Chinese Journal of Natural Medicines  2019, Vol. 17Issue (9): 672-681  DOI: 10.1016/S1875-5364(19)30081-0
0

Cite this article as: 

LI Yun-Ying, LU Xiao-Yan, SUN Jia-Li, WANG Qing-Qing, ZHANG Yao-Dan, ZHANG Jian-Bing, FAN Xiao-Hui. Potential hepatic and renal toxicity induced by the biflavonoids from Ginkgo biloba[J]. Chinese Journal of Natural Medicines, 2019, 17(9): 672-681.
[Copy]

Research funding

The work was supported by the National S & T Major Project (2018ZX09201011) and the Key Program from Sci-Tech Plan of Zhejiang Province (2018C03075)

Corresponding author

FAN Xiao-Hui, Tel/Fax:86-571-88208596, E-mail:fanxh@zju.edu.cn

Article history

Received on: 17 May., 2019
Available online: 20 Sep., 2019
Potential hepatic and renal toxicity induced by the biflavonoids from Ginkgo biloba
LI Yun-Ying1 , LU Xiao-Yan2 , SUN Jia-Li2 , WANG Qing-Qing3 , ZHANG Yao-Dan3 , ZHANG Jian-Bing3 , FAN Xiao-Hui1,2     
1 Tianjin University of Traditional Chinese Medicine, Tianjin 301617, China;
2 Pharmaceutical Informatics Institute, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China;
3 Zhejiang University-Wanbangde Pharmaceutical Group Joint Research Center for Chinese Medicine Modernization, Hangzhou 310058, China
[Abstract]: Evidence continues to grow on potential health risks associated with Ginkgo biloba and its constituents. While biflavonoid is a subclass of the flavonoid family in Ginkgo biloba with a plenty of pharmacological properties, the potential toxicological effects of biflavonoids remains largely unknown. Thus, the aim of this study was to investigate the in vitro and in vivo toxicological effects of the biflavonoids from Ginkgo biloba (i.e., amentoflavone, sciadopitysin, ginkgetin, isoginkgetin, and bilobetin). In the in vitro cytotoxicity test, the five biflavonoids all reduced cell viability in a dose-dependent manner in human renal tubular epithelial cells (HK-2) and human normal hepatocytes (L-02), indicating they might have potential liver and kidney toxicity. In the in vivo experi-ments, after intragastrical administration of these biflavonoids at 20 mg·kg-1·d-1 for 7 days, serum biochemical analysis and histo-pathological examinations were performed. The activity of alkaline phosphatase was significantly increased after all the biflavonoid administrations and widespread hydropic degeneration of hepatocytes was observed in ginkgetin or bilobetin-treated mice. More-over, the five biflavonoids all induced acute kidney injury in treated mice and the main pathological lesions were confirmed to the tubule, glomeruli, and interstitium injuries. As the in vitro and in vivo results suggested that these biflavonoids may be more toxic to the kidney than the liver, we further detected the mechanism of biflavonoids-induced nephrotoxicity. The increased TUNEL-positive cells were detected in kidney tissues of biflavonoids-treated mice, accompanied by elevated expression of proapoptotic protein BAX and unchanged levels of antiapoptotic protein BCL-2, indicating apoptosis was involved in biflavonoids-induced nephrotoxicity. Taken together, our results suggested that the five biflavonoids from Ginkgo biloba may have potential hepatic and renal toxicity and more attentions should be paid to ensure Ginkgo biloba preparations safety.
[Key words]: Biflavonoids    Ginkgo biloba    Potential toxicity    Liver    Kidney    Apoptosis    
Introduction

Ginkgo biloba is one of the most extensively used botanical dietary supplements and phytopharmaceutical drugs worldwide. In the past few decades, it has proven to be beneficial for the treatment of many chronic and acute diseases, such as Alzheimer's disease, ischemic heart disease, atherosclerosis, thrombosis, cancer, and diabetes [1-6]. Nowadays, ginkgo leaf extract preparations are available in the Asia, European and American markets in the forms of tablets, dropping pills, soft capsule, oral solutions, injectable solutions, and others. In 2012, total global sales of ginkgo products were $1.26 billion, of which $578 million were sold in China, $152 million in Germany, and $40 million to $61 million in Australia, France, Brazil and South Korea [7]. The standardized extract preparation of Ginkgo biloba leaf commonly contains 24% flavonoids, 6% terpene trilactones (TTLs), and less than 5 ppm of ginkgolic acid [8, 9], among which flavonoids and TTLs are the main active components in ginkgo leaf extract [10, 11].

Biflavonoids are one kinds of flavonoids in Ginkgo biloba and ginkgo leaf extract with the structure of flavonoid- flavonoid dimer. There are several biflavonoids existed in Ginkgo biloba, including amentoflavone, bilobetin, ginkgetin, isoginkgetin, sciadopitysin, tetrahydroamentoflavone, sequoiaflavone, and isocryptomerin [12]. Previous studies have been demonstrated that these biflavonoids possess extensively pharmacological properties, such as anti-inflammatory, antiviral, anti-tumoral, and anti-diabetic effects [13-17]. It was found that ginkgetin has dual inhibitory properties both on cyclooxygenase-2 and 5-lipoxygenase, which can enhance anti-inflammatory effects [18]. Amentoflavone could inhibit the production of prostaglandin E2 by down-regulating the expression of cyclooxygenase-2, which has certain anti-inflammatory activity [18]. Moreover, amentoflavone has a strong inhibitory effect on the respiratory syncytial virus (RSV) with IC50 of 5.5 μg·mL-1 and also has a significant inhibitory effect on herpes simplex virus (HSV)-1 with IC50 of 16.5 μg·mL-1[19]. In addition, a patent proved that sciadopitysin has a good therapeutic effect on diabetes with no obvious adverse reactions and sciadopitysin is expected to be a new generation drug for prevention and treatment of diabetes and its complications [20].

In recent years, although herbal medicines exhibit a lot of effects, their potential toxicity has been frequently reported [21-25]. Similarly, despite the good pharmacological activity and potential therapeutic value of biflavonoids, the potential toxicity induced by biflavonoids has been sporadically declared [26-29]. A clinical report said that two cases of acute renal failure were observed after ingestion of Taxus Celebica for treatment of diabetes mellitus, and by compared with six cases of flavonoid-induced acute renal failure in the literature, the authors suggested that sciadopitysin may be the cause of acute nephropathy as Taxus Celebica contains sciadopitysin [27]. In another study, it was found that amentoflavone exhibited positive mutagenicity in Salmonella typhimurium assay [29]. The inhibitory effects of amentoflavone on human cytochrome P450 3A4 and 2C9 activities were also reported [28]. Additionally, recent studies demonstrated that several natural biflavonoids such as amentoflavone and sciadopitysin could inhibit the activity of human UDP-glucuronosyltransferase (UGT) which are the most important class of detoxification enzymes and highly expressed in metabolic organs including liver, intestine, and kidney[30-32]. Taken together, although evidences from the in vitro models and clinical case have suggested the health risks induced by individual biflavonoids, the effects of biflavonoids on health are still uncertain and require further investigation.

Hence, this study was aimed to investigate the potential toxicity of biflavonoids contained in Ginkgo biloba (namely amentoflavone, bilobetin, ginkgetin, isoginkgetin, and sciadopitysin) with in vitro and in vivo experiments. Based on previous studies, liver and kidney may be the target organs of these biflavonoids [27-28, 30-32], thus the effects of these compounds on human proximal tubular cell line (HK-2) and human normal liver cell line (L-02) representing the two important organ systems were first tested in vitro. Then, an acute toxicity of orally administered these bioflavonoids was further investigated in BALB/c mice, along with the serum biochemical analysis and histopathological examinations of the main organs. Furthermore, the possible toxicity mechanisms were detected by immunohistochemistry analysis and TUNEL assay of the target organs.

Materials and Methods Chemicals and reagents

Dulbecco's modified Eagle medium/Nutrient Mixture F-12 medium (DMEM/F-12), phosphate-buffered saline (PBS), Roswell Park Memorial Institute (RPMI) 1640 medium, 0.25% trypsin-EDTA, fetal bovine serum (FBS), and penicillin-streptomycin solution 100 × (10 000 U·mL-1 penicillin and 10 000 μg·mL-1 streptomycin) were purchased from Gibco (Grand Island, NY, USA). 3-(4, 5-dimethyl-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), and trypan blue were purchased from Sigma Co. (St. Louis, MO, USA). Amentoflavone, sciadopitysin, ginkgetin, isoginkgetin, and bilobetin were obtained from Shanghai Yuan Ye Biotechnology Co., Ltd. (Shanghai, China). Sodium chloride, potassium chloride, potassium dihydrogen phosphate, sodium carboxymethyl cellulose, methanal, disodium hydrogen phosphate, and phenobarbital sodium were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). BCL-2 antibody, BAX antibody, HRP-labeled Goat Anti-mouse IgG, and HRP-labeled Goat Anti-Rabbit IgG were purchased from Servicebio Biotechnology Co., Ltd. (Wuhan, China). All the other reagents were obtained from commercial sources and were of analytical grade.

Cell culture

The human proximal tubular cell line HK-2 cells were obtained from the Cell Bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China), and cultured in DMEM/F12 supplemented with 10% heat-inactivated FBS, 100 U·mL-1 penicillin, and 100 μg·mL-1 streptomycin in a humidified atmosphere incubator at 37 ℃ with 5% carbon dioxide (CO2). Human normal liver cell line L-02 cells were obtained from Shanghai North Nuo Biotechnology Co., Ltd. (Shanghai, China) and cultured in RPMI- 1640 supplemented with 10% heat-inactivated FBS, 100 U·mL-1 penicillin, and 100 μg·mL-1 streptomycin in a humidified atmosphere of 5% CO2 at 37 ℃. The cells were detached from the flasks by treatment with 0.25% trypsin-EDTA when the cells reached 90% confluency. The cell passage ratio of these two cell lines was both set at 1 : 4.

Cell viability assay

In order to assess the cytotoxicity of the biflavonoids from Ginkgo biloba, cell viability was determined by MTT assay. HK-2 or L-02 cells were plated in 96-well plates at a density of 1 × 104 cells per well. After 24 h incubation at 37 ℃ in an atmosphere of 5% CO2, cells were treated with different concentrations of biflavonoids for 48 h. Then, the culture medium was removed and replaced by 100 μL fresh culture medium containing 0.5 mg·mL-1 MTT. After incubation at 37 ℃ for an additional 4 h, the MTT solution was removed and the formed formazan crystals were solubilized with 100 μL DMSO. The optical density was measured at 580 nm using Infinite M1000 Pro (TECAN, Germany). The cell viability of biflavonoid-treated group was quantified as a percentage compared to vehicle control.

Animal experiments and sample collection

BALB/c mice (female, body weight 18-20 g) were supplied by Shanghai SLAC laboratory Animal Co., Ltd. (Shanghai, China). Animals were housed at 25 ± 1 ℃ with a relative humidity of 50% ± 10% under a 12 h light-dark cycle. Food and water were available ad libitum. After 3 days of an acclimation period, mice were randomly divided into 6 groups according to the body weight (n = 7), including vehicle control group (0.5% sodium carboxymethyl cellulose), amentoflavone group (20 mg·kg-1), sciadopitysin group (20 mg·kg-1), ginkgetin group (20 mg·kg-1), isoginkgetin group (20 mg·kg-1), and bilobetin group (20 mg·kg-1). The exposure dose and time of these biflavonoids were determined by the previously published studies in pharmacological activities [33-36] as well as our preliminary experiment. As intragastrical administration of these biflavonoids at 20 mg×kg-1·d-1 for 7 days induced obvious changes in serum biochemistry and histopathology in the preliminary experiment, thus the same exposure dose and time were used in this study. Each biflavonoid-treated group was intragastrically administered at the volume of 0.1 mL/10 g body weight per day for 7 days as well as the vehicle control. On the eighth day, mice were sacrificed under anesthesia by intraperitoneal injection of 1.5% pentobarbital sodium. Blood samples were collected from inferior vena cava and put into anticoagulant tubes. Serum was separated by centrifugation (5810R, Eppendorf, Germany) at 3000 r·min-1 for 10 min at 4 ℃, and then stored at -80 ℃ until biochemical analysis. The major organs (heart, liver, spleen, lung, and kidney) were removed and fixed with 10% neutral-buffered formalin for histopathological examinations.

Biochemical analysis

Blood urea nitrogen (Bun), serum creatinine (CREA), alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphate (ALP), total bilirubin (TBIL), total cholesterol (TCHOL), and triglycerides (TG) of serum samples were analyzed using a Roche COBAS C8000 Automatic Analyzer with the appropriate kits.

Histopathological examinations

The tissues fixed in 10% neutral-buffered formalin were dehydrated by decreasing concentrations of ethanol, cleared in xylene, and embedded in paraffin. Then, the tissue was section with a microtome (Leica RM2016) into sequential slices of 4 μm and subjected to hematoxylin and eosin (HE) staining. HE-stained slices were employed to assess the degrees of organ injury using a light microscope (IX53, Olympus, Japan).

TUNEL assay

A Roche In Situ Apoptosis Detection Kit was used to detect cell apoptosis in kidney according to the protocol of manufacturer. Briefly, sections were incubated with proteinase K for 30 min at 37 ℃. After washing 3 times with PBS, slides were incubated with TUNEL mixture for 2 h at 37 ℃. Followed by staining with 4′, 6-diamidino-2-phenylindole for 10 min, the FITC labeled TUNEL-positive cells was obtained using a fluorescence microscope (IX53, Olympus).

Immunohistochemistry analysis

Immunohistochemistry was used to assess the expression levels of apoptosis-related proteins, i.e., BCL-2 and BAX. Paraffin-embedded sections were cut at 5 μm and deparaffinized and rehydrated with a sequence of xylene and aqueous alcohol solutions. Then, the sections were incubated in 3% hydrogen peroxide solution for 20 min. After washing 3 time with PBS, sections were incubated overnight with primary antibodies (BCL-2 dilution ratio: 1 : 2000; BAX dilution ratio: 1 : 300) at 4 ℃. Then, sections were incubated with HRP conjugated secondary antibody (1 : 200) for 50 min at room temperature after washing 3 times with PBS. Substrate was placed in sections for 50 min following 3, 3'-diaminobenzidine (DAB) staining and hematoxylin counterstaining. Finally, sections were dehydrated in ethanol and a mounting medium was used for cover-slipping. Staining intensity was analyzed under a light microscope (IX53, Olympus).

Statistical analysis

All the experimental data were presented as mean ± standard deviation (mean ± SD). Statistical significance was assessed by Microsoft Excel or Graph Pad Prism 6 software. Differences between each biflavonoid-treated group and vehicle control group in mouse body weight were determined by T test. Serum biochemical data were analyzed by one-way ANOVA, followed by Dunnett's multiple comparison test. A P < 0.05 was considered to be significant.

Results and Discussion Biflavonoids induce cytotoxicity on HK-2 cells

HK-2 cell line is a widely-used human renal proximal tubular cell line derived from the healthy kidney of an adult male, which has similar biochemical properties and activities as human proximal tubule cells in vivo [37]. A plenty of previous studies have demonstrated that HK-2 is a suitable in vitro model for assessing drug-induced nephrotoxicity in humans [38-40]. In this study, in order to investigate the cytotoxicity of biflavonoids from Ginkgo biloba on renal cells, HK-2 cells were subjected to different concentrations of ginkgetin, isoginkgetin, amentoflavone, sciadopitysin, or bilobetin (i.e., 0.01, 0.1, 1, 10, and 100 μg·mL-1) for 48 h. Cell viability was measured by MTT assay and the results were shown in Fig. 1.

Fig. 1 The cytotoxicity of the five biflavonoids on HK-2 cells. (A) amentoflavone; (B) sciadopitysin; (C) ginkgetin; (D) isoginkgetin; (E) bilobetin. Data were obtained by MTT assay from three repeated experiments and expressed as mean ± SD. *P < 0.05, **P < 0.01 vs the control group

After incubation with different concentrations of biflavonoids, amentoflavone and sciadopitysin can significantly reduce the cell viability of HK-2 at the concentrations of 1, 10, and 100 μg·mL-1 (Figs. 1A and 1B). Similarly, ginkgetin and isoginkgetin markedly inhibited the proliferation of HK-2 cells at same concentrations, and the effects of these two biflavonoids were comparable at the concentrations of 10 and 100 μg·mL-1 (Figs. 1C and 1D). Moreover, bilobetin can remarkably reduce the survival rate of HK-2 cells at 10 and 100 μg·mL-1 (Fig. 1E). Taken together, these results suggested that these five biflavonoids could induce cytotoxicity on HK-2 cells in a dose-dependent manner.

Biflavonoids induce cytotoxicity on L-02 cells

The human normal liver cell line L-02, expressing the main molecules of human hepatocytes, is a good in vitro model for investigating liver diseases as the cells have great proliferative capacity and maintain excellent hepatocyte functions [41-43]. In this study, we further investigated the cytotoxicity of the five biflavonoids on liver cells using L-02 cells. The concentration gradient setting of each biflavonoid was the same as HK-2 cell experiment. The cell viability was shown in Fig. 2.

Fig. 2 The cytotoxicity of the five biflavonoids on L-02 cells. (A) amentoflavone; (B) sciadopitysin; (C) ginkgetin; (D) isoginkgetin; (E) bilobetin. Data were obtained by MTT assay from three repeated experiments and expressed as mean ± SD. *P < 0.05, **P < 0.01 vs the control group

Interestingly, only amentoflavone, ginkgetin, isoginkgetin, and bilobetin significantly inhibit the proliferation of L-02 cells at the higher concentration (100 μg·mL-1), whereas 0.01-100 μg×mL-1 sciadopitysin treatments did not show any cytotoxicity compared with control group in this cell line, suggesting these biflavonoids may be more toxic to kidney than liver.

Biflavonoids decrease the body weight of mice

Based on the results of in vitro experiments, we further detected the potential health risk of these biflavonoids in vivo. As females are generally more sensitive to drug toxicity than males [44-45], female mice were selected to investigate the potential toxicity of these biflavonoids in this study. Ginkgetin, isoginkgetin, amentoflavone, sciadopitysin, or bilobetin was intragastically administered to mice at a dose of 20 mg·kg-1 per day for 7 days, then continuous observation and recording were performed. The body weight of the mice was changed during the administration period, and the results were shown in Fig. 3.

Fig. 3 The effect of biflavonoids from Ginkgo biloba on the body weight of mice. (A) amentoflavone; (B) sciadopitysin; (C) ginkgetin; (D) isoginkgetin; (E) bilobetin. CON means the vehicle control group. Data were expressed as mean ± SD. n = 7. *P < 0.05, **P < 0.01 vs the vehicle control group

Compared with the vehicle control group, the body weight of all the biflavonoids-treated mice decreased during the 7 consecutive days of administration, and the weight of mice in the group treated with amentoflavone or sciadopitysin decreased significantly from the third day. Moreover, the mouse body weight of ginkgetin and bilobetin groups showed a significant decrease from the fourth and fifth day of administration, respectively. Isoginkgetin-treated mice only obviously decreased body weight on the seventh day. At the same time, loss of appetite, reduction in activities, and bad hair condition were detected in the drug-administered mice. These results indicated that the biflavonoids from Ginkgo biloba indeed caused some health problems in mice.

Biflavonoids cause alterations in the levels of serum biochemical parameters in mice

As the in vitro results suggested that the five biflavonoids can cause cytotoxicity in kidney and liver cells, thus the serum biochemical parameters which are closely related to kidney and liver functions were analyzed in the in vivo experiment.

ALT, AST, and ALP activities with TBLI level are the most widely adopted biomarkers for drug-induced liver injury [46]. Among them, ALT is a gold standard for the detection of liver cell damage, whereas AST can also reflect the degree of liver injury [46, 47]. The elevation of serum ALP activity is a sensitive marker for cholestatic disease and chronic hepatitis/cirrhosis[48, 49] and increase in serum TBIL level indicates hepatobiliary diseases [50, 51]. These four serum parameters are complementarily used to identify severe liver injury [46]. In our study, it is worth noting that the serum ALP activity was markedly increased after all biflavonoid treatments compared with the vehicle control group. In contrast, no significant increase was observed in ALT and AST activities as well as TBIL level. Meanwhile, the liver lipid metabolism related two parameters, TG and TCHOL, were also detected. An enhanced level of TG was only observed in amentoflavone- and bilobetin-treated mice along with no significant alterations identified in TCHOL. These findings suggested that these biflavonoids may have weak hepatotoxicity, which were in agreement with those from our in vitro experiments where these biflavonoids only significantly inhibited liver cell viability at the higher concentration or did not show any toxicity within the test dose range.

As serum CREA and Bun are the most useful and easily measurable biomarkers for diagnose of acute kidney injury in the clinic [52], the levels of these two parameters were further observed in this study. As the results, the values of serum CREA and Bun were significantly increased in the ginkgetin-treated mice, while serum Bun level was also increased after sciadopitysin administration. It has been demonstrated that serum CREA and Bun levels increase only after a substantial decline of kidney functions [52-53]. Thus, our data indicated that ginkgetin and sciadopitysin may cause acute kidney injury.

Biflavonoids induce histopathological change in mouse liver and kidney

In order to confirm the liver and kidney injuries induced by the biflavonoids from Ginkgo biloba, histopathological examinations with HE staining were performed to the main organs of the biflavonoids-treated mice, including heart, liver, spleen, lung, and kidney. Among them, there were no obvious lesions in the heart, spleen, and lung (data not shown), but histopathological changes were detected in the liver and kidney tissues (Figs. 5 and 6), which was consistent with the data from in vitro experiments and serum biochemistry.

Fig. 4 Serum biochemistry analysis of the biflavonoids-treated mice. (A) ALP; (B) ALT; (C) AST; (D) TBIL; (E) TCHOL; (F) TG; (G) Bun; (H) CREA. Data were expressed as mean ± SD, *P < 0.05 vs vehicle control group
Fig. 5 HE staining of the liver tissues after biflavonoid treatments. Con: vehicle control group; Ame: amentoflavone; Sci: sciadopitysin; Gin: ginkgetin; Iso: isoginkgetin; Bil: bilobetin. Original magnification × 400
Fig. 6 HE staining of the kidney tissues after biflavonoid treatments. Con: vehicle control group; Ame: amentoflavone; Sci: sciadopitysin; Gin: ginkgetin; Iso: isoginkgetin; Bil: bilobetin. Original magnification × 400

A comparison of liver sections obtained from the control and biflavonoids-treated mice indicated widespread hydropic degeneration of hepatocytes was detected in the ginkgetin and bilobetin groups, and no overt sign of toxicity was found in amentoflavone, isoginkgetin, and sciadopitysin groups. Ginkgetin exposure also caused inflammatory cell infiltration in mouse liver. These data suggested that bilobetin and ginkgetin were more toxic to hepatocytes among the five biflavonoids, which were basically consistent with the in vitro results.

The HE staining of kidney tissues showed that control mice presented a normal kidney histoarchitecture with normal tubules and glomeruli. The main pathological lesions of biflavonoids-treated groups were confirmed to the tubule, glomeruli, and interstitium injuries. For instance, the mouse renal tubules of amentoflavone group had a small amount of protein casts, and the glomerular mesangial was slightly thickened, accompanied by renal interstitial hemorrhage. Bilobetin induced localized renal interstitial hemorrhage, glomerular mesangium thickening and glomerular telangiectasia. In sciadopitysin group, local renal interstitial small-scale hemorrhage occurred and glomerular capillaries were slightly hyperemia. Renal interstitial hemorrhage and glomerular capillary congestion were observed in ginkgetin group, whereas the renal tubules of isoginkgetin group were slightly edematous. Taken together, it was worth noting that these biflavonoids all can induce kidney injury and renal interstitial hemorrhage was the common pathological phenomenon, except the isoginkgetin treatment. Consistently, pervious study showed that after ingestion of Taxus Celebica which contains sciadopitysin caused acute interstitial and tubular injuries with cola-colored urine in human and the possible mechanism may be related with uptake and accumulation the drug into tubular cells [27].

Biflavonoids induce apoptosis in mouse kidney

Apoptosis is a form of programmed cell death and plays a critical role in drug-induced nephrotoxicity [54-55]. Thus, as these biflavonoids were more toxic to kidney than liver, we further detected the possible mechanism of bioflavonoids- induced nephrotoxicity by identifying the apoptosis in kidney tissue with TUNEL assay. As shown in Fig. 7, there were few TUNEL-positive cells in kidney tissue of vehicle control mice. By contrast, the TUNEL-positive cells increased obviously in biflavonoids-treated mice, especially in ginkgetin and sciadopitysin groups, indicating the biflavonoids induced apoptosis in mouse kidney.

Fig. 7 Apoptosis was observed by TUNEL assay in the kidney tissues of biflavonoids-treated mice. The images of TUNEL-positive cells were captured by a fluorescence microscope (400 ×). Con: vehicle control group; Ame: amentoflavone; Sci: sciadopitysin; Gin: ginkgetin; Iso: isoginkgetin; Bil: bilobetin

In vertebrates, the interaction of Bcl-2 family proteins initiate the apoptosis and determine the cell fate. Among this family, BAX acts as a proapoptotic protein and BCL-2 is an antiapoptotic protein. BAX can convert into homooligomers that permeabilize the mitochondrial outer membrane, triggering the apoptosis, whereas BCL-2 is able to form a heterodimer with BAX and prevent the apoptosis, indicating BCL-2 and BAX are important factors in cell apoptosis [56, 57]. Thus, we further detected the expression levels of BAX and BCL-2 proteins in the mouse kidney using immunohistochemical analysis in this study. As shown in Fig. 8A, the expression of proapoptotic protein BAX was negligible in the kidney tissues of vehicle control group, whereas biflavonoid-treated groups revealed dark brown granules in their cytoplasm throughout a plenty of the cells in kidney. Moreover, the antiapoptotic protein BCL-2 was negatively expressed in the kidneys of biflavonoid-treated and vehicle control mice (Fig. 8B). These findings indicated that biflavonoids may cause nephrotoxicity by increasing apoptosis.

Fig. 8 Immunohistochemical analysis of BAX and BCL-2 in the kidney tissues of biflavonoids-treated mice. (A) BAX; (B) BCL-2. Original magnification × 400. Con: vehicle control group; Ame: amentoflavone; Sci: sciadopitysin; Gin: ginkgetin; Iso: isoginkgetin; Bil: bilobetin
Conclusion

This is the first in vitro and in vivo study describing the potential toxicological effects of the biflavonoids contained in Ginkgo biloba. In light of the in vitro results, the biflavonoids, i.e., ginkgetin, isoginkgetin, amentoflavone, sciadopitysin, and bilobetin, all induced cytotoxicity in human proximal tubular cells and caused weaker toxicity in human normal liver cells. Consistently, oral administration of these biflavonoids to the mice for 7 days at 20 mg·kg-1 revealed acute kidney injury and mild hepatotoxicity. Furthermore, the activation of apoptosis was involved in biflavonoids-induced nephrotoxicity. The data obtained from present investigation suggests that these biflavonoids may have potential hepatic and renal toxicity. As Ginkgo biloba extract occupy a large share in the market, the safety of long-term use of Ginkgo biloba preparations which contains these biflavonoids deserves further study. In addition, further clarifying the toxicological mechanism of these biflavonoids is needed, which will aid in rational, safe, and effective use of Ginkgo biloba preparations.

References
[1]
Yao X, Chen N, Ma CH, et al. Ginkgo biloba extracts attenuate lipopolysaccharide-induced inflammatory responses in acute lung injury by inhibiting the COX-2 and NF-κB pathways[J]. Chin J Nat Med, 2015, 13(1): 52-58.
[2]
Ahlemeyer B, Krieglstein J. Pharmacological studies supporting the therapeutic use of Ginkgo biloba extract for Alzheimer's disease[J]. Pharmacopsychiatry, 2003, 36(S1): 8-14.
[3]
Pietri S, Maurelli E, Drieu E, et al. Cardioprotective and anti- oxidant effects of the terpenoid constituents of Ginkgo biloba extract (EGb 761)[J]. J Mol Cell Cardiol, 1997, 29(2): 733-742. DOI:10.1006/jmcc.1996.0316
[4]
Gohil K, Moy RK, Farzin S, et al. mRNA expression profile of a human cancer cell line in response to Ginkgo biloba extract: induction of antioxidant response and the Golgi system[J]. Free Radic Res, 2000, 33(6): 831-849. DOI:10.1080/10715760000301351
[5]
Rhee KJ, Lee CG, Kim SW, et al. Extract of Ginkgo biloba ameliorates Streptozotocin-induced type 1 diabetes mellitus and high-fat diet-induced type 2 diabetes mellitus in mice[J]. Int J Med Sci, 2015, 12(12): 987-994. DOI:10.7150/ijms.13339
[6]
Zha WB, A JY, Wang GJ, et al. Metabonomic approach to evaluating pharmacodynamics of Ginkgo biloba extract on the perturbed metabolism in hamsters with atherosclerosis by high fat diet[J]. Chin J Nat Med, 2011, 9(3): 232-240.
[7]
Mei N, Guo X, Ren Z, et al. Review of Ginkgo biloba-induced toxicity, from experimental studies to human case reports[J]. J Environ Sci Health C Environ Carcinog Ecotoxicol Rev, 2017, 35(1): 1-28. DOI:10.1080/10590501.2016.1278298
[8]
Jacobs BP, Browner WS. Ginkgo biloba: a living fossil[J]. Am J Med, 2000, 108(4): 341-342. DOI:10.1016/S0002-9343(00)00290-4
[9]
Lu XY, Chen L, Liu T, et al. Chemical analysis, pharmacological activity and process optimization of the proportion of bilobalide and ginkgolides in Ginkgo biloba extract[J]. J Pharm Biomed Anal, 2018, 160: 46-54. DOI:10.1016/j.jpba.2018.07.037
[10]
Smith JV, Luo Y. Studies on molecular mechanisms of Ginkgo biloba extract[J]. Appl Microbiol Biotechnol, 2004, 64(4): 465-472. DOI:10.1007/s00253-003-1527-9
[11]
Shan SJ, Zhang PP, Luo J, et al. Two new phenolic glycosides isolated from Ginkgo seeds[J]. Chin J Nat Medicines, 2018, 16(7): 505-508. DOI:10.1016/S1875-5364(18)30086-4
[12]
Gontijo VS, Dos Santos MH, Viegas C Jr. Biological and chemical aspects of natural biflavonoids from plants: a brief review[J]. Mini Rev Med Chem, 2017, 17(10): 834-862.
[13]
Ishola IO, Chaturvedi JP, Rai S, et al. Evaluation of amentoflavone isolated from Cnestis ferruginea Vahl ex DC (Connaraceae) on production of inflammatory mediators in LPS stimulated rat astrocytoma cell line (C6) and THP-1 cells[J]. J Ethnopharmacol, 2013, 146(2): 440-448. DOI:10.1016/j.jep.2012.12.015
[14]
Miki K, Nagai T, Suzuki K, et al. Anti-influenza virus activity of biflavonoids[J]. Bioorg Med Chem Lett, 2007, 17(3): 772-775. DOI:10.1016/j.bmcl.2006.10.075
[15]
Cao J, Tong C, Liu Y, et al. Ginkgetin inhibits growth of breast carcinoma via regulating MAPKs pathway[J]. Biomed Pharmacother, 2017, 96: 450-458. DOI:10.1016/j.biopha.2017.09.077
[16]
Lee JS, Lee MS, Oh WK, et al. Fatty acid synthase inhibition by amentoflavone induces apoptosis and antiproliferation in human breast cancer cells[J]. Biol Pharm Bull, 2009, 32(8): 1427-1432. DOI:10.1248/bpb.32.1427
[17]
Laishram S, Sheikh Y, Moirangthem DS, et al. Anti-diabetic molecules from Cycas pectinata Griff. traditionally used by the Maiba-Maibi[J]. Phytomedicine, 2015, 22(1): 23-26. DOI:10.1016/j.phymed.2014.10.007
[18]
Son JK, Son MJ, Lee E, et al. Ginkgetin, a biflavone from Ginko biloba leaves, inhibits cyclooxygenases-2 and 5-lipoxygenase in mouse bone marrowderived mast cells[J]. Biol Pharm Bull, 2005, 28(12): 2181-2184. DOI:10.1248/bpb.28.2181
[19]
Miao LP, Wang XJ, Zhou HF, et al. Advance on the pharmacological effects of biflavonoids[J]. World Clinical Drugs, 2012, 33(6): 369-375.
[20]
Chen JW, Xiang L, Ping X, et al. Application of sciadopitysin in the preparation of diabetes drugs [P]. CN201310032630.2 (2013).
[21]
Yang Z, Zhu J, Zhang H, et al. Investigating chemical features of Panax notoginseng based on integrating HPLC fingerprinting and determination of multiconstituents by single reference standard[J]. J Gins Res, 2018, 42(3): 334-342. DOI:10.1016/j.jgr.2017.04.005
[22]
Zheng J, Yu LQ, Chen W, et al. Circulating exosomal microRNAs reveal the mechanism of Fructus Meliae Toosendan-induced liver injury in mice[J]. Sci Rep, 2018, 8(1): 2832. DOI:10.1038/s41598-018-21113-6
[23]
Lu X, Ji C, Tong W, et al. Integrated analysis of microRNA and mRNA expression profiles highlights the complex and dynamic behavior of toosendanin-induced liver injury in mice[J]. Sci Rep, 2016, 6: 34225. DOI:10.1038/srep34225
[24]
Ji C, Zheng J, Tong W, et al. Revealing the mechanism of melia toosendan fruit-induced liver injury in mice by integrating microRNA and mRNA-Based toxicogenomics data[J]. RSC Adv, 2015, 10: 1039.
[25]
Zheng J, Ji C, Lu X, et al. Integrated expression profiles of mRNA and microRNA in the liver of Fructus Meliae Toosendan water extract injured mice[J]. Front Pharmacol, 2015, 6: 236.
[26]
Baron-Ruppert G, Luepke NP. Evidence for toxic effects of alkylphenols from Ginkgo biloba in the hen's egg test (HET)[J]. Phytomedicine, 2001, 8(2): 133-138. DOI:10.1078/0944-7113-00022
[27]
Lin JL, Ho YS. Flavonoid-induced acute nephropathy[J]. Am J Kidney Dis, 1994, 23(3): 433-440.
[28]
Kimura Y, Ito H, Ohnishi R, et al. Inhibitory effects of polyphenols on human cytochrome P450 3A4 and 2C9 activity[J]. Food Chem Toxicol, 2010, 48(1): 429-435. DOI:10.1016/j.fct.2009.10.041
[29]
Cardoso CR, de Syllos Colus IM, Bernardi CC, et al. Mutagenic activity promoted by amentoflavone and methanolic extract of Byrsonima crassa Niedenzu[J]. Toxicology, 2006, 225(1): 55-63. DOI:10.1016/j.tox.2006.05.003
[30]
Lv X, Zhang JB, Wang XX, et al. Amentoflavone is a potent broad-spectrum inhibitor of human UDP-glucuronosyltransferases[J]. Chem Biol Interact, 2018, 284: 48-55. DOI:10.1016/j.cbi.2018.02.009
[31]
Liu XY, Lv X, Wang P, et al. Inhibition of UGT1A1 by natural and synthetic flavonoids[J]. Int J Biol Macromol, 2019, 126: 653-661. DOI:10.1016/j.ijbiomac.2018.12.171
[32]
Wang XX, Hou J, Ning J, et al. Inhibition of sciadopitysin against UDP-glucuronosyltransferases[J]. Acta Pharm Sin, 2016, 51(5): 749-55.
[33]
Cao J, Lu Q, Liu N, et al. Sciadopitysin suppresses RANKL- mediated osteoclastogenesis and prevents bone loss in LPS- treated mice[J]. Int Immunopharmacol, 2017, 49: 109-117. DOI:10.1016/j.intimp.2017.05.029
[34]
Cao Q, Qin L, Huang F, et al. Amentoflavone protects dopaminergic neurons in MPTP-induced Parkinson's disease model mice through PI3K/Akt and ERK signaling pathways[J]. Toxicol Appl Pharmacol, 2017, 319: 80-90. DOI:10.1016/j.taap.2017.01.019
[35]
Zhang J, Yang S, Chen F, et al. Ginkgetin aglycone ameliorates LPS-induced acute kidney injury by activating SIRT1 via inhibiting the NF-κB signaling pathway[J]. Cell Biosci, 2017, 7(1): 44. DOI:10.1186/s13578-017-0173-3
[36]
Kunert O, Swamy RC, Kaiser M, et al. Antiplasmodial and leishmanicidal activity of biflavonoids from Indian Selaginella bryopteris[J]. Phytochem Lett, 2008, 1(4): 171-174. DOI:10.1016/j.phytol.2008.09.003
[37]
Ryan MJ, Johnson G, Kirk J, et al. HK-2: an immortalized proximal tubule epithelial cell line from normal adult human kidney[J]. Kidney Int, 1994, 45(1): 48-57. DOI:10.1038/ki.1994.6
[38]
Murphy RA, Stafford RM, Petrasovits BA, et al. Establishment of HK-2 cells as a relevant model to study tenofovir-induced cytotoxicity[J]. Int J Mol Sci, 2017, 18(3): 531-533. DOI:10.3390/ijms18030531
[39]
Cheng L, Ge M, Lan Z, et al. Zoledronate dysregulates fatty acid metabolism in renal tubular epithelial cells to induce nephrotoxicity[J]. Arch Toxicol, 2018, 92(1): 469-485. DOI:10.1007/s00204-017-2048-0
[40]
Gunness P, Aleksa K, Kosuge K, et al. Comparison of the novel HK-2 human renal proximal tubular cell line with the standard LLC-PK1 cell line in studying drug-induced nephrotoxicity[J]. Can J Physiol Pharmacol, 2010, 88(4): 448-455. DOI:10.1139/Y10-023
[41]
Hu X, Yang Y, Li C, et al. Human fetal hepatocyte line, L-02, exhibits good liver function in vitro and in an acute liver failure model[J]. Transplant Proc, 2013, 45(2): 695-700. DOI:10.1016/j.transproceed.2012.09.121
[42]
Xiao Y, Zeng M, Yin L, et al. Clusterin increases mitochondrial respiratory chain complex Ⅰ activity and protects against hexavalent chromium-induced cytotoxicity in L-02 hepatocytes[J]. Toxicol Res (Camb), 2019, 8(1): 15-24. DOI:10.1039/C8TX00231B
[43]
Liang Q, Xiao Y, Liu K, et al. Cr(Ⅵ)-induced autophagy protects L-02 hepatocytes from apoptosis through the ROS-AKT-mTOR pathway[J]. Cell Physiol Biochem, 2018, 51(4): 1863-1878. DOI:10.1159/000495713
[44]
Nicolson TJ, Mellor HR, Roberts RRA. Gender differences in drug toxicity[J]. Trends Pharmacol Sci, 2010, 31(3): 0-114.
[45]
Buzzetti E, Parikh PM, Gerussi A, et al. Gender differences in liver disease and the drug-dose gender gap[J]. Pharmacol Res, 2017, 120: 97-108. DOI:10.1016/j.phrs.2017.03.014
[46]
Shi Q, Hong H, Senior J, et al. Biomarkers for drug-induced liver injury[J]. Expert Rev Gastroent, 2010, 4(2): 225. DOI:10.1586/egh.10.8
[47]
Lala V, Minter DA. Liver Function Tests[M]. StatPearls, 2019.
[48]
Hirschfield GM. Diagnosis of primary biliary cirrhosis[J]. Best Pract Res Clin Gastroenterol, 2011, 25(6): 701-712. DOI:10.1016/j.bpg.2011.10.005
[49]
Elevated Alkaline Phosphatase - Levels, Causes and Treatment [M]. 2016.
[50]
Pushpavalli G, Veeramani C, Pugalendi KV. Influence of chrysin on hepatic marker enzymes and lipid profile against D-galactosamine-induced hepatotoxicity rats[J]. Food Chem Toxicol, 2010, 48(6): 1654-1659. DOI:10.1016/j.fct.2010.03.040
[51]
Wang Y, Jiang ZZ, Chen M, et al. Protective effect of total flavonoid C-glycosides from Abrus mollis extract on lipopolysaccharide-induced lipotoxicity in mice[J]. Chin J Nat Med, 2014, 12(6): 461-468.
[52]
Gobe GC, Coombes JS, Fassett RG, et al. Biomarkers of drug-induced acute kidney injury in the adult[J]. Expert Opin Drug Metab Toxicol, 2015, 11(11): 1683-1694. DOI:10.1517/17425255.2015.1083011
[53]
Endre ZH, Pickering JW, Walker RJ. Clearance and beyond: the complementary roles of GFR measurement and injury biomarkers in acute kidney injury (AKI)[J]. Am J Physiol Renal Physiol, 2011, 301(4): 697-707. DOI:10.1152/ajprenal.00448.2010
[54]
Yan M, Tang C, Ma Z, et al. DNA damage response in nephrotoxic and ischemic kidney injury[J]. Toxicol Appl Pharmacol, 2016, 313: 104-108. DOI:10.1016/j.taap.2016.10.022
[55]
Zhu S, Pabla N, Tang C, et al. DNA damage response in cisplatin-induced nephrotoxicity[J]. Arch Toxicol, 2015, 89(12): 2197-2205. DOI:10.1007/s00204-015-1633-3
[56]
Oltvai ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in-vivo with a conserved homolog, bax, that accelerates programmed cell-death[J]. Cell, 1993, 74(4): 609-619. DOI:10.1016/0092-8674(93)90509-O
[57]
Czabotar PE, Lessene G, Strasser A, et al. Control of apoptosis by the BCL-2 protein family: implications for physiology and therapy[J]. Nat Rev Mol Cell Biol, 2014, 15(1): 49-63. DOI:10.1038/nrm3722