A new irregular monoterpene acetate along with eight known compounds with antifungal potential from the aerial parts of Artemisia incisa Pamp (Asteraceae)

Abstract A new compound named as santolinylol-3-acetate (4-(2-hydroxypropan-2-yl)-2-methylhexa-1,5-dien-3-yl acetate) (3), along with seven known compounds; linoleic acid (1), benzoic acid (2), santolinylol (4), ethyl-(E)-p-hydroxy cinnamate (5), scopoletin (6), esculetin (7) isofraxidin (8) and eupatorin (9), were isolated from the aerial parts (ethanolic extract) of endangered species: Artemisia incisa Pamp (Asteraceae). The compounds’ structures were determined through modern spectroscopic techniques, and comparison of data (physicochemical constants) with the literature. The relative stereochemistry of santolinylol-3-acetate (3) was determined by comparing its data of NOESY, and specific rotation with its diol analogue; santolinylol (4), isolated from the same plant; A. incisa. The results of the antifungal activity showed that coumarins are as whole less active compounds. Compounds 3 (25 and 300 μg/mL), and 4 (12.5 and 300 μg/mL), showed good activities against Candida albicans, and Aspergillus flavus, respectively, which justifies A. incisa as a traditional medicine for curing the said fungal infections. Irregular monoterpene activity against Candida albicans infection.


Introduction
Artemisia is a very important medicinal plant, the various parts of which has been used as a food additives and traditional medicines against the yellow fever, treatment of hepatitis, malaria, cancer, inflammation and microbial infections (Aniya et al. 2000;Cha et al. 2009;Mase et al. 2010). The genus had been evaluated for different biological activities including hepatoprotective, antipyretic, antimalarial, nematicidal, anthelmintic, anticomplementary, antiviral, antioxidant, interferon-inducing and antifungal (Jang et al. 2005;Kim et al. 2008). Biological active metabolites of various classes such as coumarins, essential oils, flavonoids, polyacetylenes and sterol glycosides were reported (Rashid et al. 2014).
After the isolation of artemisinin, the magic bullet against multidrug resistant strains of malaria from Artemisia annua, the natural product chemist focused on biosynthesis and pharmacology of artemisinin. Moreover, the Artemisia genus has emerged as a potent candidate of natural occurring therapeutic agents for diabetes due to their bioactive-compounds,   including flavonoids, coumarins and caffeic acids (Okada et al. 1995;Logendra et al. 2006;Cui et al. 2009).
Artemisia is considered one of the most important medicinal plant, distributed and practiced traditionally everywhere, in Pakistan and Kashmir, but their several species are endangered and going to be extinct in the near future from Pakistan as well as several other parts of the world where recent climate changes has been noticed. Ornano et al. (2016), have done phytochemical analysis on the non-volatile fraction of Artemisia caerulescens subsp. densiflora (Viv.) (Asteraceae), an endemic species of La Maddalena Archipelago (Sardinia-Italy) which reflects the importance of polar compounds from the genus Artemisia. Recently, Giang et al. (2014), isolated three new eudesmanes from Artemisia japonica, while we have previously reported a highly polar new lipid: ceramide  from the root parts of Artemisia incia Pamp (current plant under investigation).
More than 200 species of Artemisia have been evaluated for their GC-MS analysis of essential oil (Tan et al. 1999). Although, most of the phytochemical work on Artemisia species have been done on the aerial parts for essential oil analysis, while the other non-volatile parts (hard stems and roots) are still need to be explored, phytochemically. Among the various classes of compounds, sesquiterpene and phenolics were the most common and chosen as marker for the quality evaluation, standardisation of Artemisia genus (han et al. 2008). however, numerous studies showed that some unusual compounds like irregular monoterpenes, didterpenes, oxygenated aliphatic hydrocarbons, phenolic hydrocarbons, polyacetylenes, furans, ceramides, alkamides and peptides were reported from Artemisia which might be associated with the synergistic or specific therapeutic functions, overall pharmacological potential of Artemisia .
In continuation of our exploration of Artemisia species for the characterisation of structurally new and/or bioactive metabolites, we describe here the isolation and complete structure assignment of one new irregular monoterpene ester (3), and its diol analogue (4) (Näf-Müller et al. 1981) along with six known compounds including phenolic (5), coumarins (6-8) (Tan et al. 1999;Wu et al. 2001), flavonoid (9) (Shilin et al. 1989), benzoic acid (2) and long chain fattyacids (1) (Ul'chenko et al. 2005, Figure. 1), from aerial parts of. A. incisa (one of the endangered plant species of Pakistan). We have investigated the ethno-botanical and traditional applications of the aerial parts of A. incisa used by the local people of Lower Dir, Pakistan. A. incisa extract were used as a traditional medicine for the treatments of dryness of mouth and infections caused by Candida albicans at foot, pelvic, mouth and forehead regions. The selected compounds (3-9) were evaluated for their potential against the fungal strains; C. albicans and Aspergillus flavus.

Results of the antifungal activity
The compounds 3 (27.4 and 300 μg/mL), and 4 (23.3 and 300 μg/mL) showed good activities against C. albicans and A. flavus, respectively. Among the other tested compounds, compounds 5 (700 and 400 μg/mL), 6 (500 and 1000 μg/mL) and 9 (400 and 400 μg/mL) were also found to be active. The oxygenated coumarins (compounds 7 and 8) were found to be inactive against the all tested fungal strains. The details of these results are given in Tables S1 and S2 (supplementary materials).

Plant collection and Identification
The aerial parts of A. incisa were collected in October 2010 from lower Dir, KPK, Pakistan. The vouchers specimen (Bot. 2020 (PUP)) was deposited in the herbarium of the Department of Botany, University of Peshawar, KPK, Pakistan.

Antifungal activity of the selected compounds (3-9)
The antifungal activity was performed according methods established by Tan et al. (1998). The root and leaves ethanol, hexane, ethyl acetate, chloroform, butanol and aqueous extracts were dissolved in distilled water (5 mg/mL) and the aliquots were used to test the antifungal activity. All the extracts were tested for their activity against the fungal strains of C. albicans, A. flavus. Penicillin 100 I.U/mL and streptomycin (1 mg/mL) were used as control for comparison. Test micro-organisms were maintained on stock culture SDA. From stock culture 24-h fresh culture was prepared. Inocula were prepared by diluting 24-h old culture in saline. A dilution of 1:100 was used in all the tests. The tests were run in triplicate. Petri plates (10 × 10) were prepared with SDA. The diluted culture 0.1 mL was poured on each plate and plates were dried for 30 min at 37 °C. Wells of 6 mm (approximate) diameter were cut with sterile cork borer in the inoculated agar. The wells were filled with the plant extracts and control solutions for comparison. Plates were incubated at 25 ± 1 °C for 5 days. At the end of incubation period, the clear zone of inhibition around the wells was measured in millimetre (mm). Then the procedure given above was repeated again in order to calculate the MIC μg/ mL by diluting the initial conc. of the samples by 1000 fold and then the MIC was determined using the agar dilution method with slight modifications. Varying concentrations (1000, 700, 500.0, 400.0, 300.0, 200.0, 100.0, 50.0, 25.0 and 12.5 μg/mL), of the extracts were prepared and incorporated separately into respective PDA plates. All the plates were incubated at 25 °C for 48 h and inhibition of growth was noted. To determine the MFC, plates which did not show any growth after 48 h, were further incubated for next 48 h, and after incubation, the concentration at which no visible growth was seen, noted as the MFC. The lowest concentration preventing the growth was taken as the MIC in μg/mL, for the compound after 48 h. The results are given in Tables S2 and S3. The results were obtained using the following formula .

Conclusion
The obtained results confirm the justification of a new irregular monoterpene (santolinylol-3-acetate) and its diol analogue (santolinylol) isolated from A. incisa for the treatment of fungal infections caused by C. albicans and A. flavus.