Efficacy and safety evaluation of black ginseng (Panax ginseng C.A. Mey.) extract (CJ EnerG): broad spectrum cytotoxic activity in human cancer cell lines and 28-day repeated oral toxicity study in Sprague-Dawley rats

Panax ginseng is a perennial plant belonging to genus Panax under the Araliaceae family, which consists of about 14 species including P. ginseng C.A. Mey. (Korean ginseng), P. quinquefolius L. (American ginseng), and P. notoginseng (Burkill) F.H. Chen (Chinese ginseng) [1]. Ginseng roots have been one of the most important and valuable ingredients mainly in Asian folk medicine due to their pharmacological benefits. Consistently, a line of recent studies revealed therapeutic activities against a variety of clinical symptoms and diseases such as tumorigenesis, memory loss and cognitive dysfunction, neurodegenerative diseases, diabetes, cardiovascular diseases, abnormal blood pressure, and male sexual dysfunction [1, 2].

Ginseng contains biologically active components known as ginsenosides [3]. Many ginsenosides are exclusively found in ginseng and reported to account for a large part of ginseng-mediated medicinal effects [4, 5]. Until now, approximately 200 major and minor ginsenosides have been associated with ginseng [6], of which 38 have been isolated from Korean ginseng [2].

Along with simple use of fresh ginseng roots, several processing methods have been traditionally applied to increase preservability and effectiveness; White ginseng is the dried product of peeled roots, while red ginseng is produced through steaming and drying of fresh roots without peeling skin. Preparation of black ginseng requires further effort and time of repeating the steaming and drying cycle. Interestingly, these ginseng products have been observed to be significantly different in the range of applicable diseases and efficacy according to the processing methods [7], and recent studies discovered that the kinds and concentration of ginsenosides may be responsible; white ginseng is rich in main ginsenosides including Rg1, Rb1 and Rb2, while minor ginsenosides such as Rg3, Rg5 and Rk1 become more abundant in red ginseng and, to even higher levels, in black ginseng due to accumulation during processing [8, 9].

Black ginseng has been known for its superior pharmacological activity, to which accumulation of minor ginsenosides plays as the main contributing factor [10]. The repetitive processing facilitates structural transformation of Rb1, leading to accumulation of Rg3 and Rg5 [11]. Both Rg3 and Rg5 have been shown to possess high biological activities [12], among which anti-cancer activity has been well documented in several cancer cell lines and xenograft mouse models [13‐18].

Ginseng has been generally acknowledged as safe based on its clinical use in traditional medicine and the results of toxicity studies [19‐21]. Among the processed ginseng products, safety of red ginseng has been well investigated, establishing essential toxicity parameters including LD50 and NOAEL in model systems [22‐26]. On the contrary, safety information on black ginseng has been limited despite its growing popularity in commercial markets, raising safety concerns; only one study reported acute oral toxicity for single oral administration of black ginseng [27], but to date the toxicity profile associated with its repetitive use has not been available. In this study, we therefore evaluated the subacute oral toxicity of black ginseng extract (BGE; commercially known as CJ EnerG) in SD rats. Prior to the toxicity study, we verified the pharmacological validity of BGE by examining cytotoxic activity in six human cancer cell lines in comparison with red ginseng extract (RGE). Our results demonstrated that compared to RGE, BGE had higher cytotoxic activity in all cancer cell lines tested in this study largely due to its high contents of Rg5, and did not cause recognizable test substance-associated toxicity in vivo.

Ginseng roots have been used as a valuable medicinal herb in oriental medicine from the ancient times and several processing methods have been applied for improvement of medicinal benefits as well as preservation. Since initially developed in Korea, black ginseng has been spread to other countries with recognition of its high pharmacological activities in various diseases. In this study, we investigated subacute toxicity of black ginseng extract in SD rats by oral administration for 28 days.

Prior to the in vivo toxicity study, BGE was assessed for the contents of ginsenosides as well as in vitro cytotoxic activity to confirm the pharmacological validity as a test substance. Consistently with the previous reports [8, 9, 11], HPLC analysis showed efficient accumulation of Rg3 and Rg5 in BGE through the production process. When tested for cytotoxic activity, BGE displayed higher cytotoxicity than RGE in all cell lines tested in the study; BGE showed similarly high potency across the cell lines as indicated by low IC50, whilst the cytotoxic effect of RGE was observed only at the high doses to a lesser degree than BGE.

Anti-cancer activity of black ginseng has been reported in several in vitro and in vivo studies and our results are closely in line with their findings. Black ginseng was shown to elicit cytotoxic activity against several cancer cell lines including MCF-1 human breast cancer, HT-1080 human fibrosarcoma [31], and HepG2 human hepatocellular carcinoma cells [32]. In xenograft mouse models, black ginseng significantly decreased the weight and volume of solid cancer masses derived from Lewis lung carcinoma [31], H22 [33], HepG2 [30, 32], indicating potent anti-cancer activity. Further to these findings, our data showed that BGE is effective in more types of human cancers in vitro, suggesting its potential application in treating a broad range of cancers. It is of great interest to know whether BGE also has such levels of efficacy in vivo, and further studies are warranted to investigate anti-cancer activity of BGE in rodent xenograft models.

Similarity in cytotoxicity patterns observed for Rg5 and BGE in consideration of Rg5 levels suggested that Rg5 is the main contributor to the anti-cancer activity of BGE [16], providing a plausible mechanism underlying the different anti-cancer activity between BGE and RGE. Given the degree of cytotoxicity caused by BGE, Rg3 may play an additive role to the action of Rg5. Several studies reported greater cytotoxicity for Rg5 than Rg3 in a variety of cancer cells including breast cancer cells (e.g., MDA-MB-453 and MCF-7), colorectal cancer cells (e.g., Caco-2 and HCT-8), lung cancer cells (e.g., NCI-H460), hepatocellular carcinoma cells (SMMC-7721) and gastric cancer cells (SGC-7901) [16, 34, 35]. In agreement with these findings, we observed that Rg5 consistently showed greater reduction of cell viability than 20(S)-Rg3 across all the six cell lines at the low doses, confirming its superior cytotoxic potency in a broad-spectrum of cancers. Given the structural similarity to Rg3 except for a hydroxyl group on Carbon-20, how Rg5 can possess remarkably higher therapeutic activity is an intriguing question. Rg3 and Rg5 have been shown to suppress the growth of cancers by influencing diverse aspects of tumor cell biology including cell cycle and proliferation, angiogenesis, metastasis and stemness of cancer stem cells through regulating signaling pathways such as PI3K/PKB [18, 36], STAT3 [35], MAPK [37], C/EBPβ/NF-κB [15], Wnt [38] and SNAIL [39]. Nonetheless, it is still not clear which one(s) among these pathways are responsible for the greater effect of Rg5 or whether other pathways yet to be found exist. Therefore, further investigation into comparison of the degree to which Rg3 and Rg5 activate the known pathways with continuous effort to discover novel pathways may be required to elucidate the molecular mechanism underlying the action of Rg5. Of note, a study reported that upon extracellular treatment, Rg5 accumulated intracellularly in a breast cancer cell line more readily than Rg3, suggesting a possible role of the structural difference in transmembrane transportation of ginsenosides [40].

Although toxicity information on black ginseng, as opposed to the accumulated reports on its pharmacological efficacy, has been limited, a growing body of evidence suggested its safety for oral consumption. A recent study on acute oral toxicity of black ginseng extract reported normal clinical signs, body weight gain, hematology and serum biochemistry without a test substance-associated mortality and histopathological changes, establishing the oral LD50 to be > 15 g/kg [27], which is categorized as practically non-toxic according to Hodges and Sterner Scale [41]. Rg3, one of the enriched ginsenosides in BGE, has been reported to be relatively safe for oral consumption through several toxicity studies; LD50 of 20(S)-Rg3 was determined to be > 800 mg/kg in SD rats and > 1600 mg/kg in mice, and NOAEL in SD rats was 180 mg/kg [42]. In another study using beagle dogs, NOAEL of 20(S)-Rg3 was detected to be 20 mg/kg BW after oral administration for 26 weeks [23]. Regarding Rg5, 30-day repeated intraperitoneal administration of 20 mg/kg into mice did not cause any significant deviations from normal ranges in all clinical and pathological parameters examined in the study [18]. The same research group also found that mice injected with 40 mg/kg of Rg5 showed marked size reduction of engrafted gastric tumors with normal findings in body weight gain during 30 days as well as histopathological analysis [37]. In fact, the levels of Rg3 and Rg5 in our BGE fell in the dose ranges tested in these studies, and our study consistently revealed that oral administration of up to 2000 mg/kg of BGE for 28 days was not toxic in SD rats, demonstrating the safety of BGE for repeated oral consumption. Furthermore, our findings also indicate the reliability of the BGE manufacturing process as well as other BGE components including minor ginsenosides accumulated through the processing such as compound K. Nonetheless, the toxicity profile of BGE is still incomplete and further investigation on the in vivo effect of sub/chronic exposure and genotoxicity studies may be required to understand the whole spectrum of its toxicity with characterization of target organs.

In this study, we checked the pharmacological validity of BGE in vitro and investigated subacute oral toxicity of BGE in SD rats. Our results demonstrated that BGE prepared for this study had potent and broad-spectrum cytotoxic activity with Rg5 as the main effector, and did not cause any recognizable test substance-induced in vivo toxicity in the 28-day repeated oral toxicity test with up to 2000 mg/kg BW in SD rats, establishing that NOAEL is > 2000 mg/kg. These findings, together with the previous report on its acute toxicity, demonstrated the safe dose range of BGE (CJ EnerG) in rodents, providing the essential information on safe consumption in human.

Black ginseng extract (BGE; CJ EnerG), under the conditions employed in this study, demonstrated potent cytotoxic effects to a greater extent than red ginseng extract (RGE) in all the human cancer cell lines tested with its efficacy closely resembling that of ginsenoside Rg5. Evaluation of oral safety in the 28-day repeated-dose toxicity study showed that BGE did not cause any noticeable toxicity related to the test substance in SD rats, establishing its NOAEL to be > 2000 mg/kg BW.