Rubus chingii Hu: A Review of the Phytochemistry and Pharmacology

Rubus chingii Hu (R. chingii), referred to as “Fu-Pen-Zi” in Chinese, has great medicinal and dietary values since ancient times. The dried fruits of R. chingii have been widely used in traditional Chinese medicine (TCM) for the treatment of kidney enuresis and urinary frequency for centuries. According to current findings, R. chingii has been reported to contain a variety of chemical constituents, mostly triterpenoids, diterpenoids, flavonoids, and organic acids. These compounds have been demonstrated to be the major bioactive components responsible for pharmacological effects such as anticomplementary, anticancer, antioxidant, antimicrobial, and anti-inflammatory functions. Therefore, this review focused on the up-to-date published data of the literature about R. chingii and comprehensively summarized its phytochemistry, pharmacology, quality control, and toxicity to provide a beneficial support to its further investigations and applications in medicines and foods.


INTRODUCTION
The genus Rubus, belonging to the Rosaceae family, has edible and economically important fruits and is widely distributed throughout the Northern Hemisphere (Moreno-Medina et al., 2018). This genus consists of over 700 species, about 194 of which occur in China, including R. chingii, R. idaeus, R. rosifolius, R. parvifolius, and so on (Li et al., 2015). Among them, R. chingii is an important functional food with the fruits known as "Fu-Pen-Zi" in Chinese. It is mainly cultivated in East China, especially in Jiangxi province, Anhui province, Jiangsu province, Zhejiang province, and Fujian province. Due to its rich nutritional and medicinal value, R. chingii has been frequently used in traditional Chinese medicine (TCM) for centuries (Liu and Niu, 2014). The medical properties of R. chingii have been mentioned in many landmark Chinese medical monographs, such as "Compendium of Materia Medica, " "Bencao Mengquan, " "Leigong Paozhi Lun, " and "Qianjin Yi Fang. " According to the theory of traditional Chinese herbal medical science, R. chingii is commonly used as a tonic for the treatment of enuresis, kidney deficiency, impotence and prospermia, frequency of micturition, spermatorrhea, and other illnesses (Xie et al., 2013a).
Since the universal uses of R. chingii in folk medicines, a great deal of studies concerning the chemical constituents and pharmacological activities of this medicinal plant have been carried out, which gave rise to numerous interesting and attractive results. Many in vitro and in vivo investigations have indicated that the extracts and the ingredients isolated from R. chingii possess abundant pharmacological effects, such as anticomplementary, anticancer, antioxidant, antimicrobial, antiaging and anti-inflammatory activities (Shi, 2017). These marvelous biological functions of this herb can be attributed to the presence of a broad spectrum of phytochemical constituents including triterpenoids, diterpenoids, flavonoids, organic acids, and many other compounds.
Although some brief reviews about the chemical constituents and biological activities have been conducted, these papers were written in Chinese and not studied in a systematic manner. This paper strives for a comprehensive overview of the latest information on the phytochemistry, biological activities, quality control, as well as the toxicity of this herb. More importantly, the correlation between the biological properties and the existence of the bioactive chemical components responsible for the actions has also been discussed based on the published literatures. Finally, the major achievements and shortcomings, together with the possible tendency and perspective for future food and pharmacological research of this herb, have been put forward, too. We believe that this review will highlight the significance of R. chingii and indicate new research directions of this species.

Coumarins
Coumarins are phenolic compounds characterized by a benzene ring attached to a pyrone ring. They have a fragrant smell and exist throughout the plant kingdom (Azietaku et al., 2017). To date, limited studies have been performed to investigate the coumarins in R. chingii and only five coumarins have been isolated, including two simple coumarins and three furocoumarins (Figure 6). (151), esculin (152), and imperatorin (153) from the 70% ethanol extract of the fruits of R. chingii by various chromatographic methods. You reported the isolation and structure elucidation of a new furocoumarins, 3,5,9-trihydroxy-7,8-dihydrocyclopenta[g]chromene-2,6dione(154), which they named Fu-Pen-Zi-Su (You, 2009) or rubusin A (Sun et al., 2011), from the n-butanol extract of the fruits of R. chingii. Recently, phytochemical analysis of R. chingii afforded a new chromone called rubusin B(155), which was confirmed according to the 1D and 2D NMR data and MS data (Liang et al., 2015).

PHARMACOLOGICAL ACTIVITIES OF R. CHINGII
As a well-known medicinal plant in TCM, the fruits and leaves of R. chingii are widely used for the treatment of various diseases. The major pharmacological properties such as anticomplementary, anticancer, antioxidant, antimicrobial, antiinflammatory, anti-hypotensive, anti-aging, antithrombotic, antidiabetic, neuroprotective, and anti-osteoporosis activities of this herbaceous medicine are summarized in Table 2, and the details will be further discussed below.

Anticomplementary Activity
Several studies demonstrated that the extracts of R. chingii possess anticomplementary activity. Zhang and Jiang employed a complement fixation test to assess the in vitro anticomplementary activity of the essential oils from fruits of R. chingii by three different extraction methods [steam distillation extraction (SDE), soxhlet extraction (SE) with ethanol, and SE with ether]. The results showed that the essential oils obtained by SE-ether had the strongest  anticomplementary effect, even stronger than heparin (control) (Zhang and Jiang, 2015). The flavonoids and saponins extracted from R. chingii also showed noteworthy anticomplementary activities when compared to its polysaccharides and alkaloids. The hemolysis inhibition rates of the flavonoids and saponins were 96.49% and 90.82% (at the concentration of 0.8 mg/ml), respectively, which were even higher than heparin sodium (Zhang et al., 2015a).

Anticancer Activity
The antitumor effects of the various extracts of R. chingii have been extensively investigated through a large number of in vivo and in vitro experiments. Wang et al. (2011) found that the water extract of R. chingii could inhibit the activities of matrix metalloproteinases-13 with an IC 50 value (half maximal inhibitory concentration) of 0.04 μg/ml. The results suggested that this herbal medicine may be used for the treatment of cancer. Another study showed that the water extract of R. chingii gave rise to a dose-dependent antiproliferative effect on hepatocellular carcinoma cells with an IC 50 value of 80 μg/ml (Hu, 2014). Anticancer activity was also reported for the essential oils from the unripe fruits of R. chingii by in vitro MTT cytotoxicity assay against A549 cell lines. The results showed that the essential oils extracted by SDE exhibited stronger activity than SE-ethanol, which may be due to the extract obtained by SDE, which had a higher content of unsaturated fatty acids (Zhang and Jiang, 2015). An in vitro study showed that polyphenolic composition in the fruits of R. chingii could inhibit the proliferation and induce apoptosis of human bladder cancer T24 cells remarkably in a dose-dependent and time-response manner. The IC 50 values were 73.442, 55.294, and 26.686 μg/ml for 12, 24, and 36 h, respectively . In a similar study, Zhang et al. (2015b) evaluated the anticancer activity of the polysaccharides from R. chingii via MTT assay and found that inhibitory activities on breast cancer cells' MCF-7 and liver cancer cells' Bel-7402 proliferation were also concentration-and time-dependent. From 70% ethanol extract of the fruits of R. chingii, Zhong et al. (2015) isolated three new labdane-type diterpene glycosides and in vitro tests of these compounds for anticancer activity showed that compound 29 possessed remarkable cytotoxic activity against A549 (human lung cancer cell line), with an IC 50 value of 1.81 μg/ml (2.32 μM). Furthermore, tiliroside, a representative flavonoid isolated from R. chingii, induced the apoptosis of A549 cells in a dosedependent manner, with an IC 50 value of 113.41 ± 1.89 μg/ml (190.76 ± 3.18 μM) (Zhang et al., 2015a). In 2017, Zhang et al. (2017b) investigated the antiproliferative ingredients in the fruits of R. chingii by using bio-assay guided isolation, and found that tormentic acid possessed notable cytotoxicity activities against HepG-2, Bel-7402, A549, and MCF-7 cancer cell lines with the IC 50 values of 40.57, 54.22, 62.36, and 24.23 μg/ml, respectively. All these results described above suggest that R. chingii has an exact effect on prevention of cancer. However, a common mechanism about the exact cellular and molecular targets needs to be fully elucidated and the diversity of extracts makes data interpretation difficult.

Antimicrobial Activity
Antimicrobial activity, an important effect of R. chingii, had been comprehensively studied. A moderate antibacterial activity was evident for the flavonoids from R. chingii against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Penicillium with MIC (minimum inhibitory concentration) values of 0.04, 0.08, 0.16, and 0.64 mg/ml, respectively. However, it could not inhibit the growth of Saccharomyces cerevisiae, Rhizopus, and Mucor  The ethyl acetate fraction and n-butanol fraction showed significant in vitro antioxidant activity (DPPH assay, reducing power assay and ORAC assay) Zhang et al., 2017b Flavonoids from fruits The total flavonoids displayed the best in vitro antioxidant effect (DPPH assay, reducing power assay and ORAC assay), which was very close to ascorbic acid.

Zhang et al., 2015c Polysaccharides from fruits and leaves
Anti-inflammatory potentials against LPS-stimulated murine macrophage RAW264.7 cells by decreasing NO production and increasing the TNF-α, iNOS and IL-6 gene expression (in vitro).

Zhang et al., 2015b
ANTITHROMBOTIC ACTIVITY 70% ethanol fraction from leaves Significant antithrombotic activity was observed in in vitro and in vivo tests. Han et al., 2012 NEUROPROTECTIVE ACTIVITY 80% ethanol extract from fruits Significant improvements in learning and memory were observed, especially in rats receiving the chloroform and ethylacetate fractions (in vivo). Huang et al., 2013 Different extracts from fruits The high dose water extract (24 g/kg) was found to exhibit the best anti-amnesic effects on scopolamine and sodium nitrite (NaNO 2 )-induced amnestic models, while the crude drug showed the best anti-amnesic activity on 40% ethanol-induced amnestic models (in vivo).  (Zhu, 2012). In addition, R. chingii extract combined with fluconazole displayed synergistic antifungal activity on fluconazole-resistant Candida albicans with an MIC 80 (the lowest concentration to inhibit 80% of fungal growth) value of 0.0625-16 μg/ml for fluconazole and 4.88-312.5 μg/ml for the 70% ethanol extract of R. chingii (Han et al., 2016).

Antioxidant Activity
Oxidative stress by free radicals is a significant event in the cell, which is associated with a wide range of human degenerative diseases (Bi et al., 2016). The glycoprotein from R. chingii showed significant in vitro antioxidant activity via free radical scavenging assay and reducing power assays. An in-depth in vivo study revealed that the glycoprotein could significantly increase the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-P X ) in serum, liver, and brain tissues of rats, which also confirmed the strong reducing power of the glycoprotein (Tian et al., 2010). The aqueous extract of R. chingii has also been reported to reverse tert-butyl hydroperoxide (t-BHP)-induced oxidative damage in rat hepatocytes by inhibiting lactate dehydrogenase leakage, lipid peroxidation, and the associated glutathione depletion (Yau et al., 2002). Moreover, among nine compounds isolated from the fruits of R. chingii, methyl (3-hydroxy-2-oxo-2,3dihydroindol-3-yl)-acetate, vanillic acid, kaempferol, and tiliroside displayed antioxidative capacity. Their IC 50 values were 45.2, 34.9, 78.5, and 13.7 μM, respectively (ascorbic acid, 131.8 μM) (Ding, 2011). Zeng et al. studied the in vitro antioxidant capacities of the total flavonoid contents of R. chingii by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis 3-ethylbenzothiazoline-6-sulphonic acid (ABTS) methods. The results showed that the total flavonoid content exhibited a significant correlation with antioxidant activity in the DPPH assay (r 2 = 0.758, ρ = 0.004) and the ABTS assay (r 2 = 0.788, ρ = 0.002) (Zeng et al., 2015). Zhang et al. (2015b) studied the activities of polysaccharides from R. chingii fruit (F-Ps) and leaf (L-Ps) through DPPH scavenging assay and found that the scavenging activities of F-Ps and L-Ps had almost 10 folds lower antioxidant potential than the vitamin C with half inhibition effect (IE 50 ) values of 754.33 and 671.39 μg/ml, respectively. Similarly, the polyphenolic composition in the fruits of R. chingii exhibited high DPPH scavenging effect with an IC 50 value of 33.912 μg/ml, which was half of the standard ascorbic acid . In 2017, an interesting study investigated the antioxidant effects of fruits of R. chingii by using the DPPH assay, reducing power assay and oxygen radical absorbance capacity (ORAC) assay, and the results revealed that the ethyl acetate fraction and n-butanol fraction were found to be the most potent (Zhang et al., 2017b). The polysaccharides, flavonoids, saponins, and alkaloids extracted from R. chingii were also assessed for their antioxidant activity through the same methods. The results indicated that total flavonoids displayed the best antioxidant effect, which was very close to ascorbic acid (Zhang et al., 2015a). From the results mentioned above, we can conclude that the strong antioxidant activity of R. chingii might be predominantly related to the presence of the glycoproteins and phenolic compounds, especially flavonoids. Additionally, it is worthy to note that the in vitro experiments used to test total antioxidant are not specific and prone to interferences, which may give unreliable results. Therefore, further in vivo studies are needed to validate these results.

Anti-Inflammatory Activity
Sun et al. (2013b) extracted a new compound called goshonoside-G from the fruits of R. chingii. This compound possessed notable inhibitory effect on NO production in LPS-stimulated macrophage RAW264.7 cells with an IC 50 value of 54.98 μg/ml. In bio-assay guided fractionation of the ethanol extract of R. chingii, which provided the best anti-inflammatory effect, tiliroside, astragalin, hyperoside, quercitrin, and kaempferol 3-rutinoside were isolated. Among the flavonoid glycosides, tiliroside possessed the strongest inhibitory effect on NO production in LPS-stimulated macrophage RAW 264.7 cells with the inhibitory rate of 30.4% at a concentration of 100 μg/ml, which was very close to that of dexamethasone at a concentration of 50 μg/ml. Western blot and RT-PCR showed that the underlying mechanism of the suppression of inflammatory reactions by tiliroside may be due to its modulation of a signaling mitogen-activated protein kinase (MAPK) and pro-inflammatory cytokines activities (Zhang et al., 2015c). In addition, the polysaccharides from leaves and fruits induced a dose-dependent (2-400 μg/ml) inhibition of the nitric oxide (NO) production in murine macrophage RAW 264.7 cells through suppressing the TNF-α, iNOS, and IL-6 gene expression (Zhang et al., 2015b). Therefore, flavonoid glycosides and polysaccharides along with goshonoside-G of the plant could be considered as potential antiinflammatory agents.

Antithrombotic Activity
The 70% ethanol fraction from an aqueous extract of R. chingii leaves was found to treat thrombosis through inhibiting the aggregation of blood platelets using activity tests carried out in vitro and in vivo. The bio-guided isolation of the extract yielded six compounds (salicylic acid, kaempferol, quercetin, tiliroside, quercetin 3-O-β-D-glucopyranoside, and kaempferol 3-O-β-Dglucopyranoside). Their anticoagulant activities were examined using plasma recalcification time (PRT) test. It is noteworthy that kaempferol, quercetin, and tiliroside obviously delayed PRT in blood at a concentration of 2 mg/ml, while salicylic acid, quercetin 3-O-β-D-glucopyranoside, and kaempferol 3-O-β-Dglucopyranoside demonstrated the weakest effect in the in vitro experiment (Han et al., 2012).

Neuroprotective Activity
Huang et al. investigated whether or not R. chingii was involved in attenuating learning and memory deficits on a classical model of Kidney Yang Deficiency Syndrome (KDS-Yang) in Alzheimer's disease rats induced by D-galactose combined with hydrocortisone. Morris water maze tests demonstrated significant improvements in learning and memory, especially in rats receiving the chloroform and ethylacetate fractions of R. chingii (Huang et al., 2013). The major mechanism may be that R. chingii could protect neurons in rat hippocampal CA1 region by increasing choline acyltransferase (ChAT) activity but decreasing acetylcholinesterase (AChE) activity and Tau protein expression. The possible memory-enhancing effects of different extracts of R. chingii on amnesic rats induced by scopolamine, sodium nitrite, and 40% ethanol were also studied by assessing a Morris water maze test. The results showed that the high-dose water extract (24 g/kg) exhibited the best anti-amnesic effects on scopolamine and sodium nitrite (NaNO 2 )-induced amnestic models, while the crude drug showed the best anti-amnesic activity on 40% ethanolinduced amnestic models (Li et al., 2016a). Moreover, Liu et al. (2018) demonstrated that the water extract of R. chingii could ameliorate H 2 O 2 -induced damages of brain microvascular endothelial cells (bEnd.3 cells) via regulating the expression of apoptosis-related proteins. In addition, two flavonoids (kaempferol and quercetin) isolated from R. chingii were investigated for neuroprotective activity. It was observed that at 80 μmol/L concentration, both compounds significantly inhibited the decrease of cell viability (MTT reduction), prevented membrane damage (LDH release), scavenged ROS formation, and attenuated the decrease of malondialdehyde (MDA) in H 2 O 2 -induced PC12 cells (Zhao et al., 2018). These abovementioned results of preclinical investigations show that R. chingii may be a promising herbal medicine to combat nerve injury.

Antidiabetic Activity and Hypolipidemic Activity
Xie et al. reported antihyperglycemic effects of raspberry ketone in the alloxan-induced diabetic rat model, which were beneficial for the treatment of diabetes. The study showed that raspberry ketone reduced the level of the blood glucose, protected the normal physiological function of pancreatic β cells, and stimulated insulin secretion by effectively inhibiting the oxidative stress . Another study showed that raspberry ketone could significantly promote glucose uptake in HepG2 cells by increasing the IRS-1 protein expression and decreasing SHP-1 mRNA gene expression . The hypolipidemic activity of the leaves from R. chingii was evaluated in the hyperlipidemia rats induced by a high-fat diet and adults with hyperlipidemia. The results revealed that treatment with raspberry leaves exhibited significant hypolipidemic effect, indicated by reduced level of serum total cholesterol (TC) and triacylglycerols (TGs). Therefore, it suggested that raspberry leaves could be further explored as a therapy for the treatment of hyperlipidemia diseases (Fan et al., 2007). Liang et al. (2015) isolated a novel compound, rubusin B, and six known compounds from the fruits of R. chingii, and an in vitro study showed that rubusin B, kaempferol, rubusin A, and quercetin exhibited anti-osteoporotic activities with different characteristics. Quercetin and kaempferol had a direct stimulatory effect on alkaline phosphatase (ALP) activity and bone formation, while rubusin A and B could effectively attenuate osteoclastic resorption even at a very low concentration (0.01 ppm).

Antihypotensive Activity
Recently, it was shown that the ethanol extract of R. chingii could induce the endothelium-dependent vasodilatory effect in rats, via stimulation of the NO/guanylate cyclase/cGMP pathway and the Akt-eNOS pathway (Su et al., 2014).

Anti-Aging Activity
A novel glycoprotein isolated from R. chingii exhibited notable anti-aging effect in the D-galactose-induced aging mice model by increasing the expression of anti-aging gene klotho and repairing the renal function .

Other Pharmacological Effects
In addition to the bio-activities mentioned above, some other pharmacological effects of R. chingii and its constituents were also reported. Chen et al. (1995) demonstrated that R. chingii has mitogenic effects on spleen lymphocytes. They also found that R. chingii could regulate the hypothalamus-pituitary-sex gland axis (Chen et al., 1996). Li (2017) reported that R. chingii could protect retinal ganglion cells from H 2 O 2 -induced cell death by increasing the Bcl-2 protein expression and decreasing Bax protein expression.

TOXICITY
Limited data are available concerning the safety assessments of R. chingii. In an acute toxicity test, the dose of the water extract of R. chingii leaves used in mice was 20 g/kg/day, and it did not induce any toxicity sign or death in 2 weeks (Tang et al., 2007). The potential adverse effects of R. chingii leaves were also determined by a repeated dose oral toxicity study, which was conducted on Wistar rats administered for 90 days at oral dosages of 2.5, 5, and 10 g/kg. The researchers found no significant differences between groups in body weights, food consumption, blood biochemistry, organ weights, gross pathology, and histopathology. Further study indicated that R. chingii leaves had no mutagenic or genotoxic effect using the Ames test, bone marrow micronucleus test, and sperm aberration test (Tang et al., 2007). Based on the results described above, we can conclude that R. chingii leaves are not toxic and hence reliably safe for use for pharmacological purposes. However, more in-depth investigations are still needed to explore the toxicity of the fruits of R. chingii to human health.

CONCLUSION AND FUTURE PERSPECTIVES
R. chingii is a nutritive plant commonly used as a functional food and medicine in China. It has been applied in clinical practice successfully for centuries to tonify the kidney, control nocturnal emissions, and reduce urination (Han et al., 2012). Although chemical compositions and biological activities of this medical plant are well documented, more conclusive studies are still needed to fill certain specific gaps in R. chingii science.
Firstly, and particularly, it is noteworthy that most pharmacological studies on R. chingii have only been conducted in animal models, cell models, and other in vitro experiments. Therefore, comprehensive placebo-controlled and double-blind clinical trials should be undertaken in the future to provide remarkable evidence for these positive findings on the efficacy of R. chingii. Besides, some of the pharmacological studies were carried out at too high doses that could hardly be translated to clinical practice and more in-depth investigations are needed to standardize the best dosage for these claimed bioactivities of R. chingii in ethnomedicine. In addition, the exact mechanisms of many medicinal properties of this herb still remain vague to date; thus, additional studies to better identify the functions and molecular targets seem to be necessary.
Secondly, most pharmacological activities were measured using uncharacterized crude extracts of R. chingii, and this makes it hard to reproduce the results of these investigations and elucidate the link between activity and particular compounds. Additionally, most of these phytochemicals were isolated from the fruits, and the chemical composition of other parts of this plant was largely unknown. Therefore, in-depth phytochemical investigations of all parts of R. chingii based on bio-guided isolation strategies are still needed, which may lead to the expansion of existing therapeutic potential of this miracle herb.
Thirdly, toxicological studies are important to understand the safety profile of herbal drugs, but data on toxicological aspects of R. chingii remain unexplored. The only toxicological study about R. chingii was conducted in the leaf extract, which revealed its nontoxic nature. Hence, to ensure a full utilization of the medicinal resource, further relative systematic toxicity and safety evaluation studies were quite considerable and necessary, especially in fruit extract and other effective extracts, to meet the Western standards of evidence-based medicine.
Fourthly, pharmacokinetic studies involving R. chingii are very limited and only focus on a few biological active substances present in R. chingii, which do not fully reflect the pharmacokinetic properties of this herb medicine. Thus, further investigations should be carried out to assess the absorption, distribution, metabolism, and excretion of the crude extracts of this plant in vivo. Additionally, metabolic studies of single isolated compounds in R. chingii should be strengthened, which could provide a scientific basis for clarifying the major metabolic route and action mechanism and defining the bio-active components responsible for the curative effects. Meanwhile, the identification of unknown metabolites may contribute to the drug discovery and development process.

AUTHOR CONTRIBUTIONS
GY and ZL searched the literature, collected the data, and drafted the manuscript. GY and WW contributed to analysis and manuscript preparation. YL and YZ helped check the chemical structures and formula. YS provided comments on the manuscript. All authors read and approved the final manuscript.

FUNDING
This study was supported by the Start-up fund from Beijing University of Chinese Medicine to YS (No. 1000061020044 and No. 1000041510052).

ACKNOWLEDGMENTS
We acknowledge Beijing University of Chinese Medicine for providing support and assistance for this review article.