Potent nitric oxide inhibitory sesquiterpenoids from the rhizome of Curcuma aromatica Salisb.

Abstract One new sesquiterpenoid, curcaromatin (1), together with twenty-one known compounds 2–22, were isolated from the rhizomes of Curcuma aromatica Salisb. (Zingiberaceae). Their structures were established by extensive spectroscopic (1D and 2D NMR and HR-MS) analysis. Most of the isolated compounds were investigated for nitric oxide (NO) production in lipopolysaccharide (LPS)-activated RAW264.7 cells. (−)-Xanthorrhizol (3) displayed the strongest NO inhibitory activity with an IC50 value of 4.3 μM, which was 3.7-fold more active than the reference compound, aminoguanidine (IC50 15.9 μM). The selectivity index (SI > 28.1) of compound 3 was almost 3-fold higher than that of aminoguanidine. Graphical Abstract


Introduction
The genus Curcuma belongs to the Zingiberaceae family.It comprises more than 70 species around the world, which is widely distributed in tropical and subtropical areas, especially in Asia.It has been used as a flavouring agent, yellow-orange dye, and traditional herbal medicine for centuries (Zhang et al. 2018).The rhizomes of Curcuma species are potential sources of phytochemicals obtained from both extracts and essential oils.These compounds have been reported for their various pharmacological properties, for example, anti-oxidant, anti-inflammatory, anti-cancer, anti-microbial, and anti-proliferative activities (Jena et al. 2017;Soumya et al. 2020;Chen et al. 2022;Doan et al. 2022).Curcuma aromatica Salisb., commonly known as wild turmeric or Wan Nang-kham in Thai (Smitinand 2014), is widely distributed throughout Thailand, India, China, and Indonesia (Umar et al. 2020).The rhizomes of this plant are the essential herb that has been extensively used in cosmetics and traditional medicinal applications (Sikha et al. 2015).In Thailand, rhizomes of this plant are traditionally used for the prevention and treatment of a variety of skin, cardiovascular and respiratory diseases, including flatulence, sprains, bruises, cough, rheumatism, blood circulating promotion, and blood stasis removal (Bunyapraphatsara and Chokchaicharoenporn 1999;Al-Reza et al. 2010).The potential of this plant extract and the major compounds to preserve anti-photoageing against UVB-induced keratinocyte cells has recently been reported (Pabuprapap et al. 2022).Previous phytochemical investigations on this plant revealed the presence of various types of compounds such as terpenoids, steroids, flavonoids, saponins, tannins, phenols, and glycosides (Kuroyanagi et al. 1987(Kuroyanagi et al. , 1990;;Choudhury et al. 1996;Agnihotri et al. 2014;Patil et al. 2019;Pintatum et al. 2020;Umar et al. 2020).Among these types of compounds, a wide range of pharmacological activities such as anti-inflammatory, anti-oxidant, anti-tumour, anti-fungal, anti-microbial, anti-diabetic, anti-platelet, immunological effects, and wound healing activities (Sikha et al. 2015;Pintatum et al. 2020) have been reported.Despite the interesting biological activities, the phytochemical constituents in this plant species have not much been extensively studied.We report herein the isolation and structure elucidation of one new sesquiterpenoid (1), along with twenty-one known compounds (2-22) from the rhizomes of C. aromatica.Most of the isolated compounds were evaluated for their nitric oxide inhibitory activities in LPS-stimulated RAW264.7 macrophages.

General experimental procedures
Optical rotations were measured on a JASCO-1020 polarimeter.IR spectra were recorded in the ATR mode using an FT-IR Perkin-Elmer spectrophotometer.1D and 2D NMR spectra were recorded on a Bruker ASCEND 400 FT-NMR spectrometer.HR-TOFMS spectra were measured with a Bruker micrOTOF-QII mass spectrometer, with positive-ion electrospray ionisation (ESI + ) as an ionising technique.Unless otherwise indicated, column chromatography was carried out using Merck silica gel 60 (particle sizes less than 0.063 mm) and GE Healthcare Sephadex LH-20.For thin-layer chromatography (TLC), Merck pre-coated silica gel 60 F 254 plates were used.Spots on TLC were detected under UV light and by spraying with anisaldehyde-H 2 SO 4 reagent followed by heating.

Plant material
The rhizomes of C. aromatica were collected from Sawang Daen Din District, Sakon Nakhon province, Thailand in February 2020, and the plant species was identified by Assoc.Prof. Nopporn Dumrongsiri, Ramkhamhaeng University.A voucher specimen is deposited at the Faculty of Science, Ramkhamhaeng University, Thailand (Apichart Suksamrarn, No. 095).

Extraction and isolation
The fresh rhizomes of C. aromatica (5.0 kg) were sliced, shade-dried, minced, and sequentially macerated with n-hexane, EtOAc and MeOH at room temperature (3 × 30 L for 72 h in each solvent extraction).The filtered solution of each extraction was evaporated to dryness under reduced pressure at a temperature of 40-45 °C to give the hexane extract (460.4 g), the EtOAc extract (28.6 g), and the MeOH extract (5.2 g).

Nitric oxide inhibitory activity assay
The tested compounds were evaluated for cytotoxicity and inhibition of NO production in LPS-stimulated RAW264.7 macrophages (RAW264.7 cells, ATCC, Rockville, MD, USA) according to the previously described protocol (Suebsakwong et al. 2020).

Conclusion
In summary, a new sesquiterpenoid 1 has been isolated from C. aromatica rhizomes, together with twenty-one known compounds (2-22) in this work.The NO inhibitory activity evaluation showed that the natural compound 3 exhibited 3.7-fold more potent NO inhibitory activity than that of the positive control, aminoguanidine.This compound possessed approximately 3-fold higher selectivity index (SI) than the positive control.This finding indicated that sesquiterpenoids isolated from C. aromatica may potentially be developed into anti-inflammatory agents.

Figure 1 .
Figure 1. chemical structures of compounds 1-22.the numbering pattern of the new compound 1 has been established according to the IuPac nomenclature recommendations (Favre and Powell 2013).