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Abstract

Scoparia dulcis L. is one of the edible widely distributed Scropholariaceae species in Asia, Africa and America. It is used in the treatment of respiratory and inflammatory diseases, diabetes, hypertension, cancer, hepatitis and tuberculosis. A phytochemical investigation on S. dulcis led to the isolation of two new acyclic diterpenes Acetic acid 6-hydroxy-2-(6-hydroxy-4-methyl-hex-4-enylidene)-4,8-dimethyl-undeca-4,8-dienyl ester (1) and Acetic acid 8-hydroxy-2-(6-hydroxy-4-methyl-hex-4-enylidene)-6,10-dimethyl-undeca-5,9-dienyl ester (2) in addition to eight known compounds (3–10), namely scopadulciol (3), 4-epi-scopadulcic acid B (4), dulcidiol (5), scopadulcic acid B (6), hymenoxin (7), glutinol (8), eupatilin (9) and 5-demethylnobiletin (10). The structures elucidation was performed using spectroscopic means, including 1D and 2D nuclear magnetic resonance and high-resolution electrospray ionization mass spectrum spectrometric analysis. Furthermore, the isolated compounds were investigated for their anti-inflammatory activity through the determination of inhibition of nuclear factor-kappa B activity in human chondrosarcoma (SW1353) cells, the inhibition of inducible nitric oxide synthase mouse macrophages (RAW264.7) and the decrease in cellular oxidative stress in HepG2 cells. Moreover, the cytotoxic activity was investigated against four cancer and two kidney cell lines. Among the isolates, 3, 5 and 10 showed anti-inflammatory activity in terms of inhibiting nuclear factor-kappa B and inducible nitric oxide synthase. Compounds 3–5 were the most cytotoxic towards cancer cell lines (IC₅₀: 3.8 µM to 42.3 µM) followed by 10 (IC₅₀: 30.9- > 64.4 µM). Cytotoxicity of compounds 3–5 was comparable to the activity of doxorubicin.

Keywords

anti-inflammatory activity chemical constituent cytotoxicity

Scrophulariaceae, the figwort family of flowering plants, have about 65 genera and 1700 species which are globally distributed.¹ The plants are annual and perennial herbs, as well as one genus of shrubs. The genus Scoparia of this family comprises 32 species of flowering plants.²

Scoparia dulcis L., which belongs to genus Scoparia also known as sweet broom weed, is a perennial edible herb with white flowers and serrated leaves, dispersed in tropical and subtropical area of Asia, Africa and America.³ Folklorically it is used to treat ailments like bronchitis, diabetes, hypertension, cancer, fever, haemorrhoids, stomach ache, ulcers hepatitis and tuberculosis^4,5 and also used to treat respiratory and inflammatory diseases in South America.⁶

S. dulcis has been extensively studied for its chemical constituents like triterpenes,^2,4,7,8 diterpenes,^4,7,9–15 benzoxazinoid, flavonoids, iridoid and iridoid glycosides compounds^3,16 and other constituents coixol, glutinol, glutinone, friedelin, betulinic acid, and tetratriacontan-1-ol¹⁷ are also reported.

Several compounds isolated from S. dulcis have been found to have different types of biological activities.^3,18 Compounds such as scoparic acid D, scoparic acid A and 8-hydroxytricetin-7-glucronide are reported to possess anti-diabetic activity;^18–20 scopadulin holds anti-viral;¹⁴ glutinol has analgesic;²¹ scoparinol possesses analgesic, diuretic and anti-inflammatory activity;²² scopadulciol, scopadulcic acid B, and diacetyl scopadol are inhibitor of gastric H+, K(+)-ATPase.^23,24 Iso-dulcinol, 4-epi-scopadulcic acid B, dulcidiol, scopadulciol and scopadiol have been examined for cytotoxicity.¹⁰

In this paper, we report isolation and structure elucidation of two new acyclic diterpenes Acetic acid 6-hydroxy-2-(6-hydroxy-4-methyl-hex-4-enylidene)-4,8-dimethyl-undeca-4,8-dienyl ester (1) and Acetic acid 8-hydroxy-2-(6-hydroxy-4-methyl-hex-4-enylidene)-6,10-dimethyl-undeca-5,9-dienyl ester (2) together with eight known compounds (3–10) recognized as scopadulciol (3), 4-epi-scopadulcic acid B (4), dulcidiol (5), scopadulcic acid B (6), hymenoxin (7), glutinol (8), eupatilin (9) and 5-demethylnobiletin (10). Keeping in view the previous reports, folkloric use of plant as anti-inflammatory and many of its constituents have already been reported for cytotoxicity and anti-inflammatory activity. The anti-inflammatory and cytotoxic activities of compounds 1 and 3–10 are also evaluated.

Results and discussion

A phytochemical work on acetonitrile fraction of S. dulcis led to the isolation of two new compounds 1 and 2 shown in Figure 1. During the course of isolation besides compounds 1 and 2, acetonitrile fraction of S. dulcis also yielded eight previously known compounds (3–10). These compounds were identified by interpretation of their 1D and 2D nuclear magnetic resonance (NMR), high resolution mass spectrometry (HRMS) data and by comparison of their physical and spectroscopic data with that reported in literature. Compounds 3–10 are recognized as Scopadulciol (3),²⁴ 4-epi-scopadulcic acid B (4), dulciodiol (5),¹⁰ Scopadulcic acid B (6), Hymenoxin (7),²⁵ Glutinol (8),² Eupatilin (9),²⁶ and 5-demethylnobiletin (10).²⁷ Compound 9 and 10 are reported for the first time from this plant.

Figure 1.

Structures of compound 1 to 10.

Compound 1 was obtained as a sticky material. Its molecular formula was determined as C₂₂H₃₆O₄, based on molecular ion peak at m/z 387.2504 in high resolution-electrospray ionization mass spectrum (HR-ESI-MS) and indicating five degrees of unsaturation. The ¹H and ¹³C NMR assignments of 1 are based on the COSY, HSQC, and HMBC spectra (supplemental material). The ¹³C NMR together with DEPT spectra confirmed the presence of 22 carbon atoms: five quaternary including one carbonyl, seven methylene, five methine and five methyl carbons. Analysis of ¹H NMR,¹³C NMR and HSQC spectra revealed that the signals resonating at δ_H/δ_C 5.27/126.1, 5.38/130.3, 5.07/126.1, 5.05/130.3 were ascribed for olefinic methines²⁸ and the signal resonating at δ_H/δ_C 4.25/66.4 was assigned for one oxymethine. Signals appearing at δ_H/δ_C 1.58/18.5, 1.57/16.8, 1.64/25.9, 1.58/16.4 and 2.0/21.1 were resolved for five methyls. The peaks resonating at δ_H/δ_C 2.02/26.8, 2.11, 1.93/48.5, 1.97/35.2, 1.97/39.5 2.16/26.1 were designated for five methylene moieties and δ_H/δ_C 4.55/61.5 and 3.93/58.0 were designated for two oxymethylene moieties. From the ¹³C NMR and DEPT spectra, the peaks appearing at δC 132.5, 134.0, 131.4, 135.5 and 170.8 belong to five quaternary carbons.

In COSY spectrum, it was observed that 1 has four spin systems (Figure 2). The peaks resonating at δ_H 3.93 [2H, H-1, t, J = 7.0 Hz, δ_C 58.0 (C-1)] showed cross peak in COSY spectrum with δ_H 5.27 [1H, H-2, t, J = 14.0 Hz, δ_C 126.1 (C-2)] forming a segment of the molecule. The peaks appearing at δ_H 2.16 [2H, H-5, q, J = 7.0 Hz, δ_C 26.1 (C-5)] showed cross peak in COSY spectrum with δ_H 1.97 [2H, H-4, m, δ_C 39.5 (C-4)] and δ_H 5.38 [1H, H-6, t, J = 7.0 Hz, δ_C 13.03 (C-6)] together with HMBC correlations, making another segment of the molecule.

Figure 2.

HMBC and COSY correlations of compound 1.

In a similar manner COSY cross peaks between δ_H 5.07 m (1H, H-10)/ 4.25 m (1H, H-11)/ 2.11 (1H, H_a-12, dd, J = 7.0,7.0 Hz) and 1.93 (1H, H_b-12, dd, J = 7.0,7.0 Hz) corresponding to δ_C 126.1 (C-10), 66.4 (C-11) and 48.5 (C-12), and δ_H 5.05 m (1H, H-14)/ 2.02 m (2H, H-15)/ 1.58 t (3H, H-16) corresponding to δ_C 130.3 (C-14), 26.8 (C-15) and 18.5 (C-16), respectively, together with HMBC correlations building third and fourth segment of the molecule individually. In ¹H NMR, signals at δH 4.35 d (7.0 Hz) and 4.46 t (7.0 Hz) are ascribed to two hydroxyl protons and their attachment to C-1 (δ_C 58.0) and at C-11 (δ_C 66.4) were established via HMBC correlations (Figure 2) which was in agreement with the previously reported data for oxymethylene and oxymethine, respectively.³ Occurrence of carbonyl carbon was defined by a signal appearing at δ_C 170.8 (C-21).

The oxymethylene signals δ_H/δ_C 4.55 br s (2H, H-20) /61.5 (C-20) showing J³ correlation with C-21 and the methyl signals δ_H/δ_C 2.0 s (3H, H-22)/21.1(C-22) showing J²correlation with C-21 consequently forming a methylene acetate moiety.²⁹ The cross peaks in HSQC spectrum δ_H/δ_C 1.64 s (3H, H-17)/25.9 (C-17), 1.57 s (3H, H-18) / 16.8 (C-18) and 1.58 s (3H, H-19) / 16.4 (C-19) are attributable for three vinyl methyls and their allocation at δ_C 131.4 (C-13), 132.5 (C-9) and 135.5 (C-3), respectively, were based on HMBC correlations.²⁹ The aggregate structure of 1 was established by connecting these fragments based on hetero nuclear coupling observed in HMBC spectrum.

The vinyl methyl proton δ_H 1.58 (H-19) showing J² correlation with δ_C 135.5 (C-3) and J³ correlation with 126.1 (C-2) reversely δ_H 5.27 showing J³ correlation with 16.2 (C-19) suggested C-2 and C-3 are connected with each other through an olefinic bond. Beside this δ_H 5.27 (H-2) also showing J³ correlation with 39.5 (C-4), also δ_H 1.97 (H-4) exhibiting J³ correlation with δ_C 126.1 (C-2) and δ_C 16.4 (C-19). The protons resonating at δ_H 2.16 (H-5) presented J³ connection with and δ_C 134.0 (C-7) and J² connection with δ_C 130.3 (C-6) suggesting the olefinic linkage between C-6 and C-7. Furthermore, δ_H 2.16 also giving J³ connection with δ_C 135.5 (C-3) and J² connection δ_C 39.5 (C-4). The oxymethylene protons at δ_H 4.55 of the methylene acetate moiety showing J² correlation with δ_C 134.0 (C-7) and J³ correlation with δ_C 35.2 (C-8) and δ_C 130.3 (C-6) and proton at δ_H 1.97 (H-8) and δ_H 5.38 showing J³ correlation with δ_C 61.4 (C-20) secured the position of methylene acetate moiety at C-7. Another vinyl methyl proton resonation at δ_H 1.57 (H-18) showing J² correlation with 132.5 (C-9) and J³ correlation with δ_C 126.1 (C-10) proposed olefinic bond between C-9 and C-10, J³ correlation is also observed between δ_H 1.57 (H-18) and δ_C 35.2 (C-8). The oxymethine proton δ_H 4.25 (H-11) showing J² correlation with 48.5 (C-12) and J³ correlation with δ_C 132.5 (C-9). Signals at δ_H 5.07 (H-10) showing J² correlation with δ_C 66.4 (C-11) and δ_C 132.5 (C-9) J³ connectivity with δ_C 35.2 (C-8) and δ_C 48.5 (C-12). Protons resonating at δ_H 1.64 (H-17) has already been assigned for vinyl methyl showing J² and J³ connectivity with δ_C 131.4 (C-13) and δ_C 131.4 (C-14), respectively, proposing the olefinic linkage between C-13 and C-14 and also showing J³ connectivity with δ_C 48.5 (C-12). The H-12 protons δ_H 2.11 and 1.93 both showing J³ connectivity with δ_C 130.3 (C-14). The methylene protons resonating at δ_H 2.02 (H-15) showing J² correlation with δ_C 130.3 (C-14) and J³ correlation with δ_C 131.4 (C-13). The terminal methyl proton δ_H 1.58 (H-16) showing J³ correlation with δ_C 130.3 (C-14) and J² correlation with δ_C 26.8 (C-15).

On the basis of this spectral evidences, the structure of 1 was deduced to be Acetic acid 6-hydroxy-2-(6-hydroxy-4-methyl-hex-4-enylidene)-4,8-dimethyl-undeca-4,8-dienyl ester.

Compound 2 was obtained as a colourless gummy material. Its molecular formula was established as C₂₂H₃₆O₄ on the basis of HR-ESI-MS and indicating five degrees of unsaturation. Comparison of the ¹H and ¹³C NMR spectral data (Table 1) of 2 to that of 1 indicated that both compounds 1 and 2 shared high structure similarity with each other, in fact 1 and 2 are structural isomers. The only difference was the place of substituents including olefenic bonds in parent carbon chain. The methyl signals δ_H 1.66 [3H, s, H-18, δ_C 16.2 (C-18)] having J³ correlation with δ_C 128.0 (C-10) and δ_C 48.2 (C-12) and J²correlation with δ_C 132.1 (C-11) indication that it is located at C-11 and the down field chemical shift of δ_C 128.0 (C-10) and δ_C 132.1 (C-11) suggested olefenic bond between C-10 and C-11 instead in between of C-9 and C-10 as in 1, and in 2, C-9 is a methylene carbon as evident by up field chemical shift δ_C 26.5, while the δ_H/ δ_C 4.77 m/ 65.7 (C-13) was attributed to oxymethine having hydroxyl group.²⁸ Conversely in 1, C-11 and C-13 were substituted by a hydroxyl and a methyl group, respectively. The position of two vinyl methyls δ_H/ δ_C 1.70 s/ 18.2 and δ_H/ δ_C 1.75 s/ 25.8 at C-15 were established by J³ correlation of δ_H 5.19 [1H, d, J = 7.0 Hz, H-14, δ_C 127.3 (C-14)] with C-16 and C-17 and also the down field chemical shift of C-15 (δ_C 135.0) suggested the olefenic bond between C-14 and C-15.²⁹ While in 1, C-15 is a methylene moiety. All this spectroscopic data were supporting the structure of compound 2 to be Acetic acid 8-hydroxy-2-(6-hydroxy-4-methyl-hex-4-enylidene)-6,10-dimethyl-undeca-5,9-dienyl ester.

Table 1.

¹H and ¹³C NMR data of the isolated new compounds 1 (DMSO-d₆) and 2 (CDCl₃).

Position	1		2
	¹H (J in Hz)	¹³C	¹H (J in Hz)	¹³C
1	3.93 t (7.0)	58.0	4.18 br s	59.3
2	5.27 t (7.0)	126.1	5.44 m	124.1
3	–	135.5	–	138.8
4	1.97 m	39.5	1.20 m	39.3
5	2.16 q (7.0)	26.1	2.25 m	25.9
6	5.38 t (7.0)	130.3	5.43 m	130.7
7	–	134.0	–	133.6
8	1.97 m	35.2	2.13 m	35.1
9	–	132.5	2.18 m	26.5
10	5.07 m	126.1	5.25 m	128.0
11	4.25 m	66.4	–	132.1
12	2.11 dd (7.0,7.0); 1.93 dd (7.0,7.0)	48.5	2.16 m	48.2
13	–	131.4	4.77 m	65.7
14	5.05 m	130.3	5.19 d (7.0)	127.3
15	2.02 m	26.8	–	135.0
16	1.58 t (merge in 1.58–1.57)	18.5	1.70 s	18.2
17	1.64 s	25.9	1.75 s	25.8
18	1.57 s	16.8	1.66 s	16.2
19	1.58 s	16.4	1.68 s	16.3
20	4.55 br s	61.5	4.62 s	61.8
21	–	170.8	–	171.2
22	2.0 s	21.1	2.10 s	21.0
OHs	4.46 t (7.0 Hz), 4.35d (7.0 Hz)		5.43 (merge in 5.44)

The α configuration of OH at C-11 and C-13 of compounds 1 and 2, respectively, was defined by comparing optical rotation values with that of very close structurally similar type of compounds, (S)-12-Hydroxygeranylgeraniol derived diterpenes³⁰ and bifurcatriol.²⁸

In the history of mankind, plants were the primary source of medicines. Throughout the world, a great number of plants as whole and their different parts were used as drug like entities to cure all kind of diseases. In the scientific community, the awareness that chemical entities present inside the plant material used folklorically is responsible for potential pharmacological activities either as single or in synergetic manner and these chemical entities can be isolated for their usage as a single agent came up in the 19th century in the context of the emerging natural science-based medicine and pharmacy.³¹

The biological process of inflammation is very critical for the management of body homeostasis. It is very crucial for positively fighting with pathogens and to restoration of injured tissues. The process of inflammation is also involved in several severe complaints, such as asthma, chronic inflammatory bowel diseases, rheumatoid arthritis, neurodegenerative diseases, cancer and type-2 diabetes.³²

There are many complex (humoral and cellular) mechanisms of inflammation and these mechanisms involve in the factors of gene regulation such as the nuclear factor-kappa B (NF-κB) and also involved synthesis of signalling substance produced by immune system cells such as cytokines and prostaglandins.^33,34 Usually many steroidal and nonsteroidal anti-inflammatory drugs (NSAIDs) and immunosuppressants, used for the treatment of inflammatory diseases, need long period of treatment and often have many serious side effects. The secondary metabolites produced by various medicinal plants have already been shown their potency in the management of inflammation and pain.³³

The extractives of various plants and their isolates have been examined in vitro for cytotoxicity by using a number of human cell lines such as stomach, prostate, colon, liver and so on. as well as animal cells such as kidney cells of monkey. Testing of toxicity through cell is a valuable and cost-effective and have short period testing time. This test is able to provide the information on the dose-dependent connection as well as the dose range for exposure and risks to human’s health. The cytotoxicity of plant extractive and isolates must be assessed before their influence in drug discovery while taken into consideration.³⁵

Out of the two new compounds, only compound 1 was evaluated for biological activity. It was found to inhibit nitric oxide production in lipopolysaccharide (LPS)-induced macrophages with IC₅₀ value of 21.9 µM without having any effect on NF-κB. On the other hand, it was showing cytotoxicity at 57.5 µM and >68.6 µM against SK-MEL and LLC-PK1, respectively. Cytotoxicity of LLC-PK1 is supported by close structurally similar acyclic diterpene (2E,10L9-1,12-dihydroxy-18-acetoxy-3,7,15-trimethylhexadeca-2,10,14-triene), having methylene acetate moiety as in 1 have also been reported for cytotoxicity against SK-OV-3 and SK-MEL-2 cell line with IC₅₀ value 10.20 and 6.57 µg/mL²⁹ supports our results for compound 1. Most of the other isolates (3–10) were found to exhibit anti-inflammatory activities in terms of inducible nitric oxide synthase (iNOS) and NF-κB inhibition ranging from 3.8 to 85.5 µM and several of them were effective in decreasing oxidative stress. In literature, compound 8 was reported to have anti-inflammatory activity,³⁶ and compound 9 significantly have anti-inflammatory potency in dose-dependent manner. However, compounds 3, 4 and 5 were cytotoxic ranging from 3.8 to 42.3 µM. The cytotoxicity results of compounds 3, 4 and 5 against cell lines (SK-MEL, KB, BT-549, SK-OV-3, LLC-PK1 and Vero) also in agreement with previous reports,¹⁰ in literature compounds 3, 4 and 5 have already been reported from the same source and found cytotoxic against cancer cell lines SCL, SCL-6, SCL-37'6, SCL-9, Kato-3 and NUGC-4. Compounds 3 showed cytotoxicity ranging ED₅₀ from 8.9 to 37.7 µM against all the tested cell line. It was also cytotoxic to AGS human gastric adenocarcinoma cell.⁷ Compound 4 showed activity, ED₅₀ from 19.5 to 59.3 µM against the SCL, SCL-37′6, and SCL-9 cell while the compound 5 showed cytotoxicity (ED₅₀ from 29.0 to 46.0 µM) except NUGC-4 cell line.¹⁰

Compounds 3–7 and 9–10 inhibited iNOS with IC₅₀ of 3.8–27.3 µM, respectively. Compounds 3–10 also inhibited NF-κB with 10.3 and >85.5 µM. Compound 8 was not effective towards iNOS while compound 9 was not effective towards NF-κB as shown in Table 2. Compound 10 was most effective in inhibiting iNOS and NF-κB with IC₅₀ values of 3.8 and 10.3µM, respectively. It also decreased oxidative stress (53% at 125 µg/mL). Since traditionally the fresh or dried plant has been used in a variety of ailments including inflammation and the plant is also reported to possess cytotoxic, anti-cancerous action, the activity of the plant may be attributed to the isolates tested for anti-inflammatory activity and cytotoxicity.

Table 2.

Anti-inflammatory and cytotoxic activities of the isolated compounds from S. dulcis.

Compounds	Anti-inflammatory activity			Cytotoxicity (IC₅₀ µM)
	iNOS inhibition (IC₅₀ µM)	NF-κB inhibition (IC₅₀ µM)	% decrease in oxidative stress at (125 µg/mL)	SK-MEL	KB	BT-549	SK-OV-3	LLC-PK1	Vero
1	21.9	NA	NA	57.6	NC	NC	NC	>68.6	NC
2	NT	NT	NT	NT	NT	NT	NT	NT	NT
3	6.3	28.6	49	42.3	<14.7	30.6	NC	<14.7	NC
4	27.3	15.9	NA	<14.2	<14.2	<14.2	NC	<14.2	NC
5	9.3	18.7	55	15.7	<14.6	<14.6	NC	<14.6	NC
6	22.8	22.8	56	NC	NC	>57.0	NC	<14.2	NC
7	42.7	85.5	16	NC	NC	NC	NC	NC	NC
8	NA	79.3	NA	45.3	NC	NC	NC	NC	NC
9	40.6	>145.3	NA	>72.6	NC	NC	>72.6	NC	>72.6
10	3.8	10.3	53	>64.4	30.9	64.4	NC	30.9	>64.4
Parthenolide	2.0	0.8	NT	NT	NT	NT	NT	NT	NT
Quercetin 25 µM	NT	NT	71	NT	NT	NT	NT	NT	NT
Doxorubicin	NT	NT	NT	2.75	3.6	2.9	4.5	3.3	>9.1

iNOS: inducible nitric oxide synthase; NA: no activity; NC: not cytotoxic; NT: not tested.

Conclusion

In conclusion, 10 different compounds including two new 1 and 2 were isolated from acetonitrile fraction of Scoparia dulcis and identified by 1D NMR, 2D NMR and HRMS. Moreover, compounds 9 and 10 are reported for the first time from this plant. Most of these compounds were found to exhibit anti-inflammatory activities in terms of iNOS and NF-κB inhibition. Compounds 3–5 showed promising cytotoxicity towards the selected cell lines. The obtained results confirmed the traditional use of the plant for the treatment of inflammatory diseases and cancers.

Experimental

General experimental procedures

The ESI-HRMS are measured on Agilent Technologies 6200 series mass spectrometer. ¹H NMR (700 MHz), ¹³C NMR (175 MHz) and 2D NMR spectra are measured on Bruker Avance NMR spectrometer. UV spectra are measured on UV 1601 PC (Shimadzu). Optical rotations are recorded on Jasco Polarimeter P-2000. Thin-layer chromatograph (TLC) is performed on precoated silica gel plates with UV₂₅₄ fluorescence indicator (Merck). The chromatograms are visualized under UV light (at 254 and 366 nm) CAMAG UV cabinet and also spraying with anisaldehyde-sulphuric acid reagent followed by heating with heat gun to visualize the spots. Silica gel (230–400 mesh) and LiChroprep RP-18 (40–63 μm; Merck, Darmstadt) are utilized for column chromatography (CC). High-performance liquid chromatography (HPLC) is performed on a Shimadzu system (Kyoto, Japan), consisting of two LC-6AD semi-preparative solvent delivery pumps, bus module CBM-20A, a multi wavelength photodiode array detector (SPD-M20A), columns shim-pack PREP-ODS (H) Kit (A) 250 mm × 4.6 mm I.D. with 5 μm particles (B) 250 mm × 20 mm I.D., 5 μm.

Plant material

The plant material was collected in February 2013 from Haili near Dam, Kingdom of Saudi Arabia (KSA). The collected plant material was identified by the taxonomist Dr. M. Yusuf and a plant voucher # 16026 was deposited at the herbarium of the College of Pharmacy, King Saud University, Riyadh, KSA.

Extraction and isolation

The dried and coarsely ground plant material (2.070 kg) was extracted with dichloromethane (5 L × 3) at room temperature for 72 h (24 h × 3). The extract was filtered through filter paper (Whatman No.1) and solvent was evaporated to dryness at 40 °C under reduced pressure using Buchi Rotavapour (R 215) and yielded green solid mass 193.1 g. The dichloromethane extract was partitioned between hexane and acetonitrile (presaturated), yielded acetonitrile fraction 43.2 g and hexane fraction 145.6 g. Acetonitrile fraction 40.0 g (out of 43.2 g) was undertaken for column chromatography over normal phase silica gel that provided 28 fractions (Fr 1. to Fr. 28). Further extensive chromatographic work on these fractions including reverse phase column and semi-preparative HPLC yielded compounds 1 (8.0 mg), 2 (1.5 mg), 3 (221.0 mg), 4 (25.1 mg), 5 (9.6 mg), 6 (12.2 g), 7 (47.0 mg), 8 (10.0 mg), 9 (5.2 mg) and 10 (1.5 mg).

Compound 1

Colourless sticky material; [α] ^D ₂₇ −2.4° (c 0.05, MeOH); UV (MeOH) λ_max (log ε) 339 (2.53); ¹H and ¹³C NMR, see Table 1; ESI-HRMS m/z 387.2504 calcd for C₂₂H₃₆O₄ Na, 387.2511.

Compound 2

Colouless sticky material; [α]^D₂₇ −3.0° (c 0.06, MeOH); UV (MeOH) λ_max (log ε) 249 (2.46); ¹H and ¹³C NMR, see Table 1; ESI-HRMS m/z 387.2504 calcd for C₂₂H₃₆O₄ Na, 387.2511.

Biological assays

Determination of NF-κB inhibition

Inhibition of NF-κB activity was determined in human chondrosarcoma (SW1353) cells through a reporter gene assay as described earlier.³⁷ The cells were transfected with NF-κB luciferase plasmid construct, seeded into the wells of 96-well plates (1.25 × 10⁵ cells/well) and incubated for 24 h for confluency. Test compounds at various concentrations were added, and after 30 min, phorbol 12-myristate 13-acetate (PMA, 70 ng/mL) was added. After incubating further for 6−8 h, luciferase activity was detected using a Luciferase Assay Kit (Promega, Madison, WI, USA). The decrease in luciferase activity upon treatment with the test compound was calculated in comparison to untreated cells. Parthenolide served as the positive control. A control transcription factor Sp-1 was also included to determine the general toxicity and specificity of the compound towards NF-κB as described earlier.³⁸

Determination of iNOS inhibition

The inhibition of iNOS activity was determined in mouse macrophages (RAW264.7) according to a procedure described earlier.³⁷ In brief, macrophages were plated in 96-well plates (50,000 cells/well) and plates were incubated for confluency up to 24 h. Cells were treated with various concentrations of test compounds and after incubating for 30 min, LPS (5 μg/mL) was added and further incubated for 24 h. The nitrite production in cell culture supernatant was measured by Griess reagent. Percent decrease in nitrite level was calculated in comparison to the vehicle control. Parthenolide was included as a positive control.

Determination of the effect on oxidative stress

The decrease in cellular oxidative stress as a measure of antioxidant effect was determined in HepG2 cells as described earlier.³⁹ For the assay, the cells were seeded in the wells of a 96-well plate (60,000 cells/well) and incubated for 24 h. The cells were washed with phosphate-buffered saline and treated with the test compounds diluted in serum-free medium containing 25 μM DCFH-DA, for 1 h. The media was removed and the cells were treated with ABAP (600 μM). The plate was immediately read on a SpectraMax plate reader every 5 min for 1 h (37 °C, emission at 538 nm and excitation at 485 nm). The percent decrease in oxidative stress was calculated. Quercetin served as the positive control.

Determination of cytotoxicity

The cytotoxicity of the compounds was tested against a panel of cancer cell lines (SK-MEL, KB, BT-549, SK-OV-3) and kidney cell lines (LLC-PK₁ and VERO). The cells were plated at a density of 25,000 cells/well and incubated for 24 h for confluency. After adding the test samples at various concentrations, cells were incubated for 48 hrs. The cell viability was determined by using a tetrazolium dye (WST-8). Percent decrease in cell viability was calculated in comparison to untreated cells. IC₅₀ value was calculated from the concentration response curves. Doxorubicin was included as the positive control for cytotoxicity.

Supplemental Material

Supplementary_Information_of_scoparia_revised_updated – Supplemental material for Isolation and characterization of cytotoxic and anti-inflammatory constituents from Scoparia dulcis L

Supplemental material, Supplementary_Information_of_scoparia_revised_updated for Isolation and characterization of cytotoxic and anti-inflammatory constituents from Scoparia dulcis L by Mohammad Nur-e-Alam, Sarfaraz Ahmed, Muhammad Yousaf, Shabana I Khan, Ramzi A Mothana and Adnan J Al-Rehaily in Journal of Chemical Research

Footnotes

Acknowledgements

The authors extend their appreciation to Researchers Supporting Project number (RSP-2019/119), King Saud University, Riyadh, Saudi Arabia for funding this work.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Adnan J Al-Rehaily

Supplemental material

Supplemental material for this article is available online.

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