UPLC-PDA-ESI-MS/MS Profiling of Clerodendrum inerme and Clerodendrum splendens and Significant Activity Against Mycobacterium tuberculosis

UPLC brings dramatic improvements in sensitivity, resolution and speed to analytical procedures of plant metabolites.2 In UPLC-MS, the high sensitivity of MS detection provides an important tool for detection and measurement of minor metabolites in complex plant extract samples.3,4 Several studies have applied this technology to look at metabolite profiles in closely allied plant taxa, different cultivars of individual taxa, or plants at different stages of development.5-8


INTRODUCTION
Antibiotic resistance like that of the multi-drug resistant Mycobacterium tuberculosis (TB), can be minimized through increasing awareness about the proper use of antibiotics and development of new antibacterial agents effective against this strain. 1 UPLC brings dramatic improvements in sensitivity, resolution and speed to analytical procedures of plant metabolites. 2 In UPLC-MS, the high sensitivity of MS detection provides an important tool for detection and measurement of minor metabolites in complex plant extract samples. 3,4 Several studies have applied this technology to look at metabolite profiles in closely allied plant taxa, different cultivars of individual taxa, or plants at different stages of development. [5][6][7][8] The aim of our work is to evaluate the antimycobacterial activity of the leaves of Clerodendrum inerme and Clerodendrum splendens and to define and compare their phytochemical composition using UPLC-PDA-ESI-MS/MS technique.

For UPLC-PDA-ESI-MS/MS analysis
Solvents used for UPLC-PDA-ESI-MS/MS analysis viz., water, acetonitrile and formic acid were of LC-MS grade and acquired from Fluka, Sigma-Aldrich chemicals (Germany). deposited in the Herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Cairo University. The leaves were air-dried then finely powdered.

Investigation of chemical composition by UPLC-PDA-ESI-MS/MS analysis
UPLC-PDA-ESI-MS/MS system was used for profiling of secondary metabolites. It consisted of an Orbitrap Elite mass spectrometer (Thermo Fischer Scientific, Darmstadt, Germany) coupled to an UPLC-PDA-ESI-MS/MS system (Dionex Ulti Mate 3000, Thermo Fischer Scientific), equipped with RP-C18 column (particle size 1.8 μm, pore size 100 Å, 150 mm × 1 mm i.d., Acquity HSS T3, Waters, USA) and a photodiode array detector (220-600 nm, Thermo Fischer Scientific) and operated in the negative ion mode. A mobile phase consisting of water/formic acid, 99.9/0.1 v/v (A) and acetonitrile/ formic acid, 99.9/0.1 v/v (B) was used for the separation. The following binary gradient was applied: 0 to 1 min: isocratic 95% A, 5% B; 1 to 11 min: linear from 5 to 100% B; 11 to 19 min: isocratic 100% B and 19 to 30 min: isocratic 5% B. The column temperature was maintained at 40 °C. The injection volume was 2 μL and the flow rate was 150 μL/ min. The apparatus was externally calibrated by the Pierce ESI negative ion calibration solution (product No. 88324) from Thermo Fisher Scientific. The data were evaluated using the software X-calibur 2.2 SP1. Metabolites were characterized by their UV-VIS spectra (220-600 nm), mass spectra and comparison to phytochemical dictionary of natural products database CRC Press and reported literature.

EXTRACTION PROCEDURE
For anti-mycobacterial activity Samples (10 g, each) of the air-dried powdered leaves were separately extracted using n-hexane (60-80 0 C) and the defatted mark macerated with ethanol (70%) until exhaustion. The extraction process was aided by sonication (Elma Terrasonic TS 540, Germany). The obtained ethanol extracts were filtered then dried under vacuum (rotatory evaporator Büchi, G. Switzerland).
For UPLC-PDA-ESI-MS/MS analysis Samples (5 mg, each) of the air-dried powdered leaves were separately homogenized with 1.5 mL 80% methanol containing 10 µg/mL umbelliferone (internal standard) using a Turrax mixer (11,000 rpm) for five 20 seconds periods. To prevent heating, a period of 1 min separated each mixing period. Extracts were then vortexed vigorously and centrifuged at 3000 g for 30 min to remove plant debris. An amount of 3µL of the supernatant 5 was used for UPLC-PDA-ESI-MS/ MS analysis. Chromatographic conditions and mass spectrometry parameters followed that described in previous reports 6 .

Principle of the Assay
The ethanol extracts (EEs) of C. inerme and C. splendens were investigated for antibacterial activity against Mycobacterium tuberculosis (TB) using reported methods. 1 using the Microplate Alamar Blue Assay (MABA). The latter is based on measuring the change in colour intensity of the blue colour of Alamar Blue solution by the activity of Mycobacterium tuberculosis. The colour intensity is measured at 590 nm using ELISA microplate reader. The percentage inhibition of mycobacterial growth by different concentrations of each sample was calculated as the average of triplicate measurements. The concentrations required to inhibit 90% of mycobacterial growth (MIC 90) and the lowest concentration that prevented colour change of Alamar Blue solution (MIC) were determined from the doseresponse curve. Isoniazid was used as a reference drug.

Procedure
The assay was performed according to (Franzblau et al., 1998) in a black, clear-bottomed, 96-well microplate to minimize background effect. The outer perimeter wells were filled with sterile water to prevent dehydration in experimental wells. A serial dilution (0.06 -125 µg/ mL) of each of the tested extracts and the reference drug, isoniazid, dissolved in DMSO, were prepared in the microplate then 0.1 mL of Mycobacterium tuberculosis inoculum (10 5 CFU/mL) was added to the wells. Wells containing bacteria only were used as negative control. Plates were incubated at 37 o C for 4 days, then 20 µL of Alamar Blue solution and 12.5 µL of 20% Tween 80 were added to the entire plate. The plates were re-incubated at 37 o C for 24 hours then the colour intensity was measured at 590 nm using ELISA microplate reader. The percentage inhibition of mycobacterial growth was calculated from the following formula; % inhibition = 1 -(mean of test well/mean of B well) ×100 Where mean of test well and mean of B well are the averages of triplicate determinations of the absorbance of both sample and negative control at 590 nm, respectively. MIC 90 and MIC were determined for each sample and for standard isoniazid from the dose-response curve as the concentrations required to inhibit 90% of mycobacterium growth and the lowest concentration that prevented colour change of Alamar Blue solution indicating no mycobacterium activity, respectively.

Anti-mycobacterial activity against Mycobacterium tuberculosis (TB)
The methanol extracts of the leaves of C. inerme and C. splendens were evaluated for anti-mycobacterial activity against Mycobacterium tuberculosis using the Microplate Alamar Blue Assay (MABA). 9 Results are represented by Figure 1 and recorded in Table 1. From table 1 Table 2.

Identification of phenyl-propanoid glycosides
Two types of phenyl-propanoid glycosides were detected in the methanol extract of the leaves of C. inerme. The first type, represented by verbascoside, magnoloside A or D (peaks 14 and 15). The second type, represented by markhamioside B (peak 20). The methanol extract of the leaves of C. splendens was found to contain one type of phenylpropanoid glycosides, represented by markhamioside C (peak 13). The MS/MS data and UV absorbance of the detected phenyl-propanoid glycosides were compared to reference literature for confirmation of their identities. [10][11][12][13][14][15] Identification of iridoid glycosides Melittoside (peak 2), inerminoside A1 (peak 5), p-coumaroyl melittoside (peak 12), inerminoside B (peak 17) and inerminoside A (peak 18) were detected in the methanol extract of the leaves of C. inerme only. Teucardioside (peak 6), was found in the methanol extract

Identification of diterpenoids
Trihydroxy abieta-trienoic acid (peak 32), was detected only in the methanol extract of the leaves of C. inerme. Marrubiagenin (peak 34) was detected in both methanol extracts of the leaves of C. inerme and C. splendens. For confirmation, the MS/MS data of the identified diterpenes was compared to reference literature of their identities. 21

Identification of phenolic acid derivatives
Coumaric acid hexoside (peak 4), coumaric acid pentosyl hexoside (peak 7), p-Coumaroyl melittoside (peak 12) and galloyl hexoside acetate derivative (peak 25) were detected only in the methanol extracts of the leaves of C. inerme. Caffeoyl shikimic acid (peak 10), rosmarinic acid (peak 19) and caffeic acid derivative (peak 22) were detected only in the methanol extracts of the leaves of C. splendens. For confirmation of the identities of the identified compounds, their MS data and UV absorbances were compared to reference literature. [22][23][24] Identification of fatty acid derivatives 2-Hydroxy-2-methyl butyric acid hexoside (peak 3), tri hydroxy octadecenoic acid (peak 26), monolinolein tri hexoside (peak 33) and Hispidulin methoxy tetradecenoate (peak 35) were detected only in the methanol extracts of the leaves of C. inerme. hydroxy octadecadienoic acid (peak 36) was detected only in the methanol extracts of the leaves of C. splendens.

DISCUSSION
From the previous results, the methanol extract of the leaves of C. inerme showed significant anti-mycobacterial activity against Mycobacterium tuberculosis, with MIC 90 and MIC values of 7.2 µg/mL value of 15.63 µg/mL, respectively. The methanol extract of C. splendens showed moderate anti-mycobacterial activity against Mycobacterium tuberculosis with MIC 90 and MIC values of 30.8 and 62.5 µg/mL, respectively. The UPLC-PDA-ESI-MS/MS analysis revealed a total of 36 metabolites detected and tentatively identified in the two species under investigation. Twenty-eight chromatographic peaks, belonging to various metabolite classes, were assigned and identified in C. inerme, whereas 14 chromatographic peaks were assigned and identified in C. splendens. The main classes of secondary metabolites detected were phenyl-propanoid and iridoid glycosides, flavonoids, diterpenoids, phenolic acid and fatty acid derivatives.

CONCLUSION
The methanol extract of the leaves of C. inerme exerts significant antibacterial activity against Mycobacterium tuberculosis, with MIC 90 and MIC values of 7.2 µg/mL and 15.63 µg/mL. Comparative UPLC-PDA-ESI-MS/MS metabolic analysis of C. inerme showed a chromatographic profile wealthier in components compared to that of C. splendens. Thus, the leaves of C. inerme can be utilized for treatment of Mycobacterium tuberculosis infections after further investigations and can be considered a richer source of plant metabolites compared to C. splendens.