Ultra performance liquid chromatography-tandem mass spectrometeric analysis of ethyl acetate fraction from saudi Lavandula coronopifolia Poir and evaluation of its cytotoxic and antioxidant activities

Introduction Genus Lavandula comprises 47 species belonging to the mint family (Lamiaceae). Lavandula species are very rich in volatile oils which make the Lavandula genus one of the most valuable group of aromatic and medicinal plants with immense economic value for pharmaceutical, cosmetics, perfumery, food and flavor industries and aromatherapy. L. coronopifolia is a woody perennial http://www.herbmedpharmacol.com doi: 10.34172/jhp.2020.34


Introduction
Genus Lavandula comprises 47 species belonging to the mint family (Lamiaceae). Lavandula species are very rich in volatile oils which make the Lavandula genus one of the most valuable group of aromatic and medicinal plants with immense economic value for pharmaceutical, cosmetics, perfumery, food and flavor industries and aromatherapy. L. coronopifolia is a woody perennial herb with a pleasant aroma. It is widely distributed across North Africa, Saudi Arabia and eastern Iran (1). Moreover, Lavandula contains phenolics, flavonoids, anthocyanins, sterols and tannins (2). Lavandula species have many pharmacological actions including antioxidant (3), antimicrobial (4), antidepressant (5,6) and anticancer (7) activities. A previous study using the plant growing in Saudi Arabia, showed that its volatile oil component was rich in phenol-2-amino-4,6-bis (1,1-dimethylethyl), carvacrol, n-hexadecanoic acid, trans-2-caren-4ol, 17-pentartiacontene, caryophyllene oxide and 1-hexacosanol (8). Six major phenolic compounds were identified in the methanolic extract of the Jordanian plant via high-performance liquid chromatography (HPLC) tandem mass which included caffeic acid, rosmarinic acid, rutin, quercetin, luteolin, and hesperidin (9). L. coronopifolia has antioxidant and antimicrobial activities (8,10). Our previous findings revealed that ethyl acetate fraction of L. coronopifolia had hepatoprotective effect against ethanol-induced oxidative stress and subsequent cell death in HepG-2 cells (11). However, the chemical composition of the ethyl acetate fraction is unknown. Therefore, in this study, the chemical composition of the ethyl acetate fraction of L. coronopifolia was analyzed for the first time using UPLC-ESI-MS/MS to identify its bioactive constituents. In addition, we tested the DPPH radical scavenging activity of this fraction as a measure for its antioxidant activity. The anticancer activity of L. coronopifolia against breast and hepatic cancer cell lines was also investigated.

Plant material
The plant was collected in March 2009, from Shaza Mountains in Saudi Arabia. Plant identity was proved by Professor Jakob Thomas from the College of Science, King Saud University. A voucher specimen (#15799) was prepared and deposited at the herbarium unit of Pharmacognosy Department, College of Pharmacy, King Saud University. The aerial parts of L. coronopifolia were ground after air-drying, into coarse particles till use.
Extraction of plant material Air dried aerial parts (300 g) of L. coronopifolia were extracted by 90% ethyl alcohol till complete exhaustion to afford (30 g) of dry extract. The dried extract was suspended in water, defatted with light petroleum and then fractionated with ethyl acetate to afford 3.5 g of ethyl acetate fraction.

UPLC-ESI-MS/MS analysis
The ethyl acetate fraction of L. coronopifolia was prepared as solution of 100 µg/mL using HPLC grade methanol, filtered using a membrane disc filter (0.2 μm) then subjected to LC-ESI-MS analysis as described by (12).

Antioxidant assay
The antioxidant activity of the ethyl acetate fraction of L. coronopifolia was determined at the Regional Center for Mycology and Biotechnology (RCMB) at Al-Azhar University using the free radical 2,2-diphyenylpicrylhydrazyl (DPPH) scavenging assay, as described by (13).

Characterization
We also investigated the anticancer activity of the fraction of L. coronopifolia against HepG-2 (hepatocellular carcinoma) and MCF-7 (breast carcinoma) in comparison to cisplatin, a well-known anticancer agent. As shown in Figure 4, the fraction showed dose-dependent cytotoxic activity against HepG-2 and MCF-7 cells. In addition, the IC 50 values (the concentration which inhibits 50% of the cell viability) of the fraction in HepG-2 and MCF-7 cells were 14.6 ± 0.3 µg/mL and 29.3 ± 0.9 µg/mL, respectively (Table 3).

Discussion
In this study, we analyzed the chemical composition of the ethyl acetate fraction of L. coronopifolia for the first time using UPLC-ESI-MS/MS to identify its bioactive constituents responsible for their cytotoxic and antioxidant activities. Some of the identified compounds as caffeic acid, rosmarinic acid (9), and Pimarane diterpene derivatives (26,27) were reported before in L. coronopifolia.
In ethyl acetate fraction of L. coronopifolia, phenolic acids and their derivatives were identified based on their MS fragmentation and previous studies. The MS/MS fragmentation of phenolic acids showed the presence of caffeic hydroxycinnamic acid or tartaric hydroxycinnamic acid moieties (37). Peak 3 with molecular ion peak at m/z 179 [M-H] was identified as caffeic acid (20). Peaks 2 and 39 showed M-H at m/z 473 and were identified as chicoric acid isomers (Figure 2A) (18). Peak 4 was identified as rosmarinic acid. It was found as a major component in the ethyl acetate fraction (2.88%) and was previously reported in L. pedunculata (20). Peak     Triterpenes, hydroxylated ursolic acid, and ursolic acid derivatives were also identified in ethyl acetate fraction of L. coronopifolia. Dihydroxy, trihydroxy, tetrahydroxy, carboxy, monomethoxy, monomethoxy monohydroxy ursolic acid and other derivatives were detected. Peaks 26, 30, 57 and 59 (showed [M−H] − ions at m/z 487, the MS/MS spectrum exhibited a neutral loss of carboxyl and hydroxymethyl group, leading to form the fragment ion at m/z 409 characteristic of dihydroxy ursolic acid which is known as asiatic acid (29). Dihydroxy ursolic acid was previously reported in L. canariensis (24). Peaks 13,15,18,19,20 and 58 were identified as trihydroxy ursolic acid isomers ( Figure 2E). MS 1   were identified as mono-hydroxy mono-methoxy ursolic acid, ursolic acid derivative, an ursolic acid derivative and methoxy ursolic acid, respectively. Fragmentation of peak 60 gave molecular ion peak at m/z 501 and main base peak fragment ion at m/z 455 (ursolic acid), by loss of 46 Da; it was tentatively characterized as ursolic acid derivative (29).
In the present study, one pimarane diterpene was detected in the ethyl acetate fraction. Peak16 with deprotonated molecular ion peak at m/z 329 and MS 2 fragment ions at m/z 293.3, 139.3 (100%), indicates the presence of pimarane diterpene derivative related to the previously reported in L. multifida (26,27)  The data are presented as µg/mL. These are the mean of three determinations.
9-hydroxy palmitic acid (30 (17). An antioxidant agent can reduce or inhibit oxidative damage by scavenging free radicals. These radicals and other reactive oxygen species play a significant role in the pathogenesis of many diseases (42,43). Numerous plant extracts including those belonging to the Lamiaceae family have been reported to have antioxidant activity. In this study, the ethyl acetate fraction of L. coronopifolia showed concentration-dependent antioxidant activity as demonstrated by increase in its DPPH radical scavenging activity. These results support the previous literature about the antioxidant potential of this plant and many other Lavandula species (3,9). The antioxidant activity of ethyl acetate fraction can be due to carvacrol and ursolic acid. These compounds have been reported to have antioxidant activity in previous studies (44,45).
Cancer is the second leading cause of death all over the world. Conventional cancer therapies are associated with serious side effects. Hence, there is an increasing demand to utilize alternative approaches to treat cancer. Plantsderived compounds have been reported to have activity against different types of cancer (46,47). Importantly, these compounds are relatively not associated with toxic side effects (48). In this study, the ethyl acetate fraction of L. coronopifolia showed dose-dependent cytotoxic activity against HepG-2 (hepatocellular carcinoma) and MCF-7 (breast carcinoma) cells. Consistent with our results, previous studies demonstrated the anticancer potential of Lavandula species (49,50). The cytotoxic activity of ethyl acetate fraction can be attributed to the presence of carvacrol and ursolic acid. In accordance with our results, previous studies have shown that these compounds have antitumor activity against different types of cancer including breast and liver cancers (51)(52)(53).

Conclusion
Our findings revealed that the ethyl acetate fraction of L. coronopifolia has antioxidant and cytotoxic activities. These activities may be attributed to the high percentage of phenolic compounds and polyhydroxylated ursolic acid derivatives.