Corni Fructus: a review of chemical constituents and pharmacological activities

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.

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.

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, ion-exchange chromatography and affinity chromatography. Finally, infrared spectroscopy analysis and morphological analysis were used to determine the physiochemical and structural features of the polysaccharide.

Four phenylpropanoids, two sterols, five carboxylic acids, two furans, and several mineral substances have also been determined. Among them, 5-hydroxymethylfurfural exhibits diverse biological activities. Besides, Chen, Li, and Wen identified 32, 16, and 48 volatile compounds by GC–MS, respectively [10‐12].

Diabetes mellitus (DM) is a group of long-term and chronic metabolic disorders which are associated with high serum glucose levels. Compared with the no treatment diabetic animal model group, CF extract (at 300 mg kg⁻¹ 2 day⁻¹ and 400 mg kg⁻¹ day⁻¹ p.o.), loganin, morroniside, and ursolic acid (each at 200 mg kg⁻¹ day⁻¹ p.o.) for 4 weeks can significantly decrease fasting blood glucose and alleviate polyphagia, polydipsia, polyuria, and weight loss [13, 14]. In He’s study, metformin (at 200 mg kg⁻¹ day⁻¹ p.o.) demonstrated better effect [14]. Besides, loganin, morroniside, ursolic acid, and butyl morroniside (each at 100 μmol L⁻¹) can protect the pancreatic β-cells from high glucose-induced excessive oxidative stress and apoptosis [14, 15], may further increase the insulin release. Compared with the insulin treatment, CF extract, (−)-epicatechin-3-O-gallate, and caftaric acid monomethyl ester (each at 50 μmol L⁻¹) can also significantly inhibit α-glucosidase activity to slow down the elevation of serum glucose levels [14, 16, 17] and suppress the hepatic gluconeogenesis by decreasing the protein and mRNA levels of PEPCK in vitro [15, 18].

Also, CF extract, iridoid glycosides, and the single compound can decrease 24 h urine protein and serum levels of urea nitrogen and creatinine. To be specific, loganin, morroniside, and 7-O-galloyl-d-sedoheptulose (each at 20–100 mg kg⁻¹ day⁻¹ p.o.) for 10 days and 8 weeks can significantly inhibit both AGE/RAGE formation [19‐22] and CTGF production [23] in db/db mice or STZ-induced diabetic nephropathy model. They can also significantly alleviate diabetic organ injury by decreasing the production of NF-κB and its downstream synthetases and cytokines [19‐25], increasing antioxidant enzyme production [19, 26, 27], and suppressing apoptotic cell death [27, 28].

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 Ca²⁺ 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 [31‐33] 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.

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 PGE₂ and NO levels to control excessive inflammatory responses [36‐38].

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. The study indicated that the aqueous extract inhibited the tumor cell proliferation in a dose-dependent manner at 0.11–0.337 mg mL⁻¹, exhibited oxygen free radicals scavenging activity (at 50 µg mL⁻¹), attenuated xanthine oxidase production (at 2.62 mg mL⁻¹) and lipid peroxidation (at 0.892 mg mL⁻¹) [42]. In this study, CF aqueous extract exhibited the similar effects compared with 5-fluorouracil (at 0.5, 1, 5 µg mL⁻¹).

Many compounds in CF were further tested for the neuroprotective effects. 7R-O-Methyl-morroniside, 7S-O-methyl-morroniside, 7-O-butyl-morroniside, loganin, and morroniside (each at 10 and 50 μmol L⁻¹) for 1 h significantly protected the neurons against glutamate-induced neurotoxicity up to about 78% compared with the no treatment group [43]. CF aqueous extract (at 60 µg mL⁻¹) significantly inhibited the extracellular Ca²⁺ influx to increase cell neurite outgrowth [44]. Also, cornuside, isoterchebin, and tellimagrandin II (each at 25–100 μmol L⁻¹) displayed anti-Alzheimer’s disease potential due to their synergetic inhibitory activities against BACE1 and ChE [45].

Cerebral ischemia, multiple sclerosis, and neurodegenerative disorder models are applied in animal experiments. Iridoid glycosides (at 60 and 180 mg kg⁻¹ day⁻¹ p.o.) for 1–4 weeks and morroniside (at 90 and 270 mg kg⁻¹ day⁻¹ p.o.) for 3 days significantly decreased the infarction volume, increased the number of new mature neurons and blood vessels, and improved nervous system function [46, 47]. Also, iridoid glycosides (at 50–180 mg kg⁻¹ day⁻¹ p.o.) for 3–4 weeks can significantly promote NGF and BDNF production [48‐50], and repair the abnormal functions of microglia, oligodendrocyte, and T cell to maintain the central nervous system homeostasis [48].

In hepatitis cell models, 5-hydroxymethylfurfural (at 0.2–1 and 0.79 μmol L⁻¹) for 24 h has been shown to protect hepatocytes from H₂O₂ induced-cytotoxicity by significantly decreasing NO and intracellular Ca²⁺ levels, inhibiting abnormal production of apoptosis-related proteins and recovering back to regular cell cycle [51‐53]. In hepatitis animal models, 7-O-galloyl-d-sedoheptulose (at 20 and 100 mg kg⁻¹ day⁻¹ p.o.) for 6 weeks and CF ethanol extract (at 100–500 mg kg⁻¹ day⁻¹ p.o.) for 1 week significantly decreased the serum marker enzymes of hepatic damage, weakened the oxidative stress by promoting antioxidant enzymes production and inhibiting lipid peroxidation, finally improved hepatic histological injury [54, 55].

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].