Evidence-Based Complementary and Alternative Medicine

Evidence-Based Complementary and Alternative Medicine / 2013 / Article

Research Article | Open Access

Volume 2013 |Article ID 707285 | https://doi.org/10.1155/2013/707285

Basak Ozlem Perk, Sinem Ilgin, Ozlem Atli, Hale Gamze Duymus, Basar Sirmagul, "Acute and Subchronic Toxic Effects of the Fruits of Physalis peruviana L.", Evidence-Based Complementary and Alternative Medicine, vol. 2013, Article ID 707285, 10 pages, 2013. https://doi.org/10.1155/2013/707285

Acute and Subchronic Toxic Effects of the Fruits of Physalis peruviana L.

Academic Editor: I-Min Liu
Received05 Sep 2013
Accepted27 Oct 2013
Published28 Nov 2013

Abstract

The fruit of Physalis peruviana L. (PPL) has been traditionally used as antispasmodic, diuretic, antiseptic, sedative, and analgesic all over the world. We aimed to perform qualitative content analysis of the fruits of PPL and to clarify the in vitro genotoxicity and in vivo acute and subchronic toxicity of the fruit. Lyophilized fruit juice does not induce genetic damage. In the acute toxicity studies, LD50 value of the fruit was found to be more than 5000 mg kg−1 for both sexes. According to the subchronic toxicity studies, hepatic, renal, and hematological toxic effects were not induced in both sexes. Plasma troponin I (only in the group treated with 5000 mg kg−1 of lyophilized fruit juice) and troponin T levels were significantly increased in male groups treated with lyophilized fruit juice compared to the control group. Furthermore, potassium level was significantly increased in the male group treated with 5000 mg kg−1 of lyophilized fruit juice. These findings were considered to indicate the myocardial damage particularly in the male group treated with 5000 mg kg−1 of lyophilized fruit juice. In conclusion, lyophilized fruit juice of PPL is shown to induce cardiac toxicity only at high doses and in male gender.

1. Introduction

Herbs and herbal preparations are in use for the treatment of various diseases throughout human history. According to the World Health Organization (WHO) statistics, 70–80% of the world’s population appeals to plant-derived traditional treatment methods for the solution of health problems [1, 2]. However, it is well known that consumption of plants and plant products, of which the content and the toxicity profile and safe dose were not determined, by humans and animals may cause severe toxicity problems [2].

The fruits of Physalis peruviana L. (Solanaceae) are also named as goldenberry, gooseberry, cape gooseberry, and wintercherry fruits all over the world [3, 4]. Physalis species are grown naturally and cultured in a wide range of countries including North and South African countries, India, Australia, New Zealand, Ecuador, Venezuela, Colombia, Chile, and Peru [4]. In Turkey, Physalis pubescens L. is grown in Artvin region, Physalis alkekengi L. is grown in the regions of Kutahya, Antalya, Bitlis, Istanbul, Sakarya, Samsun, and Karaman, and the fruit of Physalis peruviana L. is cultured in Antalya [5]. The fruit contains polyunsaturated fatty acids, carbohydrates, A, B, C, E, and K1 vitamins, phytosterols, essential minerals (phosphorus, iron, potassium, and zinc), physalin, and withanolides [4, 69].

The fruit of Physalis peruviana L. is used among people as antispasmodic, diuretic, antiseptic, sedative, and analgesic as well as in the treatment of throat infections and elimination of intestinal parasites and amoeba and to strengthen the optic nerve [10], but, in Turkey, fresh or dried fruits and preparations containing fruit extract of Physalis peruviana L. are frequently used, in the aim of weight loss in recent years. Case studies showed that fruits might cause side effects such as hypertension, ventricular tachycardia, and manic episode in our country [1113]. Furthermore, biological activity assay carried out on the fruit, stem, and leaves of the plant has demonstrated that it has hepatoprotective and antioxidant effects as well as cytotoxic effects on certain cancer cell lines [4, 1419].

It is interesting that the toxicity studies on the fruits of Physalis peruviana L.—a widely used plant in different geographical regions all over the world—are lacking. Our study aimed to determine the possible acute toxic effects of the fruit by using in vivo animal models and subchronic toxicity at the organ level as well as to assess the in vivo genotoxicity of the fruits of Physalis peruviana L.

2. Materials and Methods

2.1. Materials

Cultured fruits were obtained from Antalya, Turkey. The fresh fruits were separated from their calyxes and they were cut into small pieces. These pieces were homogenized. After homogenization, the pulp was filtered in the cold. Meanwhile, the unsoluble particles were seperated from the juice. The freeze-dried fruit juice was kept in a well-closed amber-colored flacon until performing the assays. During this study, totally 20 Kg fresh fruits were prepared for the experiments and 3.9 Kg extract was obtained. The extraction yield was calculated as 19.5% of the fresh fruits.

The chemicals used were obtained from the following sources: ketamine (Ketalar) (Phizer, Turkey); xylazine (Bayer, Turkey). For the measurements of lactate dehydrogenate (LDH) (BioAssay Systems, CA, USA), creatinine kinase-MB (CK-MB) (BioCheck, CA, USA), troponin I (TI) (BioCheck, CA, USA) and troponin T (TT) (Cusabio, China) levels ELISA kits were used according to the manufacturer’s instructions in plasma samples. For the measurements of potassium and sodium levels ELISA kits from Diazyme Laboratories, CA, USA, were used. Blood aspartate amino transferase (AST), alanine amino transferase (ALT), alkaline phosphatase (AP), total and direct bilirubin, albumin, total protein, urea, creatinine, and glucose levels were determined by colorimetric kits from Biolabo SA (Châtel-St-Denis, Switzerland). Genotoxicity was evaluated by umuC assay using umuC Easy CS Kit (Xenometrix AG, Gewerbertrasse, Switzerland).

2.2. Phytochemical Screening

Phytochemical tests were carried out using lyophilized fruit juice of Physalis peruviana L. to identify the constituents by using standard colorimetric methods: carbohydrates (Fehling’s and Molisch’s reactions), tannins (Ferric chloride reaction), basic alkaloids (Mayer’s, Dragendorff and Hager’s reactions), tropane alkaloids (Vitali Morin reaction), polyuronides (precipitation with acetone), proteins (Biuret and Ninhydrin’s assays), carotenoids (Carr Price’s reaction), fixed oil (Sudan red reaction), saponins (Salkowski and Lieberman-Burchard’s reactions), cyanogenetic glucoside (Sodium picrate assay), flavon glucoside (Shibata’s reaction), and cardiotonic glucoside (Baljet and Kedde’s reactions) [2023].

2.3. umuC Assay

Salmonella typhimurium strain TA1535/pSK1002 was used for the assay. TG medium (200 μL) was added to the vial to obtain homogenous suspensions of Salmonella strain. 10 μL of ampicillin (50 mg/mL) was added to 10 mL TG medium (=TGA medium) in 50 mL culture tubes. 50 μL of Salmonella suspension was mixed with 10 mL TGA medium. Negative control was devoid of Salmonella TA1535/pSK1002. The culture tubes were loosely capped, to allow aeration, and incubated in a shaker (SI-600, Jeio Tech, Korea) at 37°C, 250 rpm for 14–16 h. The “overnight grown” cultures were diluted 10 times with TG medium and the absorbance was measured at 600 nm. Positive controls were prepared with S9 (2-aminoanthracene) and without S9 (4-nitroquinoline). TGA medium and S9 fraction were added to each well of plate. Then, lyophilized fruit juice of PPL was added to wells at 5, 2.5, 1.25, and 0.63 mg/mL concentrations. The plates were incubated at 37°C, 120–150 rpm for 2 hours. During the 2 hours, a second plate was prepared with TG medium with freshly added ampicillin to all wells (for 1 plate: 28 μL ampicillin stock (50 mg/mL) to 28 mL TG medium). After 2 hours, 30 μL of the contents of the first plate was transferred to the second plate. OD600 of the second plate was read. Then, the second plate was incubated at 37°C, 120–150 rpm for 2 hours. During the 2 hours, a third plate was prepared with 150 μL B-buffer/ONPG (o-nitrophenyl-β-D-galactopyranoside) mixture (for 1 plate: 15 mL of B buffer, 40.5 μL 2-mercaptoethanol, and 1 mL ONPG solution) and prewarmed to 28°C. At the end of the 2 hours of incubation, the second plate was mixed and OD600 of the plate was read. Then, 30 μL of each well of the second plate was transferred to the third plate. The third plate was incubated at 28°C, 120–150 rpm for 30 minutes. After 30 minutes, 120 μL of stop reagent was added to each well. The plate was mixed and OD420 of the plate was read. The results were determined with umuC Easy CS Excel Programme.

2.4. Experimental Design
2.4.1. Animals

Female-male Wistar rats weighing 200–250 g were obtained from our own animal facility. Rats were housed under controlled temperature (22°C) and lighting (12/12-hour light dark cycle) with free access to food and water. The experimental protocol was approved by the Local Ethical Committee on Animal Experimentation of Anadolu University, Eskisehir, Turkey.

2.4.2. Acute Oral Toxicity Study

Acute toxicity tests were performed in accordance with the 2008 regulations of Organization for Economic Cooperation and Development (OECD Test Guideline: 423). Lyophilized fruit juice of PPL was dissolved in distilled water and orally administered to 5 male and 5 female rats at a dose of 5000 mg kg−1. The animals were monitored during a 24-hour period for clinical symptoms (aggression, sedation, somnolence, tremor, catatonia, paralysis, convulsions, changes in skin, eyes and mucosal membranes, asphyxia, salivation, and diarrhea). Following the 24 hours, number of dead animals from each group was recorded. Because there were no dead male or female rats at the end of this period, they were subjected to a 14-day observation period. After this period, the rats were killed by exposure to high dose of ether anesthesia and liver; kidney, heart, and lung tissues of the animals were evaluated macroscopically.

2.4.3. Subchronic Oral Toxicity Study

Subchronic toxicity tests were performed in accordance with the 1998 regulations of Organization for Economic Cooperation and Development (OECD Test Guideline: 408). Lyophilized fruit juice of PPL diluted with distilled water was orally administered to 10 male and 10 female rats for a period of 90 days at the doses of 100, 1000, and 5000 mg kg−1 day−1 in a volume of 1 mL/100 g. Control group ( ) received an equal volume of distilled water for the same period of time. Toxicity symptoms and mortality were recorded on a daily basis and body weights of the animals were measured weekly during this time period. At the end of 90 days, the animals were anesthetized by intraperitoneal injection of 60 mg kg−1 ketamine and 5 mg kg−1 xylazine. Blood samples for haematological and biochemical analyses were collected by withdrawing the blood from the right ventricle of the anesthetized animals by using a syringe. The animals were killed by withdrawing large amounts of blood from the heart and heart, liver, kidney, lung, spleen, and ovary or testicular tissue were removed and weighed.

2.5. Haematology and Serum Biochemistry

Hematologic toxicity was evaluated by determining the white blood cell count, the percentages of neutrophils, lymphocytes, monocytes, eosinophils, basophils, and red blood cells, hematocrit, red blood cell volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, and mean platelet volume on Coulter LH 750 Hematology Analyzer device (BeckmanCoulter, Inc) in the Hematology Laboratory of Eskisehir Osmangazi University Faculty of Medicine, Turkey.

Biochemical parameters used for the evaluation of toxic effects at the organ level were determined in plasma samples by using commercially available kits. In accordance with the manufacturer’s instructions, cardiac toxicity was evaluated by measuring LDH, CK-MB, TI, and TT levels, hepatic toxicity was evaluated by measuring AST, ALT, AP, total and direct bilirubin, albumin, and total protein levels, renal toxicity was evaluated by measuring urea, creatinine, potassium, and sodium levels, and metabolic toxicity was evaluated by measuring blood glucose level.

2.6. Statistics

The percent increase in body weights of animals was calculated and repeated measurements were performed by two-way analysis of variance (ANOVA two-way repeated measures) and multiple comparisons by Holm-Sidak post-hoc test with using SigmaStat 3.5 program. The values less than 0.05 were considered as significant. The statistical analysis for all other parameters was performed on SigmaStat 3.5 program by using Kruskal-Wallis one-way analysis of variance (one-way ANOVA) and Tukey’s post-hoc test for multiple comparisons. All comparisons were considered as significant at a level of <0.05.

3. Results

3.1. The Content of Lyophilized Physalis peruviana L. Fruit Juice

The qualitative analysis revealed that the lyophilized fruit juice of PPL contains carbohydrate, protein, fixed oils, alkaloids, polyuronides, fruit juice of PPL died during, flavone glycosides, and cardiotonic glycosides.

3.2. Genotoxicity Study

Administration of different concentrations of lyophilized fruit juice of PPL resulted in an induction ratio of less than 1.5 and the growth factor level of more than 0.5 in the presence and absence of enzyme fraction with the Salmonella typhimurium TA1535 [pSK1002] strain. This may imply that administration of lyophilized fruit juice of PPL does not induce any genetic damage.

3.3. In-Life Parameters
3.3.1. Acute Oral Toxicity Study

No death was observed 24 hours after the oral administration of 5000 mg kg−1 lyophilized fruit juice of PPL to the groups of animals consisting of 5 male and 5 female rats. No behavioral symptoms such as aggression, sedation, and drowsiness, no neurological symptoms such as tremor, catatonia, paralysis, and convulsion, no changes in skin, eyes, and mucosal membranes, and no asphyxia, salivation, and diarrhea were observed during this 24-hour period and during 14-day observation period. At the end of observation period, no macroscopic necrotic focus was observed in the heart, liver, kidney, lung, stomach, spleen, and ovary or testicular tissues of the animals.

3.3.2. Subchronic Oral Toxicity Study

It was an important finding that 50% of male animals treated with 5000 mg kg−1 day−1 lyophilized fruit juice of PPL died during the 90-day treatment period.

When the percent body weight changes in the male animals were compared according to the doses and weeks, there was a significant decrease in percent body weight in the group treated with 5000 mg kg−1 day−1 of lyophilized fruit juice of PPL compared to the control group at week 12. No significant difference was found between the control group and the male animals treated with 100 or 1000 mg kg−1 day−1 of lyophilized fruit juice of PPL (Figure 1).

When the percent body weight changes in female animals were compared according to the doses and weeks, there was a significant increase in percent body weight in the groups treated with 100 and 1000 mg kg−1 day−1 of lyophilized fruit juice of PPL compared to the control groups at weeks 8, 10, and 12. No significant difference was found between the control group and the female rats treated with 5000 mg kg−1 day−1 of lyophilized fruit juice of PPL (Figure 2).

3.4. Organ Weights

The percent organ weight/body weight ratios calculated for male and female animals treated with 100, 1000, and 5000 mg kg−1 day−1 lyophilized fruit juice of PPL were not significantly different from the control group. Similarly, there was no difference when PPL-treatment groups were compared with each other (Tables 1 and 2).


Male
Control 100 mg kg−11000 mg kg−15000 mg kg−1

Lungs (%)
Liver (%)
Heart (%)
Kidneys (%)
Spleen (%)
Testes (%)

(% organ weights/body weights (g g−1)) ( : 7).

Female
Control100 mg kg−11000 mg kg−15000 mg kg−1

Lungs (%)
Liver (%)
Heart (%)
Kidneys (%)
Spleen (%)
Ovaries (%)

(% organ weights/body weights (g g−1)) ( : 7).
3.5. Hematology/Plasma Biochemistry

Hematological biomarkers were not significantly different in male and female groups treated with 100, 1000, and 5000 mg kg−1 day−1 lyophilized fruit juice of PPL for 90 days compared to the control groups. Similarly, there was no statistically significant difference between the PPL-treatment groups. In contrast, female rats treated with 5000 mg kg−1 day−1 lyophilized fruit juice of PPL had significantly higher blood platelet levels compared to the controls. Hematological parameters in the groups are shown in Tables 3 and 4.


Male
Control100 mg kg−11000 mg kg−15000 mg kg−1

WBC (103 L−1)
Neutrophils (%)
Lymphocytes (%)
Monocyte (%)
Eosinophils (%)
Basophils (%)
RBC (106 L)
HGB (g dL−1)
Haematocrit (%)
MCV (fL)
MCH (pg)
MCHC (g dL)
RDW-CV (%)
Platelet (103 L)
MPV (fL)

WBC: white blood cell; RBC: red blood cell; HGB: haemoglobin; MCV: mean corpuscular volume; MCH: mean corpuscular Hb; MCHC: mean corpuscular Hb conc; RDW-CV: coefficient variation of red cell distribution width; MPV: mean platelet volume.

Female
Control100 mg kg−11000 mg kg−15000 mg kg−1

WBC (103 L−1)
Neutrophils (%)
Lymphocytes (%)
Monocyte (%)
Eosinophils (%)
Basophils (%)
RBC (106 L−1)
HGB (g dL−1)
Haematocrit (%)
MCV (fL)
MCH (pg)
MCHC (g dL−1)
RDW-CV (%)
Platelet (103 L−1)
MPV (fL)

WBC: white blood cell; RBC: red blood cell; HGB: haemoglobin; MCV: mean corpuscular volume; MCH: mean corpuscular Hb; MCHC: mean corpuscular Hb conc; RDW-CV: coefficient variation of red cell distribution width; MPV: mean platelet volume.
Significantly different from the control group ( ).

Plasma hepatic and renal biomarkers were not significantly different in male and female groups treated with lyophilized fruit juice of PPL compared to the control groups. Similarly, there was no statistically significant difference between the PPL-treatment groups. It was noteworthy that serum potassium level was significantly higher in male animals treated with 5000 mg kg−1 day−1 lyophilized fruit juice of PPL compared to the controls (Tables 5 and 6).


Male
Control100 mg kg−11000 mg kg−15000 mg kg−1

ALT (U L−1)
AST (U L−1)
ALP (U L−1)
DBIL (mg dL−1)
TBIL (mg dL−1)
Albumin (g dL−1)
Total protein (g dL−1)
CRE (mg dL−1)
Urea (mg dL−1)
Calcium (mg L−1)
Chloride (mEq L−1)
Potassium (mEq L−1)
Sodium (mEq L−1)
Glucose (mg dL−1)
CK-MB (ng mL−1)
LDH (IU L−1)
Troponin I (ng mL−1)
Troponin T (pg mL−1)

ALT: alanine aminotransferase; AST: aspartate aminotransferase; ALP: alkaline phosphatase; DBIL: direct bilirubin; TBIL: total bilirubin; CRE: creatinine; URE: urea; CK-MB: creatine kinase myocardial band; LDH: lactate dehydrogenase.
Significantly different from the control group ( ).

Female
Control100 mg kg−11000 mg kg−15000 mg kg−1

ALT (U L−1)
AST (U L−1)
ALP (U L−1)
BILD (mg dL−1)
BILT (mg dL−1)
Albumin (g dL−1)
Total protein (g dL−1)
CRE (mg dL−1)
Urea (mg dL−1)
Calcium (mg L−1)
Chlorine (mEq L−1)