Dereplication approach for the first time isolation of tatarinowin a and pentadecanoic acid from Acorus calamus L. by using GC-MS

Abstract In the present study, we report herein the isolation of cadinane-type sesquiterpenoid, tatarinowin A (ACH-6), and pentadecanoic acid (ACH-8) from petroleum ether extract of rhizome of Acorus calamus L. (Acoraceae) along with 6 other known compounds in this species. It is pertinent to mention here that this is the first report to stain these compounds in which dereplication approach based on GC-MS was applied to target unknown compounds ACH-6 and ACH-8 in A. calamus L. Derelpication approaches based on GC-MS is very useful technique in the area of drug discovery and have eminence potential to identify known and unknown compounds present in extracts of medicinal important plants. This technique can be used to expedite the process of purification of unknown compounds from different matrixes. The isolated compounds were identified with the help of inbuilt library search which reveals the presence of 17 known and 4 unknown compounds. Further, the structure elucidation of all isolated compounds was done using spectroscopy techniques. Also, the structure of ACH-6 was further confirmed by using the single-crystal X-ray diffraction technique. Graphical Abstract


Introduction
Indian Medicinal system is a great antiquity. Traditional systems of medicine such as Ayurveda, Unani, Siddha and Homeopathy are most developed and widely practised in India. In Ayurveda, the medicinal parts of the plant are roots and rhizome. Acorus calamus L. (Family Acoraceae) is also known as sweet flag, Vacha. It is a specie of semi-evergreen perennial hairless herb with creeping and extensively branched, aromatic rhizome. The rhizome is cylindrical in shape, up to 2.5 cm thick. The rhizome is externally purplish-brown to light brown colour and internal part is whitish to slightly pinkish. This plant is native to central Asia, North America, and Europe. In India it is growing in huge amounts in Kashmir, Himachal Pradesh, Uttarakhand, Manipur, Karnataka and Naga hills. (Rajput et al. 2014) In Ayurvedic medicine, A. calamus is a magical herb. It is used as a 'rejuvenator' for the brain and nervous system. It is also used for digestive disorders as a remedy. A. calamus (rhizome) is widely used in the treatment of appetite, fever, stomach cramps, tooth ache etc. (Divya et al. 2011). It contains large number of chemical constituents such as a-asarone, b-asarone, c-asarone, eugenyl acetate, eugenol, isoeugenol, calamenenol, a-pinene, b-pinene, calamol, camphene, calameone, calamene, azulene, methyl isoeugenol, eugenol methyl ether, p-cymene, dipentene, methyleugenol, asaronaldehyde, terpinolene, a-caryophyllene, 1,8-cineole, camphor, and hydrocarbons. (Nigam et al. 1990;Srivastava et al. 1997;Mukherjee 2002). Furthermore, this is one of wonderful plant which possesses remarkable chemical diversity in its secondary metabolite. This fact can be evident from the several literature reports which reveal the presence of different compounds such as isocalamendiol, acoradin and tropoloisoquinoline alkaloids (Li et al. 2017;Hao et al. 2021) from it. A. calamus is widely used in medical systems for the treatment of various diseases such as neurosis, anti-inflammatory, epilepsy, hysteria, loss of memory, antispasmodic, insomnia, carminative, anthelmintic (Sharma et al. 2014). In addition to this, it is also used for 'antimicrobial activity' (Radu sien _ e et al. 2007), and 'antidiabetic activity' (Wu et al. 2007) 'antibacterial activity' (Pawar et al. 2020), 'nematicidal activity' (Kundu et al. 2020), 'anti-inflammatory potential' (Verma et al. 2021).
Ethanolic extract of the plant also possess effect on central nervous system (Vohora et al. 1990). Petroleum ether extract of A. calamus is explored extensively for its different biological activities such as anti-HIV Activity (Silprasit et al. 2011), coronary vasodilator effect (Shah and Gilani 2012), antispasmodic and antidiarrhoeal effect (Shoba and Thomas et al. 2001;Gilani et al. 2006) licicidal activity (Nalamwar et al. 2009). Now days dereplication study ) based on separation and detection technique such as LC-MS and GC-MS is gaining more popularity for understanding metabolic profile of an extract or matrix. This is the unique way to target unknown compounds, thus it has significant role in drug discovery. Keeping in view the importance of the plant and higher extractive value of petroleum ether extract obtained from the plant material, it has been selected for the present study.

Results and discussion
Herein, we explored the chemistry of A. calamus plant. In the present work, we have isolated eight compounds among them two were isolated first time from this plant.

Extraction and isolation of compounds
The petroleum ether extract, chloroform extract, methanolic extract and methanol: water extract (1:1) were prepared by using NCI protocol. The petroleum ether extract was subjected to repeated column chromatography over silica gel (100-200 mesh) to afford eight compounds, i.e., a-bisabolol, methyl isoeugenol, b-asarone, a-asarone, b-sitosterol and asaraldehyde (Patra and Mitra 1981) along with tatarinowin A (Tong et al. 2010) and pentadecanoic acid which were being isolated for the first time from this plant ( Figure S0001, Supplementary information).

GC-MS analysis of petroleum ether extract
Gas Chromatography Mass Spectrometry (GC-MS) finger printing profile of the different fractions of the extract was developed. As our aim was target unknown compounds from petroleum ether extract by using GC-MS based dereplication approach. The compounds present in the extract were identified with the help of inbuilt library search which reveals the presence of 17 known and 4 unknown compounds. Compounds identified with GC-MS are given in chromatogram Figure S2-S6 (Supplementary information) and also presented in Table S1 (Supplementary information).

Characterisation of isolated compounds
Isolated compounds were characterised with the help of NMR spectroscopy along with by comparing their respective NMR values with reported literature. The compound ACH-6 was obtained as light-yellow colored oil. Its GC-MS analysis displayed a molecular ion peak at m/z 234.10, indicating unsaturation. The 1 H NMR spectrum of ACH-6 ( Figure S0007) indicated the presence of one olefinic proton, as evidenced by the resonances at d (ppm) 5.89 (s), an oxymethine proton at d (ppm) 4.17 (d), four methyl groups at d (ppm) 0.95 (t, 6H), 2.02 (s), and 1.97 (s). Further 13 C NMR spectrum analysis of ACH-6 ( Figure S0008) revealed the presences of four methyl, two methylene, five methine and four quaternary carbons. These results along with the literature value and obtained XRD report of ACH-6 indicated that it was a cadinane-type sesquiterpenoid, tatarinowin A which has been reported from Acorus tatarinowii (Tong et al. 2010).
The second compound ACH-8 isolated from A. calamus was identified by GC-MS as Pentadecanoic acid (ACH-8). GC-MS analysis displayed a molecular ion peak at m/z 242.4. 1 H NMR spectrum of ACH-8 ( Figure S0009) indicated the presence of one methyl proton as evidenced by the resonances at d (ppm) 0.90, one methylene group at d (ppm) 2.36 À 2.33 (2H), one methylene group at d (ppm) 1.65 À 1.61 (2H), twenty two methylene group at d (ppm) 1.26 (22H) while as 13 C NMR of ACH-8 ( Figure S0010) revealed the presences of one methyl, thirteen methylene and one quaternary carbons which appears at d (ppm) 180.12.
There is tremendous chemical diversity among the same species growing at different places or different species. The compounds isolated in the present specie were also reported in other species of the genus Acorus. Recently, bioguided isolation of antifungal compounds from Acorus tatarinowii was reported (Wang et al. 2020); this study also confirmed the presence of the compounds which have been isolated in this communication. Similarly isolation of asaronaldehyde was also being reported from rhizome of A. gramioneus (Kim et al. 2005).

Conclusions
In conclusion, two natural compounds, a cadinane-type sesquiterpenoid, tatarinowin A (ACH-6) and pentadecanoic acid (ACH-8) were successfully isolated for the first time from A. calamus along with other six known compounds which were purified from the petroleum ether fraction. It is important to highlight that the GC-MS has emerged as an important analytical technique to identify known and unknown compounds from the petroleum ether extract of the plant. Dereplication approach can be used to target unknown compounds which can easily be isolated by using column chromatography.