Chemical Constituents and Antioxidant, Anti-Inflammatory and Anti-Tumor Activities of Melilotus officinalis (Linn.) Pall

Two new p-hydroxybenzoic acid glycosides, namely p-hydroxybenzoic acid-4-O-α-d-manopyranosyl-(1 → 3)-α-l-rhamnopyranoside (compound 1) and 4-O-α-l-rhamnopyran-osyl-(1 → 6)-α-d-manopyranosyl-(1 → 3)-α-l-rhamnopyranoside (compound 2), and seven known compounds, compound 3, 6, 7 (acid components), compound 8, 9 (flavonoids), compound 4 (a coumarin) and compound 5 (an alkaloid), were isolated from the 70% ethanol aqueous extract of the aerial parts of Melilotus officinalis (Linn.) Pall. The structures of all compounds were elucidated by use of extensive spectroscopic methods Infrared Spectroscopy (IR), High resolution electrospray ionization mass spectrometry (HR-ESI-MS), and 1H and 13C-NMR). Sugar residues obtained after acid hydrolysis were identified by high-performance liquid chromatography (HPLC). The antioxidant activity of all the compounds was evaluated by 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS+) and 1,1-diphenyl-2-picrylhydrazyl (DPPH). The anti-inflammatory effects of the compounds were also evaluated in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. All compounds were shown to inhibit LPS-induced nitric oxide (NO) and prostaglandin E 2 (PGE 2) production by suppressing the expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2), respectively, in LPS-stimulated RAW 264.7 cells. The inhibitory effect of all the compounds on MCF-7 cells was determined by Cell Counting Kit-8 (CCK-8) method. The results showed that compounds 1, 2, 7, 8, 9 exhibited better antioxidant activity compared to the other compounds. compounds 1–9 had different inhibitory effects on the release of NO, TNF-α and IL-6 in LPS-stimulated RAW264.7 cells by LPS, of which compound 7 was the most effective against inflammatory factors. compounds 1 and 2 have better antitumor activity compared to other compounds. Further research to elucidate the chemical composition and pharmacological effects of Melilotus officinalis (Linn.) Pall is of major importance towards the development and foundation of clinical application of the species.


Introduction
Melilotus officinalis (Linn.) Pall belongs to the genus Melilotus of Fabaceae family, and is an annual herb. It is also known as yellow sweet clover. It was first published in the "European Pharmacopoeia" eighth edition [1], widely distributed around the world. It was regarded as a drug to against edema and renal vein circulation in the UK, Melilotus officinalis (Linn.) Pall as a drug against aggregation, as well as for it antioxidative and hepatoprotective properties in the Netherlands, Germany, Poland and Austria [2][3][4]. In Japan, SETUS-M, which is produced with Melilotus officinalis (Linn.) Pall, has a good effect for treating post-surgical tissue swelling. In China, Melilotus officinalis (Linn.) Pall is used for the treatment of diseases such as spleen disease, gutting, diphtheria and larvae [5]. Meanwhile, the extract of Melilotus officinalis (Linn.) Pall achieved good results as an in-hospital preparation of Jilin University and has been widely accepted by patients.
Modern research shows that the Melilotus officinalis (Linn.) Pall contains coumarins [6], flavonoids [7], steroids and saponins, phenolic acids [2], volatile components, fats, alcohols, uric acid [8] and other chemical compounds, with anti-inflammatory, swelling, and anti-tumor properties, as well as with therapeutic effects against hemorrhoids, thrombophlebitis, and varicose veins [9][10][11][12]. The coumarin, phenolic acids, flavonoids and saponins of Melilotus officinalis (Linn.) Pall have a certain anti-inflammatory effect [8], however, these studies are mainly focused on extracts of Melilotus officinalis (Linn.) Pall, which involve a few monomers of the above-mentioned compounds. Therefore, 70% ethanolic extracts of Melilotus officinalis (Linn.) Pall were used as the research object, and the isolation, purification, identification and activity study of the monomer compounds were carried out in order to provide the basis for the clinical application.
Characterizations of compound 1 included: White amorphous powder, its IR spectrum exhibited absorption bands due to -COOH at 3364, 1677 cm −1 and dihydrogen ortho aromatic ring at 1588, 1284, 1155, 856 cm −1 . The HR-ESI-MS of 1 indicated the molecular formula C 19 Na, 469.1322). In the NMR spectra (Table 1), two proton signals at δ H 8.00 (2H, d, J = 7.8 Hz) and 6.73 (2H, d, J = 7.8 Hz), and four tertiary carbon signals at δ C 130.6, 130.6, 115.9, 115.9, two quaternary carbon signals at δ C 121.1, 161.2, one carbonyl carbon signal at δ C 175.8, combining the IR and 2D NMR spectra data, suggested that a p-hydroxybenzoic acid moiety existed in the structure of compound 1. The above-mentioned 13 C-NMR data were very similar with these of p-hydroxybenzoic acid reported [19], which confirmed existence of a p-hydroxybenzoic acid moiety in the structure of compound 1. The 13 C-NMR spectrum of compound 1 showed also two six-carbon units, one was characteristic of D-mannosyl group (a methylene carbon signal at δ C 59.8 and five methines carbon signals at δ C 102.9, 71.4, 70.5, 67.6, 73.4) which was coincident with these of methyl O-α-D-mannoside reported [20], and other one was characteristic of L-rhamnosyl group (a methyl carbon signal at δ C 18.0 and five methines carbon signals at δ C 97.8, 70.0, 75.7, 71.8, 69.7) which was coincident with these of methyl O-α-L-rhamnoside reported [20], except for carbon signal at δ C 75.7 showing a significant downfield shift (∆δ = 4.6) than C-3 signal of α-L-rhamnose. The existence of D-mannosyl and L-rhamnosyl groups in the structure of was also confirmed by TLC comparing the acid hydrolysate of compound 1 with authentic samples. The proton signal at δ H 5.41 was determined to be anomeric proton of rhamnosyl group by cross peak (Figure 1 and Table 1) at δ H 5.41 (rha-H-1 )/δ C 97.8 (rha-C-1 ) in HMQC spectrum, and cross peak at δ H 5.41/δ C 161.2 (C-4) in HMBC spectrum revealed linkage of L-rhamnosyl group with 4-OH. The proton signal at δ H 5.09 (Mann-H-1) was determined to be anomeric proton of D-mannosyl group by cross peak at δ H 5.09/δ C 102.9 (mann-C-1 ) in HMQC spectrum, and cross peak at δ H 5.09/δ C 75.7 (Rha-C-3 ) in HMBC, revealed linkage of C-3 of L-rhamnosyl group with C-1 of D-mannosyl group, this illustrated the carbon signal at δ C 75.7 (Rha-C-3 ) showing a significant downfield shift (∆δ = 4.6) than C-3 signal in these of L-rhamnose reported [20]. Thus, compound 1 was determined to be p-hydroxybenzoic Characterizations of compound 2 included: White amorphous powder. Its IR spectrum was similar with that of 1. The molecular formula C 25 H 36 O 16 was derived from the positive-ion mode HR-ESI-MS [M + Na] + at m/z 615.1807. In the NMR spectra (Table 1) of compound 2, the appearance of two proton signals at δ H 7.97 (2H, d, J = 7.8 Hz) and 6.69 (2H, d, J = 7.8 Hz), and seven carbon signals at δ C 121.1, 130.69, 130.7, 116.0, 116.0, 161.2, 175.8 showed the existence of the same aglycone (p-hydroxybenzoic acid) in the structure of compound 2 as in Table 1. The 13 C-NMR spectrum of compound 2 showed three six-carbon units. Acid hydrolysis of compound 2 gave rhamnose and mannose which identified by TLC comparing with authentic samples. In comparison with the NMR data of compound 1, two of three six-carbon units were coincident with the sugars moiety of compound 1, except for carbon signal at δ C 65. 6 showing a significant downfield shift (∆δ = 5.9) than C-6 signal of D-mannosyl group of compound 1, which point to the existence of p-hydroxybenzoic acid-4-O-α-D-mannopyranosyl-(1 → 3)-α-L-rhamnopyranosyl moiety in the structure of compound 2, the 13 C-NMR data (carbon signals at δ C 100.1, 70.0, 70.4, 72.0, 68.2, 17.9) of the remainder six-carbon unit was coincident with these of methyl-O-α-L-rhamnoside reported [20], which point to one rhamnosyl group more than compound 1 in compound 2, the proton signal at δ H 4.40 was determined to be anomeric proton of the rhamnosyl group by cross peak (Figure 2 and Table 1) at δ H 4.40 (Rha-H-1 )/δ 100.2 (Rha-C-1 ) in the HMQC spectrum, the cross peak at δ H 4.40/δ C 65.7 (Mann-C-6 ) in the HMBC spectrum, revealed linkage of C-6 of D-mannosyl group with C-1 of L-rhamnosyl group, that illustrated the carbon signal at δ C 65.7 showing a significant downfield shift (∆δ = 5.8) than C-6 signal of D-mannosyl group of compound 1. Thus, the compound 2 was determined to be p-hydroxybenzoic acid-4-O-α-L-rhamnopyranosyl-(1 → 6)-α-D-manopyranosyl-(1 → 3) -α-L-rhamnopyranoside. It was a new compound.  Table 1. The 13 C-NMR spectrum of compound 2 showed three six-carbon units. Acid hydrolysis of compound 2 gave rhamnose and mannose which identified by TLC comparing with authentic samples. In comparison with the NMR data of compound 1, two of three six-carbon units were coincident with the sugars moiety of compound 1, except for carbon signal at δC 65. 6 showing a significant downfield shift (Δδ = 5.9) than C-6 signal of D-mannosyl group of compound 1, which point to the existence of p-hydroxybenzoic acid-4-O-α-D-mannopyranosyl-(1 → 3)-α-L-rhamnopyranosyl moiety in the structure of compound 2, the 13 C-NMR data (carbon signals at δC 100.1, 70.0, 70.4, 72.0, 68.2, 17.9) of the remainder six-carbon unit was coincident with these of methyl-O-α-L-rhamnoside reported [20], which point to one rhamnosyl group more than compound 1 in compound 2, the proton signal at δH 4.40 was determined to be anomeric proton of the rhamnosyl group by cross peak (Figure 2 and Table 1)

Monosaccharide Analysis
Compound 1 and compound 2 showed similar monosaccharide composition ( Figure 2). Only two monosaccharides were found, namely D-mannopyranose and L-rhamnopyranosyl.

Biological Activity
It had been reported that benzoic acid derivatives showed antioxidant activity, anti-inflammatory [21], and cytotoxic activities [22,23]. In this work, the antioxidant activity, anti-inflammatory, and cytotoxic activities of compounds 1-9 was investigated.

Anti-Inflammatory Activity
In the inflammatory response of RAW264.7 cells stimulated by LPS, compounds 1-9 had different inhibitory effects on the release of NO, TNF-α and IL-6 in LPS-stimulated RAW264.7 cells by LPS, and showed good anti-inflammatory activity in vitro, in which compound 7 was effective against inflammatory factors. The strongest inhibitory effect, the results shown in Table 2.

Monosaccharide Analysis
Compound 1 and compound 2 showed similar monosaccharide composition ( Figure 2). Only two monosaccharides were found, namely D-mannopyranose and L-rhamnopyranosyl.

Biological Activity
It had been reported that benzoic acid derivatives showed antioxidant activity, anti-inflammatory [21], and cytotoxic activities [22,23]. In this work, the antioxidant activity, anti-inflammatory, and cytotoxic activities of compounds 1-9 was investigated.

Anti-Inflammatory Activity
In the inflammatory response of RAW264.7 cells stimulated by LPS, compounds 1-9 had different inhibitory effects on the release of NO, TNF-α and IL-6 in LPS-stimulated RAW264.7 cells by LPS, and showed good anti-inflammatory activity in vitro, in which compound 7 was effective against inflammatory factors. The strongest inhibitory effect, the results shown in Table 2.

Antitumor Activity
The results of compounds of CCK-8 kit assay are displayed in Table 3, the results show that compounds 1, 2, 3, 5, 7, 8 and 9 can inhibit the growth of tumor cells MCF-7 with IC 50 value of 4.83, 5.18, 8.20, 7.85, 7.53, 8.40 and 9.24 µg/mL. However, compounds 4 and 6 did not inhibited potently the growth of MCF-7 cells. According to IC 50 values, compound 1 with the best antitumor activity was divided into three groups: low dose group (1/2 IC 50 value), medium dose group (IC 50 value), high dose group (2 × IC 50 value), the concentration of 5-FU was IC 50 , which is positive control group, and the negative control group was not given the drug (0 mg·mL −1 ). Each concentration in parallel with 3 copies, 37 • C, 5% CO 2 incubation. The number of viable cells was counted after staining with trypan blue after digestion with trypsin at the same time point. Each set of data is expressed as an average number of cells. The growth curve was plotted with the culture time as the horizontal axis and the average number of cells as the vertical axis. MCF-7 results showed that the compound 1 had a significant dose-dependent effect on the growth of MCF-7 cells, which was significantly different from that of the control group (p < 0.05), and there was no significant difference compared with the existing positive control drug 5-FU (p > 0.05) (See in Figure 3). The expression of PCNA was observed by immunohistochemical staining of MCF-7 cells; it was found that the expression of PCNA decreased gradually with the increase of concentration (See in Figure 4).

Antitumor Activity
The results of compounds of CCK-8 kit assay are displayed in Table 3, the results show that compounds 1, 2, 3, 5, 7, 8 and 9 can inhibit the growth of tumor cells MCF-7 with IC50 value of 4.83, 5.18, 8.20, 7.85, 7.53, 8.40 and 9.24 μg/mL. However, compounds 4 and 6 did not inhibited potently the growth of MCF-7 cells. According to IC50 values, compound 1 with the best antitumor activity was divided into three groups: low dose group (1/2 IC50 value), medium dose group (IC50 value), high dose group (2 × IC50 value), the concentration of 5-FU was IC50, which is positive control group, and the negative control group was not given the drug (0 mg·mL −1 ). Each concentration in parallel with 3 copies, 37 °C, 5% CO2 incubation. The number of viable cells was counted after staining with trypan blue after digestion with trypsin at the same time point. Each set of data is expressed as an average number of cells. The growth curve was plotted with the culture time as the horizontal axis and the average number of cells as the vertical axis. MCF-7 results showed that the compound 1 had a significant dose-dependent effect on the growth of MCF-7 cells, which was significantly different from that of the control group (p < 0.05), and there was no significant difference compared with the existing positive control drug 5-FU (p > 0.05) (See in Figure 3). The expression of PCNA was observed by immunohistochemical staining of MCF-7 cells; it was found that the expression of PCNA decreased gradually with the increase of concentration (See in Figure 4).
Melilotus officinalis (Linn.) Pall has anti-inflammatory [9], swelling [10] and anti-tumor [11] and other pharmacological effects. This study showed that its flavonoids and phenolic acids have good antioxidant capacity, which suggest that the flavonoids and phenols acid composition is the material basis to antioxidant of Melilotus officinalis (Linn.) Pall. The phenolic acids achieved anti-inflammatory effects by inhibiting the activity of NO, TNF-α and IL-6 in LPS-induced RAW264.7 cells, which suggest that the treatment of edema with Melilotus officinalis (Linn.) Pall is related to its antioxidant and anti-inflammatory properties. Earlier studies have shown it has good anti-tumor activity [18]. In previous studies, our group studied the purification process of its saponins, and applied for a patent; we also found that its saponins had the better inhibitory effect on MCF-7, PC3M and other tumor cell lines. In this paper, we found two new benzoic acid compounds have good inhibitory activity on prostate cancer, and the inhibitory effect is stronger than the other compounds. In clinical applications, Melilotus   Melilotus officinalis (Linn.) Pall has anti-inflammatory [9], swelling [10] and anti-tumor [11] and other pharmacological effects. This study showed that its flavonoids and phenolic acids have good antioxidant capacity, which suggest that the flavonoids and phenols acid composition is the material basis to antioxidant of Melilotus officinalis (Linn.) Pall. The phenolic acids achieved anti-inflammatory effects by inhibiting the activity of NO, TNF-α and IL-6 in LPS-induced RAW264.7 cells, which suggest that the treatment of edema with Melilotus officinalis (Linn.) Pall is related to its antioxidant and anti-inflammatory properties. Earlier studies have shown it has good anti-tumor activity [18]. In previous studies, our group studied the purification process of its saponins, and applied for a patent; we also found that its saponins had the better inhibitory effect on MCF-7, PC3M and other tumor cell lines. In this paper, we found two new benzoic acid compounds have good inhibitory activity on prostate cancer, and the inhibitory effect is stronger than the other compounds. In clinical applications, Melilotus officinalis (Linn.) Pall is mainly used to address swelling, which suggests its antitumor activity maybe has a certain correlation with therapeutic effect of its edema, and also shows that it has potential value in anti-tumor applications. Therefore, further research to elucidate the chemical composition and pharmacological effects of Melilotus officinalis (Linn.) Pall is of major importance towards the development and foundation of clinical application of the species.

Monosaccharide Analysis
A solution of each Compound (1 or 2) (5 mg) in a mixture of 1:2 (v/v) 1M H 2 SO 4 -MeOH (20 mL), was heated under reflux for 3 h in a water bath at 80 • C. The reaction mixture was evaporated to dryness in vacuo, dissolved in H 2 O (5 mL), and neutralized with NaOH. Then, the resulting samples were analyzed using high-performance liquid chromatography (HPLC) coupled with an ELSD detector according to the method of Yang [24], with some modifications. Instead of the gradient elution, an isocratic mobile phase consisting of 22:78 (v/v) mixtures of water and acetonitrile (ACN) was used.

Anti-Oxidative Activity
The methods for determining ATBS + free radical scavenging activity was as follows. About 0.2 mL tested compounds at various concentrations (0.01, 0.05, 0.1, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40 and 0.45 mg/mL) were added to 2 mL ATBS + solution, respectively. The mixture, protected from light, was reacted for 30 min. The decrease of absorbance was monitored at 734 nm. The control was 0.2 mL of distilled water and 2 mL of ATBS + solution. The same method was used in Vitamin C (Vc). The methods for determining DPPH· free radical scavenging activity was as follows. About 0.2 mL tested compounds at various concentrations (0.01, 0.05, 0.1, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40 and 0.45 mg/mL) were added to 2 mL DPPH· solution (200 µM ethanol solution), respectively. The mixture, protected from light, was reacted for 30 min. The decrease of absorbance was monitored at 517 nm. The control was the DPPH· solution. The same method was used in Vc. The half maximal inhibitory concentration (IC 50 ) was used to evaluated the ATBS + free radical scavenging activity and the DPPH· free radical scavenging activity.

Cytotoxicity Assay
The cytotoxicity assay was carried out using CCK-8 method. MCF-7 and PC-3M cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 and DMEM at 37 • C in 5% CO 2 , respectively.
The cells of logarithmic growth phase were seeded into 96-well plates with density of 1 × 10 4 cells/well in 100 µL medium, respectively. The cells were treated with the tested compounds at various concentrations (0.5, 1.0, 2.5, 5, 7.5, 10.0, 12.5 and 15.0 µg/mL) and 5-FU as positive control, each of two parallel holes are located, then incubated for 72 h. Subsequently, remove the 96 well plate, add 10 µL of CCK-8; meanwhile, two separate holes for the blank control, only added to each well with 10 µL CCK-8 in DMEM 0.1 mL. Then incubate under the same conditions for 4 h. The optical density (OD) was measured at 490 nm using a Bio-red 550 (Bio-red company, Hercules, CA, USA). Reference wavelength was 620 nm. The experiment was repeated 3 times. Calculation of the impact of drugs on cell growth inhibition rate and IC 50 values is performed with the following equation: Growth inhibition rate (100%) = (D 0 − D 1 )/D 0 × 100% where D 0 is the OD value of the control wells, and D 1 is the OD value of the samples wells.