Acute and subchronic toxicity studies of methanol extract of Polygonum minus leaves in Sprague Dawley rats

Accepted Manuscript Acute and subchronic toxicity studies of methanol extract of Polygonum minus leaves in Sprague Dawley rats Parayil Varghese Christapher, Subramani Parasuraman, Mohd Zaini Asmawi, Vikneswaran Murugaiyah PII:

S0273-2300(17)30026-0

DOI:

10.1016/j.yrtph.2017.02.005

Reference:

YRTPH 3765

To appear in:

Regulatory Toxicology and Pharmacology

Received Date: 19 February 2016 Revised Date:

5 February 2017

Accepted Date: 8 February 2017

Please cite this article as: Christapher, P.V., Parasuraman, S., Asmawi, M.Z., Murugaiyah, V., Acute and subchronic toxicity studies of methanol extract of Polygonum minus leaves in Sprague Dawley rats, Regulatory Toxicology and Pharmacology (2017), doi: 10.1016/j.yrtph.2017.02.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Acute and subchronic toxicity studies of methanol extract of Polygonum minus leaves in

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Sprague Dawley rats Parayil Varghese Christapher1, 2, Subramani Parasuraman1, Mohd. Zaini Asmawi2,

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*Vikneswaran Murugaiyah2

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Kedah, Malaysia

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11800 Penang, Malaysia

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*Corresponding author:

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Unit of Pharmacology, Faculty of Pharmacy, AIMST University, Semeling, Bedong 08100

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Discipline of Pharmacology, School of Pharmaceutical Sciences, Universiti Sains Malaysia,

Associate Professor Dr. Vikneswaran Murugaiyah

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Discipline of Pharmacology

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School of Pharmaceutical Sciences,

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Universiti Sains Malaysia,

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11800, Penang, Malaysia.

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Email: [email protected]

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Phone: +6 04 6534583

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Fax: +6 04 6570017

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Word counts

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Abstract: 209

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Text: 4454

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References: 1212

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Abstract

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Medicinal plant preparations may contain high levels of toxic chemical constituents to

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potentially cause serious harm to animals and/or humans. Thus, toxicity studies are important to

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assess the toxic effects of plant derived products. Polygonum minus is used traditionally for

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different ailments in Southeast Asia. This study was conducted to establish the acute and

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subchronic toxicity profile of the methanol extract of P. minus leaves. The acute toxicity study

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showed that the methanol extract of P. minus is safe even at the highest dose tested of 2000

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mg/kg in female Sprague Dawley rats. There were no behavioural or physiological changes and

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gross pathological abnormalities observed. The subchronic toxicity study of methanol extract of

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P. minus at 250, 500, 1000 and 2000 mg/ kg were conducted in both sexes of Sprague Dawley

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rats. There were no changes observed in the extract treated animal’s body weight, food and water

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intake, motor coordination, behaviour and mental alertness. The values of haematological and

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biochemical parameters were not different between the treated and control animals. The relative

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organ weights of extract-treated animals did not differ with that of control animals. Based on the

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present findings, the methanol extract of P. minus leaves could be considered safe up to the dose

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of 2000 mg/kg.

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Key words: Polygonum minus; methanol extract; acute toxicity; subchronic toxicity

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1. Introduction Polygonum minus Huds. (syn: Persicaria minor (Huds.) Opiz) is a medicinal plant, used

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extensively in Southeast Asian countries for flavouring food preparations or consumed as salad.

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This non-woody, shrubby and creepy plant belongs to Polygonaceae family and its leaves taste

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sour with a rich lemony aroma. These plants usually found in wet marshy places and besides

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lakes and ponds (Bunawan et al., 2011). P. minus is locally known as “kesum” in Malay and has

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various vernacular names across Asia and Europe (English: Pygmy smartweed/knotweed; Thai:

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Phak phai; German: Kleiner Knöterich; Manipuri: Chakhong-macha; Malay: Kesum, Jarak

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Belanda; Vietnamese: Nghể bé). Both wild and cultivated species of this plant are available

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locally. The present study was conducted using leaves of cultivated plants. Whole plant, aerial

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parts, root, root bark, stem, stem bark, flower and seeds of this plant are usually used but leaves

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are mostly used in many traditional medicine preparations. In Malaysia, P. minus has been used

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in herbal medicine as a cure for digestive disorders (decoction), used as a tonic after childbirth

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and to treat dandruff. In Sarawak state of Malaysia, the whole plant is pound with rice and that

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paste is used for sprains and body aches. This plant is included as one of the salad plant in

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Malaysia and usually consumed with food (Jaganath & Ng, 2000; Shanmugam, 2004; Aziman,

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Abdullah et al., 2012).

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The phytochemical studies about P. minus are scarce and the reported works showed the

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presence of flavonoids and essential oil (Yaacob, 1990). Myricetin and quercetin are the mainly

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reported flavonoids. The high flavonoids content of this plant is thought to be useful in the

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prevention and management of cardiovascular risk conditions (Mollik et al., 2009). Some of the

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pharmacological activities of this plant were studied and reported (Christapher et al., 2015). The

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inhibition of LDL oxidation activity by P. minus was measured against copper mediated

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oxidation in isolated human LDL and reported that the inhibition was dose dependent as the

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increase in concentration of the extract showed higher rate of inhibition (Saputri and Jantan,

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2011). The antiulcer activity of the bioactive fraction (Ethyl acetate: methanol = 1:1) of P. minus

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leaves at 250 mg/ kg was reported to have equal effect with omeprazole at 20 mg/kg, and is due

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to alteration in mucus production, hexoseamine levels and PGE2 synthesis in rodents (Qader et

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al., 2012). In an immunomodulatory evaluation using in vivo carbon clearance assay, the aqueous

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extract of P. minus at 200 and 400 mg/kg exhibited a dose dependent increase in phagocytic

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index which is comparable to levamisole at 2.5 mg/kg of body weight (George et al., 2014).

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Analgesic and anti-inflammatory potential as well as the antiulcer activity were studied and

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reported (Christapher et al., 2015). A randomized, double blind, and placebo-controlled parallel

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clinical study in human demonstrated the increase in sexual wellbeing effect of Eurycoma

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longifolia at 200mg daily and water extract of P. minus at 100 mg/kg (Udani et al., 2014).

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Despite of the use as food and traditional medicine, and many reported pharmacological

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activities, the toxicity information on P. minus is scarce. The present study is aimed to assess the

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toxic effects of methanol extract of P. minus leaves after single and 28-day repeated oral

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administration in Sprague Dawley rats.

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2. Materials and methods

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2.1. Preparation of methanol extract

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Fresh leaves of P. minus were harvested from the suburban region of Penang, Malaysia

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during September 2014. The plant was authenticated and a voucher specimen (USM Herbarium.

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11542) was deposited at the Herbarium, School of Biological Sciences, Universiti Sains

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Malaysia. The leaves were separated from stems and rinsed thoroughly with water to remove

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dirt. The leaves were dried in a hot air oven below 50 ºC for 3 days. The dried leaves were

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pulverized using a blender. The methanol extract was prepared by maceration method. The

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powdered leaves were macerated using methanol (raw material to solvent ratio of 1: 10 w/v) for

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5 days. The extract was decanted and replenished with fresh solvent every day and the extract

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was pooled and kept at 4 ºC. After fifth day, the pooled extract was filtered through a cotton plug

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followed by filter paper. The filtrate was then concentrated in a rotary evaporator (Yamato

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Rotary Evaporator RE300, Yamato Scientific Co., Ltd. Tokyo, Japan) and freeze-dried (Thermo

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Scientific, MA, USA) to obtain dry extract of 15.64g per 100g powdered materials. The dry

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extract was kept at 4 °C until further use. The extract was subsequently suspended in 0.5% w/v

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carboxymethylcellulose (CMC) to the required dose for administration. The extract was

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administered by oral gavage to the experimental animals.

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2.2. Experimental animals

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Female and male Sprague Dawley rats were used for the acute and subchronic toxicity

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studies. The animals were obtained from the Central Animal House of AIMST University and

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were acclimatized for a period of 7 days before the commencement of experiments. The animals

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were housed in polypropylene cages, maintained at room temperature and a 12h light/dark cycle.

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Free access to normal diet and water was allowed during the adaptation period. The food pellets

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for the experimental animals was procured from Gold Coin Holdings Sdn. Bhd. (Penang,

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Malaysia). All the experiments were conducted in accordance with the guidelines of institutional

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animal experiment guidelines and approval to use the animal was obtained from Animal Ethics

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Committee, Universiti Sains Malaysia [Ref. No. USM/Animal Ethics Approval /2014/ (94)

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(480)] and Animal and Human Ethics Committee, AIMST University [Ref. No. AUHAEC

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7/FOP/2014].

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2.3. Heavy metal analysis

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The presence of heavy metals in the methanol extract of P. minus was tested at a local

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commercial testing laboratory, North Malaya Laboratory Sdn. Bhd. (Penang, Malaysia). The

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presence of mercury (Hg), lead (Pb), arsenic (As) and cadmium (Cd) were determined using an

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inductively coupled plasma optical emission spectrometry (ICP-OES).

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2.4. Acute toxicity study

The study was conducted in accordance with OECD Guidelines for testing (test number

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420) chemicals (OECD, 1992; OECD, 2008). Five to seven week old, female Sprague Dawley

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rats (weight of 140-160g) were used for the study. In the sighting study, an overnight fasted

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female rat was administered with single oral dose of methanol extract at 300 mg/kg. The

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animal was observed closely for the symptoms of toxic effects such as colour change in skin,

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fur, eyes, mucous membrane and secretions, urination, defecation, autonomic activities, change

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in posture, gait, seizures, aggressiveness and strange behaviour for the first 4 hours. The onset

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and duration of the toxic symptoms observed were recorded systematically. The animal was

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monitored for the next 48 h for its survival and onset of any sign of toxicity. As the first rat did

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not show any sign of toxicity and survived more than 48h, the second female rat was

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administered with a higher dose at 2000 mg/kg and observed as before. The main study is

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carried out as the second animal also survived for more than 48 h. In the main study, four

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female rats were administered with 2000 mg/kg dose and the survival time and toxic signs

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were observed. The animals that survived the dosing were continued to be observed at least

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once in a day for 14 consecutive days. During this study, the body weight of the rats were

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measured and recorded on day 1, 7 and 14. At the end of the experiments, the animals were

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anaesthetized and then sacrificed by cervical dislocation for necropsy. The rats were dissected

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and the organs (brain, heart, kidney, lungs, liver, thymus, spleen, adrenal glands, sex organs,

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stomach and gut) were excised and examined.

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2.5. Subchronic toxicity study

Rats of both sexes were used for the 28-day repeated dose oral toxicity study following

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OECD Guideline for Testing Chemicals Guideline 407 (OECD, 2008). Five to seven week old

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male and female rats (initial weight of 140-160g) were divided into 10 groups (n=10; 5 females

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and 5 males). Group 1 received 0.5% CMC, while groups 2-5 received 250, 500, 1000, 2000

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mg/kg methanol extract of P. minus leaves, respectively, orally once daily for 28 days. The

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dosing time (Approx. at 11.00 am) for the animals was maintained throughout the study to

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minimise biological variation among the animals.

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2.5.1. Observational study

During the treatment, the signs of toxicity, other behavioural changes, food consumption,

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water intake and event of mortality were closely monitored. The motor activity, grooming, touch

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response, pain response, tremors, convulsion, righting reflex, writhing, urination, salivation and

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skin colour of rats were also observed throughout the study (Hor et al., 2012). The locomotor

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activity of the animals on day 1 and 28 also was measured to determine the mental alertness of

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the animals. The body weight of the animals was measured weekly.

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2.5.2. Haematological and biochemical analysis At the end of the study, the animals were anaesthetized and terminal blood sample (5 mL)

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was withdrawn through cardiac puncture. The blood was collected in EDTA-containing and non-

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heparinized tubes for the haematological and biochemical analysis, respectively. The

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haematological parameters measured were white blood cells (WBC), red blood cells (RBC),

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haemoglobin concentration (Hb), packed cell volume (PCV), mean corpuscular volume (MCV),

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mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC),

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platelets, neutrophil, lymphocyte, eosinophil and monocyte. The biochemical parameters

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measured were total protein, albumin, globulin, albumin to globulin ratio (ALB/GLO), alkaline phosphatase

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glutamyltransferase (GGT), alanine aminotransferase (ALT), glucose, total cholesterol (TC),

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triglycerides (TG), high density lipoprotein (HDL), low density lipoprotein (LDL), total

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cholesterol to high density lipoprotein ratio (TC/HDL), sodium, potassium, chloride, urea,

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creatinine, uric acid, calcium and phosphate. The haematological parameters were determined

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using automated haematology analyzer (Sys-mex-XT-1800, Hamburg, Germany) and serum

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biochemistry tests were performed using automated chemistry analyzer (Olympus 640

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Biochemistry Analyzer, Tokyo, Japan) at Gribbles Pathology Sdn. Bhd. (Sungai Petani, Kedah,

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Malaysia).

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bilirubin,

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(AST),

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total

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(ALP),

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2.5.3. Histopathology evaluation The animals were sacrificed after blood collection by cervical dislocation and organs

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were excised and weighed. The relative organ weight was calculated. Vital organs such as brain,

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kidney, liver, lung and heart were preserved in 10% neutral formalin for histopathological

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evaluation. The sections were prepared and stained with haematoxylin and eosin and mounted in

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neutral DPX medium. The slides were observed under light microscope.

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2.6. HPLC analysis

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2.6.1. Instrumentation

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Chromatographic analysis was carried out using Agilent 1260 infinity-series HPLC

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system consist of quaternary pump (G1311A Quat pump), vacuum degasser (G1379A), auto-

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sampler/auto-injector (G1313A ALS), column compartment (G1316A Colcom) and UV detector.

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Agilent Chem Station software (Revision B 04.04.03 (16)) was used for the data collection and

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analysis.

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2.6.2. Analytical conditions for fingerprinting and quantification of phytochemicals The analytical conditions for the phytochemical analysis are as following: An isocratic

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elution was carried out on a Thermo scientific Hypersil C18 column (250 10 x 4.0 mm i.d., 5

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m particle size) (Thermo Scientific, MA, USA). The mobile phase consisted of

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tetrahydrofuran (THF) and sodium dihydrogen phosphate buffer (15.6 g/L) adjusted to pH 3.0

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using phosphoric acid. The proportion of the THF and buffer is 40:60. The mobile phase was

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degassed by sonication and filtration was carried out using 0.45µm pore size filter. The mobile

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phase flow rate was set at 1mL/min, injection volume was 20 µL and the signals were detected at

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356 nm. For sample preparation, 10 mg of methanol extract of P. minus was dissolved in 10 mL

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of mobile phase to obtain 1mg/mL solution for injection. Selected reference compounds such as

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quercitrin and myricetin was also prepared using mobile phase at a concentration of 250 µg/mL.

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Retention times of the detected compounds were compared with that of authentic standards for

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the compound identification and quantification.

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2.7. Statistical analysis

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Data are expressed as mean ± SEM. Significant difference between groups were

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determined using one-way ANOVA followed by Tukey’s multiple comparison tests (IBM-SPSS

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version 20). A P<0.05 value was considered to be significant.

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3. Results

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3.1. Heavy metal analysis

The presence of mercury, lead, cadmium and arsenic in the methanol extract of P. minus

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was measured using ICP-OES. The limit of detection (LOD) for Hg, Pb, As and Cd for this

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method is less than 0.05, 1, 1 and 0.05 mg/kg of extract respectively. None of these heavy metals

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were detected in the extract, thus the levels are well below the limits (Table 1). The absence of

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the heavy metal content was confirmed with spiking of the respective heavy metal in the extract.

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3.2. Acute toxicity

In the sighting study, the animals were monitored for 14 consecutive days after dosing

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and no death was observed. The animals did not show any signs of toxicity during the study

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period. The animals used in the main study were also observed for 14 consecutive days after

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dosing and none of these animals showed any toxic signs or died. The general appearance,

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grooming, posture, gait, behaviour and all other observational parameters were normal during the

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study. The body weight of the animals was not significantly different between days 1, 7 and 14

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of the study. At the end of the study, the necropsy found no gross pathological changes in the

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organs examined. The histopathological examination was not conducted as the gross pathological

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showed no abnormality.

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3.3. Subchronic toxicity

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3.3.1. Observational study

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During the 28-day subchronic study, no death, changes in behaviour or other

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physiological changes were observed in control and extract-treated animals. The body weight

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analysis showed that weight gain in the male and female animals treated with the extract at 250,

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500, 1000 and 2000 mg/kg, which is comparable with the control (Figures 1 & 2). There was no

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significant variation in weight gain between the control and extract treated groups throughout the

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study period. Food and water consumption for all the animals was found to be normal and

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similar between control and treated groups. The animals did not show any signs of neurotoxicity

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as there were no changes in sensory and motor signs. The findings of the mental alertness study

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also showed normal mental activity pattern prior and at the end of the study. No significant

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changes were found in the locomotor activity for the control and treated group (Table 2).

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3.3.2. Haematological and biochemical analysis

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The haematological analysis on different parameters, namely WBC, RBC, Hb, PCV,

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MCV, MCH, MCHC, platelets, neutrophil, lymphocyte, eosinophil and monocyte did not show

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any significant differences between the control and extract treated groups at all doses (Table 3).

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Likewise, there was no significant change observed in the biochemical parameters between the

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control and extract treated groups (Table 4). The parameters such as total protein, albumin,

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globulin, albumin to globulin ratio, ALP, total bilirubin, AST, GGT, ALT, glucose, TC, TG,

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HDL, LDL, TC/HDL ratio, sodium, potassium, chloride, urea, creatinine, uric acid, calcium and

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phosphate were found to be in normal range for all animals at the end of the study.

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3.3.3. Histopathology

The relative organ weights of the extract treated groups were found to be not different

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with that of the control group (Table 5). The histopathological evaluation conducted on vital

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organs such as brain, kidney, liver, lung and heart showed normal architecture and no

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abnormality in the control and extract treated groups. Some minor changes were observed in the

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histological slides examined, but were minimal and also evident in the control group [data shown

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as supplementary material].

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3.4. HPLC analysis

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The chromatograms for the analysis of crude extract of P. minus and reference standards

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are presented in figure 3. The fingerprinting analysis confirmed the presence of quercitrin and

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myricetin. The amounts of these compounds in the sample were 5.5 and 0.6 µg/mg, respectively.

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4. Discussion

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Natural products including their derivatives and analogues still represent a major part of

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therapeutic alternatives. The World Health Organization estimates that 80% of the people in

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developing countries rely on traditional medicine including plants and plant extracts (Turner,

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1996). The major disadvantage with plant derived extracts or compounds are their potential

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toxicity and to date there are not many studies on their toxicity, due to the belief that the plant is

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usually safe to consume. The main purpose of a toxicity study is to establish any adverse effect

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caused by the phytochemicals, investigate any possible dose-effect relationship and to identify

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the responsible phytochemicals causing the toxicity. The pharmacological effects of P. minus

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were reported in many previous studies. The present study was conducted to assess the acute and

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subchronic toxicity study of methanol extract of this plant.

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The heavy metal contamination is one of the reasons for toxicity exhibited by the plant

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and plant derived products. Presence of heavy metals even at trace amount is dangerous to the

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health as they may cause serious damages in various systems of the body (Martin and Griswold,

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2009). The presence of mercury, lead, cadmium and arsenic were not detected in the methanol

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extract of P. minus, indicating that the extract used for this study is devoid of the heavy metal

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contamination during its cultivation and processing.

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The acute toxicity study is utilized to check the harmful effects of an agent to the

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organism given as a single or short term exposure (Krishnaraju et al., 2005). Mainly, the study

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evaluates the mortality, changes in behaviour, body weight and other spontaneous changes in

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overall well-being of the rats. In the present study, no mortality was recorded during the sighting

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as well as in main study. There were no significant changes in body weights for the pre-study

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compared to days 7 and 14 of the study. The general signs of the animals were normal in low

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(300 mg/kg) and high (2000 mg/kg) dose treated animals for the first 48 h and the same was

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observed for the following 14 days. The absence of toxicity was further confirmed during the

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necropsy. None of the organs showed any abnormality in their gross pathology examination. The

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methanol extract of P. minus at 2000 mg/kg did not exhibit any signs of acute toxicity, thus the

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LD50 is determined as greater than 2000 mg/kg. According to the Globally Harmonized System

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(GHS), a dose which is safe above 2000 mg/kg of body weight is classified as category 5 or

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unclassified (Ming et al., 2013). The 28-day repeated subchronic toxicity study is essential for the assessment of safety of

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an agent when it is administered repeatedly over a period of time. Varying doses of methanol

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extract of P. minus leaves (250, 500, 1000 and 2000 mg/kg) were administered to different group

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of animals of both sexes, once daily for 28 days. No death was recorded for any of the dose

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tested during the study period. This finding reaffirms the findings of acute study. The drastic

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decrease or increase in body weight is a well-known sign for toxicity. In our study, the change in

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body weights observed was found to be according to the normal growth pattern as it is

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comparable with the weight gain of control animals. Likewise, treated animals did not show any

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abnormality in the food consumption and water drinking pattern. These two parameters are

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important as any hesitation to consume food and drink water is a direct indication of alteration in

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metabolism (Teff and Kim, 2011). The neurological signs such as spontaneous activity,

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reactivity, touch response and pain response in animal is considered as evidence for the

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neurological activity (Petchi et al., 2014). The present study revealed that the methanol extract of

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P. minus leaves does not possess any neurotoxicity as these signs were normal. The general

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movement of the animal is an indication of its mental alertness (Kumar et al., 2011). The

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locomotor activity of the treated group of animals were compared and found no reduction or

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stimulation. Thus, the result indicates absence of any inhibitory or stimulatory activity on the

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mental activity.

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Changes in blood parameters are considered to be one of the most reliable evidence for

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the toxicity studies. Measurement of levels of blood components is relevant to assess the risk of

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toxicity in animal studies that can be translated into human values (Hor et al., 2012; Olson et al.,

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2000). The male and female animals in the treated groups did not show any appreciable changes

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in their blood parameters when compared to that of the control, suggesting that the methanol

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extract has no adverse effect on the haematopoiesis and other blood cell formations. Liver toxicity can be detected by the measurement of various liver enzymes, in particular

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the levels of ALT and AST. ALT is a cytoplasmic enzyme and found at high level in the liver.

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Elevation in the ALT level indicates the liver cell damage (Wang et al., 2007). AST is present in

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many of the vital organs and also serves as an indicator for liver function. Biliary occlusion and

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damage can be confirmed by the alteration in the serum bilirubin level. Serum proteins are

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mainly synthesized in the liver, so the levels of these proteins are considered as the indicator of

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normal synthetic functions of hepatocytes (Solomon et al., 1993). Any reduction in serum

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proteins such as albumin and globulin is a reflection of liver cell damage. In the present study,

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none of these important liver function parameters were significantly different between the extract

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treated and normal groups in both male and female animals. The histopathology studies

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confirmed and supported these findings. The study suggests that the methanol extract of P. minus

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leaves is not toxic to liver cells up to the dose of 2000 mg/kg.

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Alterations in the normal metabolism of animals can be evaluated by the measurement of

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glucose and lipids (Seiva et al., 2012). In the present study, the level of glucose, total cholesterol,

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triglycerides, LDL and HDL of the extract treated animals were not significantly different from

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those levels of control animals. Kidney function is also an important aspect to indicate the

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potential toxicity of a compound. The level of creatinine is an indicator for the kidney function.

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An elevation in its level indicates the impaired glomerular filtration and kidney damage

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(Rhiouani et al., 2008). Serum urea elevation is an indicator for various tissue injuries such as

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cardiac, renal parenchymal and renal calculi. In our present study both parameters were not

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significantly different from control values, indicating that the extract has no deleterious effect on

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the kidney function. The electrolytes such as sodium, potassium, chloride, calcium, phosphate

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and uric acid of the extract treated animals were in the normal limit when compared with control

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groups, which further substantiates the kidney function of the animals are not affected (Xing et

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al., 2014). The histopathology evaluation also did not detect any abnormality in the architecture

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of the vital organs of the extract treated groups.

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Relative organ weight measurement provides valuable information about the toxicities of

8

the compounds towards targeted organs (Wang et al., 2007). The alteration in organ weight could

9

be observed if the tested compound is potentially toxic to them. In the present study the physical

10

conditions of the organs were normal and there is no sign of swelling, atrophy or hypertrophy. In

11

addition, there is no difference in the relative organ weight between the control male and female

12

animals with the extract treated animals. Histopathology study also revealed that the microscopic

13

characters of the tissues were normal for all the vital organs tested. The findings from the present

14

study concurs with the results of a previous study conducted with aqueous extract of P. minus at

15

a maximum dose of 1000 mg/kg (Ming et al., 2013). The results of both these studies suggest

16

that the active principles present in water or methanol extract of P. minus are not toxic to rats

17

even at high concentrations.

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In the present study chronic treatment of methanol extract of P. minus up to 2000 mg/kg

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did not produce any mortality. Development of toxicity due to any herbal extracts depends on the

20

chemical constituents present in that particular herb. P. minus leaves were reported to contain

21

compounds such as quercitrin, myricetin, rutin, quercetin and kaempferol (Narasimhulu et al.,

22

2014). Phytochemical analysis confirms the presence of quercitrin and myricetin in the methanol

23

extract of P. minus. It has been reported that the identified chemical constituents like quercitrin

16

ACCEPTED MANUSCRIPT

and myricetin from our study and also other compounds reported from previous studies such as

2

quercetin, kaempferol and rutin did not cause any major toxic effects in the pre-clinical

3

experiments. Reports on the pre-clinical toxicity and clinical studies of these compounds suggest

4

that they are considerably safe to animals and humans within the accepted daily intake limit

5

(Ross & Kasum, 2002; Harwood, Danielewska-Nikiel et al., 2007; Kupradinun, Tepsuwan et al.,

6

2010; Hobbs, Swartz et al., 2015; Zhou, Yang et al., 2015). In addition, a commercially available

7

product of P. minus, standardized using quercetin 3-glucoronide and quercitrin was reported to

8

be safe for human consumption (Biotropics, 2016).

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The clinical trials conducted with plant extracts from P. minus or a mixture of plant

10

extracts containing extract of P. minus suggest that the plant has a positive effect on the sexual

11

well-being. The recommended dose of P. minus leaves extract is 100 mg daily to produce the

12

above mentioned effects in humans and it was suggested that P. minus along with Eurycoma

13

longifolia improve the sexual performance in healthy, middle aged men (Udani et al., 2014). In

14

an another trial, improved attention, short-term memory, quality of life and mood as well as

15

intelligence quotient (IQ) in good mood sub group was exhibited by P. minus at a dose of 250

16

mg daily in middle aged women (Shahar, Aziz et al., 2015). Likewise, the positive effect of

17

SuperUlam- a mixture of extract of various plants including P. minus (150mg) and vitamins on

18

mood and concentration of healthy adult was observed (Udani, 2013). All these study outcomes

19

suggest the safety as well as the efficacy of P. minus as a beneficial herb. This study also helped

20

to support the fact that the toxicity of a plant variety in a particular family of plants is not a

21

general characteristic of that family, rather the toxicity is dependent upon the phytochemicals

22

present in the individual plant. Polygonum multiflorum, a variety of Polygonum was reported to

23

possess hepatotoxic effect (Dong, Slain et al., 2014; Teschke, 2014; Frenzel & Teschke, 2016).

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In contrast, other varieties including P. minus were found to be not hepatotoxic based on the

2

present findings and previous report (Choudary, Kotak et al., 2012).

3

5. Conclusion

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Acute and subchronic toxicity studies were conducted to establish the safety of methanol

6

extract of P. minus leaves in Sprague Dawley rats. General signs and behaviour were found to be

7

normal in animals tested up to 2000 mg/kg. Likewise, in the 28 days repeated dose subchronic

8

toxicity study, there was no difference in the blood and biochemistry parameters values of the

9

extract treated and control animals. Based on these findings, the methanol extract of P. minus

10

leaves is considered safe up to 2000 mg/kg in Sprague Dawley rat. This warrants further safety

11

evaluation on chronic toxicity and other toxicities studies such as genotoxicity and

12

carcinogenicity to establish a complete toxicity profile of P. minus.

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Conflict of interest statement

15

The authors declare that there are no conflicts of interest.

16

Acknowledgment

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This work was supported in part by AIMST University Internal research Grant

18

(AURGC/18/FOP/2013). The authors would like to thank Dr. Urmila Banik, Department of

19

Pathology, Faculty of Medicine, AIMST University, Malaysia for her help in pathological study

20

interpretations and CARe Keralam Laboratory, Koratty, Kerala, India for their help in HPLC

21

analysis of the extract sample.

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Biotropics,

Malaysia,2016.

Product

information

about

http://www.biotropicsingredients.com/lineminus (accessed 20.09.16). 23

Line

Minus.

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Figure 1: Body weight of female rats in subchronic toxicity study of methanol extract of Polygonum minus leaves

3 4 5

Figure 2: Body weight of male rats in subchronic toxicity study of methanol extract of Polygonum minus leaves

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Figure 3: Chromatograms of the (A) reference standards and (B) methanol extract of Polygonum minus and (C) expanded chromatogram of methanol extract of Polygonum minus. The phytochemical analysis was performed on a Thermo Scientific hypersil C18 column (250 10 x 4.0 mm i.d., 5 m particle size) with UV detection at 356 nm.

Supplementary material-1 (Figure 4): Photomicrograph of histology of vital organs of control animals (female rats) in subchronic toxicity study of methanol extract of Polygonum minus leaves. (A) Brain; (B) Lung; (C) Heart; (D) Liver; (E) Kidney. All the organs showed normal cellular architecture. The slides were stained with H&E and viewed at 400 x magnification.

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Supplementary material-3 (Figure 6): Photomicrograph of histology of vital organs of male extract treated (2000 mg/kg) rats in subchronic toxicity study of methanol extract of Polygonum minus leaves. (A) Brain; (B) Lung; (C) Heart; (D) Liver; (E) Kidney. All the organs showed normal cellular architecture. The slides were stained with H&E and viewed at 400 x magnification.

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Supplementary material-2 (Figure 5): Photomicrograph of histology of vital organs of female extract treated (2000 mg/kg) rats in subchronic toxicity study of methanol extract of Polygonum minus leaves. (A) Brain; (B) Lung; (C) Heart; (D) Liver; (E) Kidney. All the organs showed normal cellular architecture. The slides were stained with H&E and viewed at 400 x magnification.

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Table 1: Heavy metal content of methanol extract of Polygonum minus leaves Element Result Specification Cadmium Not Detected Not more than 0.2 ppm Lead Not Detected Not more than 5.0 ppm Arsenic Not Detected Not more than 2.0 ppm Mercury Not Detected Not more than 0.1 ppm

Table 2: Locomotor activity of rats in subchronic toxicity study of methanol extract Polygonum minus leaves Female Day 1

Male

Day 28

SC

Treatment

Day 1

Control

122.80 ± 9.95

144.60 ± 8.38

250 mg/kg

119.20 ± 8.25

125.40 ± 10.03

123.60 ± 10.48

134.80 ± 12.65

500 mg/kg

123.00 ± 11.96

127.60 ± 14.32

122.40 ± 12.01

129.60 ± 14.26

1000 mg/kg

129.80 ± 8.29

129.60 ± 5.75

139.60 ± 12.17

128.80 ± 9.83

2000 mg/kg

133.40 ± 13.38

127.40 ± 14.52

127.00 ± 8.33

138.00 ± 13.09

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129.40 ± 10.44

Day 28 130.00 ± 9.81

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Each interruption of light beam due to movement of animal was considered as a score and measured for 10 minutes. All the values are reported as mean ± SEM (n=5).

1

ACCEPTED MANUSCRIPT Table 3: Haematological values of rats in subchronic toxicity study of methanol extract of Polygonum minus leaves Female Male Methanol extract of P. minus Methanol extract of P. minus 1000 mg/kg 130.00

2000 mg/kg 129.60

Control 133.60

250 mg/kg 137.40

500 mg/kg 140.20

1000 mg/kg 138.80 ±

2000 mg/kg 126.20

3.38

1.86

± 3.04

± 3.32

± 4.64

± 4.60

± 0.62

± 5.20

2.40

± 2.70

7.68 ±

7.18 ±

7.27 ±

6.78 ±

7.28 ±

7.08 ±

6.80 ±

7.04 ±

6.70 ±

0.24

0.06

0.25

0.14

0.23

0.30

0.15

0.34

0.09

0.27

c

0.40 ±

0.44 ±

0.44 ±

0.44 ±

0.41 ±

0.43 ±

0.44 ±

0.42 ±

0.44 ±

0.35 ±

0.01

0.01

0.01

0.01

0.02

0.02

0.01

0.01

0.01

0.03

55.20 ±

61.40 ±

61.20 ±

59.60 ±

59.80±

56.80 ±

59.20 ±

61.80 ±

62.00 ±

61.60 ±

2.13

0.37

1.67

4.46

1.70

1.34

1.67

1.74

1.04

1.06

18.00 ±

18.00 ±

18.2 ±

19.6 ±

19.80 ±

0.50

0.50

0.73

0.37

0.45

316.40 ±

306.69 ±

307.80

296.00

297.60

4.33

4.42

± 4.13

± 2.33

11.70 ±

9.40 ±

10.40 ±

0.48

1.22

1.03

2.18 ±

2.56 ±

3.58 ±

0.64

0.32

0.70

5.96 ±

5.18 ±

5.70 ±

0.46

0.34

0.48 ±

MCV (fL) e

MCH (pg) f

MCHC (g/L) g

WBC 9

(x10 /L) h

(x109/L) i

L

9

(x10 /L) Mj 9

(x10 /L) k

(x109/L) 9

PLT (x10 /L)

17.60 ±

18.40 ±

19.00 ±

19.40 ±

0.53

0.37

1.06

0.29

0.47

311.20

304.40

312.40

299.20 ±

296.20

± 5.14

± 5.88

± 5.90

± 4.66

6.84

± 6.92

9.36 ±

14.46 ±

10.94 ±

11.94 ±

9.32 ±

10.86 ±

13.94 ±

1.26

1.56

0.50

0.72

1.31

0.63

0.57

2.48 ±

4.56 ±

1.92 ±

3.90 ±

3.58 ±

3.92 ±

3.66

0.34

1.03

0.44

0.35

0.40

0.83

±1.24

5.38 ±

7.04 ±

5.46 ±

5.56 ±

5.40 ±

5.06 ±

6.38 ±

0.31

0.62

0.95

0.31

0.50

0.35

0.52

0.22

0.54 ±

0.52 ±

0.48 ±

0.82 ±

0.46 ±

0.88 ±

0.72 ±

0.62 ±

0.98 ±

0.15

0.14

0.15

0.08

0.19

0.10

0.17

0.12

0.12

0.14

1.01 ±

0.22 ±

0.70 ±

0.64 ±

0.68 ±

0.92 ±

0.60 ±

0.56±

0.42 ±

1.12 ±

0.06

0.09

0.18

0.14

0.12

0.12

0.09

0.16

0.11

0.09

848.80 ± 61.33

831.00 ±

824.00

799.80±

855.00

877.60

724.80

728.0 ±

912.60 ±

850.60

30.25

± 77.89

106.66

± 93.61

± 35.49

± 66.18

110.97

111.29

± 96.10

All the values are reported as mean ± SEM (n=5). Haemoglobin concentration (Hb)a, Red Blood Cells (RBC)b, Packed Cell Volume (PCV)c, Corpuscular Volume (MCV)d, Mean Corpuscular Haemoglobin (MCH)e, Mean Corpuscular Haemoglobin Concentration (MCHC)f, White Blood Cells (WBC)g, Neutrophilh, Lymphocytei, monocytej, eosinophilk and plateletsl.

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17.80 ±

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PCV (L/L)

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6.77 ±

12

SC

RBCb

E

500 mg/kg 127.60

Control 130.80 ±

Hba (g/L)

N

250 mg/kg 133.40 ±

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ACCEPTED MANUSCRIPT Table 4: Blood chemistry values of rats in subchronic toxicity study of methanol extract of Polygonum minus leaves

1.49 ± 0.12

53.20 ± 1.88 29.40 ± 1.06 21.00 ± 1.58 1.2 ± 0.04 7.09 ± 0.92

2000 mg/kg

53.20 ± 3.50 28.40 ± 1.65 23.40 ± 3.53 1.06 ± 0.06 9.00 ± 2.10

46.60 ± 1.57 23.80 ± 0.93 22.80 ± 0.67 1.08 ± 0.03 5.59 ± 0.55

42.40 ± 3.76 22.00 ± 2.58 15.20 ± 1.59 1.10 ± 0.04 10.80 ± 0.93

1.46 ± 0.09

1.79 ± 0.18

1.77 ± 0.12

1.95 ± 0.03

1.85 ± 0.09

2.75 ± 0.14 81.40 ± 9.85 82.60 ± 4.18 5.24 ± 0.37

2.79 ± 0.16 84.80 ± 7.05 93.40 ± 7.38 4.72 ± 0.24

2.71 ± 0.16 99.20 ± 5.62 119.40 ± 11.51 4.94 ± 0.12

2.87 ± 0.11 93.80 ± 4.69 106.2 ± 4.59 4.68 ± 0.28

2.91 ± 0.03 106.60 ± 4.49 118.2 ± 13.71 4.32 ± 0.34

1.26 ± 0.19 0.47 ± 0.08 0.54 ± 0.06 0.57 ± 0.10 2.39 ± 0.31

1.66 ± 0.08 0.53 ± 0.04 0.47 ± 0.04 0.81 ± 0.06 3.66 ± 0.41

1.74 ± 0.11 0.47 ± 0.08 0.50 ± 0.03 0.94 ± 0.16 3.54 ± 0.20

1.52 ± 0.13 0.41 ± 0.05 0.50 ± 0.07 0.68 ± 0.08 3.14 ± 0.23

1.40 ± 0.08 0.41 ± 0.09 0.39 ± 0.05 0.79 ± 0.11 3.79 ± 0.41

1.32 ± 0.11 0.45 ± 0.07 0.52 ± 0.06 0.51 ± 0.05 2.66 ± 0.23

187.00 ± 12.66 4.50 ± 0.18 103.20 ± 6.15 2.92 ± 0.26 11.40 ± 1.21 0.02 ± 0.01 0.63 ± 0.43 1.51 ± 0.24

161.00 ± 9.11 3.88 ± 0.13 102.60 ± 3.25 3.52 ± 0.47 17.40 ± 1.49 0.01 ± 0.00 0.24 ± 0.02 1.37 ± 0.15

171.00 ± 7.35 3.94 ± 0.22 103.60 ± 1.10 3.44 ± 0.21 13.60 ± 2.15 0.02 ± 0.00 0.23 ± 0.01 1.48 ± 0.07

168.60 ± 2.73 4.08 ± 0.15 103.20 ± 2.26 3.64 ± 0.33 13.20 ± 1.90 0.02 ± 0.00 0.23 ± 0.01 1.54 ± 0.11

182.60 ± 2.48 3.82 ± 0.12 108.80 ± 2.57 3.42 ± 0.40 11.16 ± 1.71 0.02 ± 0.00 0.23 ± 0.01 1.48 ± 0.10

170.40 ± 13.10 4.22 ± 0.29 120.20 ± 8.18 2.70 ± 0.30 10.60 ± 0.98 0.02 ± 0.01 0.23 ± 0.01 1.55 ± 0.14

2.60 ± 0.15 108.20 ± 11.97 106.40 ± 5.37 4.32 ± 0.34

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EP

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51.00 ± 2.18 27.40 ± 1.28 22.60 ± 1.06 1.12 ± 0.04 5.99 ± 0.91

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45.60 ± 6.69 22.20 ± 3.94 19.8 ± 3.85 1.16 ± 0.06 10.40 ± 0.47

Control

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Liver function Total protein 53.60 ± 52.60 ± 54.00 ± 54.00 ± (g/L) 2.91 2.95 1.55 3.38 Albumin 27.80 ± 25.40 ± 27.20 ± 25.60 ± (g/L) 3.06 1.10 0.79 1.86 Globulin 21.80 ± 23.00 ± 23.20 ± 23.80 ± (g/L) 1.24 0.87 1.67 1.62 1.18 ± 1.10 ± 1.16 ± 2.04 ± ALB/GLO 0.03 0.04 0.07 0.90 7.80 ± 6.48 ± 8.50 ± 5.74 ± ALP (U/L) 0.93 0.84 2.75 1.52 Total 1.50 ± 1.61 ± 1.66 ± 1.72 ± bilirubin 0.03 0.22 0.19 0.19 (umol/L) 2.80 ± 3.76 ± 2.79 ± 2.74 ± GGT (U/L) 0.09 1.03 0.11 0.17 90.00 ± 103.60 ± 91.00 ± 91.80 ± AST (U/L) 7.57 9.24 9.15 8.67 90.40 ± 127.60 ± 112.40 ± 97.80 ± ALT (U/L) 6.53 14.98 13.66 5.24 Glucose 5.24 ± 4.72 ± 4.94 ± 4.68 ± (mmol/L) 0.37 0.24 0.12 0.28 Lipids profile TC 1.62 ± 1.62 ± 1.62 ± 1.28 ± (mmol/L) 0.13 0.11 0.08 0.17 TG 0.56 ± 0.65 ± 0.61 ± 0.50 ± (mmol/L) 0.05 0.08 0.05 0.06 HDL 0.46 ± 0.45 ± 0.44 ± 0.42 ± (mmol/L) 0.04 0.02 0.04 0.04 LDL 0.84 ± 0.77 ± 0.76 ± 0.80 ± (mmol/L) 0.06 0.04 0.04 0.10 3.70 ± 3.59 ± 3.75 ± 3.08 ± TC/HDL 0.46 0.25 0.37 0.33 Electrolytes Sodium 160.60 ± 167.40 ± 166.80 ± 175.00 ± (mmol/L) 3.36 4.91 5.43 5.37 Potassium 4.06 ± 3.96 ± 3.84 ± 3.80 ± (mmol/L) 0.15 0.18 0.10 0.24 98.40 ± 106.20 ± 104.40 ± 101.40 ± Chloride (mmol/L) 2.27 1.17 1.06 1.75 Urea 3.50 ± 3.74 ± 3.40 ± 3.56 ± (mmol/L) 0.12 0.56 0.24 0.26 Creatinine 17.80 ± 15.40 ± 15.20 ± 13.80 ± (umol/L) 2.05 2.36 1.13 2.03 Uric acid 0.02 ± 0.03 ± 0.02 ± 0.03 ± (mmol/L) 0.01 0.01 0.01 0.00 Calcium 0.25 ± 0.23 ± 0.24 ± 0.22 ± (mmol/L) 0.05 0.01 0.00 0.02 Phosphate 1.45 ± 1.49 ± 1.55 ± 1.41 ± (mmol/L) 0.17 0.11 0.04 0.08 All the values are reported as mean ± SEM (n=5).

Male Methanol extract of P. minus 250 500 1000 mg/kg mg/kg mg/kg

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Female Methanol extract of P. minus 250 500 1000 2000 mg/kg mg/kg mg/kg mg/kg

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Table 5: Relative organ weights of rats in subchronic toxicity study of methanol extract of Polygonum minus leaves Female Male Methanol extract of P. minus Control Control Methanol extract of P. minus 250 250 500 1000 2000 500 1000 2000 mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg Brain 1.23 ± 1.19 ± 1.24 ± 1.35 ± 1.26 ± 1.27 ± 1.18 ± 1.23 ± 1.27 ± 1.31 ± 0.08 0.05 0.05 0.04 0.07 0.08 0.07 0.11 0.03 0.05 Lung 1.03 ± 1.09 ± 1.11 ± 1.13 ± 1.07 ± 1.12 ± 1.09 ± 1.11 ± 1.17 ± 1.14 ± 0.10 0.07 0.02 0.07 0.09 0.05 0.06 0.04 0.06 0.07 Heart 0.49 ± 0.41 ± 0.42 ± 0.44 ± 0.46 ± 0.43 ± 0.40 ± 0.41 ± 0.43 ± 0.41 ± 0.03 0.01 0.01 0.01 0.03 0.03 0.01 0.01 0.02 0.01 Liver 4.06 ± 3.94 ± 4.03 ± 4.02 ± 4.09 ± 4.25 ± 3.89 ± 4.05 ± 4.06 ± 4.17 ± 0.31 0.12 0.14 0.11 0.22 0.23 0.15 0.11 0.13 0.14 Spleen 0.35 ± 0.33 ± 0.34 ± 0.34 ± 0.34 ± 0.33 ± 0.34 ± 0.33 ± 0.32 ± 0.34 ± 0.04 0.02 0.04 0.02 0.03 0.02 0.02 0.02 0.03 0.04 0.12 ± 0.12 ± 0.12 ± 0.12 ± 0.12 ± Thymus 0.13 ± 0.12 ± 0.12 ± 0.12 ± 0.13 ± 0.01 0.01 0.00 0.01 0.01 0.01 0.01 0.01 0.02 0.02 Kidney 0.37 ± 0.32 ± 0.34 ± 0.35 ± 0.35 ± 0.38 ± 0.32 ± 0.36 ± 0.35 ± 0.36 ± [L] 0.02 0.01 0.02 0.02 0.01 0.02 0.01 0.02 0.01 0.00 Kidney 0.37 ± 0.32 ± 0.34 ± 0.34 ± 0.35 ± 0.38 ± 0.32 ± 0.35 ± 0.34 ± 0.35 ± [R] 0.02 0.00 0.02 0.02 0.01 0.02 0.01 0.01 0.01 0.01 Uterus/ 1.49 ± 1.45 ± 1.36 ± 1.28 ± 1.39 ± 2.98 ± 2.92 ± 2.89 ± 2.70 ± 2.73 ± testis 0.07 0.12 0.06 0.06 0.09 0.11 0.06 0.08 0.07 0.07

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All the values are reported in gram as mean ± SEM (n=5).

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ACCEPTED MANUSCRIPT

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ACCEPTED MANUSCRIPT

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Polygonum minus, a plant which is used in Malaysian traditional medicine. Toxicity profile of methanol extract of leaves of P. minus was studied. The studied extract was devoid of any heavy metals contamination. The acute and subchronic toxicity studies were conducted on Sprague Dawley rats. The toxicity studies suggest the P. minus extract is safe up to 2000 mg/kg.

AC C

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