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Toxicity evaluation of standardized extract of Gynostemma pentaphyllum Makino
Journal of Ethnopharmacology 149 (2013) 228–234
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Toxicity evaluation of standardized extract of Gynostemma pentaphyllum Makino Natthakarn Chiranthanut a,b,n, Supanimit Teekachunhatean a,b, Ampai Panthong c, Parirat Khonsung a, Duangta Kanjanapothi a, Nirush Lertprasertsuk d a
Department of Pharmacology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand Center of Thai Traditional & Complementary Medicine, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand c Department of Pharmacology & Center of Excellence for Innovation in Chemistry, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand d Department of Pathology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand b
art ic l e i nf o
a b s t r a c t
Article history: Received 13 March 2013 Received in revised form 8 May 2013 Accepted 13 June 2013 Available online 21 June 2013
Ethnopharmacological relevance: To evaluate the safety of standardized extract of Gynostemma pentaphyllum in rats. Materials and methods: The water extract of Gynostemma pentaphyllum was prepared and standardized, the dry powder yielded 6% gypenosides. In the acute oral toxicity test, the single oral dose of 5000 mg/kg of Gynostemma pentaphyllum extract was given to female Sprague–Dawley rats. In subchronic toxicity test, the oral dose of 1000 mg/kg/day of the extract was given to rats in treatment and satellite groups for 90 days. Satellite groups of both sexes were kept for additional 28 days after 90-day treatment. Control rats received distilled water. Results: Standardized extract of Gynostemma pentaphyllum did not cause death or any toxic signs in rats. The daily administration of the extract for 90 days did not produce lethal or harmful effects. Although certain hematological and blood chemistry values (i.e., neutrophil, monocyte, glucose, and serum alkaline phosphatase levels) were found to be statistically different from the control group, however; these values were within the ranges of normal rats. Conclusion: Standardized extract of Gynostemma pentaphyllum did not produce mortality or any abnormality in rats. & 2013 Elsevier B.V. All rights reserved.
Keywords: Gynostemma pentaphyllum Makino Safety Toxicity Acute Subchronic
1. Introduction Gynostemma pentaphyllum Makino (family Cucurbitaceae), commonly known in China as Jiaogulan, is a perennial liana growing widely throughout Asian countries. It is well known as medicinal plant with powerful biological effects that lead to longevity (Blumert and Liu, 1999). In traditional medicine, the aerial part of this plant is commonly used for a variety of diseases such as diabetes mellitus, cancer, gastritis, bronchitis, and hypertension (Blumert and Liu, 1999; Bansiddhi, 2005). Previous experimental studies showed that extracts of Gynostemma pentaphyllum produce various pharmacological effects, including antidiabetic (Yeo et al., 2008), hypolipidemic (Megalli et al., 2006), antioxidant
Abbreviations: ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; HPLC, high performance liquid chromatography; LDL, low density lipoprotein; WBC, white blood cell. n Corresponding author at: Department of Pharmacology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand. Tel.: +66 53 945352, +66 53 945354; fax: +66 53 945355. E-mail addresses: [email protected], [email protected] (N. Chiranthanut). 0378-8741/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jep.2013.06.027
(Müller et al., 2012) and antitumor (Schild et al., 2010) effects. From phytochemical studies, Gynostemma pentaphyllum contains saponins (Yin et al., 2006; Kao et al., 2008; Shi et al., 2012), flavonoids (Kao et al., 2008), polysaccharides (Chi et al., 2012; Yan et al., 2013), amino acids and vitamins (Zheng, 2004), and some essential elements (Nookabkaew et al., 2006). Gypenosides and dammarane-type glycosides, are the major constituents of Gynostemma pentaphyllum (Piacente et al., 1995; Cui et al., 1999; Zhang et al., 2010). Nowadays, more than 100 types of gypenosides in this plant have been found (Yin et al., 2006; Hu et al., 2010; Ky et al., 2010; Kim and Han, 2011). These compounds have been reported to possess antioxidant (Li et al., 1993; Shang et al., 2006), antitumor (Yin et al., 2006; Lin et al., 2011; Lu et al., 2012), neuroprotective (Wang et al., 2010), and antiinflammatory activities (Lin et al., 1993). Moreover, glucose lowering (Norberg et al., 2004) and hypolipidemic effects (Huang et al., 2006) of these compounds have also been demonstrated. Chronic toxicity of Gynostemma pentaphyllum with doses up to 750 mg/kg in rats has been previously reported with no obvious toxicity and mortality (Attawish et al., 2004). However, the amount of gypenosides in the water extract used in that study
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was not quantified and standardized, therefore the variability of gypenoside content and the exact doses of gypenosieds could not be accounted for. Standardized Gynostemma pentaphyllum extract with quantitative control of gypenoside content has recently been validated in our laboratory. In the present study, the standardized formulation of water extract of Gynostemma pentaphyllum was used for acute and subchronic toxicity evaluation in rats as a prelude to future clinical study. The objective of the present study was to carry out basic toxic evaluation and to establish the safety of standardized extract of Gynostemma pentaphyllum. This article focuses on acute and subchronic toxicity evaluation of the standardized Gynostemma pentaphyllum extract in rats.
2.4. Acute toxicity study
2. Materials and methods
2.5. Repeated dose 90-day subchronic toxicity study
2.1. Animals
The subchronic toxicity was conducted in accordance with the OECD test guideline no. 408 (OECD, 1998) with slight modification. Both sexes of Sprague–Dawley rats were used and divided into four groups of 10 females and ten males for each group. Since the safety of the plant extract at the chronic oral doses up to 750 mg/kg/day has been reported (Attawish et al., 2004), therefore the dose of standardized Gynostemma pentaphyllum extract used for this study was chosen to be 1000 mg/kg/day. This dose is equivalent to 60 mg/kg the daily dose of gypenosides in rats which is 20–60 times of recommended daily dose of gypenosides for glucose and lipid lowering effects in human (Blumert and Liu, 1999). Both sexes of rats in the treated group received standardized Gynostemma pentaphyllum extract daily by gavage for 90 days. Rats in the control group received distilled water. Another set of equal number of rats, served as satellite groups, were treated with the same manner as described above and were kept for additional 28 days after treatment. Individual animal was observed daily for at least 1 h following treatment for signs of toxicity. All animals were checked for mortality, and all signs of illness and any behavioral changes related to treatment were recorded. Their food intake and body weight changes were monitored weekly. At the end of the study, all surviving rats were fasted overnight before anesthetization. Blood samples were collected and assayed for hematological and biochemical values. Hematological parameters including hemoglobin, hematocrit, platelets, white blood cell (WBC) count, and WBC differential count were determined using an automated hematology analyzer (Celltac E MEK-7222, Nihon Kohden, Tokyo, Japan). Serum biochemical parameters included glucose, blood urea nitrogen (BUN), creatinine, total protein, bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), albumin, and globulin. Immediately after blood collection, all rats were sacrificed and visceral organs such as lungs, heart, livers, spleen, pancreas, kidneys, adrenal glands, and sex organs were removed and blotted free of blood. They were then weighed to determine relative organ weight and observed for gross lesions. All tissues were preserved in 10% buffer formaldehyde solution for histological examination. Any changes in vital organs compared with those of control animals were recorded. Any rat that died during the test period was also examined for pathological changes.
Both sexes of Sprague–Dawley rats (8 weeks old) were obtained from the National Laboratory Animal Center (NLAC), Salaya, Mahidol University, Nakorn Pathom, Thailand. The animals were housed in a room maintained under automatically controlled conditions of 247 1 1C and a light–dark cycle of 12 h each. They were fed a standard laboratory diet (Pokphan Animal Feed Co., Ltd., Bangkok, Thailand) and water. All rats were acclimatized for 7 days before starting the experiment. At the end of the experiment, they were fasted overnight prior to the necropsy. The experimental protocol was approved by the Animal Ethics Committee in accordance with the guide for the care and use of laboratory animals by the Faculty of Medicine, Chiang Mai University, Thailand (Protocol number 3/2550).
2.2. Plant extract Gynostemma pentaphyllum was grown in Amphoe Mae-on, Chiang Mai, Thailand. The herbarium specimen (no. 010112) of Gynostemma pentaphyllum was authenticated and deposited at the Faculty of Pharmacy, Chiang Mai University, Thailand. The standardized Gynostemma pentaphyllum extract was prepared by Thai Traditional Medicines and Medicinal Plants Development Center, Department for Development of Thai Traditional and Alternative Medicine, Ministry of Public Health, Thailand. Briefly, aerial parts of Gynostemma pentaphyllum (12.2 kg) were washed thoroughly, cut into small pieces, dried in an oven at 40 1C and ground. Thereafter, they were extracted by boiling in distilled water (350 l) at 100 1C for 15 min and then filtered. The water extracting step was repeated. The filtrates were pooled, concentrated using a Rotary Evaporator and spray dried. The yellowish-brown dried extract powder was obtained (yield 16%) and monitored according to the guidelines of the FDA of Thailand for microbial, heavy metal and pesticide contaminations. Total gypenosides in the extract was measured by high performance liquid chromatography (HPLC) and found to be 6% as shown in Fig. 1. Total saponins in the extract was also determined and found to be 14.9%. The standardized extract was then stored in a desiccator (25 1C) until use.
The acute toxicity was performed in accordance with the OECD test guideline no. 420 (OECD, 2001). Female Sprague–Dawley rats were used and divided into two groups of five rats each. The first group served as control and orally received distilled water. The second group received standardized Gynostemma pentaphyllum extract in a single oral dose of 5000 mg/kg. All rats were fasted overnight prior to substance administration. Toxicity was assessed on the basis of mortality. The number of survival rats was recorded at 24 h after dosing and then kept for a further 14 days with a once daily observation of survivors. Body weight of individual rat was recorded before dosing and at weekly intervals thereafter. At the end of the experiment, all surviving animals were sacrificed and visceral organs were examined.
2.6. Statistical analysis 2.3. Dose preparation and administration The solution of standardized Gynostemma pentaphyllum extract was freshly prepared by dissolving in distilled water. All control groups received the vehicle, distilled water. All rats were dosed by oral gavage with an equivalent volume of 1 ml/100 g body weight.
All values were expressed as mean 7S.E.M. All statistical analyses were performed using the Statistical Package for Social Sciences (SPSS) version 11.0 (SPSS Inc., IL, USA). The significant differences between groups were evaluated by one-way analysis of variance (ANOVA) and post hoc least-significant difference (LSD) test. P values o0.05 were considered significant.
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Fig. 1. HPLC chromatograms of (A) commercially available crude (ethanol and water) extract of G. pentaphyllum containing 98% of gypenosides (Huzhou N.B.C. Biological Meterial CO., Ltd., China), and (B) water extract of G. pentaphyllum used in this study. The chromatograms were obtained at 203 nm Column, Gemini-NX C18 (250 mm 4.6 mm, 5 μm); Eluent, acetonitrile/water; Flow rate, 0.4 ml/min.
3. Results 3.1. Acute toxicity Standardized Gynostemma pentaphyllum extract given at the dose of 5000 mg/kg did not produce any signs of toxicity or mortality in rats. The body weight gain of the control and treated groups observed at 14 days after dosing was similar (data not shown). All rats were sacrificed and the gross examination showed the size and color appearance of internal organs of the treated group were similar to those of the control group. 3.2. Repeated dose 90-day subchronic toxicity During the experimental period, there were no death or toxic signs and symptoms in all groups of both sexes. There were no significant differences in food and water intake and body weight gain among 90-day subchronic, 118 days satellite and the control
groups. In the satellite groups that were kept for additional 28 days after treatment, the body weight gain increased normally (Table 1). There were no significant differences in the relative weight of internal organs between the treated and control groups (Table 2). There were no differences between groups in gross and microscopic examinations of internal organs. Table 3 shows the hematological values of rats in subchronic and satellite groups. There were no significant differences in hematological values among groups of male rats. In female rats, however, the extract caused significant increases in neutrophil and monocyte levels as compared to those of the control group. Only the female rats in the satellite group that the hemoglobin and hematocrit values of the extract-treated group were significantly higher than those of the control group. However, all these hematological values were in the range of normal rats (Carpenter, 2012). As shown in Table 4, the biochemical values of all groups of female rats were not significantly different. Significant differences in blood glucose and ALP values among the experimental groups
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Table 1 Body weight and weight gain of rats after subchronic treatment with standardized Gynostemma pentaphyllum extract. Controla
Gynostemma pentaphylluma
Controlb
Gynostemma pentaphyllumb
Female Body weight (g) Day 1 Day 30 Day 60 Day 90 Weight gain (g)
203.60 73.12 225.80 75.14 258.20 74.09 256.80 73.06 53.20 72.69
211.20 76.49 227.80 79.66 259.20 75.15 267.60 75.02 56.40 76.00
203.20 7 4.16 238.007 9.72 262.007 5.91 264.007 7.09 60.80 7 4.32
206.20 7 7.15 225.20 7 10.07 258.20 7 4.79 264.80 7 4.79 58.60 7 6.95
Male Body weight (g) Day 1 Day 30 Day 60 Day 90 Weight gain (g)
221.40 79.80 321.80 710.44 383.40 710.84 410.20 714.92 188.80 711.03
227.40 77.04 330.80 79.50 387.60 711.74 403.60 714.67 176.20 712.47
221.20 7 6.45 307.20 7 8.94 389.80 7 5.24 400.40 7 8.63 179.007 6.05
211.60 7 14.52 337.60 7 15.18 374.80 7 9.36 404.40 7 14.75 191.60 7 11.55
Values are expressed as mean7 S.E.M. (n¼ 10/sex). a b
Test group received distilled water or standardized Gynostemma pentaphyllum extract 1000 mg/kg daily for 90 days. Satellite group received distilled water or standardized Gynostemma pentaphyllum extract 1000 mg/kg daily for 90 days followed by no treatment for 28 days.
Table 2 Relative organ weight (g/100 g body weight) of internal organs of rats after subchronic treatment with standardized Gynostemma pentaphyllum extract. Organ
Controla
Gynostemma pentaphylluma
Controlb
Gynostemma pentaphyllumb
Female Lung Heart Liver Spleen Pancreas Kidney Adrenal Ovary Uterus
1.25 7 0.19 0.917 0.04 6.20 7 0.31 0.58 7 0.03 0.917 0.11 0.81 7 0.02 0.047 0.00 0.067 0.00 0.50 7 0.05
1.0770.04 0.95 70.02 6.43 70.15 0.56 70.02 0.93 70.08 0.86 70.02 0.047 0.00 0.067 0.00 0.45 70.03
1.15 70.05 0.86 7 0.03 6.28 7 0.36 0.59 7 0.08 0.87 7 0.12 0.87 7 0.02 0.047 0.00 0.077 0.00 0.50 7 0.06
1.357 0.06 0.92 7 0.02 6.42 7 0.10 0.617 0.02 1.03 7 0.13 0.86 7 0.02 0.047 0.00 0.0770.00 0.50 7 0.06
Male Lung Heart Liver Spleen Pancreas Kidney Adrenal Testis Epididymis
1.517 0.16 1.25 7 0.03 9.30 7 0.39 0.767 0.04 0.96 7 0.05 1.157 0.03 0.03 7 0.00 1.86 7 0.07 0.727 0.04
1.377 0.05 1.30 7 0.04 9.82 70.41 0.727 0.03 0.94 70.06 1.157 0.03 0.047 0.00 1.90 7 0.05 0.777 0.03
1.56 70.11 1.22 70.02 9.177 0.27 0.727 0.03 0.93 7 0.06 1.077 0.02 0.047 0.00 1.717 0.05 0.687 0.03
1.46 7 0.06 1.317 0.03# 9.78 7 0.49 0.747 0.02 0.917 0.05 1.127 0.03# 0.047 0.00 1.74 70.04 0.69 7 0.02
Values are expressed as mean7 S.E.M. (n¼ 10/sex). a Test group received distilled water or standardized Gynostemma pentaphyllum extract 1000 mg/kg daily for 90 days. b Satellite group received distilled water or standardized Gynostemma pentaphyllum extract 1000 mg/kg daily for 90 days followed by no treatment for 28 days. # Significantly different from controlb: po 0.05.
were evident only in male rats. However, these blood biochemistry levels were in the range of those of normal rats. Moreover, there were no treatment-related histopathological findings in the visceral organs (including liver and kidneys) of the rats in all groups (Fig. 2).
4. Discussion In acute toxicity test in rats, a single oral administration of standardized Gynostemma pentaphyllum extract at a dose of 5000 mg/kg body weight did not produce mortality or any
hazardous effect. This indicates that standardized Gynostemma pentaphyllum extract is fairly non-toxic. In traditional medicine practice, the use of Gynostemma pentaphyllum extract for adaptogenic and therapeutic purposes is usually based on gypenoside content, with a recommended daily dose of 60–180 mg (or 1–3 mg/kg) gypenosides for blood glucose and lipid lowering effects (Blumert and Liu, 1999). In our subchronic toxicity study, the dose of standardized Gynostemma pentaphyllum extract was 1000 mg/kg/day, which contained gypenosides of about 20–60 times the human dose. The daily oral treatment of standardized Gynostemma pentaphyllum extract at this dose did not cause death or any toxic signs and symptoms. No marked changes in body weight, food intake, or rat behaviors were observed in the extract-treated rats. Moreover, no abnormal signs of internal organs were observed by gross examination. These results indicate that repeated doses of standardized Gynostemma pentaphyllum extract did not significantly alter the appetite or metabolism of the treated animals. As blood is an index of physiological and pathological status, changes of hematological parameters and serum biochemical values may indicate toxic effects of a test compound. In the present subchronic toxicity study, the significant increase in neutrophil and monocyte levels as well as hemoglobin and hematocrit values were observed only in 90-day Gynostemma pentaphyllum treated female rats and female satellite group, respectively. However, these values were within the ranges of historical normal rats (Carpenter, 2012). Other parameters were slightly but not significantly different from those of their respective control groups. These results indicate that standardized Gynostemma pentaphyllum extract is not harmful to the hematopoietic system. For serum biochemical analysis, significant differences in ALP and serum glucose values among the experimental groups were found only in male subchronic and male satellite groups, respectively. The expected hypoglycemic effect was, however, not observed in standardized Gynostemma pentaphyllum extract treated groups. This is in contrast to the blood glucose lowering and insulin stimulating effects of Gynostemma pentaphylum extract as well as its gypenosides demonstrated in earlier studies (Norberg et al., 2004; Hoa et al., 2007). However, our negative result on the hypoglycemic effect is in accordance with the study of Attawish et al. (2004). Moreover, it has been reported that Gynostemma pentaphylum does not produce hypoglycemic effect in lean animals with normal blood glucose regulation (Megalli et al., 2006). In the present study, ALP value of the subchronic group was slightly but
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Table 3 Hematological values of rats after subchronic treatment with standardized Gynostemma pentaphyllum extract. Parameter
Controla
Gynostemma pentaphylluma
Controlb
Gynostemma pentaphyllumb
Female Hemoglobin (G/L) Hematocrit (L/L) WBC ( 109/L) Neutrophil (%) Lymphocyte (%) Monocyte (%) Eosinophil (%) Basophil (%)
154.80 7 2.63 0.477 0.01 4.93 7 0713 17.50 7 2.88 81.20 7 3.27 1.007 0.45 0.20 7 0.13 1.007 1.00
160.90 7 2.16 0.497 0.01 4.92 7 0.69 26.50 7 3.34n 73.007 3.25 0.20 7 0.20n 0.30 7 0.21 0.007 0.00
157.50 73.89 0.49 70.01 2.78 70.34 24.00 73.50 57.7073.65 0.60 70.40 0.20 70.13 0.00 70.00
169.80 7 2.38# 0.53 7 0.01# 2.84 7 0.26 25.107 2.38 74.40 7 2.30 0.30 7 0.21 0.20 7 0.13 0.007 0.00
Male Hemoglobin (G/L) Hematocrit (L/L) WBC ( 109/L) Neutrophil (%) Lymphocyte (%) Monocyte (%) Eosinophil (%) Basophil (%)
162.20 7 3.70 0.50 7 0.01 7.43 7 1.04 20.80 7 2.60 77.20 7 2.56 1.707 0.42 0.20 7 0.13 0.007 0.00
156.50 7 3.94 0.50 7 0.01 11.017 2.43 25.90 7 3.87 72.90 7 3.84 0.90 7 0.50 0.30 7 0.30 0.007 0.00
148.10 72.35 0.48 70.01 3.81 70.38 20.7072.20 77.50 72.22 1.20 70.53 0.50 70.22 0.00 70.00
154.30 7 2.34 0.497 0.01 3.95 7 0.33 18.50 7 2.16 80.90 7 2.15 0.30 7 0.15 0.30 7 0.15 0.007 0.00
Values are expressed as mean7 S.E.M. (n¼ 10/sex). a
Test group received distilled water or standardized Gynostemma pentaphyllum extract 1000 mg/kg daily for 90 days. Satellite group received distilled water or standardized Gynostemma pentaphyllum extract 1000 mg/kg daily for 90 days followed by no treatment for 28 days. n Significantly different from controla: p o0.05. # Significantly different from controlb: po 0.05. b
Table 4 Blood chemistry values of rats after subchronic treatment with standardized Gynostemma pentaphyllum extract. Parameter
Controla
Gynostemma pentaphylluma
Controlb
Gynostemma pentaphyllumb
Female BUN (mg/dl) Creatinine (mg/dl) AST (U/L) ALT (U/L) ALP (U/L) Direct bilirubin (mg/dl) Total bilirubin (mg/dl) Total protein (g/dl) Albumin (g/dl) Globulin (g/dl) Glucose (mmol/L)
22.90 7 1.35 0.767 0.07 150.90 7 17.46 51.80 7 7.88 38.50 7 8.46 0.09 7 0.01 0.417 0.09 6.25 7 0.27 3.95 7 0.17 2.30 7 0.13 6.017 0.55
23.40 7 1.27 0.717 0.03 176.60 7 16.86 58.707 11.25 31.007 2.61 0.107 0.00 0.29 7 0.06 6.32 7 0.21 3.87 7 0.12 2.45 7 0.11 5.077 0.56
27.707 1.65 0.63 7 0.03 156.20 7 6.28 42.80 7 4.42 24.007 3.01 0.107 0.00 0.167 0.02 5.79 7 0.13 3.277 0.09 2.52 7 0.06 4.96 7 0.16
24.90 70.96 0.63 70.03 148.80 77.83 40.40 74.02 29.00 71.60 0.10 70.00 0.22 70.04 6.22 70.10 3.48 70.08 2.73 70.06 4.44 70.16
Male BUN (mg/dl) Creatinine (mg/dl) AST (U/L) ALT (U/L) ALP (U/L) Direct bilirubin (mg/dl) Total bilirubin (mg/dl) Total protein (g/dl) Albumin (g/dl) Globulin (g/dl) Glucose (mmol/L)
20.30 7 1.17 0.58 7 0.03 155.90 7 8.10 48.807 4.186 47.50 7 3.95 0.107 0.00 0.317 0.04 6.247 0.21 3.84 7 0.10 2.40 7 0.15 6.127 0.50
23.107 1.38 0.59 7 0.02 167.107 11.79 58.30 7 9.38 65.40 7 7.88n 0.107 0.00 0.277 0.04 6.40 7 0.17 3.81 7 0.12 2.59 7 0.09 6.88 7 0.63
17.60 7 1.04 0.60 7 0.02 126.007 6.71 39.50 7 3.10 39.107 4.59 0.107 0.00 0.217 0.03 5.86 7 0.07 3.56 7 0.04 2.30 7 0.07 7.58 7 0.62
19.90 70.64 0.58 70.02 137.30 76.64 52.00 74.64 55.60 73.41 0.10 70.00 0.13 70.02 5.66 70.09 3.41 70.07 2.25 70.05 5.73 70.33#
Values are expressed as mean 7S.E.M. (n¼ 10/sex). a
Test group received distilled water or standardized Gynostemma pentaphyllum extract 1000 mg/kg daily for 90 days. Satellite group received distilled water or standardized Gynostemma pentaphyllum extract 1000 mg/kg daily for 90 days followed by no treatment for 28 days. n Significantly different from controla: p o 0.05. # Significantly different from controlb: p o0.05. b
significantly increased when compared to that of the control group. However, this value was still in range of normal rats (Carpenter, 2012) and there was no pathological lesion in the rat livers, therefore this change may not be of clinical significance. As the blood analysis and the histopathological examination of visceral organs including the livers did not show any abnormalities related to the extract treatment, the obtained results indicate
that standardized Gynostemma pentaphyllum extract is relatively harmless. The main limitations of the present study are as follows. Firstly, this study was done in only one animal species. For preclinical evaluation of drug safety, rats are the preferred animals. However, other animal species, especially nonrodent animals, should be used. Secondly, the crude plant extract was measured for only total
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Control group
G. pentaphyllum extract treated group
233
G. pentaphyllum extract satellite group
Liver
Kidney
Fig. 2. Histological sections of rat livers and kidneys (hematoxylin–eosin stain).
gypenosides, the active component of Gynostemma pentaphyllum. Other compounds, especially toxic constituents in this extract were not determined. Recently, cucurbitacins existed in Lagenaria siceraria, which is the plant in same family of Gynostemma pentaphyllum (Cucurbitaceae family), have been reported to produce toxic effect to mammals (Ferguson et al., 1983; Sharma et al., 2012). Consumption of Lagenaria siceraria juice containing cucurbitacins caused vomiting, abdominal pain, diarrhea, and death (Sharma et al., 2012). Although other potentially toxic compounds (especially cucurbitacins) in this extract were not determined, but if they were present, their toxic effects should be revealed in the toxicity tests.
5. Conclusion The present study demonstrates that standardized Gynostemma pentaphyllum extract does not cause death or any toxic effects after acute and subchronic treatment in rats.
Acknowledgments This work is financially supported by Thai Traditional Medical Knowledge Fund, Department for Development of Thai Traditional and Alternative Medicine, Ministry of Public Health, Thailand. We would like to acknowledge Luang San Kam Phaeng Forest Industry Organization, Ministry of Natural Resource and Environment, for providing Gynostemma pentaphyllum. We also would like to express our special gratitude to Thai Traditional Medicine Development Center (TDC) for preparing the water extract of Gynostemma pentaphyllum. We wish to thank Dr. Khanittha Taneyhill, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, for her help in HPLC analysis. Special thanks are also extended to the Faculty of Medicine, Chiang Mai University, for their support to this work.
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Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Toxicity evaluation of standardized extract of Gynostemma pentaphyllum Makino Natthakarn Chiranthanut a,b,n, Supanimit Teekachunhatean a,b, Ampai Panthong c, Parirat Khonsung a, Duangta Kanjanapothi a, Nirush Lertprasertsuk d a
Department of Pharmacology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand Center of Thai Traditional & Complementary Medicine, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand c Department of Pharmacology & Center of Excellence for Innovation in Chemistry, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand d Department of Pathology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand b
art ic l e i nf o
a b s t r a c t
Article history: Received 13 March 2013 Received in revised form 8 May 2013 Accepted 13 June 2013 Available online 21 June 2013
Ethnopharmacological relevance: To evaluate the safety of standardized extract of Gynostemma pentaphyllum in rats. Materials and methods: The water extract of Gynostemma pentaphyllum was prepared and standardized, the dry powder yielded 6% gypenosides. In the acute oral toxicity test, the single oral dose of 5000 mg/kg of Gynostemma pentaphyllum extract was given to female Sprague–Dawley rats. In subchronic toxicity test, the oral dose of 1000 mg/kg/day of the extract was given to rats in treatment and satellite groups for 90 days. Satellite groups of both sexes were kept for additional 28 days after 90-day treatment. Control rats received distilled water. Results: Standardized extract of Gynostemma pentaphyllum did not cause death or any toxic signs in rats. The daily administration of the extract for 90 days did not produce lethal or harmful effects. Although certain hematological and blood chemistry values (i.e., neutrophil, monocyte, glucose, and serum alkaline phosphatase levels) were found to be statistically different from the control group, however; these values were within the ranges of normal rats. Conclusion: Standardized extract of Gynostemma pentaphyllum did not produce mortality or any abnormality in rats. & 2013 Elsevier B.V. All rights reserved.
Keywords: Gynostemma pentaphyllum Makino Safety Toxicity Acute Subchronic
1. Introduction Gynostemma pentaphyllum Makino (family Cucurbitaceae), commonly known in China as Jiaogulan, is a perennial liana growing widely throughout Asian countries. It is well known as medicinal plant with powerful biological effects that lead to longevity (Blumert and Liu, 1999). In traditional medicine, the aerial part of this plant is commonly used for a variety of diseases such as diabetes mellitus, cancer, gastritis, bronchitis, and hypertension (Blumert and Liu, 1999; Bansiddhi, 2005). Previous experimental studies showed that extracts of Gynostemma pentaphyllum produce various pharmacological effects, including antidiabetic (Yeo et al., 2008), hypolipidemic (Megalli et al., 2006), antioxidant
Abbreviations: ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; HPLC, high performance liquid chromatography; LDL, low density lipoprotein; WBC, white blood cell. n Corresponding author at: Department of Pharmacology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand. Tel.: +66 53 945352, +66 53 945354; fax: +66 53 945355. E-mail addresses: [email protected], [email protected] (N. Chiranthanut). 0378-8741/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jep.2013.06.027
(Müller et al., 2012) and antitumor (Schild et al., 2010) effects. From phytochemical studies, Gynostemma pentaphyllum contains saponins (Yin et al., 2006; Kao et al., 2008; Shi et al., 2012), flavonoids (Kao et al., 2008), polysaccharides (Chi et al., 2012; Yan et al., 2013), amino acids and vitamins (Zheng, 2004), and some essential elements (Nookabkaew et al., 2006). Gypenosides and dammarane-type glycosides, are the major constituents of Gynostemma pentaphyllum (Piacente et al., 1995; Cui et al., 1999; Zhang et al., 2010). Nowadays, more than 100 types of gypenosides in this plant have been found (Yin et al., 2006; Hu et al., 2010; Ky et al., 2010; Kim and Han, 2011). These compounds have been reported to possess antioxidant (Li et al., 1993; Shang et al., 2006), antitumor (Yin et al., 2006; Lin et al., 2011; Lu et al., 2012), neuroprotective (Wang et al., 2010), and antiinflammatory activities (Lin et al., 1993). Moreover, glucose lowering (Norberg et al., 2004) and hypolipidemic effects (Huang et al., 2006) of these compounds have also been demonstrated. Chronic toxicity of Gynostemma pentaphyllum with doses up to 750 mg/kg in rats has been previously reported with no obvious toxicity and mortality (Attawish et al., 2004). However, the amount of gypenosides in the water extract used in that study
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was not quantified and standardized, therefore the variability of gypenoside content and the exact doses of gypenosieds could not be accounted for. Standardized Gynostemma pentaphyllum extract with quantitative control of gypenoside content has recently been validated in our laboratory. In the present study, the standardized formulation of water extract of Gynostemma pentaphyllum was used for acute and subchronic toxicity evaluation in rats as a prelude to future clinical study. The objective of the present study was to carry out basic toxic evaluation and to establish the safety of standardized extract of Gynostemma pentaphyllum. This article focuses on acute and subchronic toxicity evaluation of the standardized Gynostemma pentaphyllum extract in rats.
2.4. Acute toxicity study
2. Materials and methods
2.5. Repeated dose 90-day subchronic toxicity study
2.1. Animals
The subchronic toxicity was conducted in accordance with the OECD test guideline no. 408 (OECD, 1998) with slight modification. Both sexes of Sprague–Dawley rats were used and divided into four groups of 10 females and ten males for each group. Since the safety of the plant extract at the chronic oral doses up to 750 mg/kg/day has been reported (Attawish et al., 2004), therefore the dose of standardized Gynostemma pentaphyllum extract used for this study was chosen to be 1000 mg/kg/day. This dose is equivalent to 60 mg/kg the daily dose of gypenosides in rats which is 20–60 times of recommended daily dose of gypenosides for glucose and lipid lowering effects in human (Blumert and Liu, 1999). Both sexes of rats in the treated group received standardized Gynostemma pentaphyllum extract daily by gavage for 90 days. Rats in the control group received distilled water. Another set of equal number of rats, served as satellite groups, were treated with the same manner as described above and were kept for additional 28 days after treatment. Individual animal was observed daily for at least 1 h following treatment for signs of toxicity. All animals were checked for mortality, and all signs of illness and any behavioral changes related to treatment were recorded. Their food intake and body weight changes were monitored weekly. At the end of the study, all surviving rats were fasted overnight before anesthetization. Blood samples were collected and assayed for hematological and biochemical values. Hematological parameters including hemoglobin, hematocrit, platelets, white blood cell (WBC) count, and WBC differential count were determined using an automated hematology analyzer (Celltac E MEK-7222, Nihon Kohden, Tokyo, Japan). Serum biochemical parameters included glucose, blood urea nitrogen (BUN), creatinine, total protein, bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), albumin, and globulin. Immediately after blood collection, all rats were sacrificed and visceral organs such as lungs, heart, livers, spleen, pancreas, kidneys, adrenal glands, and sex organs were removed and blotted free of blood. They were then weighed to determine relative organ weight and observed for gross lesions. All tissues were preserved in 10% buffer formaldehyde solution for histological examination. Any changes in vital organs compared with those of control animals were recorded. Any rat that died during the test period was also examined for pathological changes.
Both sexes of Sprague–Dawley rats (8 weeks old) were obtained from the National Laboratory Animal Center (NLAC), Salaya, Mahidol University, Nakorn Pathom, Thailand. The animals were housed in a room maintained under automatically controlled conditions of 247 1 1C and a light–dark cycle of 12 h each. They were fed a standard laboratory diet (Pokphan Animal Feed Co., Ltd., Bangkok, Thailand) and water. All rats were acclimatized for 7 days before starting the experiment. At the end of the experiment, they were fasted overnight prior to the necropsy. The experimental protocol was approved by the Animal Ethics Committee in accordance with the guide for the care and use of laboratory animals by the Faculty of Medicine, Chiang Mai University, Thailand (Protocol number 3/2550).
2.2. Plant extract Gynostemma pentaphyllum was grown in Amphoe Mae-on, Chiang Mai, Thailand. The herbarium specimen (no. 010112) of Gynostemma pentaphyllum was authenticated and deposited at the Faculty of Pharmacy, Chiang Mai University, Thailand. The standardized Gynostemma pentaphyllum extract was prepared by Thai Traditional Medicines and Medicinal Plants Development Center, Department for Development of Thai Traditional and Alternative Medicine, Ministry of Public Health, Thailand. Briefly, aerial parts of Gynostemma pentaphyllum (12.2 kg) were washed thoroughly, cut into small pieces, dried in an oven at 40 1C and ground. Thereafter, they were extracted by boiling in distilled water (350 l) at 100 1C for 15 min and then filtered. The water extracting step was repeated. The filtrates were pooled, concentrated using a Rotary Evaporator and spray dried. The yellowish-brown dried extract powder was obtained (yield 16%) and monitored according to the guidelines of the FDA of Thailand for microbial, heavy metal and pesticide contaminations. Total gypenosides in the extract was measured by high performance liquid chromatography (HPLC) and found to be 6% as shown in Fig. 1. Total saponins in the extract was also determined and found to be 14.9%. The standardized extract was then stored in a desiccator (25 1C) until use.
The acute toxicity was performed in accordance with the OECD test guideline no. 420 (OECD, 2001). Female Sprague–Dawley rats were used and divided into two groups of five rats each. The first group served as control and orally received distilled water. The second group received standardized Gynostemma pentaphyllum extract in a single oral dose of 5000 mg/kg. All rats were fasted overnight prior to substance administration. Toxicity was assessed on the basis of mortality. The number of survival rats was recorded at 24 h after dosing and then kept for a further 14 days with a once daily observation of survivors. Body weight of individual rat was recorded before dosing and at weekly intervals thereafter. At the end of the experiment, all surviving animals were sacrificed and visceral organs were examined.
2.6. Statistical analysis 2.3. Dose preparation and administration The solution of standardized Gynostemma pentaphyllum extract was freshly prepared by dissolving in distilled water. All control groups received the vehicle, distilled water. All rats were dosed by oral gavage with an equivalent volume of 1 ml/100 g body weight.
All values were expressed as mean 7S.E.M. All statistical analyses were performed using the Statistical Package for Social Sciences (SPSS) version 11.0 (SPSS Inc., IL, USA). The significant differences between groups were evaluated by one-way analysis of variance (ANOVA) and post hoc least-significant difference (LSD) test. P values o0.05 were considered significant.
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Fig. 1. HPLC chromatograms of (A) commercially available crude (ethanol and water) extract of G. pentaphyllum containing 98% of gypenosides (Huzhou N.B.C. Biological Meterial CO., Ltd., China), and (B) water extract of G. pentaphyllum used in this study. The chromatograms were obtained at 203 nm Column, Gemini-NX C18 (250 mm 4.6 mm, 5 μm); Eluent, acetonitrile/water; Flow rate, 0.4 ml/min.
3. Results 3.1. Acute toxicity Standardized Gynostemma pentaphyllum extract given at the dose of 5000 mg/kg did not produce any signs of toxicity or mortality in rats. The body weight gain of the control and treated groups observed at 14 days after dosing was similar (data not shown). All rats were sacrificed and the gross examination showed the size and color appearance of internal organs of the treated group were similar to those of the control group. 3.2. Repeated dose 90-day subchronic toxicity During the experimental period, there were no death or toxic signs and symptoms in all groups of both sexes. There were no significant differences in food and water intake and body weight gain among 90-day subchronic, 118 days satellite and the control
groups. In the satellite groups that were kept for additional 28 days after treatment, the body weight gain increased normally (Table 1). There were no significant differences in the relative weight of internal organs between the treated and control groups (Table 2). There were no differences between groups in gross and microscopic examinations of internal organs. Table 3 shows the hematological values of rats in subchronic and satellite groups. There were no significant differences in hematological values among groups of male rats. In female rats, however, the extract caused significant increases in neutrophil and monocyte levels as compared to those of the control group. Only the female rats in the satellite group that the hemoglobin and hematocrit values of the extract-treated group were significantly higher than those of the control group. However, all these hematological values were in the range of normal rats (Carpenter, 2012). As shown in Table 4, the biochemical values of all groups of female rats were not significantly different. Significant differences in blood glucose and ALP values among the experimental groups
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Table 1 Body weight and weight gain of rats after subchronic treatment with standardized Gynostemma pentaphyllum extract. Controla
Gynostemma pentaphylluma
Controlb
Gynostemma pentaphyllumb
Female Body weight (g) Day 1 Day 30 Day 60 Day 90 Weight gain (g)
203.60 73.12 225.80 75.14 258.20 74.09 256.80 73.06 53.20 72.69
211.20 76.49 227.80 79.66 259.20 75.15 267.60 75.02 56.40 76.00
203.20 7 4.16 238.007 9.72 262.007 5.91 264.007 7.09 60.80 7 4.32
206.20 7 7.15 225.20 7 10.07 258.20 7 4.79 264.80 7 4.79 58.60 7 6.95
Male Body weight (g) Day 1 Day 30 Day 60 Day 90 Weight gain (g)
221.40 79.80 321.80 710.44 383.40 710.84 410.20 714.92 188.80 711.03
227.40 77.04 330.80 79.50 387.60 711.74 403.60 714.67 176.20 712.47
221.20 7 6.45 307.20 7 8.94 389.80 7 5.24 400.40 7 8.63 179.007 6.05
211.60 7 14.52 337.60 7 15.18 374.80 7 9.36 404.40 7 14.75 191.60 7 11.55
Values are expressed as mean7 S.E.M. (n¼ 10/sex). a b
Test group received distilled water or standardized Gynostemma pentaphyllum extract 1000 mg/kg daily for 90 days. Satellite group received distilled water or standardized Gynostemma pentaphyllum extract 1000 mg/kg daily for 90 days followed by no treatment for 28 days.
Table 2 Relative organ weight (g/100 g body weight) of internal organs of rats after subchronic treatment with standardized Gynostemma pentaphyllum extract. Organ
Controla
Gynostemma pentaphylluma
Controlb
Gynostemma pentaphyllumb
Female Lung Heart Liver Spleen Pancreas Kidney Adrenal Ovary Uterus
1.25 7 0.19 0.917 0.04 6.20 7 0.31 0.58 7 0.03 0.917 0.11 0.81 7 0.02 0.047 0.00 0.067 0.00 0.50 7 0.05
1.0770.04 0.95 70.02 6.43 70.15 0.56 70.02 0.93 70.08 0.86 70.02 0.047 0.00 0.067 0.00 0.45 70.03
1.15 70.05 0.86 7 0.03 6.28 7 0.36 0.59 7 0.08 0.87 7 0.12 0.87 7 0.02 0.047 0.00 0.077 0.00 0.50 7 0.06
1.357 0.06 0.92 7 0.02 6.42 7 0.10 0.617 0.02 1.03 7 0.13 0.86 7 0.02 0.047 0.00 0.0770.00 0.50 7 0.06
Male Lung Heart Liver Spleen Pancreas Kidney Adrenal Testis Epididymis
1.517 0.16 1.25 7 0.03 9.30 7 0.39 0.767 0.04 0.96 7 0.05 1.157 0.03 0.03 7 0.00 1.86 7 0.07 0.727 0.04
1.377 0.05 1.30 7 0.04 9.82 70.41 0.727 0.03 0.94 70.06 1.157 0.03 0.047 0.00 1.90 7 0.05 0.777 0.03
1.56 70.11 1.22 70.02 9.177 0.27 0.727 0.03 0.93 7 0.06 1.077 0.02 0.047 0.00 1.717 0.05 0.687 0.03
1.46 7 0.06 1.317 0.03# 9.78 7 0.49 0.747 0.02 0.917 0.05 1.127 0.03# 0.047 0.00 1.74 70.04 0.69 7 0.02
Values are expressed as mean7 S.E.M. (n¼ 10/sex). a Test group received distilled water or standardized Gynostemma pentaphyllum extract 1000 mg/kg daily for 90 days. b Satellite group received distilled water or standardized Gynostemma pentaphyllum extract 1000 mg/kg daily for 90 days followed by no treatment for 28 days. # Significantly different from controlb: po 0.05.
were evident only in male rats. However, these blood biochemistry levels were in the range of those of normal rats. Moreover, there were no treatment-related histopathological findings in the visceral organs (including liver and kidneys) of the rats in all groups (Fig. 2).
4. Discussion In acute toxicity test in rats, a single oral administration of standardized Gynostemma pentaphyllum extract at a dose of 5000 mg/kg body weight did not produce mortality or any
hazardous effect. This indicates that standardized Gynostemma pentaphyllum extract is fairly non-toxic. In traditional medicine practice, the use of Gynostemma pentaphyllum extract for adaptogenic and therapeutic purposes is usually based on gypenoside content, with a recommended daily dose of 60–180 mg (or 1–3 mg/kg) gypenosides for blood glucose and lipid lowering effects (Blumert and Liu, 1999). In our subchronic toxicity study, the dose of standardized Gynostemma pentaphyllum extract was 1000 mg/kg/day, which contained gypenosides of about 20–60 times the human dose. The daily oral treatment of standardized Gynostemma pentaphyllum extract at this dose did not cause death or any toxic signs and symptoms. No marked changes in body weight, food intake, or rat behaviors were observed in the extract-treated rats. Moreover, no abnormal signs of internal organs were observed by gross examination. These results indicate that repeated doses of standardized Gynostemma pentaphyllum extract did not significantly alter the appetite or metabolism of the treated animals. As blood is an index of physiological and pathological status, changes of hematological parameters and serum biochemical values may indicate toxic effects of a test compound. In the present subchronic toxicity study, the significant increase in neutrophil and monocyte levels as well as hemoglobin and hematocrit values were observed only in 90-day Gynostemma pentaphyllum treated female rats and female satellite group, respectively. However, these values were within the ranges of historical normal rats (Carpenter, 2012). Other parameters were slightly but not significantly different from those of their respective control groups. These results indicate that standardized Gynostemma pentaphyllum extract is not harmful to the hematopoietic system. For serum biochemical analysis, significant differences in ALP and serum glucose values among the experimental groups were found only in male subchronic and male satellite groups, respectively. The expected hypoglycemic effect was, however, not observed in standardized Gynostemma pentaphyllum extract treated groups. This is in contrast to the blood glucose lowering and insulin stimulating effects of Gynostemma pentaphylum extract as well as its gypenosides demonstrated in earlier studies (Norberg et al., 2004; Hoa et al., 2007). However, our negative result on the hypoglycemic effect is in accordance with the study of Attawish et al. (2004). Moreover, it has been reported that Gynostemma pentaphylum does not produce hypoglycemic effect in lean animals with normal blood glucose regulation (Megalli et al., 2006). In the present study, ALP value of the subchronic group was slightly but
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Table 3 Hematological values of rats after subchronic treatment with standardized Gynostemma pentaphyllum extract. Parameter
Controla
Gynostemma pentaphylluma
Controlb
Gynostemma pentaphyllumb
Female Hemoglobin (G/L) Hematocrit (L/L) WBC ( 109/L) Neutrophil (%) Lymphocyte (%) Monocyte (%) Eosinophil (%) Basophil (%)
154.80 7 2.63 0.477 0.01 4.93 7 0713 17.50 7 2.88 81.20 7 3.27 1.007 0.45 0.20 7 0.13 1.007 1.00
160.90 7 2.16 0.497 0.01 4.92 7 0.69 26.50 7 3.34n 73.007 3.25 0.20 7 0.20n 0.30 7 0.21 0.007 0.00
157.50 73.89 0.49 70.01 2.78 70.34 24.00 73.50 57.7073.65 0.60 70.40 0.20 70.13 0.00 70.00
169.80 7 2.38# 0.53 7 0.01# 2.84 7 0.26 25.107 2.38 74.40 7 2.30 0.30 7 0.21 0.20 7 0.13 0.007 0.00
Male Hemoglobin (G/L) Hematocrit (L/L) WBC ( 109/L) Neutrophil (%) Lymphocyte (%) Monocyte (%) Eosinophil (%) Basophil (%)
162.20 7 3.70 0.50 7 0.01 7.43 7 1.04 20.80 7 2.60 77.20 7 2.56 1.707 0.42 0.20 7 0.13 0.007 0.00
156.50 7 3.94 0.50 7 0.01 11.017 2.43 25.90 7 3.87 72.90 7 3.84 0.90 7 0.50 0.30 7 0.30 0.007 0.00
148.10 72.35 0.48 70.01 3.81 70.38 20.7072.20 77.50 72.22 1.20 70.53 0.50 70.22 0.00 70.00
154.30 7 2.34 0.497 0.01 3.95 7 0.33 18.50 7 2.16 80.90 7 2.15 0.30 7 0.15 0.30 7 0.15 0.007 0.00
Values are expressed as mean7 S.E.M. (n¼ 10/sex). a
Test group received distilled water or standardized Gynostemma pentaphyllum extract 1000 mg/kg daily for 90 days. Satellite group received distilled water or standardized Gynostemma pentaphyllum extract 1000 mg/kg daily for 90 days followed by no treatment for 28 days. n Significantly different from controla: p o0.05. # Significantly different from controlb: po 0.05. b
Table 4 Blood chemistry values of rats after subchronic treatment with standardized Gynostemma pentaphyllum extract. Parameter
Controla
Gynostemma pentaphylluma
Controlb
Gynostemma pentaphyllumb
Female BUN (mg/dl) Creatinine (mg/dl) AST (U/L) ALT (U/L) ALP (U/L) Direct bilirubin (mg/dl) Total bilirubin (mg/dl) Total protein (g/dl) Albumin (g/dl) Globulin (g/dl) Glucose (mmol/L)
22.90 7 1.35 0.767 0.07 150.90 7 17.46 51.80 7 7.88 38.50 7 8.46 0.09 7 0.01 0.417 0.09 6.25 7 0.27 3.95 7 0.17 2.30 7 0.13 6.017 0.55
23.40 7 1.27 0.717 0.03 176.60 7 16.86 58.707 11.25 31.007 2.61 0.107 0.00 0.29 7 0.06 6.32 7 0.21 3.87 7 0.12 2.45 7 0.11 5.077 0.56
27.707 1.65 0.63 7 0.03 156.20 7 6.28 42.80 7 4.42 24.007 3.01 0.107 0.00 0.167 0.02 5.79 7 0.13 3.277 0.09 2.52 7 0.06 4.96 7 0.16
24.90 70.96 0.63 70.03 148.80 77.83 40.40 74.02 29.00 71.60 0.10 70.00 0.22 70.04 6.22 70.10 3.48 70.08 2.73 70.06 4.44 70.16
Male BUN (mg/dl) Creatinine (mg/dl) AST (U/L) ALT (U/L) ALP (U/L) Direct bilirubin (mg/dl) Total bilirubin (mg/dl) Total protein (g/dl) Albumin (g/dl) Globulin (g/dl) Glucose (mmol/L)
20.30 7 1.17 0.58 7 0.03 155.90 7 8.10 48.807 4.186 47.50 7 3.95 0.107 0.00 0.317 0.04 6.247 0.21 3.84 7 0.10 2.40 7 0.15 6.127 0.50
23.107 1.38 0.59 7 0.02 167.107 11.79 58.30 7 9.38 65.40 7 7.88n 0.107 0.00 0.277 0.04 6.40 7 0.17 3.81 7 0.12 2.59 7 0.09 6.88 7 0.63
17.60 7 1.04 0.60 7 0.02 126.007 6.71 39.50 7 3.10 39.107 4.59 0.107 0.00 0.217 0.03 5.86 7 0.07 3.56 7 0.04 2.30 7 0.07 7.58 7 0.62
19.90 70.64 0.58 70.02 137.30 76.64 52.00 74.64 55.60 73.41 0.10 70.00 0.13 70.02 5.66 70.09 3.41 70.07 2.25 70.05 5.73 70.33#
Values are expressed as mean 7S.E.M. (n¼ 10/sex). a
Test group received distilled water or standardized Gynostemma pentaphyllum extract 1000 mg/kg daily for 90 days. Satellite group received distilled water or standardized Gynostemma pentaphyllum extract 1000 mg/kg daily for 90 days followed by no treatment for 28 days. n Significantly different from controla: p o 0.05. # Significantly different from controlb: p o0.05. b
significantly increased when compared to that of the control group. However, this value was still in range of normal rats (Carpenter, 2012) and there was no pathological lesion in the rat livers, therefore this change may not be of clinical significance. As the blood analysis and the histopathological examination of visceral organs including the livers did not show any abnormalities related to the extract treatment, the obtained results indicate
that standardized Gynostemma pentaphyllum extract is relatively harmless. The main limitations of the present study are as follows. Firstly, this study was done in only one animal species. For preclinical evaluation of drug safety, rats are the preferred animals. However, other animal species, especially nonrodent animals, should be used. Secondly, the crude plant extract was measured for only total
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Control group
G. pentaphyllum extract treated group
233
G. pentaphyllum extract satellite group
Liver
Kidney
Fig. 2. Histological sections of rat livers and kidneys (hematoxylin–eosin stain).
gypenosides, the active component of Gynostemma pentaphyllum. Other compounds, especially toxic constituents in this extract were not determined. Recently, cucurbitacins existed in Lagenaria siceraria, which is the plant in same family of Gynostemma pentaphyllum (Cucurbitaceae family), have been reported to produce toxic effect to mammals (Ferguson et al., 1983; Sharma et al., 2012). Consumption of Lagenaria siceraria juice containing cucurbitacins caused vomiting, abdominal pain, diarrhea, and death (Sharma et al., 2012). Although other potentially toxic compounds (especially cucurbitacins) in this extract were not determined, but if they were present, their toxic effects should be revealed in the toxicity tests.
5. Conclusion The present study demonstrates that standardized Gynostemma pentaphyllum extract does not cause death or any toxic effects after acute and subchronic treatment in rats.
Acknowledgments This work is financially supported by Thai Traditional Medical Knowledge Fund, Department for Development of Thai Traditional and Alternative Medicine, Ministry of Public Health, Thailand. We would like to acknowledge Luang San Kam Phaeng Forest Industry Organization, Ministry of Natural Resource and Environment, for providing Gynostemma pentaphyllum. We also would like to express our special gratitude to Thai Traditional Medicine Development Center (TDC) for preparing the water extract of Gynostemma pentaphyllum. We wish to thank Dr. Khanittha Taneyhill, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, for her help in HPLC analysis. Special thanks are also extended to the Faculty of Medicine, Chiang Mai University, for their support to this work.
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