Juglans mandshurica Maxim.: A Review of Its Traditional Usages, Phytochemical Constituents, and Pharmacological Properties

Juglans mandshurica Maxim., also known as “Manchurian walnut” (Chinese) and “Onigurumi” (Japanese), is a medicinal plant widely distributed in Western and Central Asia, especially in China. It has been traditionally used to treat cancer, gastric ulcers, diarrhea, dysentery, dermatosis, uterine prolapse, and leukopenia. To date, more than 400 constituents including quinones (e.g. naphthoquinones, anthraquinones, naphthalenones, tetralones), phenolics, flavonoids, triterpenoids, coumarins, lignans, phenylpropanoids, diarylheptanoids, and steroids, were isolated and structurally identified from different plant parts of J. mandshurica. Among them, quinones, phenolics, triterpenoids, and diarylheptanoids, as the major bioactive substances, have been extensively studied and displayed significant bioactivity. Previous studies have demonstrated that J. mandshurica and a few of its active components exhibit a wide range of pharmacologically important properties, such as antitumor, immunomodulatory, anti-inflammatory, neuroprotective, anti-diabetic, antiviral, antimicrobial, and anti-melanogenesis activities. However, many investigations on biological activities were mainly based on crude extracts of this plant, and the major bioactive ingredients responsible for these bioactivities have not been well identified. Further in vitro and in vivo studies on the mechanisms of action of the pure bioactive compounds, and more elaborate toxicity studies as well as clinical studies are needed to ensure safety and effectiveness of the plant for human use. Taken together, the present review will provide some specific useful suggestions guide to further investigations and applications of this plant in the preparation of medicines and functional foods.


INTRODUCTION
Juglans mandshurica Maxim, known as Manchurian walnut and Onigurumi, is a perennial and fast-growing deciduous broad-leaf tree reaching up to 20 m in the family Juglandaceae. It is extensively cultivated and distributed on a large scale throughout China, India, Japan, Siberia, Russia, and Korean Peninsula, etc. (Son, 1995;Machida et al., 2005;Bai et al., 2010;Wang et al., 2015;Hu et al., 2016;Li et al., 2018;Zhao et al., 2018;Zhao et al., 2019). In China, as hardwood tree species together with Fraxinus mandshurica Rupr. and Phellodendron amurense Rupr., it is mainly distributed in temperate to warmtemperate zones, and thus itgrown throughout many regions of northeast China, such as Heilongjiang and Liaoning provinces (Editorial Committee of Flora of China, 1979;Wang et al., 2020a). Now, it is officially listed as a national level Ⅱ rare tree species and is also ranked as a rare and endangered tree species in China (Zhu et al., 2018). More importantly, every plant parts of J. mandshurica, including roots, stems, barks, branches, leaves, green husks, and immature fruits have important medical and health protection values, and have been used to prevent or treat multiple diseases for hundreds of years (see Figure 1; Zhao et al., 2019). As an example, "Bei-Qing-Long-Yi" (BQLY), the epicarp of immature fruits of J. mandshurica, has been used as traditional medicine for the treatment of cancer, gastric ulcers, diarrhea, dysentery, dermatosis, uterine prolapse, and leukopenia in northern China and Korea (Park et al., 2012;Liu et al., 2017;Park et al., 2017;Zhang et al., 2017;Huo et al., 2018;Zhou et al., 2019b). Currently, it is attracting increasing interest worldwide due to its various health-promoting effects. Nevertheless, overdosage or unreasonable use of BQLY can lead to some adverse reaction, such as nausea, vomiting, dizziness, dyspnea, palpitation, and even shock and death (Huo et al., 2017).
Phytochemical investigations on the different medicinal parts (roots, stems, barks, branches, leaves, and immature fruits) led to the isolation and identification of more than 400 compounds, including quinones, phenolics, flavonoids, lignans, coumarins, phenylpropanoids, triterpenoids, diarylheptanoids, and steroids. Among these compounds, quinones, phenolics, triterpenoids, and diarylheptanoids have been extensively studied and displayed the best bioactivity. As an example, naphthoquinone compounds obtained from green walnut husks of J. mandshurica were recognized as major active component that is mainly responsible for the anticancer activity, and the study on the bioactivity of these components has become a hotspot and attracted widespread attention from domestic and foreign researchers . The kernels of the nuts of J. mandshurica also have high nutritional value, containing lipids (60-66%), proteins (15-20%), carbohydrates (1-15%), vitamins, and minerals (Wang et al., 2017b;Fang et al., 2018;Wang et al., 2020a). The lipids are also considered to be the main source for bioactivities owing to their abundant polyunsaturated fatty acids (Carey et al., 2020). Recent pharmacological studies have revealed that the active components and/or crude extracts of J. mandshurica display various biological activities, such as antitumor, immunoregulatory, anti-inflammatory, neuroprotective, anti-diabetic, antiviral, antimicrobial and anti-melanogenesis activities. More importantly, most of these claimed effects are consistent with those observed therapeutic actions of J. mandshurica in folk medicine.
Until recently, scientists have made a great contribution to report the chemical constituents and biological properties of J. mandshurica. However, no systematic review covering allimportant aspects on this plant is available. In order to provide new insights for the in-depth exploration and comprehensive utilization of this plant, we systematically and critically summarize the current findings on botanical description, traditional usages, phytochemistry, pharmacology, and toxicology as well as the potential molecular mechanisms of J. mandshurica. Available information on this plant in this review enables people to explore their therapeutic potential, to highlight the gaps as well as provide the scientific basis for future study of this plant. 53 (Continued in next column) 255 1-(4-Hydro-xyphenyl)-7-(4hydroxy-3-methoxyphenyl)-4hepten-3-one EtOH Roots Zhao et al.
260 5(S)-5-hydroxy-1-(4-hydroxy-3methoxyphenyl)-7-(4hydroxyphenyl)-3-heptanone (Continued in next column)       (Zhang et al., 2018). According to another TCM monograph of "Kaibao Bencao" (Simplified Chinese: 开宝本 草) in the Song Dynasty, BQLY has the functions of nourishing lungs and relieving asthma. Moreover, the decoction of kernels, barks, roots, and immature pericarps of J. mandshurica has been used as folk remedy for treating cancer, which was consistent with their heat clearing and detoxification effects Li et al., 2003;Park et al., 2012;Yao et al., 2012;Xu et al., 2013;Gao et al., 2016;Wang et al., 2017a;Zhang et al., 2019). Interestingly, J. mandshurica is traditionally decocted together with chicken eggs to effectively prevent and treat multiple tumors in Chinese folk medicine (Wang et al., 2017a;Wang et al., 2017c). It is important that various parts of this plant, including the green walnut husks, green peels, roots, stems, barks, branches, leaves and immature fruits have a great medicinal value in indigenous medicine. The green peels were extensively used as folk remedy for removing heat and detoxication, relieving dysentery, and improving eyesight (Li et al., 2017a). The barks were commonly used to treat urinary stones, lichen planus circumscriptus, chronic bronchitis, blurred vision, shigellosis, and HIV (Xin et al., 2014;Yao et al., 2017). Its fresh rejuvenated fruit has been used traditionally as a medicine for treatment of cancer and dermatosis, and as an anodyne to relieve aches in China (Liu et al., 2004a). The nuts are extensively used as food because of its considerable nutritional value (Wang et al., 2017b;Mu et al., 2017). In Japan, several parts of this plant have been used in folk medicines and the fruits have been commonly used for the treatment of chilblains and athlete's foot (Machida et al., 2005).

PHYTOCHEMICAL CONSTITUENTS
Currently, more than 400 comounds including quinones, phenolics, triterpenoids, diarylheptanoids, flavonoids, coumarins, lignans, phenylpropanoids, and steroids, etc. have been isolated and identified from different organs of J. mandshurica Among them, quinones, phenolics, triterpenoids, and diarylheptanoids are the most important and abundant bioactive constituents, which have been considered as the promising ingredients for future evaluation. Many ingredients with significant biological activities such as juglone, juglanthraquinone C, juglonol A, juglanin B, and juglansoside C might be used as markers for quantitative validatio and quality control of the plant in the future. The chemical compounds isolated and identified from J. mandshurica are summarized in Table 1, and structures of major bioactive compounds are presented in Figure 2.

Quinones
Until now, approximately 125 quinones and their derivatives have been identified from the different plant organs of J. mandshurica. Quinones found in this plant can be (1-29), anthraquinones (30-40), naphthalenones (41-54), tetralones (55-123), and benzoquinones (124-125) based on the structural characteristics. In recent years, the study on the bioactivity of naphthoquinone compounds obtained from J. mandshurica has become a hotspot, which was recognized as major active components for the anticancer activity . However, few in vivo pharmacological activity evaluation and even clinical trials of these ingredients were still reported recently.

Phenolics
Nowadays, a total of 69 phenolics constituents (126-194) have been isolated and structurally characterized from the different parts of J. mandshurica. Nevertheless, only few bioactive phenolic compounds of this plant have been reported in recent years. To fully utilize the phenolics constituents of J. mandshurica in the development and application of cosmetic, functional foods and pharmaceutical products, more in-depth research on chemical ingredients and bioactivities are urgently needed.

Triterpenoids
To date, approximately forty-one triterpenoids (195-235) have been isolated and identified from the different parts of J. mandshurica. Among of them, dammarane-type triterpenoids isolated and identified from different medicinal parts of J. mandshurica, have captured more and more attention around the world due to their potent pharmacological activities, especially in antitumor properties (Salehi et al., 2019).

Diarylheptanoids
Diarylheptanoids own multiple pharmacological activities, raising ncreasingly attention over the last few decades (Sun et al., 2020). Currently, a total of 40 diarylheptanoids (236-275) were identified from the different parts of J. mandshurica. Among of them, compound 237-239, showed outstanding cytotoxicity against the A549 and HeLa cells .

Flavonoids
Flavonoids are widespread in the plant kingdom in free form or as glycosides, and many of them are natural drugs with various medical functions (Luan et al., 2019). Up to date, a total of 39 flavonoids (276-314) have been obtained and purified from the green peel, epicarp, stem barks, roots, green walnut husks, and pericarps of J. mandshurica. Amongst the isolated compounds, taxifolin (297) exhibited the strongest anti-HIV-1 activity against MT-4 cells (Min et al., 2002). However, pharmacological investigations on other flavonoids from J. mandshurica are Frontiers in Pharmacology | www.frontiersin.org January 2021 | Volume 11 | Article 569800 14 TABLE 2 | The pharmacological activities of bioactive compounds and extracts of J. mandshurica ("↓", decrease; "↑", increase).

Lignans
Lignans with chiral carbon atoms are usually consisted of a pair of enantiomers or several pairs of stereoisomers with different amount in nature, and the biological activities of enantiomers are not identical due to the chiral nature of the biological receptors (Pereira et al., 2011). Until now, 20 lignans (315-334) have been structurally identified from the barks, roots, and fruits of J. mandshurica.

Coumarins
Coumarins refer to the general term of o-hydroxycinnamic acid lactones with the basic skeleton of benzoben-α-pyranone parent nucleus, which is one of the main components of TCM (Jiang et al., 2020). At present, 19 coumarins (335-353) have been isolated and characterized from the stem barks of J. mandshurica, and mainly include simple coumarins and pyranocoumarins.

Phenylpropanoids
Phenylpropanoids displayed various biological effects including defending against herbivores, microbial attack, or other sources of injury. Nowadays, a total of 16 phenylpropanoids (354-369) have been isolated and structurally identified from the barks, leaves, pericarps, and green walnut husks of J. mandshurica. However, studies on biological effects of phenylpropanoids from J. mandshurica are very limited.

Alkaloids
Alkaloids is an important secondary metabolite and represent a relatively small class of compounds from this plant and possess remarkable antitumor activity. Until now, 7 alkaloids (381-387) have been isolated and structurally elucidated from the barks of J. mandshurica. However, there are not many studies on the biological activity of these alkaloids and therefore further research need to be explored.

PHARMACOLOGICAL PROPERTIES
To date, J. mandshurica have been explored for multiple pharmacological activities, such as antitumor, immunoregulatory, anti-inflammatory, neuroprotective, antidiabetic, antiviral, antimicrobial, and anti-melanogenesis activities. Next, these biological activities were discussed one by one in the following paragraphs, and the recapitulative summary was also presented in Table 2. The mechanism of the typical and representative pharmacological activities like antitumor, immune immunoregulation, antioxidant and
FIGURE 5 | Schematic representation the underlying mechanism of neuroprotective activity of J. mandshurica.
Frontiers in Pharmacology | www.frontiersin.org January 2021 | Volume 11 | Article 569800 type compounds is generally stronger than that of naphthone, naphthol and thier glycosides, and the naphthone glycosides showed the weakest antitumor activity .
In vivo in mouse models, it has been demonstrated that J. mandshurica and its secondary products showed protective activity on MCF-7 tumor-bearing mice , S180 tumor-bearing mice (Yao et al., 2009;Wang et al., 2015), and H22 tumor-bearing mouse (Wang et al., 2017c;Wang et al., 2017d). A polysaccharide, namely JRP1, purified form the fruits, at doses of 25, 50 and 100 mg/kg, i.p., for 21 days, inhibited the tumor growth with inhibition rates of 35.3%, 40.6% and 48.1%, respectively, and decreased the index of spleen and thymus and increased the serum levels of immune regulatory markers such as IL-2, TNF-α and IFN-γ with a dose-dependent manner in S180 tumor-bearing mice (Wang et al., 2015). Orally administration with JMCE (at doses of 100, 200, and 500 mg/kg) to S180 tumor-bearing mice once a day for 8 days significantly elevated the indexes thymus and spleen, inhibited the growth of tumor with inhibition rates of 48.37%, 40.81%, and 36.52%, respectively. JMCE also increased the activity of SOD and decreased the content of MDA in the serum of tumor-bearing mice (Yao et al., 2009).
In traditional Chinese medicine as described by "Zhongguo Minjian Liaofa", branches of J. mandshurica are decocted together with chicken eggs. The eggs should be initially administered and the decoction should be administered when there are no obvious side effects. Eggs decocted with J. mandshurica branches (EDJB), at doses of 0.64, 1.28, and 2.56 g/kg i.p. once a day for 10 days, suppressed the growth of tumor tissues and increased the body weights in H22 tumorbearing mouse in a dose-and time-dependent manner. Moreover, EDJB dramatically elevated the thymus index and spleen index of tumor mice, improved the peripheral red blood cells and hemoglobin numbers as well as reduced the white blood cells numbers (Wang et al., 2017c), suggested EDJB has good antitumor effect against H22 cell. In addition, total tannins (TT) obtained from J. mandshurica, at doses of 0.09 and 0.18 g/kg once a day for 10 days, prominently inhibited the growth of tumor tissues in H22 tumor bearing mouse with an inhibition rate of 34.22% and 36.92%, respectively (Wang et al., 2017d).
Multidrug resistance (MDR) is a major obstacle that hinders the treatment of cancer. Wen et al. (2017) developed a self-assembled polyjuglanin nanoparticle, namely DOX/PJAD-PEG-siRNA, and evaluated its anticancer activity both in vitro and in vivo. In vitro results showed that it improved the cytotoxicity of doxorubicin (DOX) to A549/DOX and H69/CIS cell lines with MDR. Meanwhile, at concentrations of 2, 4, and 8 μg/ml, it significantly down-regulated the mRNA expressions of Kras, P-gp, and c-Myc in a dose-dependent manner (Wen et al., 2017). Moreover, DOX/ PJAD-PEG-siRNA at 2 mg/kg for 21 days, significantly suppressed the growth of tumor, decreased the volume and weight of tumor, KI-67 positive levels and expressions of RAS and c-Myc, and increased the TUNEL positive levels and protein levels of p-JNK and p53 in drug-resistant xenografted nude mice when compared to the free DOX at same dose (Wen et al., 2017). These antitumor activities reported are consistent with the traditional usage such as the treatment of liver cancer, lung cancer, breast cancer, cervical cancer, and gastric cancer, etc.
Overall, J. mandshurica has prominent antitumor potential and has a good health benefit for human. Nevertheless, it is worth noting that most of the research conducted to study antitumor activity stay in the primary stage, and has employed in vitro-based methods and further more in-depth in vivo and mechanism of action investigations as well as clinical studies should therefore be encouraged and strengthened.
Immunoregulatory Activity Li et al. (2018) first evaluated the immunoregulatory functions of the three protein hydrolyzates (PH), namely albumin, glutelin, and globin (molecular weights: 11-35 kDa) obtained from J. mandshurica in mice. The three compounds, glutelin, albumin, and globin at doses of 200, 400, and 800 mg/kg/d, for 35 days significantly increased the thymus and spleen indexes, lymphocyte In a recent study, three peptides, namely YVLLPSPK, TWLPLPR, and KVPPLLY, obtained from J. mandshurica, at a concentration of 50 μM for 24 h, prominently inhibited the generation of ROS, increased the activity of GSH-Px and contents of ATP, and alleviated apoptosis in Aβ 25-35 -induced PC12 cells. It also promoted autophagy and affected the Akt/ mTOR signaling pathway through up-regulating the protein expression levels of Beclin-1, LC3-I, LC3-II, LAMP1, LAMP2, Cathepsin and p-Akt/Akt as well as down-regulating the protein expression level of p62 and p-mTOR/mTOR at molecule levels . Results from above studies indicated that J. mandshurica may serves as sustainable dietary supplement to further develop novel functional food to prevent or defer oxidation-incurred memory impairment damage and ageing/or age-related neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD).

Antidiabetic Activity
Recent findings have demonstrated that J. mandshurica possess significant hypoglycemic activity in vitro and the possible mechanism of this action was showed in Figure 6. The ethyl acetate fractions extracted from ethanol extract of J. mandshurica leaves (JMEE) showed significant α-glucosidase and α-amylase inhibitory activity in vitro with IC 50 of 14 and 130 μg/ml, which were stronger than that of the positive drug acarbose with IC 50 of 44 and 158 μg/ml, respectively . In insulin resistant (IR) hepatic HepG2 cells, LPLLR (Leu-Pro-Leu-Leu-Arg), a novel pentapeptide from the protein hydrolysates of J. mandshurica, at concentrations of 100 and 200 μM, increased the phosphorylation levels of insulin receptor substrate 1 (IRS-1), phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), AMPK and GSK3β, and up-regulated the expression levels of GS and glucose transporter type 4 (GLUT4), while downregulated the expression levels of G-6-Pase and PEPCK in IR hepatic HepG2 cells (Wang et al., 2020a). These findings suggested that LPLLR exerts anti-diabetic effect through increasing the glycogen synthesis and glucose uptake, as well as decreasing the gluconeogenesis. In addition, the peptide LPLLR possesses good stability under in vitro simulated gastrointestinal digestion, and the low molecular weight (610.4 Da) of LPLLR may be beneficial for its intestinal absorption. Nevertheless, more in-depth in vivo investigation is needed to explore the stability and absorption of LPLLR. Subsequently, in high glucose-induced IR and oxidative stress in HepG2 cells, three novel peptides, namely Leu-Val-Arg-Leu (LVRL), Leu-Arg-Tyr-Leu (LRYL), and Val-LeuLeu-Ala-Leu-Val-Leu-Leu-Arg (VLLALVLLR) from J. mandshurica at 12.5-100 μM, significantly improve glucose consumption, glucose uptake, GLUT4 translocation, and elevated the phosphorylation of IRS-1, PI3K, and Akt. The activities of GSH-Px, CAT, and SOD, the nuclear transport of Nrf2, and the protein expression of HO-1 were also increased. Furthermore, these peptides reduced high glucose-induced ROS overproduction and the phosphorylation of ERK, JNK, and p38 (Wang et al., 2020b). These results suggested that peptides from J. mandshurica could protect HepG2 cells from high glucose-induced IR and oxidative stress by activating IRS-1/PI3K/Akt and Nrf2/HO-1 signaling pathways.

Antimicrobial Activity
Three new juglone derivatives, namely juglonol A (71), B (72), and C (73), isolated from the immature exocarps of J. mandshurica by Yang and his colleagues (2019) and their antimicrobial activity against Gram-positive (S. aureus and E. faeculis) and Gram-negative (E. coli and K. pneumonia) bacteria, yeast (C. albicans), and fungi (F. oxysporum, F. oxysporium, C. lagenarium, and P. asparagi) were evaluated. The results showed that juglonol A (71) obviously suppressed all tested strains except for E. coli. with the MIC values ranging 8 from 64 μg/ml However, juglonol B (72) only significantly inhibited the S. aureus with MIC value of 8 μg/ml (Yang et al., 2019). Juglonol A have also been demonstrated to exhibit modestly inhibitory activity against the non-small-cell lung carcinoma (NCI-H1975), lung adenocarcinoma (HCC827), hepatocellular carcinoma (HepG2), triple-negative breast cancer (MD-AMB-231), leukemia (HL-60), mouse colon cancer (CT26) and rat glioma (C6), and IC 50 values were ranging from 9.5 to 31.6 μg/ml (Yang et al., 2019). These results suggested that the presence of juglone core or hydroxyethyl side chain is essential to the molecules' biological activity and that the position of substitution has a marked impact on the potency. Hence, juglonol A, as paninhibitors, might be cytotoxic. Min et al. (2000) found that 1,2,6-trigalloylglucose (192) and 1,2,3,6-tetragalloylglucose (193) isolated from barks of J. mandshurica showed the most potent anti-reverse transcriptase (RT) activity of HIV-1 with the IC 50 values of 67 and 40 nM, respectively. In addition, compound 192 notably suppressed the ribonuclease H (RNase H) activity with IC 50 values of 39 μM when used illimaquinone as a positive control (Min et al., 2000). Simultaneously, Min and his colleagues further found that taxifolin (297) displayed the most potent anti-HIV-1 activity against MT-4 cells with the IC 100 value of 25 μg/ml and CC 100 value of above 100 μg/ml (Min et al., 2002). However, the certain mechanism of anti-HIV-1 activity should be performed at molecule level in the future.

Anti-Melanogenesis Activity
Recently, Kim et al. (2019) obtained three phenolic ingredients from fruit of J. mandshurica and evaluated their anti-melanogenesis activity in B16F10 melanoma cells and primary human epidermal melanocytes. It was found that compound 2-[4-(3-hydroxypropyl)-2methoxyphenoxy]-1,3-propanediol (126) at concentrations of 0.5 and 1.0 μM, showed the highest inhibitory effect through reducing the melanin content, increasing the p-ERK protein expression and decreasing MITF and tyrosinase protein expressions. These effects also could immediately reverse by PD98059, which a potent ERK inhibitor, indicated compound 126 effectively curbed melanogenesis mainly through p-ERK-associated MITF degradation (Kim et al., 2019). Therefore, J. mandshurica has the potential to suppress melanogenesis and can become a useful resource for developing novel skin-whitening agents to cure hyperpigmentation disorders.

PHARMACOKINETICS
Neither systemic evidences regarding the pharmacokinetics extracts from this plant nor evaluations of its target-organ toxicity have been performed. Few investigations have studied the pharmacokinetics parameters of J. mandshurica and its bioactive compounds in animal experiments. Chen et al. (2018) first measured the gallic acid and syringic acid concentrations in rat plasma after the intragastric administration of the aqueous extracts of J. mandshurica at dose of 12 g/kg using high performance liquid chromatography (HPLC). The maximum plasma concentration (C max ) was 0.64 μg/ml, while the time to reach peak concentration (T max ) and elimination half-life (T 1/2 ) were 61.80 and 184.21 min, respectively. The area under the plasma concentration-time curve (AUC 0-t ) and AUC 0-∞ of gallic acid was 96.37 μg min/mL, and 121.59 μg min/mL. Additionally, the C max , T max , T 1/2 , AUC 0-t , and AUC 0-∞ of syringic acid was 0.43 μg/ml, 30.67 min, 99.63 min, 40.33 μg min/mL, 47.02 μg min/mL, respectively (Chen et al., 2018).
Additionally, Chen et al. (2018b) studied the chemical ingredients distribution of the ethanol extracts of exocarp from J. mandshurica after orally administrated at concentration of 1.35 g/ml to rats. The results showed that a total of 54 ingredients have been identified, including 41 archetypes and 13 metabolites. The archetypes included 17 naphthoquinones, 9 diarylheptanoids, 7 flavonoids, 5 triterpenoids, and 3 polyphenols. The metabolites comprised 4 naphthoquinones, 3 diarylheptanoids, and 1 flavonoid, etc, were detected in rats' gastric tissues by UPLC-Q-TOF/MS technology for the first time . Similarly, 24 chemical components including 12 naphthoquinones, 5 flavonoids, 3 diarylheptanoids, and 4 triterpenoids were also detected in rats' kidney tissues by UPLC-Q-TOF/MS technology after orally administration of the ethanol extract of J. mandshurica at a dose of 1.35 g/ml to rats .
Overall, these results might be contributed to explain the body's metabolic process and relative mechanism of action of various components from J. mandshurica, and provide a methodological reference for the evaluation of the safety and effectiveness of compounds in the accumulation in gastric and kidney tissues and relational adverse reactions as well as composition and tissue distribution. It also provides more comprehensive information for clarifying the substance basis of anti-tumor effects in J. mandshurica. Further investigations are required to explore the pharmacokinetics, metabolic stability, and the drug delivery system of J. mandshurica and its active components.

TOXICOLOGICAL INFORMATION
When evaluating the efficacy of drugs, toxicity and safety should be firstly taken into account. Although J. mandshurica as a popular Chinese herbal medicine is frequently used in TCM, information on the side effects and safety evaluations for this plant are seldom reported and insufficient to support their safety. Liu et al. (2004a) reported the acute toxicity of total extracts (TE), petroleum ether extracts (PEE), n-butanol extracts (nBE), aqueous extracts (AE), chloroform extracts (CE), and acetic ether extracts (AEE) from BQLY in mice by administering the increasing doses orally and intraperitoneal injection (TE, PEE, nBE, and AE at doses of 3.62, 4.25, 5.00, 5.88, and 6.29 g/kg, respectively; CE at doses of 400.2, 470.6, 553.6, 651.3, and 766.3 mg/kg; AEE at doses of 930.2, 1,094.4, 1,287.4, 1,514.7, and 1781.9 mg/kg) for 14 days. The results found that the treatment by gavage did not cause any deaths or side effects. However, the intraperitoneal injection with CE and AEE resulted in dose-dependent mortality with signs of toxicity, and the median lethal dose (LD 50 ) of CE and AEE were 575.38 mg/kg and 1,303.59 mg/kg, respectively. Simultaneously, the LD 50 of TE, PEE, nBE, and AE were more than 5 g/kg both in intragastrical and intraperitoneal administration (Liu et al., 2004b). These findings suggested that intraperitoneally injected with chloroform extracts and acetic ether extracts from BQLY were toxic to mice. Recently, Ju et al. (2019) investigated the acute toxicity of aqueous extracts from the stem-barks of J. mandshurica in mice by orally administering the at maximum dose of 227.27 g/kg daily for continuous 14 days. They found that the treatment by aqueous extracts did not cause any deaths or side effects (Ju et al., 2019). Therefore, these results further confirmed that the aqueous extracts of J. mandshurica did not present the apparent toxicity, and might be relatively safe for human.
Additionally, studies showed that BQLY contain many toxic compounds, such as juglone (Huo et al., 2017). In previous study, Westfall et al. (1961) reported that the LD 50 of juglone in mice was 2.5 mg/kg by gavage, the LD 50 of intraperitoneal injection was 25 mg/kg, and the LD 50 of rats was 112 mg/kg by gavage (Westfall et al., 1961). Chen et al. (2005) speculated that the reason for the toxicity of juglone was that it combines with blood components after entering the blood, causing a high concentration of juglone in the blood. Moreover, juglone can react with the sulfhydryl compounds in the gastrointestinal contents, resulting in low absorption of juglone during intragastric administration, which accumulates in the cardia antrum, causing toxicity. In addition, juglone and its metabolites can covalently bind to cytosolic proteins in the kidney, causing renal toxicity (Chen et al., 2005).
The toxicity studies regarding the J. mandshurica and its active components are still in the exploratory stage and mainly focused on acute toxicity study. Therefore, apart from the classical toxicological evaluation, research on chronic toxicity, toxicity mechanism, and toxicokinetics should be further conducted in several animal models and provide scientific explanations for its toxicity and safety applications in the future.

CONCLUSION AND FUTURE PERSPECTIVES
The present review systematically summarizes the findings of the latest research on the traditional usages, phytochemical constituents, pharmacological properties, and toxicities of different extracts and ingredients of J. mandshurica. As a historical herbal medicine, it has been traditionally and popularly used in indigenous populations to treat cancer in China, Japan, Korea, and India more than 2000 years. Recent investigations have focused primarily on evaluating the anticancer activities of the extracts or isolated compounds of this plant. Until now, more than 400 chemical constituents have been isolated and identified from the different parts of J. mandshurica. Through a comprehensive analysis, we found that the quinones, phenolics, triterpenoids, and diarylheptanoids are major and important active compounds of J. mandshurica with numerous pharmacological activities shown in vivo and in vitro investigations.
However, there are also some points and aspects that need to be noted and researched further: (1) The quinones from J. mandshurica with prominent antitumor activity have captured researcher's attention increasingly, and further study on these compounds should be a priority. Until recently, however, J. mandshurica was still considered as folk medicine for the treatment of cancer and the related preclinical experiments results are questioned and unpersuasive, future studies are necessary to address issues regarding composition of the extract, explicability of preclinical experiments, and lack of transformation of the preclinical results to clinical efficacy. Hence, the clinical trial evaluations of J. mandshurica, including animal models and should be conducted urgently.
(2) Although a great number of chemical ingredients had been isolated and identified from this plant, pharmacological evaluations on these compounds are limited to few compounds such as juglone, juglanstetralone A, p-hydroxymethoxybenzobijuglone, juglanthraquinone C, and juglanin. Therefore, deep phytochemical studies of J. mandshurica and its pharmacological properties, especially the mechanism of action of its bioactive constituents to illustrate the correlation between ethnomedicinal uses and biological activities will undoubtedly be the focus of further research. (3) Toxicological investigations are crucial to understand the safety of herbal drugs, but data on toxicological aspects of J. mandshurica were still rarely. Although research confirmed that many medicinal parts of J. mandshurica have little or no toxicity, BQLY has some adverse reactions, which may cause harm to human health. Thence, toxicity and safety assessment studies on BQLY extract and other effective extracts are necessary to ensure the full use of medicinal resources, to meet the Western evidence-based medicine standards, and to provide accurate evidence for clinical applications. Besides, the crude drugs should be strictly in accordance with traditional processing theories and subjected to ancient processing techniques (Pao Zhi), including cleaning, cutting, drying, and digesting, which can reduce their toxicity and exert maximal therapeutic efficacy by transforming the secondary plant metabolites. (4) Pharmacokinetics is an indispensable part of new drug development and rational clinical drug use. However, data on the pharmacokinetics of active compounds and crude extracts of J. mandshurica remain unclear.
Overall, J. mandshurica is a source for nutritional and medical compounds and is worthy of further studty owing to its health-promoting properties and its potential for further development in food industry. However, the existing healthrelated evidence on J. mandshurica is insufficient, and its clinical value has not been adequately studied. Therefore, comprehensive investigations on biological properties, especially the underlying mechanism of bioactiveties of J. mandshurica and its isolated compounds, should be conducted in order to support its ethnomedicinal uses. Besides, the development of healthcare products of J. mandshurica will undoubtedly be the focus of further research. Lastly, this study will help scientists to created additional potential health-promoting pharmaceuticals and functional foods based on J. mandshurica.

AUTHOR CONTRIBUTIONS
HL, KH, DL, and XS obtained and analyzed the literatures. FL, ZW, YJ, and YY wrote the manuscript. XH and NZ gave ideas and edited the manuscript. All authors read and approved the final version of the manuscript for publication.