Abstract: Terminalia arjuna, Terminalia ballerica, Terminalia chebula, Terminalia catappa, Terminalia kaerbachii and Terminalia microcarpa leaves were tested for their α-glucosidase inhibitory activity in vitro. The α-glucosidase activity was determined by measuring the p-nitrophenol release from pNPG at 400 nm. Terminalia kaerbachii has the highest α-glucosidase inhibitor activity with IC50 value of 0.27±0.17 μg mL¯1 and is a promising antidiabetic herbal medicine candidate. However, most of the Terminalia species are also potential as antidiabetic medicine candidates as the IC50 values are approximately 5 μg mL-1, which is near the IC50 value of 1-deoxynojirimycin, the reference compound, except for Terminalia microcarpa, which has IC50 value of 25.15±0.04 μg mL-1 (above 21 μg mL-1). From the phytochemical screening, Terminalia kaerbacchi contains alkaloids, flavonoids and cathechic tannins, but does not contain saponin, quinon, steroid/terpenoids and gallic tannins. It is estimated that there is a correlation between α-glucosidase inhibitory activity and its phytochemical content.
INTRODUCTION
Diabetes is a common metabolic disease characterized by abnormally high plasma glucose levels, leading to major complications, such as diabetic neuropathy, retinopathy and cardiovascular diseases (Gao et al., 2008). More than 171 million people worldwide are currently believed to be afflicted with type 2 diabetes and it is estimated that the number will rise to 366 million by 2030 (Shinde et al., 2008). The key enzyme which catalyses the final step in the digestive process of carbohydrates in mammalian is α-glucosidase (α-D-glucoside glucohydrolase, EC 3.2.1.20), which is located in the brush-border surface membrane of intestinal cells. Hence, α-glucosidase inhibitors can retard the liberation of D-glucose of oligosaccharides and disaccharides from dietary complex carbohydrates and delay glucose absorption, resulting in reduced postprandial plasma glucose levels and suppressed postprandial hyperglycaemia (Gao et al., 2008). Consequently, α-glucosidase inhibitors, such as acarbose (Balfour and McTavish, 1993) and miglitol (Pogano et al., 1995) have been approved for clinical use in the management of type 2 diabetes, as well as the treatment of obesity.
Therefore, the search for effective and safe α-glucosidase inhibitors from natural materials, in order to develop a physiological functional food or lead compounds for antidiabetic agents is necessary.
Terminalia is a flowering plant in family of Combretaceae. Example of traditional use of Terminalia species is mentioned as follows, Terminalia chebula is used in treatment of fevers, cough, asthma, urinary diseases, piles and worms and is also useful in treating chronic diarrhoea and dysentery, flatulence, vomiting, colic and enlarged spleen and liver (Rao and Nammi, 2006). Terminalia chebula extracts had different levels of antioxidant activity for anti-LPO, anti-superoxide radical formation and free radical scavenging activities (Rao and Nammi, 2006). Terminalia arjuna is a large tree distributed throughout India and its bark is used as a cardioprotective agent in hypertension and ischaemic heart diseases. The bark powder is reported to exert hypocholesterolaemic and antioxidant effect in humans (Masoko and Eloff, 2007).
Therefore, in order to search a new plant-based medicine to inhibit α-glucosidase in diabetes type II and to find the correlation between the phytochemical compound and its pharmacological activity, this experiment was conducted.
MATERIALS AND METHODS
Materials: Terminalia arjuna, Terminalia ballerica, Terminalia chebula, Terminalia catappa, Terminalia kaerbachii and Terminalia microcarpa leaves were collected from Bogor Botanical Garden Indonesia, in May 2008; ethanol, 1-deoxynojirimycin (Sigma, D9305), α-glucosidase (Sigma, G0660-750UN), p-nitrophenyl glucopyranoside [pNPG] (Sigma, N1377) Na2CO3 (Sigma, S7795), phosphate buffer (Sigma, P5244).
Equipment: Plant grinding machine, digital scale (Mettler, Toledo AG204), maceration equipments, electric stove, volume pippettes, measuring pippettes, rotavapor (Buchi Rotavapor R-124 Buchi Waterbath B-480), water pump, distillation equipments, glasswares, spektrophotometer UV (Hewlett Packard 8452A).
Extraction methods: One hundred grams of the dried leaves were macerated using 3x500 mL ethanol 96% for 3x24 h. The filtrate obtained was concentrated under vacuum on a rotary evaporator at 40°C and then stored at 4°C for further use.
Inhibition assay for α-glucosidase activity: The inhibition assay for α-glucosidase activity was conducted in June 2008 at Research Center for Chemistry, Indonesian Institute of Sciences, Serpong. α-glucosidase (0.075 unit) was premixed with the extracts at various concentrations (0.01 -200 μg mL-1). About 3 mM p-nitrophenyl glucopyranoside (pNPG) as a substrate in phosphate buffer was added to the mixture to start the reaction. The reaction was incubated at 37°C for 30 min and stopped by adding 2 mL of 0.1 M Na2CO3. The α-glucosidase activity was determined by measuring the p-nitrophenol release from pNPG at 400 nm. The IC50 value was defined as the concentration of α-glucosidase inhibitor to inhibit 50% of its activity under the assay conditions (Kim et al., 2004).
Phytochemical screening: The phytochemical screening of the Terminalia species was conducted according to Materia Medika Indonesia procedures (Ditjen and Depkes, 1989).
RESULTS AND DISCUSSION
Terminalia species was considered as potential α-glucosidase inhibitor as the IC50 values are below or approximately 5 μg mL-1 which was comparable to 1-deoxynojirimycin as the reference compound. The result of the α-glucosidase inhibitory activity of the Terminalia species is as seen in Table 1.
In order to prove the estimation that there is a correlation between phytochemical content of the Terminalia species and its antidiabetic activity, the phytochemical screening of Terminalia was conducted. All of the Terminalia species tested contains flavonoids and mostly contains steroids/terpenoids except Terminalia kaerbachii. The result of the phytochemical screening of Terminalia species is are shown in Table 2.
Based on the experiment result, Terminalia kaerbachii is the most active α-glucosidase inhibitor, followed by Terminalia catappa, Terminalia arjuna, Terminalia chebula and Terminalia ballerica. On the other hand, Terminalia microcarpa does not have any α-glucosidase inhibitory effect as the IC50 value is above 21 μg mL-1.
Miglitol is one of α-glucosidase inhibitors used to treat diabetes type 2. The chemical compound of miglitol is 1-deoxynojirimycin. In this in vitro experiment, 1-deoxynojirimycin is used as a reference compound as it is the most suitable reference compound. Shinde (2008) demonstrated that in in vitro α-glucosidase inhibitor activity assay using Baker’s yeast and Bacillus stearothermophilus, the reference compound 1-deoxynojirimycin had the IC50 value of 83.4±2.1 and 0.175±0.003 μg mL-1, respectively. While acarbose showed no inhibition of α-glucosidase in Baker’s yeast and B. stearothermophilus, but it has the IC50 of 210±1.2 and 233±4.0 μg mL-1, respectively for sucrose and maltase in rat intestinal enzymes. It is concluded that acarbose is appropriate as the reference compound for in vivo experiment and 1-deoxynojirimycin for in vitro experiment, due to the difference of the enzymes involved and the solvability of acarbose and 1-deoxynojirimycin in the solvent.
| Table 1: | Inhibitory concentration of Terminalia species and 1-deoxynojirimycin as reference compound |
| Table 2: | Phytochemical screening of Terminalia leaves |
| +: Present, -: Absent | |
Based on the result tabulated in Table 1, Terminalia kaerbacchii is the most active α-glucosidase inhibitor and it is estimated due to its chemical contents is different from other Terminalia species. This is an important information as Terminalia kaerbacchii has not been evaluated for its α-glucosidase inhibitory activity previously.
In order to prove the estimation that there is a correlation between phytochemical content of the Terminalia species and its antidiabetic activity, the phytochemical screening of Terminalia was conducted.
Gao et al. (2008) reported the isolation of maltase inhibitory principles, chebulanin, chebulagic acid and chebulinic acid from the fruits of Terminalia chebula. As seen in Table 1, Terminalia chebula contains tannins, hence, the finding supports Gao’s idea that the antidiabetic activity of Terminalia chebula is due to the maltase inhibitory principles.
Bajpai et al. (2005) stated that the leaves, bark and fruits of Terminalia arjuna, Terminalia bellerica, Terminalia chebula and Terminalia muelleri had high total phenolic contents (72.0-167.2 mg g-1) and high antioxidant activity (69.6-90.6%). Fruits of T. bellerica and T. chebula were a rich source of gallic acid, meanwhile bark of T. arjuna, leaves and fruits of T. bellerica and bark, leaves and fruits of T. muelleri were a rich source of ellagic acid.
Terminalia bellerica contains triterpenoids and glucosides. It has been used for the treatment of various ailments (Chadha, 1976) and has been reported to contain β-sitosterol, gallic acid, ellagic acid, ethyl gallate, galloyl glucose, mannitol, glucose, galactose, fructose and rhamnose (Nandy et al., 1989). It also contains triterpene, belleric acid and its glucoside, bellericoside and arjungenin and its glucoside (Nandy et al., 1989). The extracts of Terminalia bellerica Roxb fruits contains phenols, flavonoids, glycosides, saponin, tannin. While, the fruits of Terminalia chebula Retz. which is also with common name of Harir, contains phenols, glycosides and saponin (Farrukh et al., 2006).
Sabu and Kuttan (2002) evaluated the combination of Terminalia chebula, Terminalia belerica, Emblica officinalis, known as Triphala for their antidiabetic activity and their relation with their antioxidant activity. T. belerica, was found to be most active plant to reduce serum glucose level followed by E. officinalis and T. chebula. Triphala which is a combination of all the three produced a significant action in reducing the alloxan induced diabetic. The result is slightly different with the result in our experiment, where in our experiment, T. chebula has a higher α-glucosidase inhibitory activity compared to T. belerica. This might be due to the location where the plants grew up and the experiment by Sabu and Kuttan (2002) was conducted in vivo, while in present experiment in vitro.
In another experiment by Rao and Nammi (2006), the chloroform extract of the seeds of T. chebula were evaluated for their antidiabetic properties. They are basing their experiment on the facts that Terminalia chebula fruits and whole powder of dried ripe fruits are known for their antidiabetic properties. The evaluation data also confirmed the traditional indications. The seed extract of T. chebula indicated a potent action in short term study and a prolonged duration of antidiabetic action in long term study and this could be due to multiple sites of action possessed by the active principles of T. chebula. The study also revealed that T. chebula is more effectively inhibited the incidence of diabetic nephropathy (Rao and Nammi, 2006).
The study by Ahmed et al. (2005), indicates that Terminalia catappa leaves extracts have antidiabetic activity. Aqueous and cold extracts of Terminalia catappa exhibited significant anti hyperglycemic activities in alloxan-induced hyperglycemic rats without significant change in body weight. The number of functionally intact β-cells in the islet organ is of decisive importance the development course and outcome of diabetes mellitus. The renewal of β-cells in diabetes has been studied in several animal models. The total β-cell mass reflects the balance between the renewal and loss of these cells. It was also suggested that regeneration of islet β-cells following destruction by alloxan may be the primary cause of the recovery of alloxan-injected guinea pigs from the effects of the drug and Vinca rosea extract have also shown to act by β-cell re-generation (Ahmed et al., 2005).
The damage to pancreas in alloxan-treated diabetic control and regeneration of β-cells by glibenclamide was observed. A comparable regeneration was also shown by aqueous and cold extracts of Terminalia catappa. This effect may be due to β-carotene, which was reported to be constituents of Terminalia catappa (Duke, 1992). Photomicrographical data in the studies confirms healing of pancreas by Terminalia catappa leaves extracts, as a plausible mechanism of their anti diabetic activity (Ahmed et al., 2005).
Aqueous and cold extract of Terminalia catappa leaves exhibited significant anti hyperglycemic activities in alloxan-induced diabetic rats. These extracts showed improvement in parameters like body weight and lipid profile as well as regeneration of β-cells of pancreas and so might be of value in diabetes treatment (Ahmed et al., 2005).
As mentioned earlier, there is a correlation between phytochemical content and antidiabetic properties. In order to have thorough information of the compound responsible for the antidiabetic properties, isolation of the active phytochemical compound of the Terminalia kaerbachii is being conducted, which will be followed by the antidiabetic in vivo study.
CONCLUSION
Terminalia kaerbacchii is the most active α-glucosidase inhibitor compared to other Terminalia species, which are also known to have antidiabetic properties.