on Hodge and Sterner toxicity scale. (P = 0.032) and breathing (P = 0.001). This extract decreased motoric activity and fluctuate respiratory ACKNOWLEDGMENT rate in the treated mice compared to control. The treated mice lost the ability to return to the hanging The authors would like to thank Academic Leadership state and open the upper eyelid. Grant of Universitas Padjadjaran in 2019 for the financial support. The authors also thank to Eli There were body weight fluctuate [Figure 1]. Halimah and Sasqia Faturachman for technical Increased body weight due to the nutrient intake assistance. from food and drink consumption. Feed is a source of energy and growth of mice, so that mice’s body weight REFERENCES increase.[16] Decreased body weight was a sign of poor health, which can be caused by toxic compounds 1. Vossen H, Umali B. Plant Resources of South-East Asia No.14. Bogor: Prosea Foundation; 2002. in the Sunan candlenut seeds extract, diseases, or 2. Stuppy W, van Welzen PC, Klinratana P, Posa MC. Revision of lack of nutrient intake.[17] There were significant the genera Aleurites, Reutealis and Vernicia (Euphorbiaceae). differences in body weight to different concentration Blumea J Plant Taxonomy Plant Geogr 1999;44:73-98. of Sunan candlenut seed extract (P = 1.5 × 10−33). 3. Available from: https://www.pfaf.org/user/Plant. aspx?LatinName=Reutealis+trisperma. [Last accessed on There were no significant differences in body weight 2020 Mar 15]. in the same group, i.e., control (P = 0.34), extract 4. Sari NI. Activity Test of Ethanolic Extract and Water, n-hexane, 70 mg/kg (P = 0.10), extract 700 mg/kg (P = 0.15), Ethyl Acetate Fraction of Sunan Candlenut Seeds (Reutealis extract 1400 mg/kg (P = 0.53), extract 3500 mg/kg trisperma (Blanco) Airy Shaw) as a Stimulator of hair growth, Thesis. Jatinangor: Universitas Padjadjaran; 2018. (P = 0.36), and extract 7000 mg/kg (P = 0.40). 5. Basak S, Bakshi KP, Basu S. Keratouveitis caused by plant sap. Indian J Ophthalmol 2009;57:311-3. st On the 1 day, there was 40% death in the extract 6. Parasuraman P. Toxicological screening. J Pharmacol 3500 mg/kg BW and 60% in the extract 7000 mg/kg BW, Pharmacother 2011;2:74-9. but on days 2 through 14 there was no death [Table 1]. 7. Fransworth NR. Biological and phytochemycal screening of plants. J Pharm Sci 1996;1:55-9. Probit analysis produced the equation y = 1.661x + 8. The Indonesian Ministry of Health. Indonesian Herbal st 3.8463 so that the LD50 value of Sunan candlenut seed Pharmacopoeia. 1 ed. Jakarta: The Indonesian Ministry of Health; 2015. extract was 4954 mg/kg BW mice. The LD50 value was 6th category, i.e., relatively harmless, based on the 9. Available from: http://www.jdih.pom.go.id/Produk/ [10] PERATURAN%20KEPALA%20BPOM/PerKBPOM%20_ Hodge and Sterner toxicity scale. Nomor%207%20Tahun%202014%20tentang%20in%20vivo. pdf. [Last accessed on 2020 Mar 20]. The color, texture, size, or shape of liver and kidney 10. Available from: http://www.ccohs.ca/oshanswers/chemicals/ of the treated mice did not show abnormal changes id50.htm. [Last accessed on 2020 Mar 20].

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