Acute and subchronic oral toxicity assessment of the herbal formula Kai-Xin-San

Journal of Ethnopharmacology 138 (2011) 351–357

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Acute and subchronic oral toxicity assessment of the herbal formula Kai-Xin-San Li-Hua Mu a , Zhi-Xiong Huang a,b , Ping Liu a,∗ , Yuan Hu a , Yue Gao c a

Department of Clinical Pharmacology, General Hospital of PLA, Fuxing Road, 28, Beijing 100853, China Tianjin University of TCM, Tianjin 300193, China c Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, Beijing 100850, China b

a r t i c l e

i n f o

Article history: Received 16 April 2011 Received in revised form 5 August 2011 Accepted 14 August 2011 Available online 22 August 2011 Keywords: Herbal formula Kai-Xin-San Safety Acute toxicity Sub-chronic toxiciy

a b s t r a c t Ethnopharmacological relevance: Kai-Xin-San (KXS) is a famous traditional Chinese medicine (TCM) formula. It has been used in the treatment of diseases including neurasthenia, Alzheimer’s disease and neurosis. Aim of the study: To provide information on the potential toxicity of KXS, we evaluated the acute and subchronic toxicity in rodents. Materials and methods: In acute study, a single administration of KXS was given orally to mice at doses ranging from 19.67 to 60.04 g/kg. In the sub-chronic oral toxicity study, KXS was administered to rats at 0, 1, 3 and 9 g/kg for 13 weeks. Moreover, 30 days of post treatment (withdrawal study) was conducted. Mortalities, clinical signs, body weight changes, food and water consumption, haematological and biochemical parameters, gross findings and organ weights were monitored during the study period. Results: In the sub-chronic study in rats, daily oral administration of KXS at the dose of 9 g/kg/day result in significant increase in WBC, lymphocyte, alkaline phosphatase, blood sugar and significant decrease in bodyweight, serum Cre, CK and CHO at the last week of treatment. Recovery except for the body weight was observed after 30 days of post treatment. Conclusions: KXS is relatively safe for oral medication. The LD50 of KXS was over 32.59 g/kg for mice. The no-observed-adverse-effect-level (NOAEL) was considered to be 19.67 g/kg/day for rats. © 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Medicinal herbs are used for the prevention and treatment of diseases, and have a long history. However, the most commonly used herbal formulae have no indications of quality, safety and efficacy (Firenzuoli and Gori, 2007; Wang et al., 2009). Kai-XinSan (KXS) is a famous traditional Chinese medicine (TCM) formula, initially recorded in the Chinese ancient book Bei-Ji-Qian-Jin-YaoFang (means precious formulas for emergence) compiled by Sun Si-Mao in 652 AD. It was reported to possess various pharmacological effects such as enhancing learning and memory, anti-oxidation, and anti-depression (Huang et al., 1999; Bian et al., 2000; Shang et al., 2003; Wang et al., 2007), and has been clinically used for the treatment of neurasthenia, Alzheimer’s disease and neurosis.

Abbreviations: WBC, white blood cell; ALT, alanine amino transferase; AST, aspartate amino transferase; URE, blood urea nitrogen; ALP, alkaline phosphatase; Bil, total bilirubin; TP, total protein; GLU, glucose; Alb, albumin; Cre, creatinine; CHO, total cholesterol; CK, creatine kinase; TG, triglyceride; RBC, red blood cell; PLT, platelet counts; HCT, hematocrit; HGB, haemoglobin; MCV, mean corpuscular volume; MCH, mean corpuscular haemoglobin; MCHC, mean corpuscular haemoglobin concentration. ∗ Corresponding author. Tel.: +86 10 66936678; fax: +86 10 66936678. E-mail address: [email protected] (P. Liu). 0378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2011.08.033

The formula consists of four herbs, namely ginseng (Ren-Shen, Panax ginseng), hoelen (Fu-Ling, Wolfiporia cocos), polygala (YuanZhi, Polygala tenuifolia) and Acorus gramineus (Shi-Chang-Pu). As a principal component of KXS, ginseng has been extensively demonstrated to improve learning and memory in animals (Petkov and Mosharrof, 1987; Zhao and McDaniel, 1998; Kennedy and Scholey, 2003). The pharmacologically active ingredients in ginseng have been elucidated to be saponins and the most abundant saponins are ginsenosides Rg1, Rb1 and Re (Rg1 , Rb1 and Re). The effects of the three compounds on learning and memory have also been well demonstrated (Yamaguchi et al., 1996; Mook et al., 2001), however, there is very little information on its safety. As a part of a safety evaluation of KXS, acute and sub-chronic oral dose toxicity studies were conducted to investigate the potential toxicity after single or 13-week repeated oral dosing of KXS in Sprague-Dawley rats.

2. Materials and methods 2.1. Preparation of Kai-Xin-San (KXS) extract An aliquot of 1.5 kg ginseng (Panax ginseng), 1.5 kg hoelen (Wolfiporia cocos), 1 kg polygala (Polygala tenuifolia) and 1 kg Acorus gramineus were soaked together in 50 L water and extracted

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3 times using a circumfluence extraction method. The water extracts were filtered and evaporated under reduced pressure to obtain concentrates, which were freeze-dried to yield powder. 1 g yield powder contains 7.14 g total original herbs. Powders of KXS extract were stored at 4 ◦ C. The contents of biochemical ingredients including ginsenoside Rg1, ginsenoside Re and triterpene saponin in KXS were analyzed in prior study (Liu et al., 2005; Hou, 2008).

with free access to water prior to administration of single doses (60.04, 48.03, 38.42, 30.74, 24.59 and 19.67 g/kg) of the extract dissolved in distilled water. Treatment was given twice by gavage of 0.8 mL/mice/time with the interval of 8 h. The general behaviour of the mice was continuously monitored for 4 h after the treatment, intermittently during a 24-h period (Twaij et al., 1983), and thereafter daily up to 7 days. The LD50 was determined as previously described (Molle, 1986).

2.2. Analysis of chemical contents of KXS by HPLC 2.5. Sub-chronic oral toxicity study in rats The chemicals used for the identification and quantification of compounds in the KXS extract included the following: tenuifoliside A, 1-o-(E)-Benzoyl -[3-o-(E)-Alphatolluyl]-␤-d-fructofuranosy(2 → 1)-[␤-d-glucopyranosyl-(1 → 2)]-␣-d-glucopyranoside and ginsenoside Rb1 which are the components respectively. KXS extract (28 mg) was dissolved in 1 mL of methanol and filtered through 0.45 ␮m syringe filter before injection into the HPLC system. The HPLC system was composed of L-2200 Autosampler (Hitachi, Japan), L-2130 pump (Hitachi, Japan), ELSD 2000ES Detector (Alltech, America) and Agilent HC C18 (4.6 mm × 250 mm) column. Agradient solvent system of acetonitrile (A) and 0.65% ammonium acetate in water (B) was used as follows: 5%A/95%B (start), 15% A/85%B (8 min), 20%A/80%B (35 min), 28%A/72%B (60 min), 35%A/65%B (70 min), 100%A (74 min) and 5%A/95%B (75 min), at a flow rate of 1.0 mL/min (Fig. 1). In order to make standard calibration curves, tenuifoliside A (11–220 ␮g/mL), 1-o-(E)-Benzoyl-[3-o-(E)-Alphatolluyl]-␤-d-fructofuranosy-(2 → 1)-[␤-d-glucopyranosyl-(1 → 2)]-␣-d-glucopyranosid (13–260 ␮g/mL) and ginsenoside Rb1 (13–260 ␮g/mL) were diluted in methanol and injected into the HPLC to give individual chromatograms. The calibration curves were plotted by calculating the peak area ratio, which has a relatively smaller error than the peak height ratio. The fitting equations of each calibration curve were as follows: tenuifoliside A, y = 12,366x + 277,479 (r2 = 0.9996);1-o-(E)-Benzoyl-[3-o-(E)Alphatolluyl]-␤-d-fructofuranosy-(2 → 1)-[␤-d-glucopyranosyl(1 → 2)]-␣-d-glucopyranoside, y = 11,862x − 144,955 (r2 = 0.9992); and ginsenoside Rb1 , y = 2169.9x − 10,933 (r2 = 0.9999). The chemical contents in KXS extract were determined to be the following: tenuifoliside A 9.99 ± 0.12 mg/g, 1-o-(E)-Benzoyl-[3-o-(E)Alphatolluyl]-␤-d-fructofuranosy-(2 → 1)-[␤-d-glucopyranosyl(1 → 2)]-␣-d-glucopyranoside 16.71 ± 0.16 mg/g, and ginsenoside Rb1 5.79 ± 0.04 mg/g. 2.3. Animals and housing condition The acute toxicity test was carried out on 1.5 months old KM mice of either sex weighing between 19 and 30 g. Wistar rats of both sexes aged 2 months and weighing 140–180 g were used for the subchronic toxicity assessment. All animals were obtained from the experimental animal center of the Academy of Military Medical Sciences. Animals were housed in colony cages (5 rats or mice per cage), under standard laboratory conditions (ventilated room, 24 ◦ C, 75% humidity, 12 h light/dark cycle) and had free access to standard commercial diet and tap water. All animal experiments were conducted in accordance with the internationally accepted principles for laboratory animal use and care as described in the European Community guidelines (Official Journal of European Union L197 vol. 50, July 2007) and were approved by the Animal Ethics Committee of our hospital (0999 and 09100). 2.4. Acute oral toxicity study in mice Mice were randomly assigned to each of six groups of 20 mice (10 females and 10 males). They were fasted overnight (12 h)

Animals were randomly divided into 4 groups (I–IV) of 30 each (15 females and 15 males). The extract, dissolved in distilled water, was administered by daily gavage for 91 days, to groups I–IV (doses of 0, 1, 3 and 9 g/kg, respectively). The animals were observed for signs of toxicity and mortality throughout the experimental period. The BW, water and food consumption were recorded weekly. At the end of the 91-day experiment, 20 rats of each group (10 rats left for the withdrawal study) were sacrificed by decapitation under anaesthesia (thiopental 50 mg/kg). Blood was collected with and without anticoagulant (EDTA) for haematological and biochemical studies respectively. The organs (brain, thymus, heart, lung, liver, spleen, kidneys, adrenal glands, testicles, epididymis, ovaries, uterus) were weighted and the relative organ weight (weight of organ as proportion of the total body weight of each rat) was calculated and compared with the value of control. Organ samples (kidney, pancreas, lung and liver) were fixed in 10% formalin for histopathological examination. In 30-day of post treatment, the animals were withdrawed of KXS extract and had free access to standard commercial diet and tap water. At the end of withdrawal study, rats were treated according to the 91-day experiment. 2.6. Measurement of haematological and biochemical parameters in rats Whole blood cell (WBC), red blood cell (RBC) and platelet counts (PLT), hematocrit (HCT), haemoglobin (HGB), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC), were determined using a haematology analyzer MEK-6318K (Nihon Kohden Co. Ltd.). For biochemical analysis, blood without additive was centrifuged at 3000 × g at 4 ◦ C for 10 min. Serum was separated and stored at −20 ◦ C until determination of alanine amino transferase (ALT), aspartate amino transferase (AST), blood urea nitrogen (URE), alkaline phosphatase (ALP), total bilirubin (Bil), total protein (TP), glucose (GLU), albumin (Alb), creatinine (Cre), total cholesterol(CHO), creatine kinase (CK) and triglyceride (TG) using an automatic biochemistry analyzer (Italy SABA 18 Co. Ltd.). 2.7. Statistical analysis The data, expressed as mean ± SD, were subjected to Kruskal–Wallis one way analysis of variance (ANOVA). Inter group comparisons were made by Mann–Whitney-U-test (two-tailed) for only those responses which yielded significant treatment effects in the ANOV test. P < 0.05 was considered statistically significant. 3. Results 3.1. Acute oral toxicity of KXS in mice The effects of oral administration of single doses of KXS in mice are summarized in Table 1. There was a regular dose-dependent increase in mortality and adverse effects in both sexes. The mortality rate (0% at 19.67 g/kg) progressively rose to 100% at the

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Fig. 1. Chromatograms of standard components and Kai-Xin-San. (1) KXS extract; (2) tenuifeliside A (RT: 20.02 min) (3)1-o-(E)-Benzoyl-[3-o-(E)-Alphatolluyl]-␤-dfructofuranosy-(2 → 1)-[␤-d-glucopyranosyl-(1 → 2)]-␣-d-glucopyranosid (RT: 28.57 min); (4) ginsenoside Rb1 (RT: 55.41 min).

highest dose tested (60.04 g/kg). The no-observed-adverse-effect level (NOAEL) for the oral dose extract was 19.67 g/kg. The lowestobserved-adverse-effect level (LOAEL) (Alexeeff et al., 2002) was 24.59 g/kg. The symptoms of toxicity were similar in both sexes. Symptoms such as slow movement, decrease in aggressiveness, stopping food take and weight loss (results not shown) were observed later and at high doses. The acute toxicity data indicated that the estimated oral LD50 of KXS was >32.59 g/kg in mice. 3.2. Subchronic 91-day oral toxicity study of KXS in Wistar rats 3.2.1. Clinical signs and mortality There was no mortality attributed to any effect of KXS during the 91-day administration. There were no obviously difference in

appearance, activity and excrement as compared to other rats in the control group before death. Anatomical results showed that no abnormal organic damages were observed in the dead rats. Moreover, there were no treatment-related changes at autopsy in the KXS group or control group in either sex. 3.2.2. Body weights and food consumption Mean body weights for male and female rats at 1, 3, 9 g/kg/day were compared to control values (Fig. 2). As expected, rats gained weight with time. Male Wistar rats in 9 g/kg/day had significantly lower mean body weights comparing to the control group from the thirdly to thirteenth week (P < 0.05–0.001). The mean body weights of female rats in 3 g/kg/day and 9 g/kg/day KXS groups were significantly lower than control group from the twelfth to thirteenth

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Table 1 Acute oral toxicity of KXS in mice. Treatment (g/kg)

60.04 48.03 38.42 30.74 24.59 19.67

Mice

Effects

Sex

D/T

Mortality latency (h)

Symptoms of toxicity

M F M F M F M F M F M F

10/10 10/10 8/10 9/10 6/10 5/10 3/10 3/10 1/10 1/10 0/10 0/10

>5, <7 >6, <8 >7, <10 >5, <9 >6, <12 >8, <15 >9, <18 >7, <21 >9, <17 >10, <25

Slow movement, stopping food take, lying down Slow movement, stopping food take, lying down Slow movement, stopping food take, lying down Slow movement, stopping food take, lying down Decrease in aggressiveness, stopping food take Decrease in aggressiveness, stopping food take Decrease in aggressiveness, stopping food take Decrease in aggressiveness, stopping food take Decrease in aggressiveness and food take Decrease in aggressiveness and food take None None

The aqueous extract of KXS, dissolved in distilled water was administered as single oral doses to 6 groups of 20 mice (10 males, 10 females). All animals were carefully examined for adverse effects (behavioural changes and mortality) for 7 days. Symptoms of toxicity are described for a group (both males and females). M: male; F: female; D/T: dead/treated mice; latency: time to death after the dose; none: no toxic symptom during the observation period.

week (P < 0.05–0.01). The mean body weights of male and female rats in group IV was significantly lower comparing to the control group from the first to fourth week of drug withdrawal study (Fig. 2) (P < 0.05–0.01). During the thirteen-week study, the food consumption of Wistar rat was stable on the whole. The food consumptions of male rats in 3 and 9 g/kg/day KXS groups were significantly lower than which of control group at fourth week. Male rats in 9 g/kg/day KXS group had a significantly lower food consumptions comparing to control group at the fifth, sixth, eighth and eleventh to thirteenth week (P < 0.05–0.001). There was no significant difference of food consumption in all groups after 30 days of post treatment (withdrawal study). 3.2.3. Haematological and blood chemistry The effect of sub-chronic administration of KXS on haematological parameters is presented in Table 2. The WBC and lymphocyte of rats in group IV were significantly higher comparing with control group (P < 0.01–0.05). At the end of withdrawal study, no significant

difference in WBC and lymphocyte of both male and female rats was found between the group IV and control group (Table 3). Biochemical parameter profiles of the treated and control groups are shown in Table 4. A 91-day oral administration of KXS did not cause significant changes in serum ALT, AST, URE, BIL, TP, GLU, ALB, TG. Alkaline phosphatase (ALP) of female rats was significantly higher (P < 0.05) in the group IV as compared with the control group I and the effect was dose dependent. The serum creatinine (Cre) and creatine kinase (CK) level of female rats were lower at D91 compared to the control group in the groups III and IV (P < 0.05). The total cholesterol (CHO) in the male group IV was significantly lower than that of group I (P < 0.01). At the thirtieth day of the withdrawal study, there was no significant difference in the blood chemistry between groups III, IV and the control group (Table 5). 3.2.4. Organ parameters Absolute and relative organ weights of 91-day treated rats are shown in Table 6. In females, the absolute liver and kindey weights in group IV and the relative organ weights of liver and kindey in

Fig. 2. Effects of oral administration of KXS extract for 13 weeks and 30-day withdrawal study on mean bodyweights. Bodyweight was calculated and data are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 significantly different compared to control group.

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Table 2 Effect of oral administration of KXS for 13 weeks on haematological parameters of rats. Parameter

Males (mean ± standard error)

Females (mean ± standard error)

Group I (0 g/kg)

Group II (1 g/kg)

Group III (3 g/kg)

Group IV (9 g/kg) Group I (0 g/kg)

Group II (1 g/kg)

Group III (3 g/kg)

17.00 ± 1.81 13.1 ± 1.34

15.44 ± 1.32 11.7 ± 0.99

19.44 ± 1.99 13.9 ± 2.81

21.42 ± 3.08* 16.8 ± 2.03**

12.78 ± 1.86 9.6 ± 2.02

14.48 ± 1.85 11.0 ± 1.51

14.08 ± 2.94 10.0 ± 1.88

8.93 ± 0.43 165.6 ± 4.50

8.92 ± 0.73 169.6 ± 8.26

9.34 ± 0.62 173.6 ± 8.62

8.93 ± 0.50 173.0 ± 12.10

7.64 ± 0.44 160.4 ± 5.73

8.10 ± 0.72 161.2 ± 12.0

7.66 ± 0.53 157.8 ± 5.12

8.20 ± 0.65 162.2 ± 6.30

0.47 ± 0.01

0.48 ± 0.05

0.49 ± 0.03

0.48 ± 0.03

0.42 ± 0.01

0.44 ± 0.04

0.43 ± 0.02

0.45 ± 0.04

907.6 ± 157.2

907.4 ± 190.4

878.6 ± 61.3

877.2 ± 125.0

797.6 ± 110.5

906.8 ± 78.6

950.0 ± 117.9

831.6 ± 118.6

52.98 ± 1.54 18.5 ± 0.66 350.0 ± 6.8

53.32 ± 1.89 19.1 ± 0.69 358.0 ± 24.2

52.62 ± 0.82 18.6 ± 0.55 353.6 ± 9.2

53.76 ± 1.45 19.4 ± 1.16 360.2 ± 15.0

55.58 ± 1.35 21.0 ± 1.13 378.2 ± 13.3

54.38 ± 0.58 20.0 ± 1.87 367.6 ± 34.1

56.48 ± 1.34 20.7 ± 1.25 366.0 ± 22.3

54.56 ± 1.02 19.8 ± 1.07 363.8 ± 18.7

WBC (×109 /L) Lymphocytes (×109 /L) RBC (×1012 /L) Haemoglobin (g/L) Hematocrit (L/L) Platelets (×109 /L) MCV (fl) MCH (pg) MCHC (g/L)

Group IV (9 g/kg) 16.1 ± 1.83** 13.1 ± 2.21*

Wistar rats (n = 30 per group, 15 males and 15 females) were administered an extract of KXS by daily gavage for 91 days. Data are expressed as mean ± S.E.M. *P < 0.05; **P < 0.01 vs the corresponding group I value.

Table 3 Some of the haematological parameters of rats after 30 days of withdrawal study. Parameter

Treatment Group I (0 g/kg)

Group II (1 g/kg)

Group III (3 g/kg)

Group IV (9 g/kg)

15.84 ± 2.51 11.5 ± 1.53

16.80 ± 3.26 12.7 ± 2.69

16.12 ± 2.16 11.9 ± 2.01

15.78 ± 2.62 11.9 ± 2.19

13.56 ± 2.03 10.4 ± 1.90

14.82 ± 2.25 11.5 ± 2.17

13.74 ± 1.90 10.2 ± 1.84

13.38 ± 2.37 10.6 ± 1.82

Male (n = 5) WBC (×109 /L) Lymphocytes (×109 /L) Female (n = 5) WBC (×109 /L) Lymphocytes (×109 /L)

Wistar rats (n = 10 per group) were withdrawed of KXS for 30 days. Data are expressed as mean ± S.E.M. There was no significant difference.

Table 4 Effect of sub-chronic oral administration of KXS on serum biochemical parameters of rats. Parameter

ALT (nmol s−1 /L) AST (nmol s−1 /L) URE (mmol/L) ALP (␮mol/L) Bil (␮mol/L) TP (g/L) GLU (mmol/L) Alb (g/L) Cre (␮mol/L) CHO (mmol/L) CK (nmol s−1 /L) TG (mmol/L)

Males (mean ± standard error)

Females (mean ± standard error)

Group I (0 g/kg)

Group II (1 g/kg)

Group III (3 g/kg)

Group IV (9 g/kg)

796.8 ± 87.7

1120.2 ± 376.6

1100.2 ± 432.8

1817.0 ± 282.2

1673.7 ± 436.9

2290.5 ± 572.5

14.39 1.96 7.93 66.8 8.62 37.4 64.6 0.90 457.2 2.62

± ± ± ± ± ± ± ± ± ±

1.53 0.20 1.02 3.70 0.45 2.30 3.8 0.12 134.1 0.44

15.08 1.78 6.84 65.4 8.44 37.0 73.2 0.84 591.4 2.35

± ± ± ± ± ± ± ± ± ±

1.30 0.22 2.99 4.28 0.05 1.73 7.6 0.06 278.5 0.46

13.63 1.68 8.24 60.8 8.76 37.2 66.4 0.80 696.8 2.80

± ± ± ± ± ± ± ± ± ±

2.09 0.28 2.48 5.22 1.64 1.79 6.5 0.00 235.8 0.59

Group I (0 g/kg)

Group II (1 g/kg)

Group III (3 g/kg)

Group IV (9 g/kg)

746.8 ± 131.5

666.8 ± 116.7

930.2 ± 465.2

790.2 ± 180.9

680.1 ± 98.9

1627.0 ± 375.1

2123.8 ± 244.6

2233.8 ± 373.3

2070.4 ± 223.5

1943.7 ± 191.7

14.23 1.85 6.36 64.8 8.48 34.8 61.4 0.64 336.6 1.97

± ± ± ± ± ± ± ± ± ±

2.06 0.15 2.48 4.27 0.86 1.64 6.0 0.11** 164.3 0.54

14.99 1.03 10.60 61.2 7.52 33.8 75.6 0.68 805.2 1.89

± ± ± ± ± ± ± ± ± ±

1.62 0.12 2.03 5.72 1.12 2.39 5.2 0.08 114.6 0.74

16.53 0.97 9.71 62.0 8.62 35.4 73.0 0.68 783.6 1.66

± ± ± ± ± ± ± ± ± ±

2.43 0.07 4.20 6.71 0.78 2.61 4.4 0.08 131.6 0.98

15.68 1.24 8.89 60.6 8.20 34.8 62.2 0.70 746.8 1.42

± ± ± ± ± ± ± ± ± ±

2.29 0.42 3.38 1.52 0.69 0.84 7.7* 0.10 126.7 0.34

13.74 1.36 8.58 60.2 8.42 35.0 67.4 0.58 645.4 1.62

± ± ± ± ± ± ± ± ± ±

2.74 0.29* 2.14 2.77 0.97 0.71 5.6* 0.11 80.1* 0.62

Wistar rats (n = 30 per group, 15 males and 15 females) were administered KXS by daily gavage for 91 days. Data are expressed as mean ± S.E.M. *P < 0.05; **P < 0.01 vs the corresponding group I value.

Table 5 Some of the serum biochemical parameters of rats after 30 days of withdrawal study. Parameter

Treatment Group I (0 g/kg)

Male (n = 5) CHO (mmol/L) Female (n = 5) ALP (␮mol/L) Cre (␮mol/L) CK (nmol s−1 /L)

Group II (1 g/kg)

1.26 ± 0.17

1.16 ± 0.26

1.13 ± 0.25 78.4 ± 7.4 678.6 ± 408.5

0.92 ± 0.07 80.8 ± 11.1 774.0 ± 306.2

Group III (3 g/kg)

Group IV (9 g/kg)

1.36 ± 0.11

1.14 ± 0.21

1.08 ± 0.18 80.8 ± 6.2 431.6 ± 81.0

1.06 ± 0.06 79.2 ± 9.8 403.0 ± 73.7

Wistar rats (n = 10 per group) were withdrawed of KXS for 30 days. Data are expressed as mean ± S.E.M. There was no significant difference.

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Table 6 Effect of sub-chronic oral administration of KXS on organ weights of rats. Organ weight (g) Group I (0 g/kg) Male (n = 15) Brain Thymus Heart Lung Liver Spleen Kidney Adrenal glands Testicles Epididymis Female (n = 15) Brain Thymus Heart Lung Liver Spleen Kidney Adrenal glands Ovaries Uterus

2.01 0.31 1.30 1.89 13.40 0.83 2.59 0.074

± ± ± ± ± ± ± ±

0.05 0.04 0.21 0.59 2.63 0.13 0.26 0.016

3.24 ± 0.21 1.39 ± 0.13 1.92 0.32 0.98 1.33 6.80 0.62 1.52 0.060

± ± ± ± ± ± ± ±

0.06 0.09 0.12 0.26 0.74 0.09 0.14 0.009

0.16 ± 0.05 0.70 ± 0.22

Relative organ weight (g per 100 g body weight) Group II (1 g/kg) 2.07 0.34 1.35 1.92 12.99 0.75 2.63 0.063

± ± ± ± ± ± ± ±

0.07 0.13 0.18 0.34 2.14 0.09 0.38 0.015

2.97 ± 0.51 1.34 ± 0.11 1.82 0.26 0.88 1.26 6.39 0.57 1.48 0.059

± ± ± ± ± ± ± ±

0.09 0.08 0.11 0.13 0.18 0.04 0.08 0.009

0.17 ± 0.03 0.56 ± 0.08

Group III (3 g/kg) 1.97 0.28 1.31 1.80 12.25 0.86 2.51 0.064

± ± ± ± ± ± ± ±

0.20 0.06 0.20 0.17 1.25 0.07 0.21 0.005

3.19 ± 0.13 1.42 ± 0.10 1.79 0.30 0.88 1.48 7.79 0.65 1.64 0.070

± ± ± ± ± ± ± ±

0.11 0.08 0.09 0.16 0.80 0.04 0.12 0.018

0.18 ± 0.07 0.65 ± 0.23

Group IV (9 g/kg) 1.94 0.34 1.21 1.68 12.37 0.86 2.67 0.076

± ± ± ± ± ± ± ±

0.07 0.14 0.10 0.27 1.65 0.16 0.29 0.006

3.23 ± 0.17 1.50 ± 0.15 1.78 0.21 0.91 1.26 8.90 0.58 1.74 0.063

± ± ± ± ± ± ± ±

0.13 0.06 0.14 0.12 0.38*** 0.03 0.14* 0.013

0.14 ± 0.02 0.53 ± 0.15

Group I (0 g/kg) 0.43 0.06 0.28 0.40 2.84 0.18 0.56 0.016

± ± ± ± ± ± ± ±

0.03 0.01 0.04 0.12 0.33 0.02 0.05 0.002

0.70 ± 0.05 0.30 ± 0.03 0.68 0.11 0.34 0.47 2.40 0.22 0.54 0.021

± ± ± ± ± ± ± ±

0.02 0.03 0.04 0.08 0.19 0.02 0.04 0.003

0.058 ± 0.016 0.25 ± 0.07

Group II (1 g/kg) 0.46 0.07 0.30 0.42 2.86 0.16 0.58 0.014

± ± ± ± ± ± ± ±

0.05 0.02 0.02 0.06 0.40 0.02 0.04 0.002

0.66 ± 0.13 0.30 ± 0.04 0.68 0.10 0.32 0.47 2.37 0.21 0.55 0.022

± ± ± ± ± ± ± ±

0.02 0.03 0.03 0.06 0.06 0.02 0.03 0.003

0.064 ± 0.014 0.21 ± 0.03

Group III (3 g/kg) 0.44 0.06 0.29 0.40 2.74 0.19 0.56 0.014

± ± ± ± ± ± ± ±

0.04 0.02 0.02 0.05 0.20 0.02 0.02 0.001

0.71 ± 0.03 0.32 ± 0.03 0.69 0.12 0.34 0.57 3.00 0.25 0.63 0.027

± ± ± ± ± ± ± ±

0.04 0.03 0.04 0.06 0.31** 0.02 0.03** 0.006

0.070 ± 0.026 0.25 ± 0.10

Group IV (9 g/kg) 0.50 0.09 0.31 0.43 3.20 0.22 0.69 0.020

± ± ± ± ± ± ± ±

0.04* 0.03 0.02 0.06 0.34 0.04* 0.06** 0.002*

0.84 ± 0.05** 0.39 ± 0.06* 0.70 0.08 0.36 0.50 3.50 0.23 0.68 0.025

± ± ± ± ± ± ± ±

0.07 0.02 0.05 0.07 0.11*** 0.02 0.02*** 0.005

0.054 ± 0.005 0.21 ± 0.06

Wistar rats (n = 30 per group) were administered KXS by daily gavage for 91 days. Data are expressed as mean ± S.E.M. *P < 0.05; **P < 0.01; ***P < 0.001 vs the corresponding group I value. Table 7 Some of the organ weights of rats after 30 days of withdrawal study. Organ weight (g) Group I (0 g/kg) Male (n = 5) Brain Spleen Kidney Adrenal glands Testicles Epididymis Female (n = 5) Liver Kidney

2.06 0.89 2.78 0.074

± ± ± ±

0.17 0.12 0.44 0.016

Relative organ weight (g per 100 g body weight) Group II (1 g/kg) 1.96 0.88 2.55 0.067

± ± ± ±

0.13 0.14 0.36 0.015

Group III (3 g/kg) 2.10 0.89 2.64 0.071

± ± ± ±

0.10 0.08 0.15 0.013

Group IV (9 g/kg) 1.94 0.78 2.48 0.074

± ± ± ±

0.15 0.06 0.13 0.008

Group I (0 g/kg) 0.43 0.19 0.58 0.015

± ± ± ±

0.05 0.03 0.08 0.003

Group II (1 g/kg) 0.44 0.20 0.56 0.015

± ± ± ±

0.06 0.04 0.02 0.003

Group III (3 g/kg) 0.44 0.18 0.55 0.015

± ± ± ±

0.04 0.02 0.04 0.002

Group IV (9 g/kg) 0.46 0.18 0.58 0.017

± ± ± ±

0.02 0.02 0.03 0.002

3.17 ± 0.16 1.82 ± 0.64

3.10 ± 0.21 1.94 ± 0.83

2.75 ± 0.59 1.48 ± 0.20

3.09 ± 0.19 1.59 ± 0.19

0.66 ± 0.06 0.37 ± 0.12

0.69 ± 0.07 0.42 ± 0.13

0.57 ± 0.13 0.31 ± 0.05

0.72 ± 0.02 0.37 ± 0.03

7.70 ± 1.46 1.69 ± 0.16

7.11 ± 0.18 1.61 ± 0.09

6.88 ± 0.80 1.70 ± 0.12

7.42 ± 0.68 1.71 ± 0.05

2.71 ± 0.58 0.59 ± 0.06

2.56 ± 0.14 0.58 ± 0.05

2.48 ± 0.12 0.62 ± 0.04

2.84 ± 0.16 0.66 ± 0.03

Wistar rats (n = 10, 5 males and 5 females per group) were withdrew KXS for 30 days. Data are expressed as mean ± S.E.M. There was no significant difference.

groups III and IV were significantly increased as compared to controls (P < 0.05–0.001). In addition, significant increase of relative brain, spleen, kindey, adrenal glands, testis and epididymis weight was found in the male rats of group IV compared to the control group (P < 0.05–0.001). Absolute and relative organ weights of rats in 30-day withdrawal study are shown in Table 7. There was no significant change in the organ weights of the treated compared to the control groups. 4. Discussion and conclusion Estimation of safety of drugs and plant products is currently performed in animals. A good correlation has been reported between toxicological insults in rats and humans, correlation is weaker between humans and mice (Olson et al., 2000). Therefore numerous studies investigate the acute effects of high doses in mice and the chronic effects of lower doses in rats including the doses potentially usable in humans (Lahlou et al., 2008; Rhiouani et al., 2008). Kai-Xin-San (KXS) is a famous traditional Chinese medicine (TCM). Many studies have reported the pharmacological efficacy of KXS, but there is no information on its safety, such as acute and sub-chronictoxicity.

In the oral acute toxicity study, mice administered doses up to 19.67 g/kg did not exhibit any sign of adverse effect (NOAEL). Higher doses induced mortality and symptoms of pronounced adverse effects. The LD50 for the oral administration of KXS was estimated to be >32.59 g/kg BW of mice, a dose much higher than the medicinal dosage. Thus, referring to the Hodge and Stemer scale (CCOHS, 2005), the orally administered aqueous extract of KXS could be considered practically non-toxic or at worst slightly toxic. There were no treatment-related toxicological changes following the administration of KXS at the dose of 1 g/kg/day for 13 weeks to male and female rats respectively. The serum Cre in female rats in the 3 g/kg/day groups and Cre, CK, CHO in rats in the 9 g/kg/day were significantly lower comparing to the control groups. The changes were regarded as toxicologically irrelevant because they were not treatment-related incidental and within the normal ranges (Kim et al., 2002; Burn et al., 2006; Derelanko, 2008). The significant decreases in body weight found in rats in the 9 g/kg/day groups had no toxicological significance because the highest dose of KXS decreased the food consumption and the low body weight led to the increase of the relative organ weight. Recovery except for the body weight was observed after 30 days of post treatment (Tables 3, 5 and 7).

L.-H. Mu et al. / Journal of Ethnopharmacology 138 (2011) 351–357

In humans, the reported single dose of KXS is about 18 g dried herb (Chinese ancient book Bei-Ji-Qian-Jin-Yao-Fang), which is equivalent to 2.521 g of the extract (yield of extract = 14.0% of the raw material). Considering the average body weight of an adult as 60 kg, the dose for a 60 kg human is 42 mg of KXS extract/kg. To convert the human dose to rat dose equivalent, the amount would be 264.6 mg/kg (42 mg/kg × 6.3) according to Van Miert (1986). The dose equivalent per kg for rat (6.3) was derived by dividing the Km factor, body surface area (m2 ) to body weight (kg) ratio, for humans with the Km factor for rat (Van Miert, 1986). The amount of KXS extract, 264.6 mg, is a value less than the NOAEL (19.67 g/kg in mice). In conclusion, the currently recommended doses of KXS as an herbal extract of 264.6 mg/kg/day (an average individual weighing 60 kg) are about 73-fold lower than the NOAEL in mice. The present studies demonstrate that at doses consumed in traditional medicine, the aqueous extract of KXS may be relatively safe, as they had convertibly low toxicity in high dose administration and no toxicity in low dose anministration to rats in oral sub-chronic. Acknowledgement This work was supported by funds from the National Science and Technology Major Project of the PR China (2008ZXJ09004-028). References Alexeeff, G.V., Broadwin, R., Liaw, J., Dawson, S.V., 2002. Characterization of the LOAEL-to-NOAEL uncertainty factor for mild adverse effects from acute inhalation exposures. Regulatory Toxicology and Pharmacology 36, 96–105. Bian, H.M., Huang, Y.F., Guo, H.Y., Zhang, J.Y., 2000. The effect of Kai-Xin-San on monamine neurotransmitter and the activity of cholinesterase in scopolamine model rat brain. Pharmacology and Clinics of Chinese Materia Medica 16, 5–7. Burn, C.C., Peters, A., Day, M.J., Mason, G.J., 2006. Long-term effects of cage-cleaning frequency and bedding type on laboratory rat health, welfare, and handleability: a cross laboratory study. Laboratory Animals 40, 353–370. CCOHS, 2005. What is an LD50 and LC50, Canada’s National Occupational Health and Safety Resource: Canadian Centre for Occupational Health and Safety. http://www.ccohs.ca/oshanswers/chemicals/ld50.html. Derelanko, M.J., 2008. The Toxicologist’s Pocket Handbook, second ed. Informa Healthcare, UK. Firenzuoli, F., Gori, L., 2007. Herbal medicine today: clinical and research issues. Evidence Based Complement and Alternative Medicine 4, 37–40. Huang, Y.F., Bian, H.M., Gong, J.N., Liu, X.F., 1999. The effect of Kai-Xin-San on the content of SOD and MDA in four kinds of rat models. Journal of Nanjing University TCM 15, 151–152.

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