International Journal of Drug Development and Research

Key words

phenolic compound, medicinal plant, antioxidant, antidiabetic, DPPH radical, α -amylase.

Introduction

Since prehistoric era herbs and medicinal plants have been used as a traditional healer for nearly all medicinal therapy until the synthetic drugs were developed in the nineteenth century [1,2]. Even today plant based medicine continues to play an important role in health care system. It has been estimated by World Health Organization [3] that about 80% of world’s population of the developing countries relies mainly on traditional medicines (mostly plant derived) for primary health care and the remaining 20% are also dependent on plant products.

Medicinal and herbal plants are a potential source of natural antioxidant compound such as flavonoids, anthocyanins, tannins, dietary glutathionine, vitamins and endogenous metabolites which can interfere with the production of free radicals and inactivate them by quenching singlet and triplet oxygen, decompose hydrogen peroxide and inhibit enzymes. These free radicals are implicated in etiology of many diseases including cancer, atherosclerosis and neurodegenerative diseases. Moreover because of the dietary habits, that is devoid of nutritionally rich and functionally healthy plant foods, leads to the emergence of type-2 diabetes [4]. Medicinal and herbal plants are capable of treating diabetes by retarding the absorption of glucose through the inhibition of carbohydrate hydrolyzing enzymes 6-amylase and 6-glucosidase in the digestive tract.

Swertia chirata (Family: Gentianaceae) is a prized herb which is commonly available in India, Nepal and China. The plant is found at an altitude of 1200– 3000 and available throughout the year. It is generally consumed by the older people and/or people with type 2 diabetes mellitus as it is useful for lowering the blood glucose level. Besides the plant also found to posses anti-viral [5], anti-inflammatory [6], anthelmintic [7], and anticarcinogenic [8] activities. Early studies also documented the presence of flavonoids, xanthones, terpenoids, iridoid and secoiridoid glycosides in the Swertia chirata plant [9].

It has been widely accepted that efficacy of the extraction yield and the antioxidant properties largely depend on nature of the solvents as different antioxidant compounds and phenolics respond differently towards the solvent [10]. Therefore several solvent systems combined with different procedures have been used for the extraction of plant phenolics and antioxidant compounds considering their chemistry and uneven distribution in plant matrix. For example, soluble phenolics are found in outer tissues (epidermal and sub-epidermal layers) in fruits and grains than in the inner tissues (mesocarp and pulp) [11]. In general, aqueous mixtures of ethanol, methanol, and acetone are commonly employed polar solvents for the extraction of plant phenolics while choloform, ethyl acetate, hexane are used as a non-polar solvent.

Based on the above rationale, the study was undertaken to evaluate the efficacy of the four different solvents for the extraction of phenolic contents and antioxidant activities of S. chirata. In addition in vitro alpha amylase activity was performed to ascertain their therapeutic use as an antidiabetic plant.

Materials and methods

Chemicals and reagents

Sodium carbonate, disodium hydrogen phosphate, potassium dihydrogen phosphate, Folin–Ciocalteu’s phenol reagent, sodium salicylate, trichloroacetic acid, methanol, ethanol and iron (II) sulfate-7- hydrate were purchased from Merck Chemicals. 1,1- diphenyl-2-picrylhydrazyl (DPPH) was purchased from Sigma Aldrich. All the reagents are analytical and HPLC grade

Extraction and preparation of sample

The plant sample was collected from Chawk bazar, Dhaka in November 2011. The plant sample was identified by the taxonomist of the National Herbarium of Bangladesh, Mirpur, Dhaka, Bangladesh (accession number - 34531). The stem of the plant was cleaned in running tap water followed by distilled water and dried. It was then ground to 80 mesh and extracted for one hour with 25 mL of distilled water and different concentrations (hundred percent, seventy five percent and fifty percent) of methanol, ethanol and acetone. The extracts were filtered through Whatman No. 1 filter paper and used directly for the estimation of total phenolic compound and assessment of antioxidant and 6- amylase inhibition activities through various chemical assays.

Total phenolic content

The total phenolic content (TPC) of the extracts was determined according to the Folin–Ciocalteu method [12]. The total phenolic content was expressed as gallic acid equivalents in mg/100g of sample dried weight.

Ferric reducing antioxidant power

The ferric reducing antioxidant power (FRAP) assay reported by Chu et al.[13] was adopted. FRAP of extracts was expressed mg ascorbic acid equivalent/100g of sample dried weight.

Total antioxidant capacity

The total antioxidant capacity (TAC) of extracts was evaluated by the method of Prieto et al. [14]. TAC of extracts was expressed mg ascorbic acid equivalent/100g of sample dried weight.

DPPH radical scavenging activity

The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity reported by Ranilla et al.[15] was adopted. Radical-scavenging ability was calculated as IC50.

α-amylase inhibition assay

The 6-amylase inhibitory activity was determined according to the method by Ranilla et al.[15]. % Inhibition = A540 (control) - A540 (sample) / A540 (control)*100

Statistical analysis

Three replicates of each sample were used for statistical analysis and the values were reported as mean ± SD. Data were also subjected to the analysis of variance and mean values were compared by Duncan’s post-hoc multiple comparison tests. Differences at P<0.05 were considered to be significant.

Results

Total phenolic content

Table shows the TPC of S. chirata with different solvent extractions. There was a wide range of phenolic concentration in the S. chirata extractions. According to the results obtained, it was evident that water had the highest level of phenolic content (221 ± 12 mg GAE/100g) while ethanol 100% (20 ± 2 mg GAE/100g) was the least effective solvent for phenol extraction. Fifty percent fractions of acetone, ethanol and methanol also showed high level of phenolic content with the value of 206 ± 12, 202 ± 2, and 189 ± 18 mg GAE/100g respectively. No significant variation (P<0.05) was found among the 75% fractions of acetone, methanol and ethanol where the values were 108 ± 10, 113 ± 5, and 100 ± 3 mg GAE/100g respectively. TPC dropped further when the solvent concentration reaches to 100%. Acetone, ethanol and methanol in 100% fractions showed the value of 23 ± 3, 60 ± 1 and 20 ± 2 mg GAE/100g respectively.

Ferric reducing antioxidant power

As shown in the Table there were large variations in ferric reducing antioxidant power among the extraction methods indicated that extraction solvent influenced significantly (p < 0.05) S. chirata reducing power evaluation. Similar as observed in TPC, water extraction showed the highest reducing power of 162 ± 4 mg AAE/100g. No significant (P<0.05) variation was found among the 50% fractions of ethanol (142 ± 13 mg AAE/100g), acetone (144 ± 6 mg AAE/100g) and methanol (146 ± 16 mgAAE/100g). Similar results have been found between acetone 100% (18 ± 4 mg AAE/100g) and ethanol 100% (22 ± 4 mg AAE/100g) while methanol 100% displayed significantly (P<0.05) higher reducing power (55 ± 9 mg AAE/100g) than the 100% fractions of ethanol and acetone. Overall the strongest antioxidant properties when measured with the FRAP assay had water > methanol 50% K acetone 50% K ethanol 50% > methanol 75% K ethanol 75% K acetone 75% > methanol 100% > ethanol 100% K acetone 100%.

Total antioxidant capacity

The extracts were arranged from the lowest to the highest activity which ranged from 139 ± 17 mg AAE/100g (acetone 100%) to 707 ± 71 mg AAE/100g (ethanol 50%) (Table). Besides water (640 ± 38 mg AAE/100g), acetone 50% (625 ± 5 mg AAE/100g) and methanol 50% (592 ± 45 mg AAE/100g) also displayed very high antioxidant capacity. Based on the level of significance the extracts can be divided in five groups where acetone 100% and ethanol 100% were in the very low group; methanol 100%, acetone 75% and methanol 75% were in the low group; ethanol 75% was solely in the intermediate group; water, acetone 50% and methanol 50% comprised high group and ethanol 50% fell in the very high group.

DPPH radical scavenging activity

Fig 1 shows the DPPH radical scavenging activities of the S. chirata at various concentrations in different extracts. The results clearly indicated dose dependent radical scavenging activities of the extracts. The scavenging activity of acetone, ethanol, methanol and water extracts on DPPH radicals increased from 3 to 15 mg/mL. From the results it was evident that acetone 50% showed the highest radical scavenging activities (%) (61.80 ± 0.43), while ethanol 100% displayed the lowest (16.66 ± 1.02) at 15 mg/mL concentrations. Ethanol 50% and methanol 50% also showed considerable scavenging activities of 55.17 ± 1.90 and 57.14 ± 0.29 respectively at the same concentration

α-amylase inhibitory activity

In this study in vitro 6-amylase inhibition activity was determined in three different concentrations-7, 14 and 21 mg/mL. S. chirata extracted in water, ethanol 50% and methanol 50% showed diminished 6-amylase inhibition activity in a sharp contrast to the results of TPC, FRAP, TAC and DPPH radical scavenging activity (Fig 2). However acetone 50% showed 6-amylase inhibition at 7 and 14 mg/mL while at 21 mg/mL its result was comparable to water, methanol 50% and ethanol 50%. Surprisingly in acetone 50%, inhibition activity decreased with the increase of the concentration of the extract. However besides these three extracts, all other extracts showed dose dependent 6-amylase inhibition activity (%) ranged between 1.2 ± 0.72 (methanol 100%) to 10.16 ± 1.04 (acetone 50%) at 7 mg/mL, 1.66 ± 0.47 (methanol 75%) to 9.66 ± 1.52 (acetone 100%) at 14 mg/mL, 7.36 ± 0.55 (ethanol 75%) to 18.66 ± 2.08 (acetone 100%) at 21 mg/mL.

DISCUSSION

Total phenolic content

The total phenolic content was determined by the Folin-Ceucalteau procedure which is convenient, simple, and reproducible. The mechanism behind the Folin-Ceucalteau procedure is involved the reduction of the molybdenum component in the phosphotungstic- phosphomolybdic complex reagent in the presence of the antioxidant [16].

Phenolic components are potential antioxidants that can donate hydrogen to free radicals and thus break the chain reaction of lipid oxidation at the first initiation step. Phenolic compounds by their hydroxyl groups scavenge radicals such as singlet oxygen, superoxide and hydroxyl radicals [16]. From the result it was evident that TPC increased with the addition of water to the solvent which was in accordance with the earlier study by Wijekoon et al. [17]. However they found water to be the least effective solvent for the extraction of phenolic content. Our results are comparable with the earlier studies by Zhou et al. [18] and Turkmen et al. [19] wherein acetone 50% was reported to be the most effective solvent. These results suggested that the extractability of phenolic compounds is influenced by the polarity and viscosity of the solvents used [19, 20].

Ferric reducing antioxidant power

As a convenient and rapid screening method for measuring the antioxidant potential, the reducing power of each extract of S. chirata, which has been widely used in Bangladeshi traditional medicine, was assessed. FRAP measures the ability of the antioxidant to donate electron to reduce Fe3+ to Fe2+. The presence of reductants (antioxidants) in the tested sample is monitored by the formation of Perl’s Prussian blue color which is measured at 700 nm wavelength [16].

In general solvent extracts of the tested plant materials with greater TPC is concomitant with their reducing power [21].The reducing power of this plant could be due to the presence of flavonoids and phenolic acids that can capable of reducing ferric ion (Fe3+) to ferrous ion (Fe2+). These findings are also in agreement with Sulaiman et al. [22] where they found significant variation in the FRAP of different vegetables extracted with 70% fractions of acetone, methanol and ethanol. These observations suggested that both solvent polarity and phenolic compounds with diverse structure greatly influence the determination of antioxidant activity.

Total antioxidant capacity

The phosphomolybdenum method based on the reduction of Mo (VI) to Mo (V) by the antioxidant compound and the formation of green phosphate/Mo(V) complex with a maximum absorbance at 695 nm. Being simple, accurate and rapid it was decided to extend its application in this study. There are numerous studies concerning the antioxidant properties of medicinal plants used this method.

Several authors considered 9.2 mg AAE/g as high antioxidant capacity. However the antioxidant capacity of the S. chirata found herein was not surprising because they are found in very high altitude and reported to have many compounds with antioxidant ability [23]. In previous study [24] high yield variety of rice showed comparatively lower antioxidant capacity than S. chirata.

Phosphomolybdenum assay is a quantitative method to evaluate water-soluble and fat-soluble antioxidant capacity (total antioxidant capacity) and from the results it is evident that all the extracts had electrondonating capacity and thus they may act as radical chain terminators, transforming reactive free radical species into more stable non-reactive products [25].

DPPH radical scavenging activity

DPPH is a stable free radical which is scavenged by proton donating substrate such as antioxidant and the absorbance is reduced. The decrease in absorbance is a measurement of the radical scavenging. Therefore DPPH is widely used as a measurement of antioxidant capacity of the natural compounds [26].

Interestingly DPPH radical scavenging activities of the water extracts did not correlate with the corresponding water extracts of TPC, and FRAP where water extracts showed the highest phenolic content as well as antioxidant properties. The extraction of phenolic compounds from a sample is directly related to the compatibility of the compounds with the solvents [27]. Based on these results it is assumed that S. chirata contains diverse phenolic compounds with different polarity. Previous studies by Thoo et al. [28] and Durling et al. [29] showed that extraction of phenolic compounds maximized at low concentrations of ethanol which contain higher proportions of hydrophilic compounds. In this study, scavenging activities of S. chirata of different extracts increased considerably when the concentrations of the solvents decreased gradually. Kim et al.[30] also found similar phenomenon in mulberry leaves. Thus it is revealed that solvents with different polarity significantly alter the DPPH radical scavenging activity.

-amylase inhibitory activity

Inhibition of enzyme like 6-amylase which is involved in the hydrolysis of carbohydrates has been exploited as the therapeutic approach for controlling postprandial hyperglycemia. 6-amylase catalyses the breakdown of 6-1,4-glucosidic linkages of starch, glycogen and oligosaccharides into simpler monosaccharides which is readily available for the intestinal absorption. Therefore their inhibition is considered to be effective to control diabetes by suppressing the absorption of glucose composed from starch [31].

Our results are in accordance with Ranilla et al. [15] where they studied 6-amylase inhibition of commonly used medicinal plants in hyperglycemia and hypertension and found diminished activity in some of the plant extracts and also with those of previous studies [32-34] which showed that plant derived phytochemicals were capable of lowering 6- amylase inhibitory activity and these potential inhibitors would likely to offer therapeutic approaches to the treatment of postprandial hyperglycemia. These results suggested that the extracts might rich with flavonoids and other phenolic acids and therefore have potential to contribute to the management of type 2 diabetes.

CONCLUSION

In conclusion the present study clearly demonstrated that extraction solvent greatly affected the extraction of phenolic compound as well as the antioxidant activity evaluation measured by FRAP, TAC and against DPPH radical. Solvent extraction also found to have influence on 6-amylase inhibition activity. Water extracts showed the highest phenolic content as well as the reducing power while ethanol 50% showed the highest total antioxidant capacity and acetone 50% displayed stronger DPPH radical scavenging activity compared to the other extracts. In 6-amylase inhibition activity water and 50% fractions of methanol and ethanol did not show any activity but 100% fractions of methanol, ethanol and acetone showed some activity. Therefore it is very difficult to pick one solvent for the extraction of phenolic compound and antioxidant activities. Hence further research is warranted to explore the individual or major phenolic acids and bioactive compounds in the S. chirata and their contribution to health.

Declaration of interest

The authors declare no conflict of interest

Tables at a glance

Table 1

Figures at a glance

Figure 1 Figure 2

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