In vitro anti-diabetic effects and phytochemical profiling of novel varieties of Cinnamomum zeylanicum (L.) extracts

Collection of plant materials and preparation of extracts

Dried commercial Cinnamomum zeylanicum quills (1 kg) were collected from the Dassanayake Walauwa Cinnamon plantation, Nape, Kosgoda, Southern Province in Sri Lanka. Sri Wijaya and Sri Gemunu Cinnamon quills (2 kg per each) were collected from Cinnamon Research Station, Palolpitiya, Thihagoda, Southern Province, Sri Lanka. The collected samples were transported in sealed, sterilized polythene bags to the laboratory at University of Kelaniya, Sri Lanka. The samples were stored in a refrigerator (2−8 °C) until use.

Cinnamon accessions were authenticated by a botanist at the Department of Botany, University of Kelaniya. The voucher specimen of “Sri Wijaya” and “Sri Gemunu” accessions were deposited at the publicly available herbarium, Department of Plant and Molecular Biology, the University of Kelaniya, Sri Lanka under the family Lauraceae (Deposition numbers are CIN-SW-001 and CIN-SG-002 respectively for “Sri Wijaya” and “Sri Gemunu” accessions).

Cinnamon quills (10 g) were pulverized using a 0.50 mm mesh. For the microwave digestion (MD), the sample (10 g) was digested with distilled water (80 mL) for 30 min using a microwave digester (mass 6 instrument, vessel type Mars Xpress). Pulverized Cinnamon quills (10 g) were extracted with pressurized water (200 mL at 0.098 MPa, for 10 min) for the preparation of pressurized water extract (PWE). Quills of Cinnamon (40 g) were extracted by traditional steam distillation (SD) and 10 g were extracted by solvent extraction (SE) using 75% ethanol (Wong, 2014; Lee et al., 2018). For the preparation of decoction water extract (DWE), Cinnamon quills (10 g) were boiled with water (200 mL) until the volume was reduced to 1/8. Ten grams of Cinnamon powder was mixed with boiled water (200 mL) and that was allowed to stand for five minutes to obtain the infusion water extract (IWE). The MD, PWE, DWE, and IWE were filtered through Whatman 1 filter paper and concentrated under vacuum at 45 °C and further dried by passing a stream of N₂ air. The percentage yield was calculated and stored at −20 °C. The volatile compounds from SD were separated from the aqueous layer three times using hexane (30 mL). The volatiles were concentrated by using rotary evaporator (IKA^® RV 10 basic, Germany). The percentage yield of the resultant oleoresin was calculated and the oleoresin was stored at −20 °C.

α-glucosidase inhibitory activity

The α-glucosidase inhibition assay (Apostolidis & Lee, 2010) was used to determine the in vitro anti-diabetic properties of Cinnamon extracts. Varying concentrations (12.5 µg/mL – 400 µg/mL) of Cinnamon quill extract (100 µL) and 0.1M phosphate buffer, pH 6.8 (100 µL) with α-glucosidase enzyme solution (1 Unit/mL) were incubated in 96 well plates at 37 °C for 10 min. After pre-incubation, 2.5 mM 4-Nitrophenyl β-D-glucopyranoside (pNPG) solution (20 µL) in 0.1M phosphate buffer, pH 6.8 was added to each well. The reaction mixture was incubated at 37 °C for 20 min. After the incubation, the absorbance at 405 nm was recorded by the micro plate reader (Spectra Max M5, Molecular Devices, CA, USA). Acarbose was used as the positive control (12.5 µg/mL – 400 µg/mL). The solvent alone was used as the blank in the assay. The IC₅₀ values were calculated as follows;

Inhibition (%) $=1-\left\{\frac{A_{sample}}{A_{control}}\right\}\times 100$

where, A_sample and A_control were defined as absorbance of the sample and the control (blank) respectively.

α- Amylase inhibitory activity

The α-amylase inhibition assay (Ranilla et al., 2010) was also used to evaluate the anti-diabetic properties of Cinnamon extracts. Various concentrations (12.5 µg/mL - 400 µg/mL) of the extract (100 µL) and 0.02 M sodium phosphate buffer, pH 6.9 (100 µL), amylase enzyme solution (0.5 mg/mL, 10 µL) were incubated at room temperature (28 ± 2 °C) for 10 min in a test tube. After pre-incubation, 100 µL of 1% starch in 0.02 M sodium phosphate buffer, pH 6.9 was added to each tube. The reaction mixtures were incubated at room temperature (28 ± 2 °C) for 10 min. The reaction was quenched by adding Dinitrosalicylic acid color reagent (100 µL). The test tubes were boiled in a water bath until the yellowish orange color was developed and then the tubes were allowed to cool. The reaction mixture was diluted with distilled water (5.00 mL), and a 250 µL aliquot of the reaction mixture was transferred into a 96 well micro titer plate. The absorbance at 540 nm was measured using a micro plate reader (Spectra Max M5, Molecular Devices, CA, USA). Acarbose was used as the positive control (12.5 µg/mL – 400 µg/mL).

The α-amylase inhibitory activity of each extract is expressed as percent inhibition which was calculated as follows:

Inhibition (%) $=1-\left\{\frac{A_{sample}}{A_{control}}\right\}\times 100$

where, A_sample and A_control are defined as absorbance of the sample and the control respectively. Control was conducted without adding the extract.

Total phenolic content (TPC) determination assay

The Folin-Ciocalteu method (Wang et al., 2012) was used to determine the TPC. The plant extract (3.00 mg extract dry weight) was mixed with 10% Folin-Ciocalteu reagent (5.00 mL) and the mixture was incubated at room temperature (28 ± 2 °C) for five minutes. Sodium carbonate (4.00 mL, 7.5% v/w) was added and the mixture was allowed to stand for one hour at room temperature (28 ± 2 °C). The absorbance was measured at 765 nm using a UV-visible spectrophotometer. A calibration curve for Gallic acid in concentrations from 0.02 mg/mL to 1.00 mg/mL (R² = 0.99) was used to interpolate results of the TPC and the results were expressed as gallic acid equivalents (GAE) mg/g dried extract.

Proanthocyanidine content (PC) determination assay

Vanillin assay was used to determine the PC. The extract (3.00 mg extract dry weight) of Cinnamon quills was mixed with 1% w/v vanillin solution in 7 M H₂SO₄ (4.00 mL) and they were incubated at room temperature (28 ± 2 °C) for 15 min. After the incubation, the absorbance was measured at 500 nm. Catechine was used as the standard. A calibration curve for catechin in concentrations from 0.05 mg/mL to 0.25 mg/mL (R² = 0.99) was used to interpolate the results of the PC and the results were expressed as catechin equivalents (Toda, 2005).

GC-MS analysis

Crude extract (4.00 mg) was dissolved in 1.00 mL of hexane and the samples were filtered using a nylon filter (0.45 µm pore size). A 0.7 µL aliquot of the above was injected in the split less mode into a GC/MS 7890B Gas Chromatograph (Agilent, American) equipped with a 5977B mass spectrometer (Agilent, American). A fused silica capillary Agilent Technology HP-5 (5% phenyl-methyl polysiloxane) column (30 m ×  0.25 mm ×  0.25 µm) was used for the separation. The injector temperature was 250 °C. The initial temperature was kept at 40 °C, for 3 min and the temperature was gradually increased to 220 °C at the rate of 4 °C min⁻¹ and was then held for 2 min at 220 °C. Again the temperature was gradually increased to 230 °C at the rate of 8 °C min⁻¹ and held for 3 min at 230 °C. The Post run was at 235 °C held for 3 min. The Total GC running time was 54.25 min. Helium was used as the carrier gas at a constant flow rate of 1.0 mL min⁻¹ at the split-less mode. EI was used as the ion source, and the ion source temperature was 230 °C. The sector mass analyzer was set to scan from 40 to 650 amu The volatile components of the extracts were identified using mass spectral data, with computer assisted matching with WILEY 275 and National Institute of Standards and Technology (NIST6.0) libraries.

Statistical analysis

All results are expressed as mean ± SD of triplicate assays. Statistical analyses were performed using Microsoft Office Excel (2013) and Graph Pad Prism 7 statistical package (GraphPad Software, USA). Significant differences among the data were analyzed by the SPSS Statistical computer package (IBM^® SPSS^® Statistics Version 23, USA). The results were analyzed using one-way ANOVA test followed by Dunnett/Tukey test for multiple comparisons, paired sample t-test and determination of significance level. Group means were considered to be significantly different at P <  0.05.

Extract yield analysis

The yield of Cinnamon quill extracts ranged from 0.42% ± 0.03% to 2.19% ± 0.25% for SW; 0.39% ± 0.02% to 1.90% ± 0.05% for SG and 0.15% ± 0.00% to 1.26% ±   0.08% for CC (Fig. 1). The results indicated that there was significant difference (p < 0.05) in the yield of the extracts depending on the Cinnamon accessions and the extraction methods. The highest yield was obtained from SW, PWE (2.19% ±  0.25%). Microwave digestion gave the lowest yield in all Cinnamon varieties (0.42% ±  0.03% for SW; 0.39% ±  0.02% for SG and 0.15% ±  0.00% for CC).

α-glucosidase and α-amylase enzyme inhibitory activity

The α-glucosidase and α-amylase enzyme inhibitory activities of cinnamon extracts are expressed in terms of IC₅₀ values, and the lower IC₅₀ is an indicative of a stronger inhibition. SW, PWE exhibited the highest inhibitory activity (P < 0.05) for α -glucosidase and α-amylase (42 ± 8 µg mL⁻¹ and 78 ± 7 µg mL⁻¹, respectively) followed by SW, DWE (116 ± 17 µg mL⁻¹ and 132 ± 11 µg mL⁻¹, respectively). The inhibition of these extracts were comparable with the positive control, Acarbose (173 ± 7 µg mL⁻¹ and 95 ± 4 µg mL⁻¹ respectively) (Table 1).

TPC and PC analysis

Total phenolic content was expressed as mg GAE/g and PC was expressed as mg of catechin equivalent/g. In comparing the TPC and the PC of extracts, the highest TPC and the highest PC were obtained for the DWE (2.24 ±  0.00 mg GAE/g and 40.05 ±  0.10 mg of catechin equivalent/g respectively) for SW. The extracts obtained by SD had the lowest TPC and PC for SW (0.21 ±  0.01 mg GAE/g and 1 ±  0.01 mg of catechin equivalent/g) (Table 2).

GC-MS analysis

(E)- Cinnamaldehyde, was the predominant compound in CC, SW and SG extracts prepared using steam distillation, Infusion and solvent extraction respectively (Table 3). In pressured water extracts of SW and SG; Benzoic acid (1.6%, 15.49%), (E)-Cinnamaldehyde (5.8%, 5.46%), Cinnamyl alcohol (32.44%, 40.08%), and 4-Allyl-2,6-dimethoxyphenol (10.2%, 12.71%) were the major compounds present (Table 3).

Benzoic acid (2.58%, 22.51%), (E)-Cinnamaldehyde (34.6%, 5.58%), Trans-cinnamic acid (16.86%, 4.51%), O-Methoxy-cinnamaldehyde (2.04%, 2.14%), Cinamyl alcohol (21.3%, 42.48%), and 4-Allyl-2,6-dimethoxyphenol (3.24%, 8.79%) were the major compounds present in SG and SW decoction water extracts. Benzoic acid and Cinnamyl alcohol were not detected in CC, PWE and DWE. 4-Allyl-2,6-dimethoxyphenol (1.79%, 1.05%) was seen as a minor constituent in CC extracted using pressured water and as a decoction (Table 3).

Twenty seven chemical components were identified for the first time in SW; PWE. Benzcatechin (1.14%), 1-Methoxy-7-methyl-3,4-dihydrobenzo[c]pyran (1.31%), (+)-(1S,3R,4S)-4(a)-Methyladamantane (2.14%), 1-(2′-hydroxyphenyl)prop-2-en-1-ol (0.71%), 1,3,5-benzenetriol (1.11%), 4-propyl-1,2-Benzenediol (1.615%), 3,4,5-trimethoxyphenol (0.54%), 4-((1E)-3-Hydroxy-1-propenyl)-2-methoxyphenol (1.00%), and Vanillylmandelic acid (0.78%) were present only in PWE of SW. These compounds and 4-Allyl-2,6-dimethoxyphenol were detected for the first time in any Cinnamon accessions (Table 3).