Withaninsams A and B: Phenylpropanoid Esters from the Roots of Indian Ginseng (Withania somnifera)

Withania somnifera (L.) Dunal (Solanaceae), known as Indian ginseng or ashwagandha, has been used in Indian Ayurveda for the treatment of a variety of disorders, such as diabetes and reproductive and nervous system disorders. It is particularly used as a general health tonic, analgesic, and sedative. As part of continuing projects to discover unique bioactive natural products from medicinal plants, phytochemical investigation of the roots of W. somnifera combined with a liquid chromatography–mass spectrometry (LC/MS)-based analysis has led to the isolation of two novel phenylpropanoid esters, Withaninsams A (1) and B (2), as an inseparable mixture, along with three known phenolic compounds (3, 4, and 6) and a pyrazole alkaloid (5). The structures of the new compounds were elucidated using a combination of spectroscopic methods, including one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) and high-resolution electrospray ionization mass spectroscopy (HR-ESIMS). Withaninsams A (1) and B (2) are phenylpropanoid esters that contain a side chain, 4-methyl-1,4-pentanediol unit. To the best of our knowledge, the present study is the first to report on phenylpropanoid esters with 4-methyl-1,4-pentanediol unit. The anti-inflammatory activity of the isolated compounds (1–6) was evaluated by determining their inhibitory effects on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages, where compound 3 inhibited LPS-induced NO production (IC50 = 33.3 μM) and TNF-α production, a pro-inflammatory cytokine (IC50 = 40.9 μM). The anti-inflammatory mechanism through the inhibition of transcriptional iNOS protein expression was confirmed by western blotting experiments for the active compound 3, which showed decreased iNOS protein expression.


Introduction
Withania somnifera (L.) Dunal (Solanaceae), commonly known as Indian ginseng or ashwagandha, is a perennial shrub distributed in India, Morocco, Egypt, Israel, Jordan, South Africa, and the Mediterranean region. Currently, it is cultivated on a small scale in South Korea as well [1][2][3]. This plant has been used in Indian Ayurveda for over 3000 years for the treatment of a variety of disorders, such as diabetes and nervous and reproductive disorders. It is particularly used as a general health tonic, analgesic, and sedative [4]. The name, Indian ginseng is botanically not related to Korean ginseng (Panax ginseng). The similarity in the name arises from its similar bioactivity [5]. Roots of W. somnifera Roots of W. somnifera have recently gained popularity as a functional food that promotes longevity through delaying aging, increasing immunity against extrinsic factors, and strengthening the body [1,6]. In practice, extracts of the roots are consumed in several forms, such as powder, liquid, tablets, and capsules. Moreover, roots are consumed as a dietary supplement.
Previous pharmacological studies on W. somnifera have revealed that extracts of W. somnifera exhibit a protective role against bromobenzene-induced oxidative damage in the rat liver [7], as well as increase the exercise performance [8]. Similarly, a recent study on W. somnifera extracts reported its therapeutic role in stroke repair through its anti-apoptotic and anti-oxidant properties [9]. Chemically, it is a rich source of withanolides, which possess diverse pharmacological properties, including anti-inflammatory, anti-microbial, anti-tumor, hepatoprotective, and immunosuppressive effects [10][11][12][13][14][15][16]. In addition, alkaloids, steroidal saponins, lignanamides [17], and phenolics [18] have also been reported, and some of them were found to have anti-tumor activities [19].
As part of a continuing program to determine structurally and/or biologically novel natural products from medicinal plants [20][21][22][23], we conducted a chemical investigation of the methanol (MeOH) extract of roots of W. somnifera. In our recent study, chemical analysis of the MeOH extract, combined with a liquid chromatography/mass spectroscopy (LC/MS)-based analysis, we identified six new withanolides, namely withasilolides A to F, and seven known withanolides [3], some of which exhibited cytotoxicity against several human cancer cell lines (A549, SK-OV-3, SK-MEL-2, and HCT-15). In the present study, we focused on other constituents of W. somnifera rather than withanolides. The phytochemical analysis of the MeOH extract led to the isolation of two new phenylpropanoid esters (1 and 2) as an inseparable mixture, along with four known compounds (3-6) ( Figure 1). The structures of the new compounds (1 and 2) were elucidated using a combination of one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy and high-resolution electrospray ionization mass spectroscopy (HR-ESIMS) data. Further, we evaluated the inhibitory effects of the isolates on nitric oxide (NO) production in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. In the present study, we describe the isolation and structural characterization of isolated compounds, as well as the evaluation of their NO inhibitory effects on LPS-activated RAW264.7 macrophages.

Isolation of Compounds
The dried roots of W. somnifera were extracted with 80% MeOH under reflux to yield the methanol extract, which was sequentially applied to solvent-partitioning with hexane, dichloromethane, ethyl acetate, and n-butanol to obtain each solvent fraction. Chemical analysis of hexane-soluble and dichloromethane-soluble fractions was performed using repeated column chromatography and high-performance liquid chromatography (HPLC) along with LC/MS-based analysis. These techniques were combined with our house-built UV library to determine other types of minor constituents, rather than withanolides. The analysis led to the isolation of two new

Isolation of Compounds
The dried roots of W. somnifera were extracted with 80% MeOH under reflux to yield the methanol extract, which was sequentially applied to solvent-partitioning with hexane, dichloromethane, ethyl acetate, and n-butanol to obtain each solvent fraction. Chemical analysis of hexane-soluble and dichloromethane-soluble fractions was performed using repeated column chromatography and high-performance liquid chromatography (HPLC) along with LC/MS-based analysis. These techniques were combined with our house-built UV library to determine other types of minor constituents, rather than withanolides. The analysis led to the isolation of two new phenylpropanoid esters (1 and 2) as an inseparable mixture, along with three known phenolic compounds (3, 4, and 6) and a pyrazole alkaloid (5) (Figure 1). The novel compounds resulted in only one peak in HPLC performed using Phenomenex Luna C18 column (MeOH/H 2 O, 7:3 to 1:0).

Inhibitory Effects of Compounds 1-6 on LPS-Induced NO Production in RAW 264.7 Cells
To determine whether the isolated compounds 1 to 6 had anti-inflammatory properties, we performed NO assay using supernatants from LPS-stimulated RAW 264.7 cells. All compounds tested exhibited no cytotoxicity up to each highest concentration ( Figure 3A). Of these, compound 3 exhibited an inhibitory effect on NO production in LPS-stimulated RAW 264.7 cells (IC 50 = 33.3 µM) ( Figure 3B). The effect of compound 3 on the LPS-induced TNF-α production showed similar patterns to those of NO production (IC 50 = 40.9 µM) ( Figure 3C). To confirm whether NO inhibitory effect of 3 was related to reduced expression of nitric oxide synthase (iNOS), enzyme involved in the synthesis of NO, we evaluated the effect of compound 3 on iNOS expression. Similar to the results of NO production, compound 3 reduced iNOS expression in LPS-stimulated RAW 264.7 cells (Figure 4). These results indicated that compound 3 exerted anti-inflammatory effect on macrophages by reducing LPS-induced NO production through transcriptional inhibition of iNOS.

Plant Material
The roots of W. somnifera were purchased from Seonggeosan Farm, Cheonan, Korea, in October 2016, and the plant was identified by one of the authors (K.H.K.). A voucher specimen of the material (IDG-2016) was deposited in the herbarium of the School of Pharmacy, Sungkyunkwan University, Suwon, Republic of Korea.

Plant Material
The roots of W. somnifera were purchased from Seonggeosan Farm, Cheonan, Korea, in October 2016, and the plant was identified by one of the authors (K.H.K.). A voucher specimen of the material (IDG-2016) was deposited in the herbarium of the School of Pharmacy, Sungkyunkwan University, Suwon, Republic of Korea.

Plant Material
The roots of W. somnifera were purchased from Seonggeosan Farm, Cheonan, Korea, in October 2016, and the plant was identified by one of the authors (K.H.K.). A voucher specimen of the material (IDG-2016) was deposited in the herbarium of the School of Pharmacy, Sungkyunkwan University, Suwon, Republic of Korea.

Cell Viability Assay
RAW 264.7 cells (6.0 × 10 4 cells/well) were seeded into a 96-well plate and incubated overnight for adhesion. Following incubation, the cells were treated with compounds for 24 h. Next, Ez-CytoX solution (1/10 volume of the culture medium, Daeil Lab., Seoul, Korea) was added to each well and cells were further incubated for 1 h. The cell viability was assessed by measuring the absorbance at 450 nm.

NO Production Assay
RAW 264.7 cells (6.0 × 10 4 cells/well) were seeded into a 96-well plate and incubated overnight for adhesion. Following incubation, the cells were treated with compounds and LPS. After 24 h incubation, supernatants were collected and treated with Griess reagent for evaluating NO concentration in the reactants. The absorbance was measured at 540 nm and NO production was calculated by referring to the nitrite standard curve.

TNF-α ELISA
Culture supernatants were applied to ELISA experiments for measuring the production of TNF-α in LPS-stimulated RAW 264.7 cells. ELISA was performed by manufacturer's instructions (Ebioscience, San Diego, CA, USA). Each step was followed by washing with 1 × PBST 5 times. Briefly, plate was coated with coating antibody solution overnight at 4 • C and then blocked with 1 × assay diluent for 1 h at room temperature (RT). Supernatants were applied to the plate for 2 h at RT and the plate was incubated with biotinylated secondary antibody for 1 h at RT. After reacting the plate with horseradish peroxidase (HRP)-streptavidin for 40 min at RT, the plate was reacted with 4-nitrophenyl phosphate disodium salt in diethanolamine buffer as a substrate for 10 min at dark condition and the reaction was stopped by adding 1 N NaOH. Absorbances of each well at 405 nm were applied to standard curve for calculating the quantity of TNF-α at supernatants.

Western Blotting
RAW 264.7 cells (2.0 × 10 5 cells/well) were seeded into 6-well plates and incubated overnight for adhesion. Following incubation, the cells were treated with compounds 3 and 4 in the presence of LPS for 24 h. Total cell lysates were obtained and loaded onto sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were subsequently transferred onto nitrocellulose (NC) membrane. Membranes were incubated with primary antibodies against iNOS and GAPDH, following which the membranes were incubated with appropriate secondary antibodies. Finally, the membranes were blotted and protein band intensities were analyzed using Imager 680 (GE Healthcare; Chicago, IL, USA).

Statistical Analysis
The data were statistically analyzed using Student's t-test. To prove statistical significance, experiments were conducted in replicates as follows: nine for cell viability, NO assay, TNF-α ELISA and three for western blotting. A p-value < 0.05 was considered statistically significant.

Conclusions
In the present study, phytochemical analysis of the MeOH extracts of roots of W. somnifera led to the isolation of two novel phenylpropanoid esters, namely Withaninsams A (1) and B (2) as an inseparable mixture, along with three known phenolic compounds (3, 4, and 6) and a pyrazole alkaloid (5). Withaninsams A (1) and B (2) are phenylpropanoid esters that contain a side chain, 4-methyl-1,4-pentanediol unit. To the best of our knowledge, the present study is the first to report on phenylpropanoid esters with 4-methyl-1,4-pentanediol unit. All isolated compounds were evaluated for their anti-inflammatory effects on nitric oxide (NO) production in LPS-stimulated RAW 264.7 macrophages. Compound 3 exhibited NO and TNF-α inhibitory properties without cytotoxicity. The active compound 3 inhibited NO production by reducing iNOS protein expression.

Conflicts of Interest:
The authors declare no conflict of interest.