Phytochemical Analysis, Anti-inflammatory, and Anticancer Activities of the Halophyte Herb Bassia indica

Bassia indica (Wight) A.J. Scott, family Amaranthaceae, is a halophyte herb growing in extreme environments and hence deemed as a potential economic source of bioactive chemicals with functional properties. In our study, 25 compounds were obtained from B. indica. We aimed to assess the inhibitory effect of the methanol extract of B. indica and its isolated compounds on COX-2 and cytotoxicity activity against MCF-7, OVK-18, HepG2, and HCT116 tumor cells. Among the isolates, the triterpene oleanane saponin (23) displayed promising anti-inflammatory activity with an IC50 = 3.05 ± 0.15 μg/mL. Additionally, N-trans-feruloyl tyramine (11) exhibited significant cytotoxicity to OVK-18 with IC50 = 1.74 ± 1.56 μg/mL, whereas 6,7-dihydroxy coumarin (7) exhibited potent inhibition against the MCF-7 cell line with IC50 = 1.47 ± 0.22 μg/mL. Interestingly, compounds 1 and 25 exhibited remarkable cytotoxicity against HepG2 and HCT116 cells with IC50 < 0.1 μg/mL, while compounds 2, 4, 5, 6, and 9 exerted potent cytotoxicity against HepG2. Finally, B. indica is a potential source of candidate compounds for the development of anti-inflammatory and antitumor therapies.


Introduction
Cancer is a great burden to public health and a serious cause of death worldwide. [1][2][3] Recently, it has been reported that cancerrelated mortality is due to lung, colorectal, breast, and liver cancers. 4 The currently available drugs for cancer treatment are conventional chemotherapy; however, the development of drug resistance is one of the complications caused by chemotherapy. 5 It is worth noting that a wide variety of plant-based anticancer therapies are in clinical use, and they have exhibited significant efficacy. 6 On the other hand, inflammation has played a key role in cancer metastasis. Many studies have demonstrated that the possibility of cancer occurrence and development is associated with inflammation. 7 Currently, there is a growing demand for anti-inflammatory and anticancer drugs that are highly safe with good effects.
Halophytes are plants with salt-tolerant capacity and well-known adaptation to extreme environments. 8 They have been reported as potential sources of bioactive compounds with substantial economic value. 9 The halophytic plants have also been deemed to possess antitumor, 10,11 anti-inflammatory, 12,13 anticholinesterase, 14 and anti-tyrosinase 15 properties. Previous research on halophytes revealed their content of various classes of biologically interesting lead compounds, such as steroids, 16 saponins, 11,17 flavonoids, and alkaloids. 18 Bassia indica (Wight) A.J. Scott (Amaranthaceae) is a halophytic herb widely grown in the Egyptian ecosystem and other regions of the world. 19 In relation to ethnomedicine, the plant has been traditionally reported as an antitumor, cardiotonic, and anti-oxidant. 20 Till now, the research work on B. indica has been very limited particularly regarding its use as an antitumor remedy, and thus there is still a need to explore the chemicals and biological properties of this plant. Although several effective strategies are available for cancer and inflammation treatment, halophytic plants are deemed significant in providing a variety of chemical entities possessing bioactivities. To emphasize the utilization of halophyte extracts as potential sources of pharmaceutical candidates, 21,22 this study was conducted to provide a more detailed view of the anti-inflammation and antitumor activities of B. indica. Thus, we evaluated the possibility of cytotoxicity and anti-inflammatory activities of 25 compounds obtained from this species.
Considering the revealed biological properties of halophytes, the methanol extract of B. indica was screened for antiinflammatory activity against COX-2 and cytotoxic activity toward MCF-7, OVK-18, HepG2, and HCT-116. As a result, the methanol extract exhibited pronounced anti-inflammatory activity. Moreover, the extract displayed cytotoxic activity in OVK-18 cell lines. Hence, the isolates from B. indica were also investigated for their capacity as anti-inflammatory and antitumor candidates. In this context, the isolation, structural elucidation, and evaluation of anti-inflammatory and cytotoxicity properties of isolated compounds are reported.

Anti-inflammatory Activity of B. indica and Isolated Compounds
The formation of prostaglandins is regulated by either COX-1 or COX-2 from arachidonic acid and subsequently leads to the inflammatory process. COX-2 is upregulated in inflammatory conditions. 44 Thus, the discovery of potent anti-inflammatory compounds from natural plant sources through the inhibition of COX-2 is an effective approach in the pharmaceutical field. In the present study, the COX-2 enzyme inhibiting activity of crude extract of B. indica, as well as the isolated triterpenoid saponins, were evaluated. Based on previously published literature data, the saponin content of the n-butanol extract of halophytes is responsible for the anti-inflammatory potential. 12 Therefore, the isolated saponins (23-25) from B indica were investigated for their possible anti-inflammation activity via inhibition of the COX-2 enzyme. The methanol extract of B. indica exhibited promising anti-inflammatory activity by inhibiting COX-2 with an IC 50 = 7.97 ± 0.40 µg/mL. Importantly, compound 23 exhibited significant inhibitory activity on COX-2 with an IC 50 value of 3.05 ± 0.15 µg/mL compared to the activity of celecoxib (IC 50 = 1.75 ± 0.09 µg/mL). Furthermore, compounds 24 and 25 showed moderate inhibition activities against COX-2 with IC 50 values of 24.89 ± 1.26, and 19.65 ± 0.99 µg/ mL, respectively.
By reviewing the literature, previous reports demonstrated that saponins could contribute to the anti-inflammatory potential of plant crude extracts. Previously triterpene oleanane saponins isolated from the halophyte Anabasis setifera displayed a potential antiinflammatory activity. 12 Also, phenolics and flavonoids are suggested to contribute to anti-inflammatory activity. 12,45,46 The extract of the herb Suaeda fruticosa exhibited high activity against inflammation by inhibiting the release of nitric oxide (NO) owing to its phenolic content. 45 Additionally, polyphenols, mainly flavonoids, have anti-inflammatory modifying properties by modulating the expression of the iNOS gene and consequently inhibiting the production of NO. 47 Thus, the anti-inflammatory potential of B. indica might be explained due to its content of saponins and phenolic derivatives.

Cytotoxic Activity of B. indica and Isolated Compounds
The isolated chemicals  were tested for their antitumor activity by MTT assay 48 Table 1.
By reviewing the literature, it is to be noted that β-sitosterol stimulates apoptosis and inhibits proliferation through induction of caspase-3 and the Bax/Bcl-2 ratio. 49 The furofuran lignan (syringaresinol) showed cytotoxicity to a number of cell lines including Hep-2 (larynx epidermoid carcinoma), HeLa (human cervix carcinoma), and C6 (rat glioma) cell lines with IC 50 values ranging from 0.23 to 0.63 µg/mL. 50 The amide alkaloid, N-trans-feruloyl tyramine, showed cytotoxic activity against human lung (A549 and K562) cancer cell lines with IC 50 = 37.20 and 41.52 µmol L −1 , respectively. Also, aesculetin (6,7-dihydroxy coumarin) induced apoptosis in HeLa cells through a ROS-mediated mitochondrial dysfunction pathway. 51 Moreover, the stimulation of the mitochondrial pathway and induction of DNA damage produced by methyl caffeate lead to apoptosis. 52 Therefore, the cytotoxic activity of B. indica might be attributed to its content of diverse metabolites with significant antitumor activities.

Conclusions
To sum up, the halophyte herb B. indica has been deemed a promising and cheap source of bioactive compounds. Importantly, the current study marks the first report of the antiinflammatory and antitumor activity of the constituents of B. indica growing in Egypt. Besides, the findings of our study highlight the potential utilization of halophytes as a source of pharmaceutical candidates through the determination of key candidate compounds from B. indica responsible for antitumor and anti-inflammatory activities. In the future, further studies to elucidate the exact mechanisms of B. indica as an antiinflammatory and antitumor will be needed.

Material for Chromatography Experiments
Normal-phase silica (75-150 µm), reversed-phase silica (38-63 µm), and solvents for the extraction and isolation of phytochemicals were obtained from Wako Pure Chemical Corporation, Osaka, Japan. Sephadex LH-20 was used for purification and obtained from Sigma Aldrich, USA. Diaion HP-20 was purchased from Mitsubishi Chemical Corporation, Japan. Biotage selekt operated with a RP-C18 (Sfär C18 D Duo column, 30 μm, 30, and 60 g) was used for fractionation and purification of compounds (obtained from Biotage Japan Ltd, a subsidiary of Biotage Uppsala, Sweden). The medium-pressure liquid chromatograph was connected to an UV and ELSD detector used for further purification (obtained from Buchi, Switzerland). A HPLC preparative column (ODS-3, 5 µm, 20× 250 mm), obtained from GL Sciences Inc., Japan was used to achieve further purification of compounds.

Analysis of the Isolated Compounds
A JASCO P-2000 polarimeter obtained from JASCO, Tokyo, Japan, was used for measurement of optical rotations. 1D and 2D NMR spectra were obtained from a DRX-600 spectrometer (Bruker Daltonics, USA). The high-resolution mass of isolates was detected on an Agilent QTOF-LC-MS, Agilent Technologies, USA.

Plant Material
The aerial parts of B. indica were collected from desert areas of Egypt near 6 th October City (El-Wahat Road; 29°58 ′ 16"N 31°0 1 ′ 25"E) in September 2019, and identified by Prof. Ibrahim A. El-Garf, Department of Botany, Faculty of Science, Cairo University, Egypt. A voucher specimen (BIC-2019-2) has

In Vitro Assays for COX-2 Inhibition and Cytotoxicity
See Supplemental information.