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Rom J Morphol Embryol 2016, 57(2 Suppl):663–673 R J M E RIGINAL APER Romanian Journal of O P Morphology & Embryology http://www.rjme.ro/

Influence of hyperforin on the morphology of internal organs and biochemical parameters, in experimental model in mice

SIMONA NEGREŞ1), CORINA SCUTARI2), FLORIANA ELVIRA IONICĂ3), VEACESLAV GONCIAR2), BRUNO ŞTEFAN VELESCU1), OANA CRISTINA ŞEREMET1), ANCA ZANFIRESCU1), CRISTINA ELENA ZBÂRCEA1), EMIL ŞTEFĂNESCU1), EMILIA CIOBOTARU4), CORNEL CHIRIŢĂ1)

1)Department of and Clinical Pharmacy, Faculty of Pharmacy, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania 2)Department of Pharmacology and Clinical Pharmacy, Faculty of Pharmacy, “Nicolae Testemiţanu” State University of Medicine and Pharmacy, Chişinău, Republic of Moldova 3)Faculty of Pharmacy, University of Medicine and Pharmacy of Craiova, Romania 4)University of Agronomic Sciences and Veterinary Medicine, Bucharest, Romania

Abstract Background and Aims: Hyperforin (HY) is used to treat depression and skin irritation and has been shown a number of pharmacological activities. The literature does no cite data on changes that may occur in the body after HY intake (ethylene diammonium salt – EDS) in long-term administration. From this point of view, the present work is a key to determining the pharmacotoxicological profile of the HY-EDS, in long-term administration. Materials and Methods: In present research, the influence of toxic doses of HY-EDS was investigated on the biochemical serum parameters and the histopathological changes in internal organs on the experimental mice model. For acute toxicity determination, the HY-EDS was tested in doses of 2000–5000 mg/kg, administered once per day orally. For subacute toxicity, the HY-EDS was tested in three groups of mice, in doses of 50, 75 and 100 mg/kg/day, administered once daily, for 28 consecutive days. Results and Conclusions: As concern acute toxicity, a lethal effect has not occurred at any of the two tested doses and HY-EDS was classified as Class V toxic: median lethal dose (LD50) >5000 mg/kg, p.o. After 14 days of follow-up in acute toxicity, the experimental results showed a statistically significant increase of aspartate transaminase (AST) and alanine transaminase (ALT), compared to the control group. There were no changes in creatinine and serum compared to the control group. After the administration of repeated doses, it was observed an increase of serum transaminases and alkaline phosphatase. Histological examination revealed that the injuries were in an initial stage, making them reversible in case of HY-EDS treatment discontinuation. There was no evidence of kidney damage to any of the doses of HY-EDS. Keywords: hyperforin toxicity, histopathological changes, motor activity, perforatum (St. John’s wort) extract.

 Introduction To our knowledge, there are no data available on the changes that occur in the body with long-term hyperforin Hyperforin (HY) is the main active ingredient of the intake. Regarding this, it was decided to determine the medicinal plant (St. John’s wort), pharmacotoxicological profile of HY-EDS (hyperforin which is commonly use for the treatment of depressions ethylene diammonium salt), administered both in a single and skin irritations, such as atopic dermatitis (Figure 1). dose and over long term. Beside these properties, HY has several pharmacological activities, including antibacterial and antitumor properties [1]. Its antitumor effect in vivo was comparable to that of with absence of any signs of acute toxicity [2]. HY has been shown to be neuroprotective against OH O ischemic damage in the acute phase of ischemic stroke. Previous research has demonstrated that HY up-regulates the vascular endothelial growth factor (VEGF) levels in O O central nervous system (CNS) tumor cells and induces endothelial proangiogenic behavior [3, 4]. Recent study reported DNA-protective activities of HY [5]. The herb is the main source of HY and , which are interesting active pharmaceutical ingredients [6]. One recent study Figure 1 – Hyperforin: chemical structure. reported that HY-loaded gold nanoparticles used in the treatment of experimental autoimmune encephalomyelitis  Materials and Methods (EAE) in animal model of multiple sclerosis significantly Method for determination of acute toxicity reduced clinical severity of EAE, and decrease the number of inflammatory cell infiltration in the spinal cord [7]. The method of toxicity class [according to Organization

ISSN (print) 1220–0522 ISSN (online) 2066–8279 664 Simona Negreş et al. for Economic Co-operation and Development (OECD) at 45–50%. With one hour before administering the tested Test Guidelines No. 423] was employed, consisting in use substance, the food was removed and water was given of predefined doses which allow for the substances to be ad libitum. placed within a toxicity class, depending on the presence Doses, route of administration and duration of or absence of lethality (deciding for each dose whether treatment to be tested on other three animals or to switch to testing the following higher or lower dose). Based on evaluated maximum tolerated dose, HY-EDS When literature data suggest a broad safety limit for the selected doses were 50, 75 and 100 mg/kg/day. Aqueous active substance, the limit test is employed, considering suspension of HY-EDS was administered by gastric the following: intubation (oral, p.o.) per kg/day, once daily, for 28 days. ▪ administration of a 2000 mg/kg (three animals/group) The suspensions were prepared daily prior to admi- dose; if no lethality is recorded, the test is repeated with nistration. In the suspension formulation, Tween 80 was the same dose on another group of three animals; used as a suspending agent in order to ensure uniformity ▪ lack of lethality in previous determination leads to of dispersion of the administered dose. administration of a 5000 mg/kg (three animals/group) dose. Experimental groups Animal stocks After three days of accommodation, the animals were We used 24 white mice, NMRI (Naval Medical divided into four groups (20 animals/group) treated as Research Institute) strain, males, 24.194±1.14 g average following: control group, treated with distilled water, 0.1 mL/ weight, coming from Animal Facility of “Carol Davila” 10 g bw/day dose, p.o.; HY-EDS group, 50 mg/kg/day University of Medicine and Pharmacy, Bucharest, Romania. dose, p.o.; HY-EDS group, 75 mg/kg/day dose, p.o., HY- HY-EDS was semi-synthesized in the labs of “Nicolae EDS group, 100 mg/kg/day dose, p.o. Testemiţanu” State University of Medicine and Pharmacy, After administration, the animals were followed-up Chişinău, Republic of Moldova. for 28 days regarding lethality, body weight, feeding Experimental groups behavior, motor behavior, aggressiveness, appearance (furring, mucous). Initially, to a group of six animals was administered From the day 9 of treatment, the animals showed a dose of 2000 mg/kg body weight (bw), p.o. (per os), clinical signs of agitation, which is likely due to a CNS aqueous suspension of 20% HY-EDS. As no lethality was stimulating effect by HY-EDS. Because of this, we decided registered, 48 hours after the first administration, another to investigate the motor behavior of animals, in all four group of six animals was treated with the same dose of research groups, at day 15 and day 22 of treatment. HY-EDS as in the previous experiment. Lack of lethality in previous stages of the research Determination of motor activity led to administration of a 5000 mg/kg bw dose, p.o., Motor activity was determined initially and at days suspension of 50% HY-EDS. 15 and 22 of the experiment, using the Activity Cage Concomitantly, two control groups were treated with system (Ugo Basile, Italy). This system records motor distilled water (in which Tween 80 suspension agent was activity on the vertical axis and the horizontal axis. The added), administered in 0.2 mL/10 g bw, p.o., respectively, device is connected to a computer that inputs the experi- 0.5 mL/10 g bw, p.o., corresponding to the tested two mental data through Ugo Basile software and export them doses of hyperforin. into Excel documents. After HY-EDS administration, animals were observed We choose a monitoring interval of five minutes to for four hours, then daily for 14 days. They were observed collect data beyond initial exploration period, when normal for signs of toxicity: pathological changes in the skin animals are investigating any new space in which they and/or mucous membranes; changes in appearance; patho- are placed. logical impairment in organs and systems; changes in The animal is introduced in the Activity Cage and behavior (eating, sexual). At the end of 14 days follow-up, recording starts. Each determination shall receive an the biochemical parameters were determined: liver trans- identification number in order to process later the data aminases, creatinine, serum glucose. sent to the computer. In the five minutes, it takes each The animals were given free access to food and water individual determination, the animal is protected from in an environment of constant: 0 the external stimuli that could affect its behavior (i.e., ▪ temperature and humidity: 21–24 C and 40–60%, sounds, sudden changes in light intensity, etc.). respectively; After five minutes, the recording automatically stops ▪ artificial light source: 12 hours light/dark cycle. and the mouse is removed. The total amount of movements Method for determination of subacute toxicity on the two axes is obtained by adding the values registered in each of the five minutes of test. Before introducing a Animal stocks new animal into the cage, this must be cleared of manure We used a community of 80 white mice, NMRI strain, or other traces left by the previous animal, because the males, 25.3±1.1268 g average weight, coming from Animal evolution of the next animal should not be influenced by Facility of “Carol Davila” University of Medicine and these factors. Pharmacy, Bucharest. The animals were brought from the Biological tests Animal Facility and left for three days with food and water ad libitum for accommodation. The temperature was At the end of the observation period, animals were maintained constant at 22±10C and the relative humidity sacrificed and biological samples were collected (whole

Influence of hyperforin on the morphology of internal organs and biochemical parameters, in experimental model… 665 blood, serum, organs: liver, kidneys, brain), that were sent to determine the biochemical parameters of interest and histopathological changes. Measurements of transaminases, creatinine, bilirubin and alkaline phosphatase were per- formed with the automatic analyzer Hitachi 911. Blood counts parameters were determined using the automatic analyzer Pentra 80. Histopathological technique In order to remove the organs, the mice were euthanized by anesthesia with chloroform extended until exitus. Organs were fixed in 10% solution and prepared by the histological technique of inclusion in paraffin, and stained by Masson’s trichrome method. When the internal organs were harvested, we have performed their macro- scopic examination. There were no changes compared to the control group, in terms of color or appearance of internal organs. Cross-sections processing was performed Figure 2 – Variation of biochemical parameters for the at the Department of Pathology of the University of groups treated with distilled water, 0.2 mL/10 g bw Agricultural Sciences and Veterinary Medicine, Bucharest. and HY-EDS at a dose of 2000 mg/kg, p.o. HY-EDS: Hyperforin ethylene diammonium salt; ALT: Alanine Ethics rules transaminase; AST: Aspartate transaminase; SD: Acute and subacute toxicity studies for the HY-EDS Standard deviation. was conducted in accordance with Directive 2001/83/EC of the European Parliament and of the Council of Europe (November 6, 2001), establishing a Community Code relating to medicinal products for human use. The experi- ment was conducted in accordance with Directive 86/ 609/EEC of November 24, 1986 and in accordance with the Order No. 143/April 1, 2002 (Ministry of Agriculture, Food and Forestry), Order No. 400/May 20, 2002 (Ministry of Water and Environmental Protection) and Order No. 84/August 30, 2005 (National Sanitary Veterinary and Food Safety Authority – ANSVSA), on the protection of animals for research purposes. Statistical analysis The statistical analysis was carried out using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego California, USA, www.graphpad.com). Normality was establish using the D’Agostino & Pearson test. In Figure 3 – Variation of biochemical parameters for the order to compare the “n” groups, the tests used were the groups treated with distilled water, 0.5 mL/10 g bw ANOVA parametric test (compare “n” groups) followed by and HY-EDS at a dose of 5000 mg/kg, p.o. HY-EDS: Dunnett’s post-hoc test (when compared to the baseline Hyperforin ethylene diammonium salt; ALT: Alanine response). The value of p<0.05 was considered significant. transaminase; AST: Aspartate transaminase; SD: Standard deviation.  Results Acute toxicity The experimental results revealed that the lethal effect has not appeared at any of the doses tested experimentally: 2000 mg/kg, and 5000 mg/kg respectively, administered p.o. The biochemical parameters values are revealed in Figure 2, for the group treated with the dose of 2000 mg/kg and in Figure 3, for the group treated with the dose of 5000 mg/kg.

Subacute toxicity Figure 4 – Variation of body weight of animals from control group (distilled water 0.1 mL/10 g bw, p.o.), The animals that received the HY-EDS in doses of HY-EDS 50 mg/kg p.o., HY-EDS 75 mg/kg p.o., and 50, 75 and 100 mg/kg, p.o., showed body weight gain, HY-EDS 100 mg/kg p.o. HY-EDS: Hyperforin ethylene similar to that observed in the control group (Figure 4). diammonium salt.

666 Simona Negreş et al. In this experiment, the research data showed that the Table 1 – Evolution of horizontal motor activity (HMA) HY-EDS, one of the active principles contained in St. and vertical motor activity (VMA) for control group John’s wort (SJW) extract, induces an increase in body and for HY-EDS treated groups weight similar to the one registered for control group. The recorded results, that revealed evolution of motor Control activity on horizontal and on vertical (Figures 5 and 6) Distilled HY-EDS HY-EDS HY-EDS have been statistically processed using ANOVA test, water 50 mg/kg 75 mg/kg 100 mg/kg 0.1 mL/10 g followed by Dunnett’s post-hoc test, due to normal Parameter Motor activity distribution of response in collectivity as evaluated by D’Agostino and Pearson test (Table 1). M±SD / 530.7± 564.79± 548.47± 521.56± basal 50.32 86.5 61.34 90.39 After 28 days of HY-EDS administration, animals D’Agostino were sacrificed and biological samples were collected, and ND ND ND ND which were sent to determine the following biochemical Pearson parameters: AST, ALT, alkaline phosphatase (Table 2), test M±SD / 510.8± 559.7± 531.8± 494.2± creatinine, bilirubin and blood count (Figure 7). Day 15 66.72 91.40 64.76 102.5 D’Agostino and ND ND ND ND Pearson test ANOVA 0.0913 test Dunnett’s post-hoc ns ns ns ns test / basal Effect % -3.74 -0.90 -3.03 -5.24 vs. basal M±SD / 445.1± 391.1± 432.6± 414.2±

Horizontal motor activity (HMA) Day 22 58.60 111.2 89.69 104.6 ND ND ND ND ANOVA 0.0761 test Dunnett’s post-hoc ns ns ns ns test / basal Figure 5 – Variation of horizontal motor activity for Effect % -16.12 -30.75 -21.12 -20.58 control group and groups treated with hyperforin. vs. basal HY-EDS: Hyperforin ethylene diammonium salt. M±SD / 54.36± 60.57± 69.48± 71.31± basal 16.42 15.11 17.39 16.13 D’Agostino and ND ND ND ND Pearson test M±SD / 68.75± 57.95± 67.80± 67.50± Day 15 15.81 15.74 19.12 17.16 D’Agostino and ND ND ND ND Pearson test ANOVA 0.0748 test Dunnett’s post-hoc ns ns ns ns test / basal Effect % 26.47 -4.32 -2.41 -5.34 vs. basal M±SD / 38.90± 37.74± 45.11± 34.45± Day 22 9.386 12.83 9.695 11.51

Vertical motor activity (VMA) D’Agostino Figure 6 – Variation of vertical motor activity for and ND ND ND ND control group and groups treated with hyperforin. Pearson HY-EDS: Hyperforin ethylene diammonium salt. test ANOVA 0.0428* When examining the cross-sections at the microscope, test we observed the following parameters: for brain – neuron Dunnett’s integrity, vascular changes, presence/absence of edema, post-hoc ns * * * any other inflammatory reaction; for liver – appearance test / basal Effect % -28.44 -37.69 -35.07 -51.68 of hepatocyte, changes in the Disse space, circulatory vs. basal changes, eventual necrosis, other relevant comments; for HY-EDS: Hyperforin ethylene diammonium salt; M: Mean; SD: Standard kidney – aspect of renal glomeruli, proximal convoluted deviation; ND: Normal distribution; ns: Statistically non-significant; tubule aspect, appearance of renal interstitial space. *p<0.05.

Influence of hyperforin on the morphology of internal organs and biochemical parameters, in experimental model… 667

Table 2 – Statistically significant alterations of the bio- Control chemical parameters: AST, ALT, alkaline phosphatase Distilled HY-EDS HY-EDS HY-EDS for the animals in groups treated with HY-EDS com- Test water 50 mg/kg 75 mg/kg 100 mg/kg parative with control group 0.1 mL/10 g Parameter Control Effect % / 78.29 107.02 158.10 Distilled HY-EDS HY-EDS HY-EDS control

Test water 50 mg/kg 75 mg/kg 100 mg/kg 118.4± 405.2± 433.3± 421.0± 0.1 mL/10 g M±SD Parameter 7.876 70.08 53.59 83.87 27.24± 167.9± 159.1± 173.4± ANOVA M±SD <0.0001*** 5.339 47.37 97.90 87.50 test ANOVA Dunnett’s 0.0002** test post-hoc *** *** *** Dunnett’s test /

AST post-hoc control ** ** ** test / Alkaline phosphatase Effect % / 242.29 265.96 255.57 control control Effect % / 513.35 484.06 533.43 HY-EDS: Hyperforin ethylene diammonium salt; M: Mean; SD: Standard control deviation; AST: Aspartate transaminase; ALT: Alanine transaminase; 33.87± 60.39± 70.12± 87.42± *p<0.05; **p<0.001; ***p<0.0001. M±SD 12.79 26.26 54.18 92.06 For the control group, histopathological findings are ANOVA 0.02411* summarized in Table 3 and Figure 8. For the groups treated test with HY-EDS, the results are shown in Tables 4–6 and ALT Dunnett’s Figures 9–11. post-hoc * * * test / control

Figure 7 – Mean values for control group and for groups treated with hyperforin: (a) Leukocyte count [×103/μL]; (b) Thrombocyte count [×103/μL]; (c) Erythrocyte count [×106/μL]; (d) Hemoglobin [g/dL]. HY-EDS: Hyperforin ethylene diammonium salt.

668 Simona Negreş et al.

Figure 7 (continued) – Mean values for control group and for groups treated with hyperforin: (e) Hematocrit [%]; (f) Total bilirubin [mg/dL]; (g) Serum creatinine [mg/dL]. HY-EDS: Hyperforin ethylene diammonium salt.

Table 3 – Histopathological exam results for control group

Animal No. Liver Kidney Brain

1. Preserved morphological Preserved morphological Cerebral hemispheres and other architecture, discrete vacuolization architecture, focal oxyphilic and components: preserved morphological of hepatocyte cytoplasm in liver discrete vacuolization of architecture; cerebellum – oxyphilic perivascular areas, respecting the nephrocytes cytoplasm; normal cytoplasm of Purkinje neurons; centrality of the nucleus; appearance. Purkinje neurons necrosis. granulovacuolar hepatosis. 2. Normal morphological architecture; Normal morphological architecture; Normal morphological architecture; normal appearance. normal appearance. normal appearance. 3. Normal morphological architecture; Normal morphological architecture; Cerebral hemispheres (small pyknotic sporadic hepatocytes with pyknotic normal appearance. neurons, oxyphilic cytoplasm, focal nuclei and oxyphilic cytoplasm; located); focal neuronal necrosis. normal appearance. 4. Normal morphological architecture; Normal morphological architecture; Normal morphological architecture; normal appearance. normal appearance. normal appearance. 5. Normal morphological architecture; Normal morphological architecture; Cerebral hemispheres (sporadic small normal appearance. normal appearance. pyknotic neurons, oxyphilic cytoplasm); normal appearance. 6. Normal morphological architecture; Normal morphological architecture; Cerebral hemispheres (sporadic small normal appearance. normal appearance. pyknotic neurons, oxyphilic cytoplasm); normal appearance. 7. Normal morphological architecture; Normal morphological architecture; Normal morphological architecture; normal appearance. normal appearance. normal appearance. Observations: Normal appearance – 6/7; Normal appearance – 7/7. Normal appearance – 5/7; Granulovacuolar hepatosis – 1/7. Neuronal necrosis – 2/7.

Influence of hyperforin on the morphology of internal organs and biochemical parameters, in experimental model… 669

Figure 8 – Histopathology – control group: (a) Liver – normal aspects; (b) Liver – oxyphilic hepatocyte; (c) Liver – granulovacuolar hepatosis; (d) Kidney – normal aspects; (e) Cerebellum – oxyphilic Purkinje neurons. Masson’s trichrome staining: (b and c) ×200; (a, d and e) ×400. HY-EDS: Hyperforin ethylene diammonium salt.

Table 4 – Histopathology results for group treated with HY-EDS of dose 50 mg/kg, p.o.

Animal No. Liver Kidney Brain

1. Preserved hepatic architecture, Normal morphological Cerebral hemispheres; normal hepatocyte with obvious vacuolated architecture; normal morphological architecture; isolated cytoplasm and nucleus keeping the appearance. presence of glial nodules (phagocytosis of central position, on very large areas; neurons); normal appearance. granulovacuolar hepatosis. 2. Preserved hepatic architecture, Normal morphological Normal morphological architecture; normal hepatocyte with obvious vacuolated architecture; normal appearance. cytoplasm, and nucleus keeping the appearance. central position, homogeneous; granulovacuolar hepatosis. 3. Hepatocyte with obvious vacuolated Not available. Normal morphological architecture; normal cytoplasm, and nucleus keeping the appearance. central position, central located; granulovacuolar hepatosis. 4. Hepatocyte with obvious vacuolated Normal morphological Normal morphological architecture; normal cytoplasm, and nucleus keeping the architecture; normal appearance. central position, central located; the appearance. presence of cell clusters that discrete dissociated hepatic lobe architecture – possibly islands of extramedullary hematopoiesis – normal in rodents; focal granulovacuolar hepatosis. 5. Hepatocyte with vacuolated cytoplasm, Not available. Normal morphological architecture; normal and nucleus keeping the central position, appearance. central located; focal granulovacuolar hepatosis. 6. Can be seen areas with lipid Normal morphological Cerebral hemispheres; sporadic presence accumulation, which deforms the cells, architecture; normal of glial nodules (phagocytosis of neurons); and moving the nucleus: eccentric appearance. glial nodules; sporadic presence of nucleus; diffuse granulovacuolar nonpurulent perivascular sleeves – hepatosis; hepatic lipidosis. nonpurulent encephalitis (inflammatory lesion with viral etiology, parasitic). 7. Focal granulovacuolar hepatosis. Not available. Normal morphological architecture; normal appearance. 8. Diffuse granulovacuolar hepatosis. Normal morphological Normal morphological architecture; normal architecture; normal appearance. appearance. Observations: Granulovacuolar hepatosis – 8/8. Normal appearance – 8/8. Normal appearance – 7/8; Nonpurulent encephalitis – 1/8.

670 Simona Negreş et al.

Figure 9 – Histopathology – group treated with HY-EDS of dose 50 mg/kg: (a) Liver – granulovacuolar hepatosis (animal No. 1); (b) Liver – granulovacuolar hepatosis (animal No. 2); (c) Liver – extramedullary hematopoiesis (animal No. 4); (d) Liver – hepatic lipidosis (animal No. 6); (e) Liver – hepatic lipidosis (animal No. 6); (f) Brain – perivascular sleeves, nonpurulent encephalitis (animal No. 6); (g) Brain – glial nodules (animal No. 6). Masson’s trichrome staining: (a) ×100; (d and g) ×200; (b, c, e and f) ×400. HY-EDS: Hyperforin ethylene diammonium salt.

Table 5 – Histopathology results for group treated with HY-EDS of dose 75 mg/kg, p.o. Animal No. Liver Kidney Brain 1. Diffuse granulovacuolar Normal morphological architecture; Normal morphological architecture; normal hepatosis. normal appearance. appearance. 2. Normal appearance; Normal morphological architecture; Normal morphological architecture; normal extramedullary hematopoiesis. normal appearance. appearance. 3. Focal hepatic lipidosis; Normal morphological architecture; Cerebral hemispheres; sporadic presence extramedullary hematopoiesis. normal appearance. of glial nodules (phagocytosis of neurons); oxyphilic Purkinje neurons from cerebellum. 4. Normal appearance; Normal morphological architecture; Cerebral hemispheres; sporadic presence extramedullary hematopoiesis. normal appearance. of glial nodules (phagocytosis of neurons). 5. Regional hepatic lipidosis on Normal morphological architecture; Cerebral hemispheres; sporadic presence the periphery of hepatic lobe; normal appearance. of glial nodules (phagocytosis of neurons). extramedullary hematopoiesis. 6. Normal appearance; Normal morphological architecture; Normal morphological architecture; normal extramedullary hematopoiesis; normal appearance. appearance. focal hepatic lipidosis. 7. Normal appearance; Normal morphological architecture; Normal morphological architecture; normal extramedullary hematopoiesis. normal appearance. appearance. 8. Normal appearance; Normal morphological architecture; Normal morphological architecture; normal extramedullary hematopoiesis. normal appearance. appearance. Observations: Normal appearance – 5/8; Normal appearance – 8/8. Normal appearance – 5/8; Granulovacuolar hepatosis – 1/8; Neuronal necrosis – 2/8; Hepatic lipidosis – 3/8. Glial nodules – 1/8.

Figure 10 – Histopathology – group treated with HY-EDS of dose 75 mg/kg: (a) Liver – normal appearance (animal No. 4); (b) Liver – hepatic lipidosis (animal No. 5). Masson’s trichrome staining: (a and b) ×200. HY-EDS: Hyperforin ethylene diammonium salt.

Influence of hyperforin on the morphology of internal organs and biochemical parameters, in experimental model… 671 Table 6 – Histopathology results for group treated with HY-EDS of dose 100 mg/kg, p.o. Animal No. Liver Kidney Brain 1. Necrotic hepatocytes in small clusters Normal morphological architecture; Normal morphological architecture; and marked of phagocytic cells; diffuse normal appearance. normal appearance. granulovacuolar hepatosis. 2. Normal appearance; extramedullary Normal morphological architecture; Normal morphological architecture; hematopoiesis normal appearance. normal appearance. 3. Diffuse granulovacuolar dystrophy. Normal morphological architecture; Not available. normal appearance. 4. Diffuse granulovacuolar dystrophy. Normal morphological architecture; Normal morphological architecture; normal appearance. normal appearance. 5. Focal granulovacuolar dystrophy; Normal morphological architecture; Normal morphological architecture; discreet lipidosis. normal appearance. normal appearance. 6. Diffuse granulovacuolar hepatosis. Normal morphological architecture; Normal morphological architecture; normal appearance. normal appearance. 7. Diffuse granulovacuolar dystrophy. Normal morphological architecture; Normal morphological architecture; normal appearance. normal appearance. Observations: Normal appearance – 1/7; Normal appearance – 7/7. Normal appearance – 7/7. Granulovacuolar dystrophy – 4/7; Lipidosis – 1/7; Necrotic hepatocytes – 1/7.

Figure 11 – Histopathology – group treated with HY-EDS of dose 100 mg/kg: (a) Liver – normal appearance (animal No. 6); (b) Liver – necrotic hepatocyte, phagocytosis (animal No. 6); (c) Liver – vacuolar hepatosis (animal No. 6). Masson’s trichrome staining: (a) ×100; (c) ×200; (b) ×400. HY-EDS: Hyperforin ethylene diammonium salt.

 Discussion food intake, and accordingly in body weight, may also suggest a depressive event [9]. The aim of our study was to investigate toxicological In our present study, we found that weight gain is profile of hyperforin, one of the main derivatives of statistically significant from day 3 of the study and it is SJW extract, because there are no data especially about similar to that observed in the control group; therefore, possible changes that occurred in the organs after “well- hyperforin did not influence body weight of treated established use” as drugs or “traditional use” as dietary animals. It has been observed an increase in intestinal supplements, for a long time. Therefore, we investigated transit of the animals treated with HY-EDS, from the both acute and subacute toxicity. fourth day of dosing. Nevertheless, there has not been Acute toxicity detected weight loss of the animals treated. Research results concur with other literature data showing that for In 14 days of follow-up after acute toxicity, the ovariectomized female rat, H. perforatum extract (HPE) experimental results showed a statistically significant does not influence statistically significant the food intake increase of AST and ALT, compared to the control group. and the amount of adipose tissue (total, mesenteric or There were no changes in the creatinine and serum glucose visceral) [10]. In Vieira et al. study, no significant compared to the control group. differences in body weight reported in rats treated with According to OECD Test Guidelines No. 423 on SJW extract during gestation, suggesting that this mixture determining the acute toxicity of the HY-EDS, adminis- of natural compounds was not toxic [9]. tered p.o., in single dose, the HY-EDS was classified as Motor activity Class V toxic: LD50>5000 mg/kg, p.o. [8] [as shown in Global Harmonized System (GHS) – Environment (ENV) Horizontal motor activity for the animals in control Directorate, Joint Meeting (JM) of the Chemicals Committee group and in groups treated with HY-EDS has decreased and the Working Party on Chemicals, Pesticides and Bio- compared to initial statistically non-significant at day 15 technology/Monography – ENV/JM/MONO(2001)6, 2001]. evaluation, probably because of accommodation with research cage (Figure 5). By contrast, vertical motor activity Subacute toxicity for the animals treated with HY-EDS was reduced statistically significant at day 22 evaluation, suggesting Body weight a depressant effect of the tested substance on CNS Body weight decrease in treated animals is suggestive (Figure 6). Althought Buchholzer et al. [11] reported for treatment toxicity on organism, but modifications in that locomotor activity in mice treated intraperitoneally

672 Simona Negreş et al. with high-dose hyperforin (10 mg/kg) spontaneously a potent noncompetitive inhibitor of CYP2D6 activity decreased in their study whilst the lower dose of hyperforin and a competitive inhibitor of CYP2C9 and CYP3A4 (1 mg/kg) was ineffective in this respect. These researchers activity [29, 30]. Therefore, the effects of the SJW extract, presented the first data on HY interactions with cholinergic hypericin and HY on CYP1A2 and CYP2D6 expression system, and found that HY inhibited choline reuptake in are still unclear. rat synaptosomes, which create premises for the investi- Kidney and brain damages gation of the use of HY in cognitive disorders. In other study, in mice treated with SJW extract during gestation, There was no evidence of kidney damage to any of the there is no interference with motor and behavioral deve- doses of HY-EDS. lopment, as assessed immediately after weaning [9]. Brain injuries are yielded by other etiological factors CNS depressant effect for total extract of H. perforatum than those used in the experiment (viruses, protozoa). [12, 13] or of hyperforin [14] was also highlighted in other Biochemical parameters non-clinical experiments by potentiating the depressant action of diazepam and , postulating an Research results revealed that HY-EDS administered interaction with GABA-ergic receptors [13] or a reduction in repeated doses (for 28 consecutive days) induced sig- in cerebral GABA (γ-aminobutyric acid) concentration nificant changes compared to the control group: increased [14]. The results of Uzbay et al. study indicate that the levels of serum transaminases and alkaline phosphatase. caffeine-induced locomotor activity was blocked by The effects appears not to be dose-dependent probably pretreatment with SJW extract in mice [15]. because of a limiting process involved in absorption and, Liver damage and SJW-drug interactions consecutively, in bioavailability of hyperforin (Table 2). We have not found information in the literature about In our study, histopathologically diagnosed liver injuries the hyperforin action on these biochemical parameters. were dependent on concentration, generally in an initial The blood count parameters (Figure 7, a–e) did not stage, making them reversible in case of HY-EDS discon- vary significantly against the control group. tinuation. Literature data suggest that in therapeutic con- We found no significant changes in bilirubin levels centrations, the extract of H. perforatum has a protective in treated groups compared with control group (except the effect on the liver of lab animals [16, 17], although no dose of 100 mg/kg HY-EDS; Figure 7f). Creatinine levels considerations are made on the effect of different active (Figure 7g) in treated groups were similarly with control principles contained in the total plant extract. Nevertheless, group, except the group of dose of 100 mg/kg HY-EDS. clinical data highlights toxic liver effects for several species of Hypericum [18, 19] taken as nutritional supplements,  Conclusions alone or associated with other plant products. It is well known that SJW is one of the most commonly Hyperforin is classified in Class V of toxicity, according used herbal extract, and has been described as a strong to literature. Subacute toxicity studies have shown that inducer of [20, 21]. This is administration of the HY-EDS induces in all treated groups a major safety concern, because its interactions with statistically significant increase in serum transaminases involving liver drugs metabolizing leading to and alkaline phosphatase (p<0.0001), compared with the alteration of the and/or clinical controls. Histopathological examination revealed early response of a variety of drugs. SJW extract and hyperforin liver damage that occurs in a dose-dependent manner, induce the expression of cytochrome P450 enzymes reversible upon discontinuation. There was no evidence (particularly hepatic and intestinal CYP3A4), induce the of renal or brain damage from any of the HY-EDS tested expression of P-glycoprotein (P-gp), and activate the doses. pregnane X [22, 23]. The use of St. John’s wort decreases the bioavailabilities and increases the metabolism Conflict of interests of a variety of drugs. SJW may interact with oral contra- The authors declare that they have no conflict of ceptives, anticoagulant drugs like warfarin or phenpro- interests. coumon, the cardiac medication like digoxin, the asthma Author contribution drug theophylline, the hypercholesterolemia medication Simona Negreş and Corina Scutari equally contributed 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) to the manuscript. reductase inhibitors, the anti-HIV (human immunodefi- ciency virus) drug , and the immunosuppressant Acknowledgments drug cyclosporine [24–26]. CYP3A4 induction with SJW The paper was supported by bilateral Project No. extract was associated with HY content, suggesting that 136/2012 “Carol Davila” University of Medicine and this component plays a major role in clinically significant Pharmacy, Bucharest, Romania / “Nicolae Testemiţanu” drug interactions [27]. 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Corresponding author Floriana Elvira Ionică, Lecturer, Pharm, PhD, Faculty of Pharmacy, University of Medicine and Pharmacy of Craiova, 2 Petru Rareş Street, 200349 Craiova, Romania; Phone +40251–523 929, e-mail: [email protected]

Received: January 18, 2016

Accepted: August 30, 2016