Corni Fructus: a review of chemical constituents and pharmacological activities

Cornus officinalis Sieb. et Zucc. is part of the genus Cornus of the family Cornaceae. Ripening and dry fruits (Corni Fructus) are recognized as an essential herb medicine in the traditional Chinese medicine (TCM) and have been widely used for over 2000 years. This review provides a comprehensive summary of Corni Fructus (CF), including the botany, phytochemistry, traditional use, and current pharmacological activities. According to the basic theory of TCM, CF usually participates in various Chinese medicinal formulae to exert the essential roles in replenishing liver and kidney, arresting seminal emission and sweat. Based on modern pharmacological studies, about 90 compounds have been isolated and identified from CF. In vivo and in vitro experimental studies indicate that CF exhibits extensive pharmacological activities including hypoglycemic, antioxidant, anti-inflammatory, anticancer, neuroprotective, hepatoprotective, and nephroprotective activities. However, only about 18% of chemical constituents in CF were tested. It means the potential pharmacological activities and clinical values of CF need to be further investigated.

Primordial Qi and stop Blood [5]. The fourth part of symptoms contains profuse cold sweating, pale complexion, cold limbs, and a feeble pulse. For patients with the Yang depletion syndrome, Ginseng Radix et Rhizoma, Aconiti Lateralis Radix Praeparata, and CF are applied in Laifu Tang (来复汤) to restore Yang from collapse. Medical practices indicate that CF can be combined with either Yin-tonifying or Yang-invigorating herbs to act as the sovereign drug or adjuvant drug in Chinese medicinal formulae and treat different types of TCM syndromes. Besides, CF is primarily made into the honey bolus to treat chronic diseases while is usually made into the decoction to treat acute conditions.

Chemical constituents
About 90 compounds have been isolated and identified from CF, including terpenoids, flavonoids, tannins, polysaccharides, phenylpropanoids, sterols, carboxylic acids, furans, and mineral substances. Chemical constituents are listed in Table 1. Among them, iridoids, tannins, and flavonoids are the major components. Their chemical structures are shown in Figs. 2, 3, 4 and 5.

Terpenoids (1-26) and flavonoids (27-39)
Most terpenoids and flavonoids in CF shared two similar isolation processes. Firstly, CF was percolated with ethanol to acquire the solvent which was then evaporated under reduced pressure. The resulting extract was suspended in water and then partitioned with ethyl acetate for several times. Finally, the extract was subjected to column chromatography over silica gel to yield compounds. Secondly, CF was grounded into powder and then subjected to supercritical carbon dioxide to yield extract. The resulting extract was subjected to GC-MS to identify the chemical components. So far, 26 terpenoids and 13 flavonoids have been isolated and identified from CF. Among terpenoids, the pharmacological activities of sweroside (1), loganin (5), cornuside (23), ursolic acid (24), and oleanolic acid (25) have been further assayed, and a wide range of pharmacological activities has been revealed. Furthermore, two types of flavonoids namely kaempferol (28), quercetin (33), and their derivatives are the essential flavonoids.

Tannins (40-69)
During the isolation process, CF was firstly homogenized in acetone and then filtered to acquire an aqueous solution which was sequentially extracted with diethyl ether and ethyl acetate. The extract was subjected to column chromatography to give compounds. Finally, the chemical structure and molecular weight were determined using nuclear magnetic resonance (NMR) spectroscopy. To date, 30 tannic acids have been isolated from CF. Tsutomu HATANO identified 28 of them. Many tannic acids in this Chinese herb have the large molecular weight, e.g., the molecular weight of Cornusiins A-F and Camptothins A-B are even larger than 1000 Da [6,7], because dimers and trimers exist in these types of tannic acids.

Polysaccharides (70-79)
Wu and Yin identified most polysaccharides in CF [8,9]. In their isolation process, hot water or petroleum ether was initially used for combining with assistant ultrasonic and microwave to break the cell wall to isolate polysaccharides. Further separation and purification were achieved by the combination of several techniques, e.g., fractional precipitation, ethanol precipitation, ionexchange chromatography and affinity chromatography. Finally, infrared spectroscopy analysis and morphological analysis were used to determine the physiochemical and structural features of the polysaccharide.

Pharmacological activities
Although just a few chemical constituents from CF are assayed for their biological activities, these components displayed diverse pharmacological activities. Detailed biological activities are summarized in Table 2.

Antioxidant activity
Long-term oxidative stress will generate excessive ROS to oxidize protein, lipids, DNA and then cause cell death, tissue damage, and organ dysfunction. Ideal antioxidant drugs are required to regulate the defense system and scavenge excessive ROS. Studies indicated that morroniside (at 1, 10, 100 μmol L −1 ) for 24 h and total saponins (at 60 and 120 mg kg −1 day −1 p.o.) for 4 weeks regulated Ca 2+ and NO release [29,30], the aqueous extract (at 0.25-2.0 mg mL −1 ) for 20 h modulated GSH redox cycle [31], the aqueous extract, the ethanol extract (at 0.01-0.1 mg mL −1 ), morroniside (at 0.05-2 µg mL −1 ), and ursolic acid (at 0.05-2 µg mL −1 ) for 24 h promoted antioxidant enzymes syntheses [31][32][33] to inhibit lipid peroxidation [29], 5-hydroxymethylfurfural (at 100-400 μmol L −1 ) for 3 days decreased ROS release [34], morroniside (at 100 μmol L −1 ) for 2 days recovered cell cycle to normal state [35]. Mentioned effects significantly together reduced the oxidative stress-induced damages compared with the no treatment group.   Iridoid glycosides STZ-induced rat as diabetic nephropathy model Suppress over-deposition of fibronectin and laminin in the kidney. Reduce protein and mRNA levels of TGF-β1 in serum and glomeruli [25] Iridoid glycosides Triterpene acids Db/db mice as obesity-associated type 2 diabetic nephropathy model Improve the histological injury of kidney and pancreas. Ameliorate the structural alterations in mesangial cells and the podocytes in the renal cortex. Inhibit ECM accumulation in the kidney. Decrease 24 h urine protein and serum levels of urea nitrogen and creatinine. Increase insulin release, and decrease fasting blood glucose and levels of TC, TG, and GSP. Attenuate food consumption, water intake, and urine volume. Reduce the production of RAGE, NF-κB, SphK1, and TGF-β [22] CF extract STZ-induced rat as diabetic nephropathy model Inhibit AGE formation in the kidney. Attenuate hyperglycemia and proteinuria. Reduce the production of RAGE, NF-κB, TGF-β1, and CML [20]

5-Hydroxymethylfurfural
High glucose-induced HUVECs as in vitro oxidative stress model Decrease levels of ROS, IL-8, JNK1, and JNK2/3. Increase P-Akt production [34] Total saponins STZ-induced rat as a diabetic oxidative stress model Regulate NO release and endothelium-dependent relaxation on the mesenteric artery. Reduce blood glucose levels [30] Aqueous extract Hypoxanthine and xanthine oxidase-induced bovine PAECs as in vitro oxidative stress model Regulate GSH redox cycle. Increase the intracellular GSH production and the activity of GSH peroxidase and GSH disulfide reductase. Reduce the intracellular level of GSH disulfide. Increase CAT and SOD activity and inhibit the production of hydrogen peroxide and superoxide anion [31] Ethanol extract LPS-induced RAW 264.7 macrophage cells as in vitro oxidative stress model Attenuate xanthine oxidase activity and ROS production. Induce the production of antioxidant enzymes, e.g., CAT, GSX, Cu/Zn-SOD, and Mn-SOD [33] Anti-inflammatory activity  Morroniside MCAO-induced rat as focal cerebral ischemia model Decrease the infarction volume and improve neurological function.
Increase GSH expression and SOD activity. Decrease the production and activity of MDA and caspase-3 in ischemic cortex tissues [47] 5-Hydroxymethylfurfural Hydrogen peroxide-induced rat hippocampal neurons as in vitro neurodegenerative disorder model Enhance Bcl-2 production and suppress expressions of Bax, caspase-3, and p53 [84] Iridoid glycosides MCAO-induced rat as focal cerebral ischemia model Improve neurological function. Increase the number of BrdU-positive cells and nestin-positive cells in the subventricular zone, and the number of new mature neurons and blood vessels in the striatum. Increase protein and mRNA levels of VEGF and Flk-1 [46] Iridoid glycosides Fimbria-fornix transected rat as cerebral ischemia model Decrease neuron loss in the hippocampus and improve memory deficits. Increase the production of BDNF, NGF, Bcl-2, SYP, Trk A, and GAP-43, and decrease the production of Bax and Cyt c    [60] Aqueous extract Ovalbumin-induced BALB/c mice as allergic asthma model Inhibit eosinophil infiltration and ameliorate allergic airway inflammation and airway hyperresponsiveness. Decrease the production of IL-5&13 and OVA-specific IgE [61] Vasorelaxation activity Cornuside Phenylephrine-contracted rat aorta and HUVEC Dilate vascular smooth muscle in the rat and increase cGMP production in vitro [62] Antiviral activity Aqueous extract CVA16 infected Vero cells as in vitro HFMD model Inhibit CVA16 replication [63] Anti-inflammatory activity Prolonged and incurable inflammation may cause many diseases, e.g., atherosclerosis, cancer, ulcerative colitis. In LPS and TNF-α-induced cell inflammation models, compared with the no treatment group, CF aqueous extract (at 0.2, 1, 5 mg mL −1 ) and cornuside (at 1, 10, 50 μmol L −1 ) for 24 h significantly inhibited NF-κB p65 translocation, down-regulated COX-2 and iNOS production, finally decreased PGE 2 and NO levels to control excessive inflammatory responses [36][37][38].

Anticancer activity
CF aqueous extract significantly enhanced both the cytotoxicity and superoxide anion scavenging activity of vitamin C at 0.5 and 36 µg mL −1 , respectively. Together with CF aqueous extract, vitamin C further inhibited proliferation and induced apoptosis in several human oral squamous cell carcinoma cell lines. Compared with no treatment, the proliferation inhibition rate was at 1.3-71.0% [39]. Furthermore, the aqueous extract (at 1.0 mg mL −1 ) for 2 days significantly exhibited anti-ER + human mammary carcinoma activity by inhibiting cell anchorage-independent growth, regulating the metabolism of E2 and E3 [40], and influencing cell cycle progression and cellular apoptosis [41]. Finally, the aqueous extract has been tested for its cancer inhibitory effect in several hepatocellular carcinomas and leukemic cell lines.

Other pharmacological activities
In addition to the mentioned pharmacological activities, CF has also been reported to exert multiple bioactivities. Firstly, sweroside (at 7.5 µg mL −1 ) for 1 week significantly promoted the proliferation and differentiation of osteoblasts via the regulation of osteocalcin [56]. Also, CF extract (at 0-100 µg mL −1 ) for 4 days significantly inhibited osteoclast differentiation in a dose-dependent manner via the inhibition of the signaling cascades NF-κB/c-Fos/NFATc1 to improve osteoporosis [57]. Secondly, CF methanol extract (at 3.125-12.5 µg mL −1 ) for 3 days significantly up-regulated synthesis and activity of tyrosinase, raised TRP-1&2 translation associating with increasing transcription of MITF-M, finally promoted melanogenesis by 36.1% [58]. Thirdly, CF aqueous extract possesses immunomodulatory activity. In C57BL/6 mice that were transplanted with a skin graft from Balb/C donors, CF extract significantly prolonged skin allograft survival synergistically by suppressing Th1 response, promoting regulatory T cell generation, and enhancing its suppressive function [59]. Fourthly, CF shows lung-protective activity via two studies. In the cellular test, oleanolic acid (at 10 and 100 μmol L −1 ) and ursolic acid (at 100 μmol L −1 ) for 30 min' pretreatment significantly down-regulated MUC5AC mucin whose excessive level would impair airway defenses to cause serious airway diseases [60]. In an animal experiment, CF aqueous extract (at 50 and 200 mg kg −1 3 day −1 p.o.) for 5 weeks significantly decreased the production of inflammatory mediators and reduced eosinophil infiltration, finally attenuated allergic airway inflammation and airway hyperresponsiveness [61]. Fifthly, cornuside significantly dilated vascular smooth muscle in phenylephrine-contracted rat aorta via the up-regulation of cGMP level to show its vasorelaxation activity [62]. Finally, among in vitro screening of antiviral drugs for treating hand, foot, and mouth disease (HFMD) infection, CF aqueous extract (at 0.4 µg mL −1 ) for 2 days significantly inhibited CVA16 replication in cellular level [63].

Conclusion
CF is recognized as a fundamental constituent part of tonifying Yin and Yang prescription because of its harmonious and complementary features according to the basic theory of TCM. It possesses the properties of sour and astringent. Firstly, sour and sweet herbs can be combined to nourish Yin, it can act as the sovereign and ministerial drug among Radix Rehmanniae Praeparata, Dioscoreae Rhizoma, Lycii Fructus, Ligustri Lucidi Fructus, Schisandrae Chinensis Fructus. Also, sour and astringent properties exhibit their function of astringing and storing. It also behaves as the sovereign and the ministerial drug that combines with Euryales Semen, Sepiae Endoconcha, Mantidis Oötheca, Rubi Fructus, Paeoniae Radix Alba to treat spermatorrhea, urorrhagia, metrorrhagia and metrostaxis, and excessive perspiration. Finally, CF can be as the adjuvant and conductant drug to alleviate warm and dry features of Yang-reinforcing drugs.
Chemical constituents from terpenoids, flavonoids, tannins, and furans exhibited diverse biological activities, including hypoglycemic, neuroprotective, heart-protective, hepatoprotective, nephroprotective, testis-protective activities. Pharmacological activities are outlined in Fig. 6. In these studies, bioactive components from CF mainly alleviated the damage of target organs by antioxidant activity, anti-inflammatory activity, and antiapoptosis activity, i.e., up-regulating the expressions and activities of antioxidant enzymes, down-regulating the levels of cytokines and chemokines, and modulating the abnormal expressions of apoptotic death associated proteins.
Hypoglycemic activity and alleviating diabetic target organs damage are critical pharmacological activities among the broad spectrum of pharmacological activities of CF. Morroniside, loganin, oleanolic acid, ursolic acid, and 7-O-galloyl-d-sedoheptulose exhibited the similar efficacy compared with the conventional oral hypoglycemic drugs (acarbose and metformin). In vivo studies, they reduced serum glucose levels and alleviated unusual symptoms caused by diabetes. In cellular assays, they protected pancreatic β cell from oxidative damage, increased insulin release, improved insulin resistance, displayed α-glucosidase inhibition activity, and  [64][65][66][67]. Diverse anti-diabetes and anti-diabetic complication pharmacological activities make CF a potential herb to become the complementary drug for treating DM. Another significant biological activity is the neuroprotection. In cerebral ischemia rat model and neurodegenerative disorder cellular model, iridoid glycosides (e.g., morroniside) and 5-hydroxymethylfurfural increased the number of new mature neurons and blood vessels and exerted anti-oxidative stress, anti-inflammation, and anti-apoptosis properties. In cerebral ischemia rat model and multiple sclerosis rats and mice models, iridoid glycosides also enhanced the levels of brain-derived neurotrophic factor and nerve growth factor. Current studies showed that the pathogenic mechanisms of neurodegenerative diseases have the close relationship with autophagy deficiency and abnormal proteins aggregate clearance dysfunction [68,69]. In addition to the antiapoptotic activity, pharmacological activities of CF on the regulation of autophagy can be further explored. Furthermore, many classic Chinese medicinal formulae have been used to treat neurological disorders belonging to liver and kidney deficiency [70][71][72]. For example, Liuwei Dihuang Wan treats insomnia, Zuogui Wan (左 归丸) treats epilepsy and vertigo, Dabu Yinjian (大补阴 煎) treats a headache, Zuogui Wan and Dihuang Yinzi (地 黄饮子) treats stroke, and Huanshao Dan (还少丹) treats dementia. CF plays a vital role in nourishing liver and kidney Yin in these Chinese medicinal formulae.
However, about 90 compounds have been isolated and identified from CF, only 18% compounds are further assayed for their pharmacological activities in vivo and in vitro. It indicates that pharmacological activities of the remaining 90% chemical components are still unknown yet. Moreover, current studies do not provide enough evidence to verify the drug binding sites of active ingredients of CF. For example, it is difficult to judge whether these active ingredients bind the G protein coupled receptor, ion channels, transmembrane receptor kinases, or nuclear receptors to work. Therefore, more systematic and detailed pharmacological studies on CF need to be fulfilled in the future.