Isolation and Characterization of Oligomeric Procyanidins Rich PolyphenolFraction of Cinnamomum Zeylanicum Bark and it’s In Vitro Antidiabetic Studies

Introduction

The bark of Cinnamomum zeylanicum Blume, commonly known as dalchini is a common spice in the Indian household. Cinnamon is indigenous to Sri Lanka; and hence, called Ceylon Cinnamon. It is cultivated in Brazil, Mauritius, India, Jamaica, and other countries. The essential oil obtained from the bark has proven to have hypoglycemic1 , anti-hypertensive² , anti-fungal, and antimicrobial properties.^3,4 Cinnamaldehyde (both cis and trans) is the major constituent of the essential oil.⁵ The powdered bark of C. zeylanicum forms a component of several ayurvedic formulations, such as Sitopaladichurna and Chyawanprash.⁶ Other parts of the plant, such as fruit stalks, buds, and root bark are also of pharmacological significance.^7,8 However, recent studies have indicated that the polymeric procyanidins from the bark may have been responsible for anti-diabetic activity.⁹ Therefore, the present study has been aimed at isolating the polyphenol rich fraction of C. zeylanicum bark and in vitro testing for the potential anti-diabetic activity.

Materials and Methods

General

Glucobay (acarbose 25 mg tablets, Bayer Pharmaceutical Pvt Ltd, India), hemoglobin from bovine blood (SigmaAldrich, USA), L-ascorbic acid (Sigma-Aldrich, USA).

Extraction and isolation of procyanidins

The dried bark of C. zeylanicum Blume was acquired and identified by Sami Labs Pvt Ltd, Bangalore, India. The bark of C. zeylanicum Blume (3.5 kg) was pulverized using mechanical grinder. The pulverized bark was extracted thrice with a mixture of acetone, water, and acetic acid (3 × 3.5 L) in the ratio of 70:29.5:0.5. The extract was evaporated completely (206 g) and dissolved in water. The aqueous portion was fractionated with hexane to remove any remaining non-polar components. The aqueous layer was then fractionated with butanol. The butanol layer (polyphenol portion) was evaporated to dryness (175 g) using rotary flask evaporator. This polyphenol fraction was confirmed by high-performance thin layer chromatography (HPTLC) and further analyzed by liquid chromatography–mass spectrometry (LC-MS) to confirm the presence of procyanidin dimers.

HPTLC conditions:

Stationary phase                     : Pre-coated silica gel

                                                 aluminium plate 60 F₂₅₄

                                                 (10 × 10, 20 × 20) HPTLC plates

Chamber saturation time         : 20 minutes

Slit width                                  : 6 mm × 0.45 mm

Temperature                             : 25^o C ± 2^o C

Humidity                                   : 60% ± 5%

Thickness                                  : 200 μm

Syringe                                      : 100 μL Hamilton syringe

Development chamber             : CAMAG twin trough chamber

Detection                                  : UV-VISIBLE

                                                  spectrophotometer

Marker compound                    : Epicatechin

Application volume                   : 10 μL

Mobile phase                             : Toluene:Ethyl acetate:Acetic acid:Water (12:6:6:0.5)

Detection wavelength                : 280 nm

Mobile phase                             : Toluene:Ethyl acetate (93:7)

Detection wavelength                : 365 nm

Sample (10 mg/mL of polyphenolic fraction) and standards (1 mg/mL of epicatachin) were prepared in methanol.

In vitro anti-diabetic activity

1. Non-enzymatic hemoglobin glycosylation¹¹

Glucose (2%), hemoglobin (0.06%), and gentamycin (0.02%) solutions were prepared in phosphate buffer saline (0.01 M, pH 7.4) and 1 mL each of the above solutions were mixed. Different concentrations of the extract were dissolved in dimethyl sulfoxide (DMSO). To this 1 mL of each concentration was added to the above glucose-hemoglobin mixture and incubated in dark for 72 hours. The degree of glycosylation was measured at 520 nm. Gallic acid was used as standard drug.¹⁰. Percentage glycosylation was calculated using the formula:

           % glycosylation = (T-C)/C × 100

          where, T is absorbance of test; C is absorbance of control

2. α-Amylase inhibition assay¹²

One mL of substrate (potato starch 1%w/v) was added to 1 mL of various concentrations of polyphenol extract. To this, 1 mL of 3% w/v of α-amylase solution and 2 mL of acetate buffer (0.1 M, pH 7.2) was added and incubated at room temperature for half an hour. Starch solution, enzyme solution, and drug solutions were prepared in acetate buffer. After incubation, 0.1 mL iodine-potassium iodide indicator was added to the above mixture. The absorbance of this complex was measured at 565 nm.

Results

In vitro anti-diabetic activity of the extract showed good activity in non-enzymatic hemoglobin glycosylation with a half minimal inhibitory concentration (IC₅₀) value of 102.63 µg/mL. In α-amylase inhibition assay, the polyphenol extract showed an IC₅₀ value of 540.95 µg/ mL (Table 1).

The HPTLC chromatogram showed presence of Epicatachin (data presented in Table 2, Figure 1) which was further confirmed by LCMS data (Figure 2). The initial polyphenol extract (Figure 1) showed the presence of epicatechin (288), procyanidin dimer (576), and other polymers.

Discussion

The present study was aimed at isolating the polyphenol rich fraction of C. zeylanicum bark and in vitro testing for the potential anti-diabetic activity. Recent studies have demonstrated the presence of OPCs in C. zeylanicum bark.¹⁰ OPCs are high molecular weight polymers prone to breakdown resulting loss in activity. Therefore, moderate acidic medium was created by the addition of acetic acid to prevent any damage to the procyanidins during extraction.¹³ A 30% aqueous acetone was therefore, used for extracting polyphenol.¹⁴ The LCMS data of the polyphenol fraction showed the presence of catechin/epicatechin (288), dimer (576), and trimer (864).

Anti-diabetic activity of polyphenol extract was investigated by estimating prevention of the nonenzymatic glycosylation of hemoglobin. In diabetes mellitus, advanced glycosylation end products (AGEs) are thought to be one of the major causes for oxidative stress.¹⁵ Oxidative stress is a potential risk factor for diabetes which may aggravate the condition.¹⁶ Prevention of glycosylation prevents the formation of AGEs; thus, the assay is a good estimate of anti-diabetic activity. Our study demonstrates promising activity of polyphenol extract in preventing the binding of glucose to surface proteins of erythrocytes mostly due to the antioxidant property of polyphenols. OPCs are well known antioxidants as the activity could be due to these anthocyanidins. α-amylase hydrolyses α-bonds of large α-linked polysaccharides, such as glycogen and starch to yield glucose and maltose. This assay is based on the formation of starch-iodine complex due to inhibition of α-amylase. The polyphenol extract from the bark showed a poor result in α-amylase inhibition assay, suggesting that C. zeylanicum bark might not be effective in breaking down starch to minimized glucose availability. The results infer that the polyphenol rich extract may have minimal effect in maintaining postprandial glucose concentration.