Antioxidant activity, α-glucosidase inhibition and phytochemical profiling of Hyophorbe lagenicaulis leaf extracts

Collection of plant material

The plant material was collected from Lahore, Pakistan and was identified from the Department of Botany, GC University Lahore.

Green extract preparation

Quenching of fresh leaves was done in liquid nitrogen and grinded to a fine powder to enhance the surface area. The obtained powder was lyophilized using a freeze-dryer (Christ Alpha 1-4 LD; Osterode am Harz, Germany) and subjected to hydroethanolic solvent compositions (Ethanol, 100%, 80%, 60%, 40%, 20%) for 48 h. Mixtures were sonicated at soniprep 150 disintegrator below 10 °C. Samples were shaken for 2 h and filtered. The excess solvent from the filtrate was removed under the vacuum on rotary evaporator at 40 °C. The extracts were again freeze dried for 48 h. Extract yields (%) were calculated and extracts were stored at −80 °C till further use.

Determination of total phenolic and flavonoid contents

Total phenolic contents (TPC) of freeze dried leaf extracts were determined by Folin Ciocalteu reagent method with slight modification in previously reported scheme (Zhishen, Mengcheng & Jianming, 1999). The plant extract (one mg) was dissolved in methanol (one mL) and 0.25 μL of this was added to one mL of Folin Ciocalteu reagent. Then two mL of 10% solution of Na₂CO₃ followed by addition of two mL distilled water. The resultant mixture was stayed for 120 min at ambient conditions of temperature. The absorbance was noted at 765 nm. Standard curve of gallic acid was also drawn. Results were expressed as gallic acid equivalent (GAE) mg/g dried extract (Zengin et al., 2010).

Total flavonoid contents (TFC) were determined by AlCl₃ colorimetric method. The 0.1 mg of plant extract was dissolved in methanol (two mL) and added by five mL of distilled water. Then 0.5 mL of NaNO₂ (5%) was added to the mixture followed by the addition of 10% AlCl₃ solution. After 10 min NaOH (1 molar) was added to resultant mixture and after vigorous shaking, the absorbance was measured at 510 nm. The results were expressed as rutin equivalent mg/g dried extract (RE mg/DE) (Zhishen, Mengcheng & Jianming, 1999)

Antioxidant activities

Antioxidant potential of extracts was determined by DPPH scavenging assay as reported previously with little modification (Fki, Allouche & Sayadi, 2005). The one mg plant extract was dissolved in methanol. The 50–200 ppm of these dilutions were added to 10 mL of DPPH solution (0.001 molar). After 30 min incubation in the dark at room temperature, absorbance was measured at 520 nm. Butylated hyroxyanisole (BHA) was used as standard for comparison. All the measurements were carried out in triplicate and standard deviation was applied.

Phosphomolybdenum complex formation method was used as per previously reported method with minute modifications (Prieto, Pineda & Aguilar, 1999). Initially, 250 μg/mL of each extract was mixed with a solution composed of sulphuric acid (0.6M), ammonium molybdate (four mM) and 28 mM sodium phosphate. The mixtures and blank solution both were incubated at 95 °C for 90 min at water bath. After cooling, absorbance was noted at 695 nm wavelength. Ascorbic acid standard curve was drawn and butylated hyroxyanisole was used as standard antioxidant. The results were represented as ascorbic acid equivalent per gram dried plant extract (AE/g DE).

Anti-α-glucosidase activity

Inhibition potential of extracts against α-glucosidase was measured to evaluate in vitro antidiabetic potential. Various concentrations of extarcts (200 ppm) were added to phosphate buffer (70 μL of 50 mM) followed by addition of α-glucosidase (one unit/mL). After 10 min incubation at 37 °C, five mM of ρ-nitrophenolglucopyranoside was added and absorbance was noted at 405 nm after 30 min. Acarbose was used as standard reference and results were represented as IC₅₀ (μg/mL) values for each extract (Jabeen et al., 2013).

Where, Ab is absorbance of blank, As is absorbance of Sample.

The α-amylase inhibition activity

The 250 μL of each extract (1.0–10 mg/mL) were added to 0.02M sodium phosphate buffer containing porcine α-amylase (0.5 mg/mL). The reaction mixture was incubated for 10 min at 25 °C. The dinitrosalicylic acid was added to the mixture to stop the reaction. The reaction mixtures were further incubated for a time period of 5 min and diluted with distilled water to note absorbance at 540 nm. A control (no extract) was also run and acarbose was used as standard enzyme inhibitory substance (Kazeem, Adamson & Ogunwande, 2013). The % inhibition was calculated by following relationship; $$\%\text{Inhibition}=\frac{\text{Ab}-\text{As}}{\text{Ab}}\times 100$$

Where, Ab is absorbance of blank, As is absorbance of Sample.

Results were represented as IC₅₀ (μg/mL) values for each extract.

UHPLC-Q-TOF-MS/MS analysis

Plant extract was extracted with suitable solvent and filtered with plastic filter having 0.45 μm pore size. The filtered extract sample was subjected to UHPLC-Q-TOF-MS/MS (AB Sciex 5600-1, equipped with Eksigent UHPLC system). The scanning range of 50–1,200 m/z for MS/MS (negative ionization mode), column Thermo Hypersil Gold (100 mm× 2.1 mm × 3 μm), gradient mobile phase composition (water and acetonitrile) each having 0.1% formic acid and five mM ammonium formate was used. Gradient programming started from 10% acetonitrile to 90% acetonitrile with mobile phase flow rate of 0.25 mL/min. Desolvation temperature (TEM) was 500 °C and ion spray voltage was −4,500 V.

Statistical analysis

The experimental findings were evaluated for statistical significance by using Statistix 10.0 software. Analysis of variance was used to compare variations in treatment means to assess efficacy of treatments.

Extract yields (%), TPC and TFC

The extract yields, TPC and TFC with respect to solvent system used for extraction are given in Table 1. It was revealed by the findings that solvent influenced extract yields significantly. Maximum extract yield 20.46% ± 0.25^a% was given by 60% ethanol and second highest yield 18.05% ± 0.13^b% was obtained with 80% ethanol. Solvent composition not only affected the extract yields but also TPC and TFC. Maximum TPC of 178.56 ± 1.47^a mg GAE/g DE were recovered with 60% ethanol. Similarly highest TFC of 133.96 ± 1.19^a mg Rutin/g DE were extracted with 60% ethanol. Efficiency of 60% ethanolic extract was significantly higher than other solvent fraction for extract yield, TPC and TFC respectively (ρ < 0.05).

Antioxidant activities

The DPPH radical scavenging in terms of IC-50 value (μg/mL) by plant extracts in comparison with BHA is represented as Fig. 1. Maximum radical scavenging among extracts was exhibited by 60% ethanolic extract with minimum IC-50 value of 43.11 ± 0.96 μg/mL.

Total antioxidant power of extracts was determined by phosphomolybdenum methods. This method involved the reduction of Mo (VI) to Mo (V) with characteristic color change due to complex formation. This assay is widely used to evaluate the total antioxidant power of plant extracts and compounds (Prieto, Pineda & Aguilar, 1999; Rani et al., 2018).

The results of assay were represented in Fig. 2. Findings unveiled that 60% ethanolic fraction exhibited maximum antioxidant power with value of 239.33 ± 3.78^b (ASE/g PE) followed by 80% ethanolic extract (189.33 ± 2.51^c ASE/g DE). Statistical analysis indicated that antioxidant power of BHA was significantly higher than all extracts (ρ < 0.05). However 60% ethanolic extract was most potent among all plant extracts (ρ < 0.05) regarding antioxidant potential.

Anti-α- glucosidase activity

Inhibition of α-glucosidase enzyme reflects the in vitro antidiabetic potential which is determined spectrophotometrically. The IC-50 values (μg/mL) of plant extracts and standard drug acarbose are represented in Fig. 3. The comparison of extracts showed that 60% ethanolic extract among all fractions possessed maximum α-glucosidase inhibition with IC-50 value of 41.25 ± 1.25 μg/mL. The standard drug acarbose exhibited lowest IC-50 value of 25.50 ± 0.45 μg/mL which was significantly lower than all extracts (ρ < 0.05). Statistical comparison further indicated that 60% ethanolic extract was significantly better than remaining extracts (ρ < 0.05).

The α- amylase inhibition

The results of α-amylase inhibition are given in Fig. 4. The 60% ethanolic extract (IC-50 60.58 ± 3.24 μg/mL) exhibited highest α-amylase inhibition followed by 80% ethanolic extract with IC-50 value of 77.57 ± 2.25 μg/mL. The 20% ethanolic extract exhibited least inhibition of enzyme activity as indicated by IC-50 value (114.00 ± 1.88 μg/mL). The standard compound acarbose showed highest α-glucosidase inhibition with IC-50 value of 43.37 ± 0.75 μg/mL. Statistical analysis revealed that 60% ethanolic extract was the most potent α-glucosidase inhibitory fraction.

UHPLC-Q-TOF-MS/MS analysis

The efficacy of 60% ethanolic extract regarding antioxidant and α-glucosidase inhibition emphasized to explore this fraction for metabolite identification. So the 60% ethanolic extract was subjected to UHPLC-Q-TOF-MS/MS analysis and identified compounds along with their retention time (Rt), fragment ions and molecular formula are listed in Table 2. The main chromatogram of UHPLC separation is shown as Fig. 5. The fragmentation pattern of identified compounds is represented in Fig. 6. Citric acid appeared at Rt 1.603 min with characteristic parent ion peak at m/z 191[M-H]⁻ and daughter ion peak at m/z 111[M-CO₂-H₂O]⁻. Trimethoxy flavone derivative arrived at Rt 8.972 min with peaks at m/z 635, m/z 609 and m/z 300. Kaempferol was recorded at Rt 9.110 min with m/z 285. The fragment ion peaks at m/z 151 and m/z 93 due to removal of fragment of 134 amu and phenoyl moiety respectively. Rutin was recorded at Rt 9.27 min with parent ion peak m/z 609. The fragment ion having m/z 300 appeared due to removal of moiety of mass 309 amu. The fragment ion of m/z 271 was produced due to removal of 29 amu from fragment ion m/z 300. Peak at Rt 9.689 was identified as kaempferol-coumaroyl-glucoside with parent peak at m/z 593. The removal of coumaroyl glucoside produced fragment ion peak at m/z 285. Leuteolin 3-glucoside was identified at Rt 9.724 with parent ion peak at m/z 447. Removal of glucose from parent ion generated leuteolin characteristic peak at m/z 285. Hesperetin 5-O-glucoside was identified at Rt 9.433 with parent peak at m/z 463[M-H]⁻ and fragment ions at m/z 301[M-glucose-H]⁻, m/z 271[m/z 301-CH₂O-H]⁻ m/z 255[m/z 301-C₂H₂O-H]⁻, m/z 149[m/z 255-C₆H₂O₂-H]⁻ respectively. Isorhamnetin 3-O-rutinoside appeared at Rt 9.995 with m/z 623. The further collision resulted in fragment ions m/z 315[M-318-H amu]⁻, m/z 300 [m/z 315-CH₃-H]⁻ and m/z 284[m/z 315-CH₃O-H]⁻.