RP-UHPLC-MS Chemical Profiling, Biological and In Silico Docking Studies to Unravel the Therapeutic Potential of Heliotropium crispum Desf. as a Novel Source of Neuroprotective Bioactive Compounds

Heliotropium is one of the most important plant genera to have conventional folklore importance, and hence is a potential source of bioactive compounds. Thus, the present study was designed to explore the therapeutic potential of Heliotropium crispum Desf., a relatively under-explored medicinal plant species. Methanolic extracts prepared from a whole plant of H. crispum were studied for phytochemical composition and possible in vitro and in silico biological properties. Antioxidant potential was assessed via six different assays, and enzyme inhibition potential against key clinical enzymes involved in neurodegenerative diseases (acetylcholinesterase (AChE) and butyrylcholinesterase (BChE)), diabetes (α-amylase and α-glucosidase), and skin problems (tyrosinase) was assayed. Phytochemical composition was established via determination of the total bioactive contents and reverse phase ultra-high performance liquid chromatography mass spectrometry (RP-UHPLC-MS) analysis. Chemical profiling revealed the tentative presence of 50 secondary metabolites. The plant extract exhibited significant inhibition against AChE and BChE enzymes, with values of 3.80 and 3.44 mg GALAE/g extract, respectively. Further, the extract displayed considerable free radical scavenging activity against DPPH and ABTS radicals, with potential values of 43.19 and 41.80 mg TE/g extract, respectively. In addition, the selected compounds were then docked against the tested enzymes, which have shown high inhibition affinity. To conclude, H. crispum was found to harbor bioactive compounds and showed potent biological activities which could be further explored for potential uses in nutraceutical and pharmaceutical industries, particularly as a neuroprotective agent.


Total phenolic contents
The total phenolic content was determined as previously described in the literature [1] with some modifications. Sample solution (1 mg/mL; 0.25 mL) was mixed with diluted Folin-Ciocalteu reagent (1 mL, 1:9, v/v) and shaken vigorously. After 3 min, Na2CO3 solution (0.75 mL, 1%) was added and the sample absorbance was read at 760 nm after 2 h incubation at room temperature [2].

Total flavonoid contents
The total flavonoids content was determined using AlCl3 method [3]. Briefly, sample solution (1 mg/mL; 1 mL) was mixed with the same volume of aluminum trichloride (2%) in methanol. Similarly, a blank was prepared by adding a sample solution (1 mL) to methanol (1 mL) without AlCl3. The sample and blank absorbances were read at 415 nm after 10 min incubation at room temperature. The absorbance of the blank was subtracted from that of the sample [4].

UHPLC-MS secondary metabolites analysis
UHPLC Accurate-Mass Q-TOF (Agilent 1290 Infinity LC system coupled to Agilent 6520) mass spectrometer with dual ESI source was used. Column specifications were as: XDB-C18 Agilent Zorbax Eclipse, narrow-bore 2.1 x 150 mm, 3.5 micron (P/N: 930990-902). The temperature of the column was maintained at 25 °C, while the auto-sampler temperature was 4 °C. The following two mobile phases used were: A (0.1% formic acid in water), B (0.1% formic acid in acetonitrile) at a flow rate of 0.5 mL/min. Injection volume was 1.0 μL. Run time was 25 min and post-run time was 5 min.
MS analysis full scan was carried out over a range of m/z 100-1000 employing electrospray ion source in the negative ionization mode. The flow rate for nitrogen as nebulizing and drying gas was 25 and 600 L/hour, respectively with the drying gas temperature of 350 °C. The fragmentation voltage was optimized to 125. Capillary voltage for analysis was 3500 V [5].
The absorbance of the blank was subtracted from that of the sample. CUPRAC activity was expressed as milligrams of trolox equivalents (mg TE/g extract) [3,6].

Phosphomolybdenum
For phosphomolybdenum method: Sample solution (1 mg/mL; 0.3 mL) was combined with 3 mL of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The sample absorbance was read at 695 nm after a 90 min incubation at 95•C. The total antioxidant capacity was expressed as millimoles of trolox equivalents (mmol TE/g extract) [8].

Metal chelating
For metal chelating activity assay: Briefly, sample solution (1 mg/mL; 2 mL) was added to FeCl2 solution (0.05 mL, 2 mM). The reaction was initiated by the addition of 5 mM ferrozine (0.2 mL).
Similarly, a blank was prepared by adding sample solution (2 mL) to FeCl2 solution (0.05 mL, 2 mM) and water (0.2 mL) without ferrozine. Then, the sample and blank absorbances were read at 562 nm after 10 min incubation at room temperature. The absorbance of the blank was subtracted from that of the sample. The metal chelating activity was expressed as milligram [3,6].

Cholinesterases
For AChE inhibitory activity assay: Sample solution (1 mg/mL; 50 μL) was mixed with DTNB (5,5- (25 μL). Similarly, a blank was prepared by adding sample solution to all reaction reagents without enzyme (AChE or BChE) solution. The sample and blank absorbances were read at 405 nm after 10 min incubation at 25•C. The absorbance of the blank was subtracted from that of the sample and the cholinesterase inhibitory activity was expressed as galanthamine equivalents (mg GALAE/g extract) [9].
The reaction mixture was incubated 10 min at 37•C. The reaction was then stopped with the addition of HCl (25 μL, 1 M). This was followed by addition of the iodine-potassium iodide solution (100 μL).
The sample and blank absorbances were read at 630 nm. The absorbance of the blank was subtracted from that of the sample and the α-amylase inhibitory activity was expressed as acarbose equivalents (mmol ACE/g extract) [10].
Similarly, a blank was prepared by adding sample solution to all reaction reagents without enzyme (tyrosinase) solution. The sample and blank absorbances were read at 492 nm after a 10 min incubation at 25•C. The absorbance of the blank was subtracted from that of the sample and the tyrosinase inhibitory activity was expressed as kojic acid equivalents (mg KAE/g extract) [12].