Nutrition Composition and Single, 14-Day and 13-Week Repeated Oral Dose Toxicity Studies of the Leaves and Stems of Rubus coreanus Miquel

The leaves and stems of the plant Rubus coreanus Miquel (RCMLS) are rich in vitamins, minerals and phytochemicals which have antioxidant, anti-hemolytic, anti-inflammatory, anti-fatigue and anti-cancer effects. However, RCMLS is not included in the Korean Food Standards Codex due to the lack of safety assurance concerning RCMLS. We evaluated single and repeated oral dose toxicity of RCMLS in Sprague-Dawley rats. RCMLS did not induce any significant toxicological changes in both male and female rats at a single doses of 2500 mg/kg/day. Repeated oral dose toxicity studies showed no adverse effects in clinical signs, body weight, food consumption, ophthalmic examination, urinalysis, hematology, serum biochemistry, necropsy findings, organ weight, and histopathology at doses of 625, 1250, and 2500 mg/kg/day. The LD50 and LOAEL of RCMLS might be over 2500 mg/kg body weight/day and no target organs were identified. Therefore, this study revealed that single and repeated oral doses of RCMLS are safe.

It is hard to compare the absolute content of the mixture of RCMLS as there are no study results analyzing the nutritional composition of the mixture. However, when compared to the previous studies [24] separately analyzing the leaves and stems, the mixture of RCMLS can be estimated to have similar ingredients.

Single Oral Dose Toxicity Study
To investigate the toxicity and determine the lethal dose (LD 50 ), RCMLS was administrated orally to male and female Sprague-Dawley (SD) rats at single doses of 0 (vehicle control) and 2500 mg/kg. During the 14 days experimental period, mortality, clinical signs, body weight and necropsy results were observed. No animal deaths or abnormal clinical signs were observed. In addition, there were no significant changes in body weight in the RCMLS-treated groups compared with the vehicle control group. No abnormalities were found during necropsy. Therefore, LD 50 of RCMLS can be estimated to be over 2500 mg/kg body weight (b.w.).

14 Days Repeated Oral Dose Toxicity Study
To determine the dosage of RCMLS for the 13 weeks repeated oral dose toxicity tests, male and female SD rats were orally fed with RCMLS for 14 days at doses of 0 (vehicle control), 625, 1250 and 2500 mg/kg/day. During the experimental period, mortality, clinical signs, body weight, food consumption, ophthalmic examination, urinalysis, hematology, serum biochemistry, necropsy, organ weight and histopathology were observed.
In the results, no animal deaths or abnormal clinical signs related to the administration of RCMLS were observed (Table 3). In addition, there were no significant body weight changes in RCMLS-treated groups compared with the vehicle control group (Table 4). Ophthalmic examination showed no abnormality in any test rats. Urinalysis and urine color observation showed no significant difference between male and female rats. Hematology tests showed no significant change caused by feeding RCMLS (Table 6).
Serum biochemistry test showed that K + decreased in the males of the 2500 mg/kg/day group and Ca 2+ and Na + increased and Cl´decreased in the females of the 2500 mg/kg/day group (Table 7). However, the range was very slight and within the biological change range [26]. Therefore, it was not considered toxicologically significant.  Absolute and relative organ weight showed no significant change in both male and females of all test groups (Tables 9 and 10). Necropsy and histopathology test results showed miniaturized bilateral testes in the male of the 1250 mg/kg/day group that were confirmed as seminiferous tubules degeneration and the enlarged spleen in a female of the 2500 mg/kg/day group were confirmed to be increased cellularity of white pulp. However, it was not considered toxicologically significant because it was an isolated case and a natural occurrence.
In conclusion, when RCMLS was orally administered repeatedly for 14 days to SD rats, no systemic toxicological change in relation to RCMLS was observed. Therefore, setting a dosage as high as 2500 mg/kg/day in the subsequent 13 weeks repeated administration test seems appropriate.

Mortality, Clinical Signs, Body Weight and Food Consumption
There were no animal deaths during the experimental period, but a female of the 625 mg/kg/day group showed a subcutaneous mass in the right dorsal neck after 13 weeks of RCMLS administration until nucropsy (Table 3). However it was concluded as unrelated to RCMLS administration by necropsy and histopathology examination Weight changes showed that on the 11-13 weeks, the weight of males in the 2500 mg/kg/day group decreased significantly compared to the vehicle control group (Table 5). These findings are concluded to be an effect of RCMLS but are within biological fluctuation range and other test items showed no support for adverse effect of RCMLS. Therefore, it was not considered toxicologically significant. Food consumption measurement results showed 6-8 weeks period food consumption in the male of the 2500 mg/kg group and 12 weeks period in the female of the 2500 mg/kg group decreased significantly compared to the vehicle control group. However, these changes were transient and appeared to be unrelated to the doses or to the treatment with RCMLS.

Ophthalmic Examination, Urinalysis, Hematology and Serum Biochemistry
No abnormalities were found in any groups in the ophthalmic examination and urinalysis. Hematopoietic parameters are some of the most sensitive ones used to assess the toxicity of drugs in humans and animals, and a blood profile usually gives vital information on the response of the body to injury or stress [27,28]. In the present study, the hematology test results showed a significant decrease in mean platelet volume (MPV) in the males of the 2500 mg/kg/day group compared to the vehicle control group. Additionally, neutrophil (NE) and percent of neutrophil (NEP) in the females of the 625 mg/kg/day group significantly increased, whereas the percent of lymphocyte (LYP) decreased significantly. However, the difference was slight and within the biological fluctuation range [26], with no dose-dependency. Thus, it was concluded that RCMLS did not influence it.
Serum biochemistry result showed dose-dependency in total bilirubin and Mg 2+ of the females of the 2500 mg/kg/day group and it significantly decreased when compared to the vehicle control group. Na + in females of the 1250 and 2500 mg/kg/day group decreased significantly in a dose dependent manner (Table 8). But the changes were considered incidental, because they were very slight changes, not sex or dose-related, and unaccompanied by any correlative finding.

Autopsy, Organ Weights and Histopathology
One case of cyst in the pituitary gland was observed in a male of the vehicle control group and one case of a mass in the right subcutaneous neck was observed in a female of the 625 mg/kg/day group. However, considering the histopathology test results, these findings were non dose-dependent and a natural occurrence or temporary and therefore, it was not considered toxicologically significant.
Absolute liver weights in the males of the 2500 mg/kg/day group, absolute right ovary weights in the females of the 2500 mg/kg/day group, relative both testes weights in males of the 625 and 1250 mg/kg/day group, and relative left adrenal gland weights in the males of the 1250 mg/kg/day group, decreased significantly when compared with those in the vehicle control group (Tables 9  and 10).     Data are presented as mean˘standard deviation; * Significantly different from corresponding control value at p < 0.05; ** Significantly different from corresponding control value at p < 0.01; RCMLS: Rubus coreanus Miquel leaves and stems.
However, it was slight and within the biological fluctuation range and did not show dose-dependency. Additionally, no clear relative change in absolute and relative organ weight was found. Therefore, it was not considered toxicologically significant. At the histopathology test, in a male of the vehicle control group, the cyst observed in the pituitary gland was confirmed as a nerve cyst. Also, the mass observed in the subcutaneous dorsal neck in a female of the 625 mg/kg/day group was confirmed as an adenocarcinoma in the mammary gland. However, no metastasis findings were observed, there was no dose-dependency, and they are sometimes naturally observed in the female mammary gland.
The histopathology test of the lungs showed inflammation of the terminal bronchiole and alveolar duct and foreign body granuloma. Inflammation was found to be somewhat dose-dependent in both the male and female test substance administered groups. However, the vehicle control group also showed inflammation. Considering that it was found near terminal bronchiole and alveolar duct, and the foreign body granuloma covered in sporadic brown foreign substance was found in some individuals, and administered dosage was at 20 mL/kg, which was quite high and formed much froth on preparation, the findings were evaluated to have caused by aspiration during administration. Therefore, it does not seem to be a side effect of RCMLS consumption. Including the osseous metaplasia of the lungs found in a male of the vehicle control group, the rest of the female and male of the vehicle control group, and 2500 mg/kg/day group findings were either observed in the control group, were sporadic, or were natural occurrences. Therefore, it was not considered toxicologically significant.
In conclusion, as the result of repeated 13 weeks oral administration of RCMLS, no systemic toxicological changes were observed. Therefore, the no observed adverse effect level (NOAEL) of RCMLS was set at 2500 mg/kg/day and no target organs were observed.

Preparation of the Leaves and Stems of Rubus coreanus Miquel
RCMLS was collected in May 2014 from the Gochang Bokbunja Research Institute, Gochang, South Korea. The dried leaves and stems were mixed in the same percentage and pulverized using a Pin-type Mill (Seichin, Tokyo, Japan). The pulverized sample was stored at 4˝C prior to use. RCMLS was prepared by suspending the powder with sterile water for injection according to the doses assigned to each group in the single, 14 days and 13 weeks repeated oral dose toxicity studies.

Nutrition Composition
Proximate composition analyses of RCMLS were done for moisture, ash, crude protein, crude fat, carbohydrate and fiber contents according to the methods described by the Korean Food Standards Codex [29] and Korean Health Functional Food Code [30]. Moisture was determined by oven-drying at 105˝C to constant weight. Ash content was determined gravimetrically in a muffle furnace by heating at 550˝C to constant weight. Crude fat content was determined using the decomposition method. Crude protein (6ˆ6.25) was determined on a LECO ® TruSpec ® CN (Carbon/Nitrogen Determinator, St. Joseph, MI, USA). Carbohydrate content was calculated as the difference between 100 and the sum of the moisture, crude protein, crude fat and ash contents. The dietary fiber content was obtained as an indigestible residue after enzymatic digestion of non-dietary fiber components.
Mineral, vitamin contents and fatty acid composition of RCMLS were done according to the methods described by the Korean Health Functional Food Code using inductive coupled plasma (ICP, Teledyne Leeman Labs, Hudson, NH, USA), Nanospace SI-2 HPLC system (Shiseido Co., Ltd., Tokyo, Japan) and Agilent 7890 GC system (Agilent Technologies, Santa Clara, CA, USA), respectively.

Experimental Animals
Male and female specific pathogene free SD rats (160-220 g) were obtained from the animal facility of Orient Bio (Sungnam, Korea). In case of the females, only ones that were not pregnant or had ever given birth were selected. The rats used in this study are widely used in micronuclear tests, as it is a suitable test animal in toxicology experiments. Also this strain of rat has much accumulated base test data, which can be used in test result interpretation and evaluation. The animals were starved, with exception to water, from the night before the forced oral administration using a probe to empty stomach contents. After 3-4 h food was redistributed. The animals were housed in a room maintained at a temperature of 23.2˘0.3˝C and 23.2˘0.8˝C, and a relative humidity of 51.4%˘3.4% and 51.4%˘5.4% for the 14 days single dose and repeated dose toxicity, respectively. The animals were housed in a room maintained at a temperature of 22.9˘0.7˝C and a relative humidity of 47.9%˘3.9% during the 13 weeks repeated dose toxicity study. They had a 12 h dark/light cycle. Procedures involving animals and their care were approved by the ethics committee, Korea Conformity Laboratories, 2014.

Study Design Overview
This study was conducted in compliance with the Good Laboratory Practice (GLP) and Test Guidelines of the Organization for Economic Cooperation and Development (OECD) [31,32], and the Korea Food and Drug Administration (KDFA) at the GLP institute approved by the KFDA [33,34].

Single Oral dose Toxicity Study
SD rats of both male and female were divided into two groups (I, II) of 22 rats (11 males and 11 females) matched for weight. All animals had free access to water and food throughout the experimental period. Group II was administrated RCMLS at single oral doses of the 2500 mg/kg, and Group I served as the vehicle control, receiving the same volume of sterile water. Animals were starved, with exception to water, from the night before the forced oral administration using a probe to empty stomach contents, and food was offered approximately 4 h after administration. Dosing volume was set to 20 mL/kg for toxicity study, based on the most recent body weight.
Clinical signs of animals were continuously monitored during the first 24 h and then daily thereafter, for a total period of 14 days. Changes in the normal activity of rats were monitored and the time at which signs of toxicity or death appeared was documented. On administration day, test animals were observed after 30 min of administration and every 6 h thereafter. Clinical signs observation was conducted until 14 days after administration. Changes in the body weights of the rats were monitored. At the end of the observation period, all surviving animals were fasted overnight.
After 14 days of administration, all surviving rats were anesthetized with CO 2 gas to open their abdomen. Then they were killed by severing the caudal vena cava and the abdominal artery for bloodletting to observe the organs with the naked eye.

14 Days Repeated Oral dose Toxicity Study
Four groups of 22 animals (11 males and 11 females) were used. Groups II-IV received RCMLS at daily doses of 625 mg/kg/day, 1250 mg/kg/day and 2500 mg/kg/day, and Group I served as the vehicle control receiving the same volume of distilled water, for a period of 14 days. Animals underwent forced oral administration using a probe to empty stomach contents. Dosing volume was set to 20 mL/kg/day for the toxicity study, based on the most recent body weight. During the period of administration, the animals were weighed and observed daily to detect any clinical signs of toxicity. After 14 days, all surviving animals were investigated in the same way used in the single oral dose toxicity study.
Additionally, food consumption measurements were taken at initial administration and once weekly. Method of measurement was, food distribution amount on the day before weigh-in was recorded and then the remaining food amount on the day of weigh-in was used to calculate 1 day food consumption amount. Average consumption volume (g/rat/day) was calculated per individual animal. Appearance of animals' eyes was observed at group division and on the last week of administration precedents of the vehicle control group and high dosage group (Group IV, 2500 mg/kg/day) were observed by the naked eye.
During the last administration week, five rats from each test group were transferred to a metabolic cage, where fresh urine was collected and tested for following items using urine screening test (Siemens) and urine auto-analyzer (CliniTek 50, Siemens/Bayer, Munich, Germany; MAI-050-01). Urine tone was observed with the naked eye.
Hematology test was conducted using a blood analyzer (ADVIA 2120, Siemens; MAI-105-01). Planned autopsy rats were starved overnight, anesthetized with CO 2 gas, then blood was collected from ventral aorta through laparoscopy. EDTA-2K anticoagulant was used.
For serum biochemistry test, blood chemical testing device (Hitachi 7180, Hitachi, Tokyo, Japan; MAI-059-01) was used. The serum used derived from the blood collected from abdominal aorta at planned autopsy was centrifuged for 10 min at 3000 rpm.

13 Weeks Repeated Oral dose Toxicity Study
Four groups of 88 rats (44 males and 44 females) were used. Groups II-IV received RCMLS at daily doses of 625, 1250 and 2500 mg/kg/day, and Group I served as the vehicle control receiving distilled water at the same volume, for a period of 13 weeks. During the period of administration, the animals were weighed and observed daily to detect any signs of toxicity. After 13 weeks, all surviving animals were investigated in the same way that was used in the subchronic toxicity study of 14 days repeated oral dose.

Statistical Analysis
Data are expressed as the means˘standard deviation and analyzed by a one-way analysis of variance (ANOVA). When significant differences existed, Duncan's multiple range test was used to compare the means. SPSS 12.0 K (SPSS Inc., Chicago, IL, USA) was used for all statistical analysis. A p< 0.05 or 0.01 were considered as statistically different.

Conclusions
To investigate the possible application of RCMLS as food materials, we carried out an analysis of the nutritional composition and toxicity of RCMLS following 14 days single and repeated oral administration and 13 weeks of repeated oral administration to SD rats. The proximate composition of RCMLS was 76.81% carbohydrate, 10.07% crude protein, 5.57% moisture, 5.17% crude ash and 2.20% crude fat, respectively. RCMLS contained high contents of minerals and vitamins such as K and E, respectively. The major fatty acid components were 30.48% α-linolenic acid, 23.68% palmitic acid and 15.51% linoleic acid. Single and repeated oral administration of RCMLS produced no significant toxic effects in SD rats. Under the present experimental conditions, the approximate LD 50 of RCMLS might be over 2500 mg/kg b.w for both male and female, the NOAEL might be over 2500 mg/kg/day for both male and female, and no target organs were identified.