Phytochemical Study of Stem and Leaf of Clausena lansium

Clausena lansium Lour. Skeels (Rutaceae) is widely distributed in South China and has historically been used as a traditional medicine in local healthcare systems. Although the characteristic components (carbazole alkaloids and coumarins) of C. lansium have been found to possess a wide variety of biological activities, little attention has been paid toward the other components of this plant. In the current study, phytochemical analysis of isolates from a water-soluble stem and leaf extract of C. lansium led to the identification of 12 compounds, including five aromatic glycosides, four sesquiterpene glycosides, two dihydrofuranocoumarin glycosides, and one adenosine. All compounds were isolated for the first time from the genus Clausena, including a new aromatic glycoside (1), a new dihydrofuranocoumarin glycoside (6), and two new sesquiterpene glycosides (8 and 9). The phytochemical structures of the isolates were elucidated using spectroscopic analyses including NMR and MS. The existence of these compounds demonstrates the taxonomic significance of C. lansium in the genus Clausena and suggests that some glycosides from this plant probably play a role in the anticancer activity of C. lansium to some extent.


Introduction
The genus Clausena (Rutaceae) is comprised of approximately 30 species that are scattered throughout the subtropical and tropical regions, including China, Vietnam, Indonesia, Malaysia, and the Philippines [1,2]. There are approximately 10 species as well as 2 varieties in China, which appear in Southern China. Clausena lansium Lour. Skeels (Rutaceae), belonging to the genus Clausena of the family Rutaceae, is a fruit tree and a species of strongly scented evergreen tree growing in South China [1,2]. C. lansium is famous for their fruits, which are usually very popular tropical, health-promoting fruits, while their roots, stems, leaves, and seeds have also been extensively applied in folk medicine or traditional Chinese medicine for the treatments of abdominal pain, malaria, cold, dermatopathy, and snake bites [2,3]. Various biological studies, on the alkaloids, coumarins, and sesquiterpenes from this plant have reported the neuroprotective [4,5], antitumor [6,7], hepatoprotective [8], anti-inflammatory [9], antifungal [10], antioxidant [11], antiobesity [12], nematicidal [13], antimicrobial [14], and hypoglycemic [15] effects of the C. lansium. In our previous studies, some carbazole alkaloids [16] and coumarins [17] were separated from the stem and leaf of C. lansium. As a part of our ongoing research into the natural products possessing structural and biological diversity from C. lansium, a systematic phytochemical study on the stem and leaf of C. lansium
As mentioned in the introduction, as the characteristic components of the genus Clausena, the carbazole alkaloids and coumarins have been found to possess a variety of structures and biological activities [42,43]; however, little attention has been paid toward the other components of this plant, especially the water-soluble components. The current study demonstrates the presence of the aromatic glycosides, sesquiterpene glycosides, and coumarin glycosides. The carbazole alkaloids are considered to be the main anti-cancer components of the genus Clausena [6,7,[44][45][46]. However, several reports demonstrated that the analogs of aromatic glycosides [47,48] and sesquiterpene glycosides [49] also possessed a certain degree of anti-cancer activity. Therefore, it is suggested that some glycosides from this plant probably play a role in the anticancer activity of C. lansium to some extent. At the same time, this also provides some clues about testing anticancer activity and expanding the biological scope of this work for our future research.

Plant Material
The stems and leaves of C. lansium (three-year-old) were collected by pruning and air-dried in Qingyuan (Latitude N 23 • 70 ; Longitude E 113 • 03 ; altitude 71 m), Guangdong Province, China, in September 2015, which were identified by Professor Zhang Zhi-Yong (a botanist) of College of Agriculture, Jiangxi Agricultural University, Nanchang, China. A voucher specimen (no. 2015912) has been deposited in College of Agriculture, Jiangxi Agricultural University.

Determination of Absolute Configurations of Sugars
Compounds 1,6,8,9 (each compound 2-3 mg) were dissolved in 1 N HCl (4 mL) and heated at 90 • C under condition of reflux for 6 h. The reaction product was dissolved in H 2 O after evaporation and partitioned with CH 2 Cl 2 . The aqueous layer containing sugars was concentrated, and then was mixed with L-cysteine methyl ester hydrochloride. Anhydrous pyridine (1 mL) was added to the mixture and heated at 60 • C for 2 h. The product was added to isothiocyanate (3 mg) and heated at 60 • C for another 2 h. The final reaction mixture was analyzed by HPLC under the following conditions: an Agilent 1260 chromatograph equipped an Eclipse XDB-C18 column (5 µm, 4.6 × 250 mm); column temperature: 35 • C; mobile phase: isocratic elution of 25% CH 3 CN-H 2 O (V:V) in 50 mmol/L HCl; flow rate: 0.8 mL/min; injection volume: 10 µL; and UV detection wavelength: 250 nm. The standard d-glucose and d-apiose were subjected under the same conditions. After the comparison of the retention times of monosaccharide derivatives, the samples were confirmed to comprise of d-glucose (19.25 min) and d-apiose (30.34 min), respectively [19,20].

Conclusions
In conclusion, 12 compounds were isolated from C. lansium. They were obtained from the genus Clausena for the first time, including four new glycosides. The existence of these compounds demonstrates the taxonomic significance of C. lansium in the genus Clausena and suggests that some glycosides from this plant probably play a role in the anticancer activity of C. lansium to some extent.
Supplementary Materials: The 1D-and 2D-NMR spectroscopic data of compounds 1-12 in the paper are available online.