BioMed Research International

BioMed Research International / 2013 / Article
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Bioactive Natural Matrices and Compounds

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Research Article | Open Access

Volume 2013 |Article ID 303162 | https://doi.org/10.1155/2013/303162

Chatchai Chaotham, Songpol Chivapat, Anan Chaikitwattana, Wanchai De-Eknamkul, "Acute and Chronic Oral Toxicity of a Partially Purified Plaunotol Extract from Croton stellatopilosus Ohba", BioMed Research International, vol. 2013, Article ID 303162, 12 pages, 2013. https://doi.org/10.1155/2013/303162

Acute and Chronic Oral Toxicity of a Partially Purified Plaunotol Extract from Croton stellatopilosus Ohba

Academic Editor: Lillian Barros
Received06 Aug 2013
Accepted22 Aug 2013
Published30 Sep 2013

Abstract

Plaunotol, an acyclic diterpenoid with highly effective antigastric ulcer properties, has been commercially isolated from leaves of Croton stellatopilosus Ohba. This Thai medicinal plant was traditionally used in the form of crude extracts, suggesting that it is possible to administer these plaunotol-containing extracts without toxicity. To confirm its safety, the oral toxicity of a partially purified plaunotol extract (PPE) was evaluated in vivo. The PPE was simply prepared by 95% ethanol reflux extraction followed by hexane partition. The obtained extract was analyzed and found to contain 43% w/w of plaunotol and another compound, likely a fatty acid-plaunotol conjugate that is considered a major impurity. Oral administration of PPE to ICR mice and Wistar rats was conducted to evaluate acute and chronic toxicity of the plaunotol extract, respectively. The acute toxicity study demonstrated that PPE was practically nontoxic based on its high median lethal dose value (  g/kg). The chronic toxicity studies also showed the absence of mortality and clinical symptoms in all rats treated with 11–1,100 mg/kg/day of PPE during a 6-month period. Histopathological and hematological analyses revealed that altered liver and kidney function and increased blood platelet number, but only at the high doses (550–1,100 mg/kg/day). These results suggest that PPE is potentially safe for further development as a therapeutic agent in humans.

1. Introduction

Croton stellatopilosus Ohba, a Thai medicinal plant (Plau-noi), has been acknowledged for its traditional remedy in the treatment of helminthes and topical infection [1]. A major constituent isolated from leaves of Plau-Noi is plaunotol [2]. This natural acyclic diterpene has been used as an antigastric ulcer medication based on its pharmacotherapeutic effects in inducing prostaglandin E2 (PGE2) and eradicating Helicobacter pylori bacteria [36]. However, in addition to its application as a single compound that requires a complicated process of extraction and purification [7], plaunotol used in form of a partially purified plaunotol extract (PPE) is also of interest. This is due to the wide distribution of Plau-Noi plant in Thailand [8] and the reliability of pharmacological benefits of natural terpenoid provides in human healthcare [9, 10]. Furthermore, the use of PPE can minimize the cost of production and increase the access to an effective drug at a low price.

At a dose of 240 mg/day or 4.8 mg/kg/day, plaunotol has been recommended to achieve antigastric ulcer effects in humans [11]. Because equivalent pharmacological activities could potentially come from higher amounts of PPE than those of pure plaunotol, it is necessary to assess toxicity profiles of such a high doses of PPE. Therefore, this study aims to evaluate oral toxicity of PPE in order to generate a safety data in animal models for extrapolation to human toxicity profiles.

2. Materials and Methods

2.1. Preparation of PPE

PPE was prepared from dried leaf powder of Croton stellatopilosus Ohba by modifying a previously described method [7]. The leaf powder was refluxed with 95% ethanol (1 : 1 ratio by weight) at 70°C for 1 h. After filtration, the extract was concentrated by using a rotary evaporator, followed by partitioning with hexane (ratio of 1 : 1) to remove highly nonpolar constituents. With plaunotol remaining in ethanol layer, the ethanol was evaporated in a vacuum to obtain PPE, which was then collected and stored at 4°C. For use in this toxicity study, PPE was suspended in a 0.5% tragacanth solution and adjusted to various concentrations as indicated.

2.2. Plaunotol Content Analysis

Quantitative analysis of plaunotol in PPE was conducted using the method of thin-layer chromatography (TLC) reported previously [12, 13]. Briefly, a silica gel 60 F254 TLC plate (Merck, Darmstadt, Germany) was spotted with PPE and standard plaunotol (obtained from Thai Sankyo Co., Ltd., which is currently under Tipco Biotech Co. Ltd., Prachuab Khiri Khan, Thailand). The plate was then developed using a solvent system of chloroform:n-propanol (96 : 4). The developed TLC plate was scanned by a densitometer at 220 nm using a Camag TLC Scanner 3 with Wincats software V 1.3.4 (Camag, Muttenz, Switzerland). Structural analysis of each constituent was carried out using gas chromatography-mass spectrometry (GC-MS: Agilent Model 6890N-5973N, Agilent Technologies, CA, USA). A DB-5 capillary column (30 × 0.25 mm) with helium as a carrier gas at a flow rate of 13.8 mL/min was heated from 150°C up to 300°C with an increasing temperature rate of 15°C/min. Temperatures of both detector and the injector were set at 250°C [12]. Compound identification was performed by comparing the resulting mass spectra with those available in the Wiley Registry of Mass Spectral Data 7th edition (McLafferty, 2000), Agilent Part no. G1035B.

2.3. Animals and Housing

All ICR (Imprinting Control Region) mice and Wistar rats were obtained from the National Laboratory Animal Center (Mahidol University, Nakorn Pathom, Thailand). They were housed in the conventional hygienic animal rooms of the Laboratory Animal Center, Department of Medical Sciences, Nonthaburi, Thailand. Room conditions were maintained at 25°C, 60% humidity and 12 hour-light-dark cycle. The animals were given a commercial pellet diet 082 CP feed (Perfect Companion Group, Thailand) and clean water ad libitum. Prior to the experiment, the animals were allowed two weeks to acclimate to the environment.

2.4. Acute Toxicity Study

Five male and five female ICR mice were treated with either water for the control group or PPE at 2.5, 5, 10, and 20 g/kg for each treatment group. All animals were food restricted for 2 h prior to oral administration of single doses of the substances. Observations of clinical presentation and mortality were conducted at 15 min, 30 min, 3 h, 6 h, and 24 h following treatment and daily for 14 days thereafter. Finally, the mean lethal dose value (LD50) was calculated. This study was authorized by the Institutional Animal Care and Use Committee, Department of Medical Sciences as Permission no. 52-002.4.

2.5. Chronic Toxicity Study

The protocol used for the chronic toxicity study was approved by the Institution of Animal Care and Use Committee, Department of Medical Sciences as Permission no. 53-014. Wistar rats were randomized into six groups, each with 15 male and 15 female rats. Four experimental groups were administered the PPE suspension orally at doses of 11, 110, 550, and 1100 mg/kg/day for six months. Two control groups received distilled water and 0.5% tragacanth solution orally at the volume of 10 mL/kg. During the experimental period, animals were observed daily for general appearance and signs of toxicity. Body weight and food consumption were measured weekly. At the end of the study, animals were fasted overnight and were then killed using diethyl ether inhalation. Blood samples were collected from the posterior vena cava for hematological and biochemical value measurements.

Hematological analysis was performed using the hematological analyzer Cell Dyn 3500 (Abbot Laboratories Ltd., IL, USA). The parameters examined included red blood cells (RBC), hematocrit (Hct), hemoglobin, mean cell volume (MCV), mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), white blood cells (WBC), lymphocytes, neutrophils, eosinophils, basophils, monocytes, and platelets. Biochemical values were measured using Cobas integra 400 (Hoffmann-La Roche Ltd., Basel, Switzerland), which assessed levels of alkaline phosphatase (ALP), alanine aminotransaminase (ALT), aspartate aminotransaminase (AST), total protein, albumin, bilirubin, blood urea nitrogen (BUN), creatinine, glucose, uric acid, triglyceride, cholesterol and sodium, potassium, and chloride ions.

A complete necropsy was performed to assess gross lesions of visceral organs. Brain, lung, heart, liver, kidney, stomach, spleen, testis, uterus, urinary bladder, and adrenal glands were isolated and weighed using Mettler (Mettler Toledo PB 153 balance, Mettler Toledo Intl. Inc., Zurich, Switzerland). Relative organ weight was calculated. The above mentioned organs, including trachea, were fixed in 10% phosphate-buffered formalin and subjected to conventional histological processing and stained with hematoxylin for further histopathological examination [14].

2.6. Statistical Analysis

A one-way ANOVA was used to evaluate significant differences between groups via the Bonferroni test. Fisher’s exact was applied to compare histopathological differences between groups. was considered statistically significant.

3. Results

3.1. Plaunotol and a Plaunotol-Like Compound Are Major Constituents of PPE

By modifying the conventional method of plaunotol extraction and limited purification, PPE was obtained in 2% yield (w/w) as a brownish-yellow translucent oil. The extract appeared to contain plaunotol and an unknown compound as the two major constituents and a few minor impurities, as shown by the chemical profiles in the TLC (Figure 1(a)) and GC (Figure 2(a)) chromatograms. The UV absorption spectrum (Figure 1(b)) and mass spectrum (Figure 2(b)) of the unknown compound were similar to those of plaunotol, suggesting that the major impurity in PPE was a plaunotol-like compound. Because fatty acid conjugates of plaunotol are known to be present in the leaves of C. stellatopilosus [15], it is likely that the major impurity was one of the fatty acid-plaunotol conjugates that were extracted with plaunotol. Quantitative analysis indicated that plaunotol was present at approximately 43.0% w/w in the PPE preparation.

3.2. Acute Oral Toxicity and LD50

All of the observed PPE-induced acute toxicity symptoms are summarized in Table 1. The results were based on oral doses of PPE at 2.5, 5, 10, and 20 g/kg, calculated to be 200, 400, 800, and 1,600 times the recommended dose of plaunotol for human therapeutic use, respectively [11]. Acute diarrhea was detected early, 6 h after extract administration, at all doses and remained until 72–96 h. With the high doses of 10 and 20 g/kg, other clinical symptoms were observed, including dyspnea (shortness of breath), dullness, and stomach cramp.


Dose (g/kg)Symptoms/Time (Onset to recovery)Mortality
DyspneaDullnessAbdominal crampDiarrheaDeath/TotalLatency period

ControlNoneNoneNoneNone0/10
2.5NoneNoneNone6–72 h0/10
5NoneNoneNone6–72 h0/10
1015 min–24 h15 min–24 h15 min–24 h6–96 h4/1024 h
2015 min–24 h15 min–24 h15 min–24 h6–72 h10/1024–72 h

Mortality was not observed in mice treated with water (control) or PPE at 2.5 and 5 g/kg; however, it was observed in four (one male and three females) and 10 (five males and five females) mice between 24 and 72 h at the 10 and 20 g/kg doses, respectively. Probit analysis revealed that the LD50 of PPE was 10.25 g/kg with 95% confidence limit between 7.37 and 14.63 g/kg.

3.3. Chronic Oral Toxicity
3.3.1. Effect of PPE on Body Weight, Food Consumption, and Health Status

In this chronic toxicity study, rats were fed equal volumes of PPE to minimize the effect of administration differences. PPE was diluted in 0.5% w/w of tragacanth to achieve the desirable concentrations used for gavage feeding the rats. The selected concentrations of PPE were 11, 110, 550, and 1,100 mg/kg/day, which are equivalent to 1, 10, 50, and 100 times the therapeutic dose of plaunotol, respectively. Mortality and significant clinical symptoms were not observed in any of the rats fed with PPE, water, and 0.5% w/w tragacanth (Figure 3). It is important to note that, for male rats given the high dose of PPE at 1,100 mg/kg/day, a trend in altered food consumption was not apparent even though there was a significant decrease in body weight (Figure 4). Similarly, the body weight and food consumption among female rats showed no significant difference.

3.3.2. Effect of PPE on Relative Organ Weight Values

The values of relative organ weight, expressed as g/1000 g body weight, are summarized in Tables 2 and 3. For male rats, relative organ weight values of the heart, liver, stomach, spleen, and kidney were significantly increased at doses of 110, 550, and 1,100 mg/kg/day, compared with the water and tragacanth control groups (Table 2). Furthermore, relative weights of the brain, lung, bladder, adrenal gland, and testis were also higher in the male rats given 550 and 1,100 mg/kg/day of PPE. Table 3 summarizes the values of relative organ weight for female rats. It can be seen that PPE treatment at high doses (550 and 1,100 mg/kg/day) caused an increase in the relative weight of the liver and kidney compared to both control groups. An additional organ that appeared to be affected in the female group was the stomach, which showed significantly higher relative weights in the 550 and 1,100 mg/kg/day PPE treatment groups.


OrgansDose of PPE administered (mg/kg/day)
ControlTragacanth111105501100

Brain
Heart
Lung
Liver
Stomach
Spleen
Right kidney
Left kidney
Right testis
Left testis
Right adrenal
Left adrenal
Bladder
Initial body weight
Final body weight

The values are expressed as the mean ± SD.
Significantly different from water .
Significantly different from 0.5% tragacanth .
Died from feeding extract into respiratory tract.

OrgansDose of PPE administered (mg/kg /day)
ControlTragacanth111105501100

Brain
Heart
Lung
Liver
Stomach
Spleen
Right kidney
Left kidney
Right adrenal
Left adrenal
Bladder
Uterus
Right ovary
Left ovary
Initial body weight
Final body weight

The values are expressed as the mean ± SD.
Significantly different from water .
Significantly different from 0.5% Tragacanth .
Died from feeding extract into respiratory tract.
3.3.3. Effect of PPE on Altered Hematological Values

After feeding PPE at 11 and 110 mg/kg/day for 6 months to both male and female rats, no significant alterations in any of the hematological values were observed (Tables 4 and 5). At the high doses of 550 and 1,100 mg/kg/day, male rats had significantly higher platelet numbers but a lower percentage of eosinophils compared to both controls. The hematocrit, RBC, and hemoglobin values were significantly higher only in the 1,100 mg/kg/day treatment group, although their MCHC was significantly lower.


ParametersDose of PPE administered (mg/kg/day)
Water0.5% tragacanth111105501100

Hematocrit (%)
RBC
(×106 cells/mm3)
Hemoglobin
(g/dL)
MCV
(µm3/red cell)
MCH
(pg/red cell)
MCHC
(g/dL RBC)
WBC
(×103 cells/mm3)
Neutrophil (%)
Eosinophil (%)
Lymphocyte (%)
Monocyte (%)
Basophil (%)