New Phenylpropanoid and Coumarin Glycosides from the Stems of Hydrangea paniculata Sieb

A new phenylpropanoid glycoside (1), and two new coumarin glycosides (2, 3), together with two known compounds (4, 5), have been isolated from the stems of Hydrangea paniculata Sieb. Their structures have been determined by spectroscopic and chemical methods. Furthermore, compound 1 (50 μM) exhibited significant hepatoprotective activity against N-acetyl-p-aminophenol (APAP)-induced HepG2 cell damage in vitro assays.


Introduction
Hydrangea paniculata Sieb.(Saxifragaceae), widely distributed in southern part of China, has been employed to bring down fever, relieve sore throat, and treat malaria in folk medicine [1].Our previous studies have found that the effective fraction of H. paniculata is used to make pharmaceutical compounds for the prevention and/or treatment of renal insufficiency, hypertension, and diabetic nephropathy [2,3].In addition, further phytochemical investigations of this plant have led to the identification of coumarin glycosides, iridoid glucosides and secoiridoids, which showed diverse biological activities, such as neuroprotective effects, hepatoprotective and suppressive diabetic nephropathy [4][5][6].As part of our ongoing research to identify bioactive constituents, a detailed phytochemical investigation on the water extract of the stems of H. paniculata was carried out.We reported herein the isolation and structural characterization of a new phenylpropanoid glycoside and two new coumarin glycosides (1-3), along with two known constituents, coniferinoside (4) and isoconferinoside (5) (Figure 1).The evaluations of their hepatoprotective activity were also displayed in this paper.

Introduction
Hydrangea paniculata Sieb.(Saxifragaceae), widely distributed in southern part of China, has been employed to bring down fever, relieve sore throat, and treat malaria in folk medicine [1].Our previous studies have found that the effective fraction of H. paniculata is used to make pharmaceutical compounds for the prevention and/or treatment of renal insufficiency, hypertension, and diabetic nephropathy [2,3].In addition, further phytochemical investigations of this plant have led to the identification of coumarin glycosides, iridoid glucosides and secoiridoids, which showed diverse biological activities, such as neuroprotective effects, hepatoprotective and suppressive diabetic nephropathy [4][5][6].As part of our ongoing research to identify bioactive constituents, a detailed phytochemical investigation on the water extract of the stems of H. paniculata was carried out.We reported herein the isolation and structural characterization of a new phenylpropanoid glycoside and two new coumarin glycosides (1-3), along with two known constituents, coniferinoside (4) and isoconferinoside (5) (Figure 1).The evaluations of their hepatoprotective activity were also displayed in this paper.

Structure Identification of the Known Isolates
The two known compounds were identified as coniferinoside (4) [10] and isoconferinoside (5) [11] based on the gCOSY, HSQC, and HMBC spectra, as well as on comparison with those previously reported physicochemical values in the literature.

Plant Material
The stems of H. paniculata were collected from the County of Jinxiu, Guangxi Zhuang Autonomous Region, China, in May 2009, and was identified by Mr. Guangri Long (Liuzhou Forestry Bureau of Guangxi).A voucher specimen (ID-4645) has been deposited with the Institute of Materia Medica, Chinese Academy of Medical Sciences, Beijing.

Extraction and Isolation
The air-dried stems of H. paniculata (20 kg) were crushed to a coarse solid and refluxed with water (2 × 90 L, each for 3 h).The combined H 2 O extract was passed through a macroporous resin (HPD100, 30 L) column eluted with H 2 O (90 L) and 17% ethanol (150 L) successively.The ethanolic portion was dried with a rotary vacuum evaporator and the residue was refluxed with 95% ethanol.

Acid Hydrolysis and Sugar Analysis
Each compound (2 mg) was dissolved in 2 mL 2 M HCl and refluxed at 80 • C for 5 h.The reaction mixture was extracted with EtOAc (3 × 1 mL), and the aqueous layer was evaporated to give a mixture of monosaccharides.The residue was further dissolved in anhydrous pyridine (1 mL) followed by the addition of 2 mg L-cysteine methyl ester hydrochloride (J&K Scientific Ltd., Beijing, China; 99%).After heating at 60 • C for 2 h, the solvent was eliminated under N 2 , and 0.2 mL trimethylsilylimidazole (J&K Scientific Ltd., 99%) was added.Then, the mixture was heated at 60 • C for another 2 h and partitioned with n-hexane and water (2 mL each).The organic layer was analyzed by GC.The authentic samples were treated with the same method, D-glucose and D-apiose in a ratio of 2:1 were detected from 1 to 3.

Hepatoprotective Activity Assay
Human HepG2 hepatoma cells were cultured in DMEM medium supplemented with 10% fetal calf serum, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 • C in a humidified atmosphere of 5% CO 2 + 95% air.The cells were then passaged by treatment with 0.25% trypsin in 0.02% EDTA.The MTT assay was used to assess the cytotoxicity of test samples.The cells were seeded in 96-well multiplates.After an overnight incubation at 37 • C with 5% CO 2 , 50 µM test samples and APAP (final concentration of 8 mM) were added into the wells and incubated for another 48 h.Then, 100 µL of 0.5 mg/mL MTT was added to each well after the withdrawal of the culture medium and incubated for an additional 4 h.The resulting formazan was dissolved in 150 µL of DMSO after aspiration of the culture medium.The plates were placed on a plate shaker for 30 min and read immediately at 570 nm using a microplate reader (µquant Bio-tek, Bio-tek instruments, inc., Winooski, VT, USA) to dertermine the OD value [12].

Conclusions
In summary, this work described the isolation and structure identification of one new phenylpropanoid glycoside (1) and two new (2, 3) coumarin glycosides along with two known compounds (4, 5) from the stems of Hydrangea paniculata Sieb., and their hepatoprotective activities against APAP-induced toxicity in HepG2 cells.Compound 1, phenylpropanoid glycoside including D-apiofuranosyl moiety, was found in this plant for the first time.In addition, it was unusual that sugar chains of compounds 2 and 3 contained α-D-glucose in the plant.

Figure 1 .
Figure 1.Structures and key HMBCs of compounds 1-3 from the stems of Hydrangea paniculata Sieb.

Figure 1 .
Figure 1.Structures and key HMBCs of compounds 1-3 from the stems of Hydrangea paniculata Sieb.