Isolation and Characterization of Four Terpenoidal Compounds with Potential Antimicrobial Activity from Tarconanthus camphorantus L. (Asteraceae).

OBJECTIVES: The objective of this study is to describe the isolation, characterization, and antimicrobial activity of isolated compounds from Tarconanthus camphorantus.
MATERIALS AND METHODS: Bioactive compounds such as trifloculoside, parthenolide (sesquiterpene lactones), lupeol, and erythrodiol (pentacyclic triterpens) were isolated from n-hexane extract of T. camphoratus, and their antimicrobial activity against Candida albicans, Escherichia coli, Psuedomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus, and Mycobacterium smegmatis was evaluated. The compounds were characterized using chromatographic and spectroscopic techniques.
RESULTS: Trifloculoside, lupeol, and erythrodiol are being reported for the first time from T. camphoratus. The isolated compounds sesquiterpens and lupeol exhibited prodigious antimicrobial activity against B. subtilis and S. aureus with minimum inhibitory concentration values in the range of 25-1000 µg/mL but no activity was observed against other tested organisms, and erythrodiol showed no antimicrobial activity against any of the tested organisms.
CONCLUSION: The findings of this study revealed that the new compounds trifloculoside, parthenolide, and lupeol isolated from T. camphoratus exhibited effective antimicrobial potential. It was inferred that T. camphoratus can be effectively used in traditional medicine. Copyright:

Introduction

In recent years, infectious and noncommunicable diseases are the major health concerns throughout the world, especially in developing countries.[12] In the twentieth century, antimicrobial agents were of great importance to save people from life-threatening bacterial infections. During the past decade, incidences of drug-resistant organisms have reached unprecedented levels around the globe, leading to thousands of deaths annually.[3] The gradual increase in resistance of several important pathogens, including gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus), gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, and Mycobacterium smegmatis), and fungi (Candida albicans), poses a serious threat to public health at a terrifying rate.[4] There are great challenges to combat microbial infections and the development of novel antimicrobial agents to prevent the infectious diseases.

It is worth noting that medicinal plants have been continuous source of new molecules for millennia. The diversity of plant species encourages scientists to search new agents, especially ones used in folklore medicine and known to be effective. There are various examples of development of new drugs from plant sources and which undoubtedly revolutionized medicines, such as antibiotics.[5]

Tarconanthus camphorantus L. (Asteraceae), an aromatic dioecious shrub, is distributed in the southern regions of Saudi Arabia, Yemen, and Africa. It is the only plant belonging to the genus Tarconanthus.[6] Traditionally, the plant is used in the treatment of urinary tract infections, for wound healing, and as a remedy for toothache. Moreover, it is reported to relieve bronchitis, spasmodic asthma, headache, inflammation, and abdominal pains. The infusion of the leaf has diaphoretic, narcosis, and tonic effects and is also used for the relief of inflammation, abdominal pain, and spasmodic asthma.[78] In the present study, an effort is exerted toward the bioassay-guided fractionation of the leaf extracts of T. camphoratus with a view to isolate and characterize potential compounds as antimicrobial agents.

Materials and Methods

Collection and identification

The plant was collected from Sana’a-Yemen, and identification was done by Priv. Doz. Dr. Peter Koenig, at the Botanical Garden, Ernst-Moritz-Arndt-University, Greifswald, Germany. A voucher specimen (MAPTMRI-H/W/2/97) was deposited at the Pharmacognosy Department, Faculty of Pharmacy, Sana’ University.

Extraction, isolation, and structure elucidation

Powder of the air-dried leaves of the plant (150g) was exhaustively extracted with n-hexane using a soxhlet apparatus. n-hexane extract was concentrated under reduced pressure using rotary evaporator where 18g of hexane extract was obtained. The extract was subjected to column chromatography using silica gel with particle size 0.04–0.063mm as stationary phase and petroleum ether:ethyl acetate as mobile phase in the range of 15:1 to 5:1. The structures of the isolated compounds were identified by nuclear magnetic resonance (NMR).

General

Chromatographic analysis was carried out using column apparatus, precoated thin-layer chromatography plates (Merck, Germany), and detection was done at 254nm and by spraying with p-anisaldehyde/H2SO4 reagent. All chemicals were purchased from Sigma (St. Louis, MO, USA). Ultraviolet spectra were recorded on Thermo Scientific-Evolution 160 spectrophotometer, whereas Infrared (IR) spectra were recorded on Perkin-Elmer, FTIR model, 1600 spectrophotometer. NMR spectra were recorded on Bruker AMX 500 spectrometer (500 MHz) in CDCl3 with tetramethyl silane (TMS) as internal standard. Chemical shifts are given in ppm (δ) relative to TMS internal standard, and scalar coupling constants (J) are reported in Hertz. Gas chromatography-mass spectrometry (GC-MS) was done on Shimadzu QP-class-500 spectrophotometer.

Microorganisms used

The antimicrobial assay was performed using gram-positive bacteria (B. subtilis, S. aureus), gram-negative bacteria (E. coli, P. aeruginosa, and M. smegmatis), and fungi (C. albicans). All the microorganisms were maintained on trypticase-soy agar slants and recovered for testing by growth in trypticase-soy broth for 24 hours.

Antimicrobial activity

Antimicrobial evaluation of the different isolated compounds was carried out using the agar dilution method.[9] For testing at 1,000 µg/mL, isolated compounds were dissolved in dimethyl-sulfoxide (DMSO). Trypticase-soy agar was prepared and sterilized by autoclaving. Prior to congealing, 10mL of the agar medium was added to each Petri dish containing isolated compounds, and the Petri dishes were swirled carefully until the agar began to set. The microorganisms, gram-positive and -negative bacteria, and fungi, were maintained on trypticase-soy agar slants and recovered for testing by growth in trypticase-soy broth for 24 hours. Furthermore, the minimum inhibitory concentration (MIC) of the active isolated compounds was determined.

Results and Discussion

Spectral data

(1) Parthenolide: Melting point 117 °C; m/z = 248.317, corresponding to the molecular ion C15H20O3; 1H-NMR (CDCl3, 500 MHz), 13C-NMR (CDCl3, 500 MHz) [Supplementary Tables 1 and 2].

Supplementary Table 1

1H-NMR (500 MHz) spectral data of terpenoidal compounds 1-4 (CDCl3 with tetramethyl silane (TMS) as internal standard)

Position	Compound (1)	Compound (2)	Compound (3)	Compound (4)
1	*5.21 (dd,12)	2.35 (d, 8)	2.40 (m)	-
2	H2-α*2.09-2.24 (m)	*1.71-2.02 (m)	3.21(m)	-
	H2-β *2.43 (m)
3	H3-α 1.25 (m)	*1.23-1.52 (m)	-	*3.20
	H3-β *2.09-2.24 (m)
4	-	-	-	-
5	2.79 (d, 8.5)	*2.83 (dd)	-	-
6	3.86 (dd, 8.5)	4.16 (dd, 9.5)	-	-
7	2.9 (m)	*3.17 (dddd)	-	-
8	H8-α *2.09-2.24 (m)	*2.25 (m)	-	-
	H8-β 1.73 (m)
9	H9-α *2.09-2.24 (m)	2.43-2.50 (m)	-	-
	H9-β 2.37 (m)
10	-	-	-	-
11	-	-	-	-
12	-	-	-	*5.16
13	H13-α 6.34 (dd, 3.5)	6.11 (dd, 4)	-	-
	H13-β 5.46 (dd, 2.5)	5.57 (dd, 3.5)
14	1.72 (s)	H14-α 5.12 (s ,br)	-	-
		H14-β 4.97 (s ,br)
15	1.36 (s)	1.17 (s)	-	-
16	-	-	-	-
17	-	-	-	-
18	-	-	-	-
19	-	-	1.97 (m)	-
20	-	-	-	-
21	-	-	-	-
22	-	-	-	-
23	-	-	0.98 (s)	0.95 (3H, s)
24	-	-	0.79 (s)	0.81 (s)
25	-	-	0.86 (s)	0.90 (3H, s)
26	-	-	1.07 (s)	0.91 (3H, s)
27	-	-	0.97 (s)	1.02 (3H, s)
28	-	-	0.81 (s)	H28-α3.23 (d, J= 10.5)
				H28-β 3.56 (d, J= 11)
29	-	-	H29-α 4.71 (s)	0.96 (3H, s)
			H29-β 4.59 (s)
30	-	-	1.68 (s)	-

Supplementary Table 2

13C-NMR (125 MHz) spectral data of terpenoidal compounds 1-4 (CDCl3 with tetramethylsilane (TMS) as internal standard, *Overlapped signals)

Position	Compound (1)	Compound (2)	Compound (3)	Compound (4)
1	125.3	54.0	38.7	38.6
2	24.1	27.1	27.4	27.2
3	36.3	29.3	79.0	79.0
4	61.6	153.1	38.8	38.8
5	66.4	53.1	55.3	55.2
6	82.5	84.4	18.3	18.4
7	47.7	45.6	34.2	32.6
8	30.7	25.8	40.8	39.8
9	41.2	31.5	50.4	47.6
10	134.6	75.4	37.1	36.9
11	139.2	142.9	20.9	23.5
12	169.4	172.4	25.1	122.3
13	121.3	119.9	38.0	144.2
14	16.9	109.0	42.8	41.7
15	17.3	18.48	27.4	25.5
16	-	-	35.5	22.0
17	-	-	43.0	36.9
18	-	-	48.3	42.3
19	-	-	48.0	46.5
20	-	-	150.9	150.9
21	-	-	29.7	34.1
22	-	-	40.0	31.0
23	-	-	27.9	28.0
24	-	-	15.3	15.5
25	-	-	16.1	15.5
26	-	-	15.9	16.7
27	-	-	14.5	25.9
28	-	-	18.0	69.6
29	-	-	109.3	33.1
30	-	-	19.3	23.5

(2) Trifloculoside: Faint yellowish residue, IR (broad absorption at 3,425cm-1 [OH group] and 1,750 [carbonyl group]), m/z = 248.0, corresponding to the molecular ion C15H20O3; 1H-NMR (CDCl3, 500 MHz), 13C-NMR (CDCl3, 500 MHz) [Supplementary Tables 1 and 2].

(3) Lupeol: White powder; melting point 213 °C; 1H-NMR (CDCl3,500 MHz), 13C-NMR (CDCl3, 500 MHz) [Supplementary Tables 1 and 2].

(4) Erythrodiol: White residue; melting point 230 °C; m/z = 442.8, corresponding to the molecular ion C30H50O2; 1H-NMR (CDCl3,500 MHz), 13C-NMR (CDCl3,500 MHz) [Supplementary Tables 1 and 2].

Structure elucidation of the isolated terpenoidal compounds

Bioactivity guided fraction of n-hexane extract led to isolation and identification of four terpenoidal compounds (1)–(4) [Figure 1]. Structure of the isolated compounds were established using NMR spectral data. All the peaks appeared at their expected chemical shift values with appropriate multiplicities. NMR data [Supplementary Tables 1 and 2] were used to identify the compounds trifloculoside, lupeol, and erythrodiol, and X-ray crystallography led to identification of parthenolide [Supplementary Figure 1 and Supplementary Table 3]. Trifloculoside, lupeol, and erythrodiol are reported for the first time from the T. camphoratus [Figure 1].

Figure 1

Structure of the reported compounds—parthenolide, trifloculoside, lupeol, and erythrodiol

Supplementary Table 3

X-ray crystallographic data of parthenolide

Chemical formula	C15H20O3
Mr	248.31
Crystal system, space group	Orthorhombic, P212121
Temp (K)	100
A, b, c (Å)	11.8177 (7), 11.9875 (6), 18.8455 (11)
V (Å3)	2669.7 (3)
Z	8
Radiation type	MoKa
µ (mm-1)	0.09
Crystal size (mm)	0.30 x 0.30 x 0.05
Defractometer	Agilant Technologies Supar Nova Dual defractometer with Altas detector
Absorption correction	Multi scan CrysAlis PRO (Agilent 2012)
Tmin, Tmax	0.975, 0.996
No of measured, independent and observed	14549, 3457, 2946
Rint	0.044
(Sin θ/ λ) max (Å1)	0.651
R[F2>2 σ(F2)], wR (F2), S	0.041, 0.103, 1.04
No of reflection	3457
No of parameters	3.27
No of restraints	0
h-atom treatment	H-atom parameters constrained
Δ ρmax, Δ ρmin (e Å-3)	0.21, -0.17

Proton at position one in compounds (1), (2), and (3) appeared as double doublet, doublet, and multiplet at δ values of 5.21 ppm (J = 12.0 Hz), 2.35 ppm (J = 8.0 Hz), and 2.40 ppm and carbon at δ values of 125.3 ppm, 54.0 ppm, and 38.7 ppm. Proton-H2α, H2β, H3α, H3β, H8α, H8β, H9α, and H9β in compounds (1) and (2) were seen in NMR spectrum as multiplet. H5 in compound (1) appeared as a doublet, but in compound (2), it was observed as double doublet, at δ values of 2.79 ppm (J = 8.5 Hz) and 2.83 ppm. Double doublet was observed for H-6 in both the compounds (1) and (2) at δ values of 3.86 ppm (J = 8.5 Hz) and 4.16 ppm (J = 9.5 Hz) and carbon at δ values of 82.5 ppm and 84.4 ppm. At position, H13α and H13β in compounds (1) and (2) double doublets at δ values 6.34 ppm (J = 3.5 Hz), 5.46 ppm (J = 2.5 Hz), 6.11 ppm (J = 4 Hz) and 5.57 ppm (J = 3.5 Hz) were observed. For protons at positions 14 and 15, singlet was found in case of both compounds 1 and 2 at δ values of 1.72 ppm, 1.36 ppm and 5.12 ppm, 4.97 ppm, and carbon at δ value of 16.9 ppm and 17.3 ppm. These protons were seen as singlet because of the absence of any interacting protons in the surrounding environment. In compound (4), proton at positions 28α and 28β, doublet was observed at δ value 3.23 ppm (J = 10.5 Hz) and 3.56 ppm (J = 11 Hz). Owing to the presence of an adjacent proton, this peak was observed as a doublet. It was split into a doublet as per (n + 1) rule of multiplicity. However, in compound (3) at positions 29α and 29β, singlet was observed at δ value 4.71 ppm and 4.59 ppm, respectively [Supplementary Tables 1 and 2].

Antimicrobial activities of isolated compounds

Currently much attention has been directed toward biologically active compounds isolated from popular plants, and these plants play an important role in covering the basic health needs, especially in the developing countries against infective microorganisms.[10] The World Health Organization estimates 80% of the world populations rely on traditional medicine.[11] In this study, agar dilution method was used to evaluate antimicrobial activity of the different species. It is a quick screening method to identify the antimicrobial potential of the isolated compounds. After inoculation and incubation, active compounds displayed a zone of inhibition in which there was no bacterial growth. Investigated data from this study revealed isolated compounds parthenolide,[12] trifloculoside, lupeol,[13] and erythrodiol[14] were used as antimicrobial agents whereas trifloculoside was reported as antimicrobial for the first time. Parthenolide shows excellent antimicrobial activity against B. subtilis and S. aureus at a concentration level of 25 µg/mL, and against C. albicans at 300 µg/mL. Trifloculoside and lupeol show antimicrobial activity against B. subtilis and S. aureus at concentration levels of 200 µg/mL and 1mg/mL, respectively [Table 1]. These findings support the traditional use of this plant in the treatment of infectious diseases. Overall results of the present study reveal that parthenolide, trifloculoside, and lupeol exhibit antibacterial activity with MIC values ranging from 25 µg/mL to 1,000 µg/mL, whereas erythrodiol is deprived of antimicrobial activity

Table 1

Antimicrobial activity of isolated compounds—trifloculoside, parthenolide (sesquiterpene lactones), lupeol, and erythrodiol (pentacyclic triterpens)

Compound name	Organism tested
	Bacillus subtilis	Staphylococcus aureus	Escherichia coli	Pseudomonas aeruginosa	Mycobacterium smegmatis	Candida albicans
Parthenolide	MIC = 25 µg/mL	MIC = 25 µg/mL	-ve at 1mg/mL	-ve at 1mg/mL	-ve at 1mg/mL	MIC = 300 µg/mL
Trifloculoside	MIC = 200 µg/mL	MIC = 200 µg/mL	-ve at 1mg/mL	-ve at 1mg/mL	-ve at 1mg/mL	-ve at 1mg/mL
Lupeol	MIC = 1mg/mL	MIC = 1mg/mL	-ve at 1mg/mL	-ve at 1mg/mL	-ve at 1mg/mL	-ve at 1mg/mL
Erythrodiol	-ve at 1mg/mL	-ve at 1mg/mL	-ve at 1mg/mL	-ve at 1mg/mL	-ve at 1mg/mL	-ve at 1mg/mL

MIC = minimum inhibitory concentration. Samples were tested first at 1mg/mL if it is positive, MIC will be determined. Dimethyl-sulfoxide was used as solvent

Conclusion

The findings of this study revealed that the new compounds trifloculoside, parthenolide, and lupeol isolated from T. camphoratus exhibited effective antimicrobial potential. It was inferred that T. camphoratus can be effectively used in traditional medicine.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

Labelled ORTEP diagram of parthenolide with 50 % thermal probability ellipsoids