PHENOLIC GLYCOSIDES FROM THE STEMS OF CLERODENDRUM INERME GAERTN. COLLECTED IN

Five phenolic glycosides, clerodenoside A (1), seguinoside K (2), cistanoside D (3), verbascoside (4), and isoverbacoside (5) were isolated from the ethyl acetate soluble fraction of methanolic extract obtained from the stems of Clerodendrum inerme growing in Viet Nam. Their structures were determined by spectroscopic analysis and comparison of spectral data with those reported in the literature. Compound 3 was isolated from this plant for the first time.


INTRODUCTION
Clerodendrum inerme Gaertn., belonging to the Verbenaceae family, is a mangrove shrub found extensively near the seashore from the north to the south of Viet Nam [1]. This plant has been used as a folk medicine in Thailand, India, China, and Viet Nam for treating skin diseases [2]. Previous phytochemical studies have shown that it contains flavonoids [3], diterpenoids [4,5], phenylethanoid glycosides [6,7], megastigmane and iridoid glycosides [8]. In a preceding paper, we have reported the isolation and structural determination of andrographolide and lupeol hexacosanoate from the ethyl acetate fraction of the methanol extract of C.inerme leaves [9]. This paper deals with the isolation and structural elucidation of five phenolic glycosides comprising clerodenoside A (1), seguinoside K (2), cistanoside D (3), verbascoside (4), and isoverbacoside (5) from the ethyl acetate soluble fraction of the methanol extract of C. inerme stems collected in Thai Binh province. The Cistanoside D (3) was isolated for the first time from this plant.

Plant materials
The stems of C. inerme (Verbenaceae) were collected from the semi-mangrove areas of Thai Binh province in May 2018 and identified by Prof. Tran Huy Thai, Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology (VAST). A voucher specimen has been kept in the laboratory of the Organic Department, Hanoi University of Science and Technology (HUST), Viet Nam.

Extraction and isolation
The dry powdered stems (5.0 kg) of the C.inerme were extracted three times with 80 % aqueous methanol at 50 o C using sonicator. After evaporation of the solvent under reduced pressure, the residue (185.0 g) was suspended in water (3 L) and defatted with n-hexane. The aqueous layer was further extracted with ethyl acetate (EtOAc) to give EtOAc (17.0 g) and water (95.5 g) residues, after removal of the solvents.

RESULTS AND DISCUSSION
The separation of ethyl acetate soluble fraction (17.0 g) of the C. inerme stems using chromatography methods yielded five compounds 1-5 ( Figure 1). Their structures were identified by comparison of spectral data with those reported in the literature. and other proton signals from 3.14 to 4.25 ppm. The 13 C NMR and DEPT spectra showed twenty six carbon signals comprising an ester carbonyl carbon at  C 165.3, twelve carbon signals of two aromatic rings, two methoxy signals at  C 55.3 and 55.6, and two sets of sugar signals with two anomeric carbon signals at  C 99.9 and 108.4. Two sugar units were identified as -D-glucose and -D-apiose by comparing the 1 H and 13 C-NMR data with those published [10]. The HMBC spectrum of 2 showed a cross peak between the anomeric proton signal of -D-glucose at  H 4.73 and C-1 of aglycone at  C 150.4, indicating that the aglycone moiety (C-1) was attached to C-1' of glucose unit via O-glycoside bond. The correlation peaks between the anomeric proton signal of -D-apiose at  H 5.38 and C-2' of Dglucose at  C 76.0, those between H 2 -5" of apiose ( H 4.25 and 4.20) and the ester carbonyl carbon at  C 165.3 were also observed in HMBC of 2, indicating that the apiosyl group was attached to C-2' of glucose and the benzoyl group was attached at C-5" of apiose. The position of two methoxy groups at C-3 of aglycone moiety and C-3"' of benzoyl moiety were indicated by the HMBC correlations of 3-OCH 3 /C-3 and 3"'-OCH 3 /C-3"'. Thus, by spectroscopic analysis of the compound 2 and comparison of its spectral data with those reported in the literature [11], the structure of the compound 2 was determined as seguinoside K. This compound was previously isolated from the Myrsine seguinii and Millettia speciosa [10,11].  2) were observed in the HMBC spectrum, indicating the linkage of rhamnosyl and feruloyl groups with C-3' and C-4' of the glucose moiety and the position of two acetoxyl groups at C-2" and C-3" of the rhamnose moiety. Thus, the structure of compound 1 was identified as clerodenoside A by comparison of its NMR spectral data with those published [6].
The compound 3 was isolated as a yellow amorphous powder. The molecular formula was determined as C 31 H 40 O 15 on the basis of the ESI-MS and NMR spectroscopic data. The 1 H and 13 C NMR data of 3 was very similar to that of phenylethanoid glycoside 1, excepting the absence of two acetoxyl groups. On the basis of spectral analysis and comparison with the literature [12], the structure of compound 3 was determined to be cistanoside D.
The compound 4 was obtained as a yellow amorphous powder. The ESI-MS and NMR data suggested the molecular formula of this compound to be C 29 H 36 O 15 . The NMR spectral data of 4 were very similar to those of 3, excepting the absence of two methoxy groups. The structure of compound 4 was identified as verbascoside when compared with published data [13].
The compound 5 was isolated as a yellow amorphous powder. It gave the same molecular formula (C 29 H 36 O 15 ) as the compound 4 from ESI-MS and NMR data. The NMR spectra of 5 were very similar to those of 4, except that the low field shifts of two carbon signals in the 13 C NMR spectrum, one assigned to the C-3' of glucose, shifted to  C 84.0 ( + 2.4 ppm) and the other assigned to the C-6' of glucose, shifted to  C 64.6 ( + 2.2 ppm). The correlations between H 2 -6'of glucose ( H 4.52 and 3.38) and C-' of caffeic acid ( C 169.1), and those between proton anomeric H-1" of rhamnose ( H 5.20) and C-3' of glucose ( C 84.0) were also observed in the HMBC spectrum of 5. Thus, the structure of 5 was shown to be isoverbascoside [14], in which the rhamnosyl and caffeoyl groups are attached to C-3' and C-6' of the glucose moiety, respectively. This is the first report on isolation of the phenolic glycoside 3 from the C. inerme, whereas the compounds 1, 2, 4 and 5 were previously reported from the same plant growing in Thailand and China [6,8].

CONCLUSIONS
From the ethyl acetate soluble fraction of the methanol extract of the C.inerme stems, five phenolic glycosides including clerodenoside A (1), seguinoside K (2), cistanoside D (3), verbascoside (4), and isoverbacoside (5) have been isolated and characterized. The Cistanoside D (3) was isolated from this plant for the first time.
Declaration of competing interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.