Acute and subacute toxicities of the saponin mixture isolated from Schefflera leucantha Viguier

Journal of Ethnopharmacology 89 (2003) 115–121

Acute and subacute toxicities of the saponin mixture isolated from Schefflera leucantha Viguier P. Witthawaskul a , Ampai Panthong a,∗ , D. Kanjanapothi a , T. Taesothikul a , N. Lertprasertsuke b a

Department of Pharmacology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand b Department of Pathology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand Received 29 January 2003; received in revised form 22 July 2003; accepted 25 July 2003

Abstract Acute toxicity of the bronchodilator saponin mixture isolated from Schefflera leucantha Viguier leaves was investigated in comparison with the methanol and the water extract of this plant. Oral doses of 5000 mg/kg of the methanol extract, the water extract and the saponin mixture did not produce mortality or significant changes in the general behavior and gross appearance of internal organs of rats. Subacute toxicity of the saponin mixture was evaluated with the dose of 1000 mg/kg, orally for 14 days. An extra group (satellite group) was given saponin mixture and kept for a further 14 days after treatment. All animals did not show signs of toxicity during the experimental period. Liver weights of the saponin-treated and the satellite male groups were higher whereas testis weight were lower than those of the control group which received distilled water. However, the histological examination of various organs revealed that there were no differences between the control and the treated rats. BUN, Cr, AST, ALT and ALP levels increased in saponin-receiving rats. It is possible that the saponin mixture directly impacts on the liver and the kidney functions. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Acute toxicity; Subacute toxicity; Saponin; Schefflera leucantha Viguier

1. Introduction Schefflera leucantha Viguier, family Araliaceae (Thai local name—Hanumarnprasaankai or Waan Sungoranee), is an ornamental plant that is widely used in Thailand and China as a single herbal drug for the treatment of chronic asthma and for the prevention of asthmatic attack (Panthong et al., 1986; Hiranramdej, 1992). The plant has been prove to possess various pharmacological activities such as antibacterial (Laopaksa et al., 1988), hypoglycemic (Satayavivad et al., 1996) and insect repellent activities (Sinchaisri et al., 1988). From phytochemical work, three saponins were obtained from this plant by purification of the n-butanol fraction from the methanol extract of the fresh leaves (Pancharoen et al., 1994). These saponins were found to possess bronchodilator effect when tested on histamine- and methacholine-induced bronchoconstriction of guinea pig tracheal muscle both in in vitro and in vivo experiments (Tipsuwan, 1992). At present, there are many forms of preparation of Schefflera leucantha available in the local markets such as tea, tablets ∗ Corresponding

author. Tel.: +66-53-945351; fax: +66-53-217144. E-mail address: [email protected] (A. Panthong).

and capsules. However, no toxicity study of this plant has ever been ever performed. The aims of this study were to evaluate the acute subacute toxicity of the saponin mixture in rats as well as the acute toxicity of the methanol and water extracts of the fresh leaves of Schefflera leucantha. In the subacute toxicity study the effect on biochemical, hematological and histopathological parameters was investigated. 2. Material and methods 2.1. Plant material The fresh leaves of Schefflera leucantha were collected in Chiang Mai, Thailand. The material was identified and a voucher specimen (QBG. 13036) was deposited at the Herbarium of the Queen Sirikit Botanical Garden, Chiang Mai, Thailand. 2.2. Water extraction The water extract was prepared according to the method mentioned in Thai traditional use (Utokpach, 1975). Ten

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fresh compound leaves were boiled in 250 ml of water for 15–30 min until the 75 ml of extract remained. The extract was lyophilized and dark stored at 0 ◦ C. The yield was approximately 3.1% (w/w). 2.3. Methanol extraction The fresh leaves of Schefflera leucantha were milled and macerated in methanol for 24 h. The extract was filtered through the filter paper using a vacuum pump. Maceration was repeated two times. The filtrates were pooled and evaporated under reduced pressure and controlled temperature (60 ◦ C) by using a vacuum rotary evaporator. The residue obtained was then lyophilized and dark stored at 0 ◦ C. The yield of this extract was approximately 7.3% (w/w). 2.4. Saponin mixture The saponin mixture (provided by Professor W.C. Taylor, Department of Organic Chemistry, University of Sydney, Australia) was obtained by purification of the n-butanol fraction from the methanol extract of the fresh leaves of the plant (Pancharoen et al., 1994). The percentage of the saponin mixture from the methanol extract was approximately 0.2% (w/w). The three major bronchodilator saponins present in the saponin mixture are two derivatives of betulinic acid and one derivative of oleanolic acid. The content of these three saponins in the saponin mixture is approximately 40% (w/w). Test solutions of the saponin mixture, the methanol extract and the water extract were prepared by dissolving in distilled water. 2.5. Experimental animals Male and female Sprague–Dawley rats weighing 160–190 g were purchased from the National Laboratory Animal Center, Nakorn Pathom, Thailand. All animals were kept in a room maintained under environmentally controlled conditions of 24 ± 1 ◦ C and 12-h light:12-h dark cycle. The animals had free access to water and standard diet. Rats were deprived of food but not water 16–18 h prior to administration of the test substances. 2.6. Acute toxicity The procedure was according to the Organization of Economic Cooperation and Development (OECD) guideline for testing of chemicals (1981). Three groups of 10 Sprague–Dawley rats (5 males and 5 females) were given single oral doses of 5000 mg/kg body weight of the saponin mixture, the methanol extract and the water extract. The control group received distilled water at the same volume.

Observations were made and recorded systematically 1, 2, 4 and 6 h after test substance administration. The visual observations included changes in the skin and fur, eyes and mucous membranes, and also respiratory, circulatory, autonomic and central nervous system as well as somatomotor activity and behavioral pattern. The number of survivors was noted after 24 h and these were then maintained for a further 14 days with a once daily observation. At the conclusion of the experiment, all surviving animals were sacrificed and the internal organs such as heart, lungs, livers, kidneys, spleen, adrenals, sex organs and brain were examined. 2.7. Subacute toxicity Two groups of 12 rats (6 males and 6 females) received the saponin mixture at the dose of 1000 mg/kg/day or distilled water (control) for a period of 14 days. An extra group (satellite group) was given orally the saponin mixture at the dose of 1000 mg/kg/day for a further 14 days and kept for other 14 days after treatment to observe for reversibility, persistence, or delayed occurrence of toxic effects. During the period of administration, the animals were weighed and observed daily to detect signs of toxicity. Daily visual observations were made and recorded systematically similar those performed as in the case acute toxicity study. Rats died during the test period were examined pathologically. At the end of the period, all surviving animals were fasted overnight before anesthetization. Blood samples were collected from a common carotid into heparinized and dry non-heparinized centrifuge tubes. The heparinized blood was used for hematological study included WBC and differential leukocyte count, platelet, hematocrit and hemoglobin estimation. The serum was separated from the non-heparinized blood and was assayed for glucose, serum urea (BUN), creatinine (Cr), total protein (TP), albumin (Alb), globulin (Glob), total billirubin (TBil), direct bilirubin (DBil), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP). After blood collection animals were sacrificed for tissue studies. The organs such as heart, lungs, livers, kidneys, spleen, adrenals, and sex organs were removed, blotted free of blood and weighed immediately on an electronic balance for subsequent analysis. Eyes, brain, thymus, intestine, uterus, epididymis, seminal vesicles, prostate glands, thoracic spines and muscle with sciatic nerve were also observed. Histological examinations were performed on the preserved tissues with particular emphasis on those which showed gross pathological changes. 2.8. Statistical analysis Results were expressed as mean ± S.E.M. Statistical significance was determined by one-way ANOVA and

P. Witthawaskul et al. / Journal of Ethnopharmacology 89 (2003) 115–121 Table 1 Body weights of rats in acute toxicity of saponin mixture, methanol extract and water extract from Schefflera leucantha Body weight (g) Day 0

Day 14

Weight gain

Female Control Saponin Methanol extract Water extract

171.20 164.40 160.00 170.60

± ± ± ±

3.06 3.19 2.85 6.29

216.60 216.80 209.80 197.00

± ± ± ±

3.52 4.59 7.76 16.33

26.63 31.86 32.45 18.89

± ± ± ±

2.51 0.58 4.99 7.96

Male Control Saponin Methanol extract Water extract

165.60 174.80 169.80 170.00

± ± ± ±

1.91 4.41 2.65 3.52

279.80 284.20 289.80 265.40

± ± ± ±

3.17 6.41 2.33 4.35∗

69.01 62.96 70.77 56.66

± ± ± ±

2.85 5.21 1.83 3.03∗

Values are expressed as mean ± S.E.M., n = 5. ∗ Significantly different from control: P < 0.05.

post hoc least-significant difference (LSD) test. The data obtained from acute toxicity study were analyzed using Student’s t-test. P-values less than 0.05 were considered significant.

3. Results In the acute toxicity test doses of 5000 mg/kg of the saponin mixture, the methanol extract and the water extract did not cause death of rats during 14-day observation. The rats did not show any signs of toxicity or change in general behavior or other physiological activities. The body weight gain of the rats treated with the water extract was slightly lower compared with that of the control group (Table 1). The pathological examination of the internal organs revealed that there were no signs of abnormalities. There were no differences in the weight and the color of the internal organs of rats in the control and the treated groups. In the subacute toxicity study, the saponin mixture at a dose of 1000 mg/kg, given orally for 14 days, did not

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produce death of the rats. No signs of toxicity were observed during the experimental period. The body weight gains of the saponin-treated and the satellite male rats were higher than in the control rats. The satellite group, which was kept for a further 14 days, showed an increased weight gain on day 28 (Table 2). As shown in Table 3, the organ weights of the saponin-treated female rats did not differ from those of the control group except that heart weight of the satellite group was slightly less than that of the control group. The liver weights of the saponin-treated and the satellite male groups were higher than in the control group. The lung weight of male rats in the satellite group was less than that of the control group. The weights of testis of the saponin-treated and the satellite groups were lower than in the control group. The histological examination of various organs from male and female rats was performed in both control and treated groups. All the sampling tissue sections were within normal limits. Neither degenerative nor infiltrative lesions were observed. The results obtained revealed that there were no significant changes in the histology of the internal organs of the treated rats compared to the control rats. The hematological values of treated female rats were not significantly different from those of the control group (Table 4). HBG and HCT values of the satellite male group were slightly higher than those of the control group whereas platelet values were lower. The differential white blood cell count in the female treated group was not different from that of the control group except the lymphocyte value of the satellite group was slightly lower than that of the control group (Table 5). The number of neutrophils of the saponin-treated male group was slightly higher than that of the control group. Blood chemistry values of female and male rats are shown in Tables 6 and 7, respectively. Cr, Tp, Glob, AST and ALT values of both saponin-treated and satellite groups were significantly increased when compared with those of the control group. In the satellite group, BUN, Alb and ALP values were higher than those of the control group.

Table 2 Body weights of rats in subacute toxicity of saponin mixture 1000 mg/kg from Schefflera leucantha Body weight (g) Day 0

Day 14

Day 28

Weight gain on day 14

Female Control Saponina Satelliteb

165.83 ± 2.01 167.50 ± 1.12 169.17 ± 5.23

198.67 ± 4.29 202.33 ± 3.29 215.17 ± 6.12∗

– – 240.00 ± 13.56

32.83 ± 2.77 34.83 ± 2.96 46.00 ± 3.62∗

Male Control Saponina Satelliteb

180.67 ± 6.59 187.50 ± 4.03 185.83 ± 2.01

228.17 ± 5.06 257.33 ± 2.99∗ 258.67 ± 4.25∗

– – 305.00 ± 4.46

41.78 ± 5.78 69.83 ± 3.72∗ 72.83 ± 2.64∗

Values are expressed as mean ± S.E.M., n = 6. a A group was given 1000 mg/kg daily for 14 days. b A separate group was given 1000 mg/kg daily for 14 days followed by no treatment for 14 days. ∗ Significantly different from control: P < 0.05.

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Organ weight (g/100 g body weight) Lung Female Control 0.49 ± 0.02 (0.44–0.59) Saponina 0.51 ± 0.03 (0.43–0.64) Satelliteb 0.45 ± 0.02 (0.40–0.53)

Heart

Liver

0.43 ± 0.01 (0.40–0.45) 3.18 ± 0.16 (2.73–3.82) 0.41 ± 0.01 (0.38–0.45) 3.33 ± 0.15 (3.01–3.83) 0.39 ± 0.01 (0.36–0.42)∗ 3.51 ± 0.20 (3.15–3.71)

Male Control 0.44 ± 0.01 (0.39–0.50) 0.39 ± 0.01 (0.36–0.42) Saponina 0.42 ± 0.01 (0.40–0.46) 0.38 ± 0.01 (0.35–0.41) Satelliteb 0.36 ± 0.01 (0.33–0.36)∗ 0.37 ± 0.01 (0.34–0.41)

Spleen

Adrenal

Kidney

Ovary

0.29 ± 0.01 (0.26–0.32) 0.02 ± 0.00 (0.01–0.02) 0.43 ± 0.03 (0.35–0.57) 0.03 ± 0.00 (0.02–0.03) 0.29 ± 0.01 (0.27–0.32) 0.02 ± 0.00 (0.01–0.02) 0.45 ± 0.02 (0.40–0.52) 0.03 ± 0.00 (0.02–0.03) 0.28 ± 0.00 (0.27–0.30) 0.02 ± 0.00 (0.01–0.02) 0.41 ± 0.02 (0.36–0.50) 0.03 ± 0.00 (0.02–0.04)

2.97 ± 0.13 (2.56–3.46) 0.25 ± 0.01 (0.20–0.28) 0.01 ± 0.00 (0.01–0.02) 0.44 ± 0.03 (0.35–0.52) 0.70 ± 0.01 (0.66–0.75) 3.38 ± 0.01 (3.16–3.74)∗ 0.28 ± 0.01 (0.25–0.33) 0.01 ± 0.00 (0.01–0.01) 0.47 ± 0.02 (0.42–0.52) 0.59 ± 0.03 (0.47–0.65)∗ 3.41 ± 0.01 (3.07–3.68)∗ 0.26 ± 0.01 (0.22–0.30) 0.01 ± 0.00 (0.01–0.01) 0.42 ± 0.01 (0.35–0.43) 0.57 ± 0.01 (0.54–0.64)∗

Values are expressed as mean ± S.E.M. Parentheses are expressed as range values, n = 6. a A group was given 1000 mg/kg daily for 14 days. b A satellite group was given 1000 mg/kg daily for 14 days followed by no treatment for 14 days. ∗ Significantly different from control: P < 0.05.

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Table 3 Organ weights of rats in subacute toxicity of Schefflera leucantha

HGB (g%)

HCT (%)

MCV (ft)

MCH (pg)

MCHC (g%)

Platelet (105 /mm3 )

Female Control Saponina Satelliteb

13.47 ± 0.43 (12.00–14.90) 13.45 ± 0.59 (10.60–14.50) 13.20 ± 0.10 (12.90–13.60)

38.67 ± 1.01 (36.00–43.00) 39.33 ± 0.71 (37.00–42.00) 37.67 ± 0.21 (37.00–38.00)

55.00 ± 0.37 (54.00–56.00) 55.83 ± 0.17 (55.00–56.00) 54.67 ± 0.33 (54.00–56.00)

19.00 ± 0.37 (18.00–20.00) 19.00 ± 0.63 (16.00–20.00) 19.17 ± 0.17 (19.00–20.00)

34.50 ± 0.43 (33.00–36.00) 34.17 ± 1.30 (28.00–37.00) 34.83 ± 0.31 (34.00–36.00)

8.80 ± 0.24 (7.88–9.44) 8.35 ± 0.30 (7.43–9.33) 8.24 ± 0.16 (7.68–8.67)

Male Control Saponina Satelliteb

14.17 ± 0.32 (13.10–15.30) 14.18 ± 0.32 (13.20–15.00) 15.15 ± 0.33 (14.00–16.10)∗

41.87 ± 1.04 (39.00–46.20) 41.33 ± 0.76 (39.00–43.00) 44.67 ± 0.67 (43.00–47.00)∗

57.67 ± 0.21 (57.00–58.00) 58.50 ± 0.50 (57.00–60.00) 56.50 ± 0.43 (55.00–58.00)

19.33 ± 0.33 (18.00–20.00) 19.33 ± 0.67 (16.00–20.00) 19.17 ± 0.31 (18.00–20.00)

34.00 ± 0.52 (32.00–35.00) 33.17 ± 1.08 (28.00–35.00) 33.83 ± 0.40 (32.00–35.00)

9.15 ± 0.17 (8.54–9.61) 9.67 ± 0.24 (8.67–10.32) 7.47 ± 0.18 (6.95–8.28)∗

Values are expressed as mean ± S.E.M. Parentheses are expressed as range values, n = 6. a A group was given 1000 mg/kg daily for 14 days. b A satellite group was given 1000 mg/kg daily for 14 days followed by no treatment for 14 days. ∗ Significantly different from control: P < 0.05.

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Table 4 Hematological values of rats in subacute toxicity of Schefflera leucantha

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Table 5 Differential white blood cell count of rats in subacute toxicity of saponin mixture from Schefflera leucantha WBC (103 /mm3 )

Neutrophil (%)

Eosinophil (%)

Monocyte (%)

Lymphocyte (%)

Female Control Saponina Satelliteb

2.10 ± 0.23 (1.60–3.00) 1.88 ± 0.38 (0.90–3.50) 2.40 ± 0.27 (1.60–3.30)

12.17 ± 2.15 (4.00–18.00) 14.33 ± 1.63 (9.00–21.00) 12.83 ± 1.45 (8.00–17.00)

1.00 ± 0.26 (0.00–2.00) 1.83 ± 0.70 (0.00–5.00) 2.50 ± 0.81 (1.00–6.00)

0.00 0.00 0.67 ± 0.49 (0.00–3.00)

86.83 ± 2.12 (81.00–95.00) 83.83 ± 1.74 (78.00–89.00) 78.50 ± 3.66 (63.00–88.00)∗

Male Control Saponina

4.15 ± 0.38 (2.90–5.40) 4.15 ± 0.35 (3.40–5.80)

8.25 ± 1.73 (3.50–16.00) 15.33 ± 1.80 (10.00–21.00)∗

1.00 ± 0.52 (0.00–3.00) 1.17 ± 0.40 (0.00–2.00)

87.75 ± 2.38 (78.50–94.00) 83.00 ± 2.02 (77.00–90.00)

Satelliteb

3.98 ± 0.50 (2.80–5.60)

9.17 ± 1.01 (5.00–12.00)

0.50 ± 0.34 (0.00–2.00)

0.00 0.50 ± 0.34 (0.00–2.00) 0.67 ± 0.33 (0.00–2.00)

83.33 ± 1.15 (80.00–88.00)

Values are expressed as mean ± S.E.M. Parentheses are expressed as range values, n = 6. a A group was given 1000 mg/kg daily for 14 days. b A satellite group was given 1000 mg/kg daily for 14 days followed by no treatment for 14 days. ∗ Significantly different from control: P < 0.05.

Table 6 Blood chemistry values of female rats in subacute toxicity of saponin mixture of Schefflera leucantha Saponina

Control Glucose (mg/dl) BUN (mg/dl) Creatinine (mg/dl) Total protein (g/dl) Albumin (g/dl) Globulin (g/dl) Total bilirubin (mg/dl) Direct bilirubin (mg/dl) AST (U/l) ALT (U/l) ALP (U/l)

143.33 17.00 0.43 5.00 1.55 3.45 0.20 0.00 77.50 30.00 80.83

± ± ± ± ± ± ±

5.35 0.68 0.02 0.07 0.03 0.06 0.03

(128.00–160.00) (16.00–20.00) (0.40–0.50) (4.80–5.30) (1.50–1.70) (3.30–3.60) (0.10–0.30)

± 4.67 (59.00–91.00) ± 2.10 (23.00–39.00) ± 10.55 (64.00–130.00)

107.00 22.33 0.48 5.45 1.62 3.83 0.20 0.03 106.00 25.00 77.50

± ± ± ± ± ± ± ± ± ± ±

Satelliteb 8.48 1.26 0.03 0.06 0.03 0.04 0.05 0.02 4.58 13.7 7.94

(87.00–139.00)∗ (19.00–27.00)∗ (0.40–0.60) (5.30–5.70)∗ (1.50–1.70) (3.70–4.00)∗ (0.10–0.40) (0.00–0.10) (93.00–122.00)∗ (22.00–31.00) (60.00–113.00)

160.83 31.83 0.67 6.43 1.92 4.52 0.22 0.00 97.83 39.50 146.00

± ± ± ± ± ± ±

5.53 1.30 0.03 0.12 0.04 0.09 0.02

(142.00–176.00) (26.00–35.00)∗ (0.60–0.80)∗ (6.10–6.80)∗ (1.80–2.10)∗ (4.20–4.70)∗ (0.20–0.30)

± 5.15 (75.00–111.00)∗ ± 2.38 (31.00–48.00)∗ ± 10.15 (128.00–187.00)∗

Values are expressed as mean ± S.E.M. Parentheses are expressed as range values, n = 6. a A group was given 1000 mg/kg daily for 14 days. b A satellite group was given 1000 mg/kg daily for 14 days followed by no treatment for 14 days. ∗ Significantly different from control: P < 0.05.

Table 7 Blood chemistry values of male rats in subacute toxicity of saponin mixture of Schefflera leucantha Group

Control

Glucose (mg/dl) BUN (mg/dl) Creatinine (mg/dl) Total protein (g/dl) Albumin (g/dl) Globulin (g/dl) Total bilirubin (mg/dl) Direct bilirubin (mg/dl) AST (U/l) ALT (U/l) ALP (U/l)

118.50 20.50 0.40 4.82 1.37 3.37 0.24 0.01 99.00 31.50 95.33

Saponina ± ± ± ± ± ± ± ± ± ± ±

9.23 1.28 0.03 0.15 0.02 0.10 0.04 0.01 5.14 2.66 7.85

(92.00–143.00) (15.00–24.00) (0.30–0.50) (4.40–5.30) (1.30–1.40) (3.10–3.70) (0.10–0.30) (0.00–0.03) (80.00–119.00) (20.00–39.00) (72.00–127.00)

105.83 23.00 0.48 5.25 1.45 3.80 0.25 0.02 141.33 37.67 143.67

± ± ± ± ± ± ± ± ± ± ±

Satelliteb 7.97 2.03 0.03 0.03 0.02 0.03 0.06 0.02 9.23 2.01 9.08

(76.00–128.00) (18.00–29.00) (0.40–0.60) (5.10–5.30)∗ (1.40–1.50)∗ (3.70–3.90)∗ (0.10–0.40) (0.00–0.10) (104.00–170.00)∗ (33.00–46.00) (117.00–181.00)∗

Values are expressed as mean ± S.E.M. Parentheses are expressed as range values, n = 6. a A group was given 1000 mg/kg daily for 14 days. b A satellite group was given 1000 mg/kg daily for 14 days followed by no treatment for 14 days. ∗ Significantly different from control: P < 0.05.

141.67 32.17 0.58 6.40 1.75 4.65 0.28 0.00 135.83 54.17 156.33

± ± ± ± ± ± ±

4.26 1.35 0.03 0.10 0.03 0.07 0.02

(127.00–158.00)∗ (27.00–36.00)∗ (0.50–0.70)∗ (6.10–6.70)∗ (1.70–1.90)∗ (4.40–4.80)∗ (0.20–0.30)

± 6.20 (114.00–157.00)∗ ± 3.40 (46.00–67.00)∗ ± 8.82 (128.00–185.00)∗

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4. Discussion and conclusion The present study found that single administrations of the saponin mixture, the methanol extract and the water extract by the oral route up to a dose of 5000 mg/kg did not produce any mortality or alter the behavioral pattern of rats as compared with the control group. Weight gains of treated male and female rats were not different from those of the control groups. At the same age, the rate of growth of male rats is usually higher than that of female rats (Waynforth, 1980). According to the OECD guideline for testing of chemicals, the results of acute toxicity test in this study indicate that the saponin mixture, the methanol extract and the water extract isolated from leaves of Schefflera leucantha are fairly non-toxic. A subacute toxicity study was done because the Thai traditional use of the leaves of Schefflera leucantha claims that the preparations sold in the local market are safe for the chronic treatment of bronchitis and asthma (Utokpach, 1975). As the saponin mixture isolated from this plant was found to possess a bronchodilator effect (Tipsuwan, 1992), it was selected for subacute toxicity study. The dose of Schefflera leucantha used per day by an asthmatic patient contains about 5 mg of the saponin mixture (unpublished results). According to the OECD guideline, if an acute toxicity test at one dose level of at least 5000 mg/kg body weight produce no observable toxic effects then a full study using three dose levels is considered not to be necessary and a dose of 1000 mg/kg given once daily for 14 days can be used to evaluate subacute toxicity. The present work indicated that the saponin mixture at a high dose of 1000 mg/kg daily for 14 days did not cause death of the rats, although it caused higher body weight gain of male rats. Liver weight of male rats was higher whereas testis weight was lower than those of the control group. An effect of the saponin mixture on these organs may be suggested. However, the histology of these organs was found to be normal. Although the weights of some internal organs were found to be statistically significantly different between the treated and the control groups, the weight range of those organs of each group overlapped. Some of the hematological and the differential white blood cell values of the saponin-treated rats differed slightly from those of the control group, but some values were in the ranges of the control. These effects may therefore be due to variations in the animals. BUN and serum creatinine values of the saponin-treated and of the satellite groups increased significantly but still were not higher than onefold when compared with those of the control group. For liver inspection, the two enzymes most often associated with hepatocellular damage are AST and ALT. Although both AST and ALT are commonly thought of as liver enzymes because of their high concentrations in hepatocytes, only ALT is markedly specific for liver function since AST is widely present in myocardium, skeletal muscle, brain and kidney. In general with liver disease, serum levels of AST and ALT rise and fall in parallel (Sacher and

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McPherson, 1991). In the present study both AST and ALT levels increased slightly in the satellite group while only the AST level rose slightly in the saponin-treated group. The enzyme most often measured to indicate bile duct obstruction is ALP. High levels of ALP exist in cells that are rapidly dividing or are otherwise metabolically active. These cells include the epithelium of the biliary tract and liver, osteoblasts laying down new bone, granulocytes of circulating blood, intestinal epithelium, proximal tubules of the kidney, placenta, and lactating mammary glands. ALP levels reach spectacular heights in primary biliary cirrhosis, in conditions of disorganized hepatic architecture (cirrhosis), and in diseases characterized by inflammation, regeneration, and obstruction of intrahepatic bile ductules (Sacher and McPherson, 1991). In this study, ALP levels were significantly increased in the female satellite group, the male saponin-treated group and the satellite group (Table 7). However when the bilirubin values were considered, it revealed that the saponin mixture did not encourage bile duct obstruction in rats. It is possible that the saponin mixture directly impact the liver and the kidney functions. An additional chronic toxicology evaluation is recommended. Acknowledgements We are thankful to The Thailand Research Fund for the award of The Royal Golden Jubilee (RGJ) Ph.D. program scholarship to P.W. Thanks also extend to Professor W.C. Taylor for providing the saponin mixture. References Hiranramdej, S.P., 1992. Handbook of Thai Folkfore Medicine for Health. Bangkok, p. 230. Laopaksa, A., Umnouypol, S., Jongbunpasert, V., 1988. The study of antibacterial activity of herbs in respiratory tract infection. Part I. The Thai Journal of Pharmaceutical Sciences 13, 23–36. OECD Guideline for Testing of Chemicals, 1981. Organization for Economic Cooperation and Development. Pancharoen, O., Tuntiwachwuttikul, P., Taylor, W.C., 1994. Triterpenoid glycosides from Schefflera leucantha. Phytochemistry 35 (4), 987–992. Panthong, A., Kanjanapothi, D., Taylor, W.C., 1986. Ethnobotanical review of medicinal plants from Thai traditional books. Part I. Plants with anti-inflammatory, anti-asthmatic and antihypertensive properties. Journal of Ethnopharmacology 18, 213–228. Sacher, R.A., McPherson., R.A., 1991. Widmann’s Clinical Interpretation of Laboratory Test. Pennsylvania, USA, pp. 416–443. Satayavivad, J., Bunyapraphatsara, N., Thiantanawat, A., Kositchaiwat, U., 1996. Hypoglycemic activity of the aqueous extract of Schefflera leucantha Viguier in rats. Thai Journal of Phytopharmacy 3, 1–5. Sinchaisri, N., Roongsook, D., Areekul, S., 1988. Botanical repellent against the diamondback moth, Plutella xylostella L. The Kasetsart Journal 22, 71–74. Tipsuwan, S., 1992. Bronchodilator Activity of Saponin Mixture from the Leaves of Schefflera leucantha Viguier. M.Sc. Thesis. Department of Pharmacology, Faculty of Medicine, Chiang Mai University. Utokpach, C., 1975. Manual of Use and Treatment of Thai Folklore Medicine in Topical Diseases. Paepitaya, Bangkok, pp. 82–116. Waynforth, H.B., 1980. Experimental and Surgical Technique in the Rat. London, UK, p. 245.