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INTRODUCTION
Essential oils (EO) are complex, odoriferous and naturally occurring compounds characterized by high volatility [1]. Pharmacologically, essential oils have been used as insecticide, antimicrobial, antioxidant, pesticide, deodorants and as aroma-therapeutic agents; therefore, the possibility of exploring them as anti-diabetic agent is important [2].
Diabetes mellitus (DM) is a chronic endocrine disorder that affects the metabolism of carbohydrates. It includes a group of metabolic diseases characterized by hyperglycemia (HG), in which blood sugar levels are elevated either because the pancreas do not produce enough insulin or cells do not respond to production of insulin [3, 4, 5]. Out of about 382 million people living with Diabetes world-wide it is estimated that over 20 million people are living with the disease in sub-Saharan Africa. Nigeria has the highest number of people with diabetes with an estimated 3.9 million people of adult population [6]. An effective therapeutic approach to manage diabetes is by decreasing postprandial hyperglycemia (PPHG). It can be achieved by the regulation and/or inhibition of carbohydrate hydrolyzing enzymes like α-amylase and α-glucosidase which are utilized in the digestion of carbohydrates [7]. α-amylase is involved in the breakdown of long chain carbohydrates while α-glucosidase breaks down starch and disaccharides to glucose. These inhibitors are the potential targets utilized in the development of lead compounds for the treatment of diabetes. α-glucosidase, is a very important enzyme in carbohydrates digestion [8]. It catalyzes the 1,4- α-bonds of the unabsorbed oligo- and disaccharides, and converts them into monosaccharides, which are absorbed in the upper jejunum, resulting in PPHG [9, 10]. α-amylase catalyzes the initial hydrolysis of starch and other carbohydrate polymers into shorter oligosaccharides through cleavage of α-1,4- bonds. The salivary isozyme provides an initial partial cleavage into shorter oligomers [11, 12]. Once these partially digested saccharides reach the gut, they are extensively hydrolyzed by the α-amylase synthesized in the pancreas and excreted in the lumen into simpler oligosaccharides, such as maltose, maltotriose and α-limit dextrins [13]. These sugars are eventually broken down into glucose by α-glucosidases (intestinal brush border), which is in turn absorbed from the intestinal mucosa into the portal blood, by means of the glucose transporter and sodium-glucose co-transporter, leading to postprandial hyperglycaemia [14]. Impaired regulation of PPHG constitutes a common feature in type 2 diabetes mellitus (T2DM), the most prevalent form of diabetes and accounting for about 90 % of all diabetes cases [15, 16]. After a meal, α-amylase synthesized in the pancreas and released in the duodenum, catalyzes the hydrolysis of α-1,4 glycosidic linkages in partially hydrolyzed starch (amylopectin and amylose). From this reaction, intermediate unbranched, such as maltose and maltotriose, and branched (α-limit dextrins) oligosaccharides are formed. α-glucosidase present in the brush border of the intestinal epithelium (enterocytes) is responsible for the final step of carbohydrates digestion, prior to their absorption. This enzyme converts the disaccharides and oligosaccharides into glucose, which is then transported by sodium/glucose co-transporter 1 (SGLT1) from the intestinal lumen to the cytosol of enterocytes. In turn glucose transporter 2 (GLUT2), found in the basolateral membrane of enterocytes, transports glucose from cytosol to blood via facilitated diffusion. The pancreatic α-amylase activity has been targeted for inhibition by means of the so-called starch blockers in order to mitigate PPHG [17]. Acarbose is the most widely prescribed α-amylase and α-glucosidase inhibitor, and in spite of its efficiency in the control of PPHG, the administration of this drug is accompanied by gastrointestinal adverse effects in diabetic patients such as; abdominal distention, flatulence and diarrhea [18]. Thus, the search and development of effective and safer therapeutic agents, able to control glucose levels without deleterious side effects is urgent for the management of type II Diabetes mellitus [19].
Medicinal plants have been extensively applied in the treatment of diverse disease conditions, especially in developing economies where resources, affordability and access to modern treatment is a challenge. Volatile and non-volatile phytochemicals in medicinal plants possess several pharmacological and biological properties which have been the focus of researches targeted at prospection of reliable, affordable and potent drugs [20, 21, 22]. These constituents could be found in plant extracts or essential oils with great activity for different therapeutic applications [23, 24]. Thus, plant based natural inhibitors of α-amylase and α-glucosidase could be developed as phyto-therapeutic agents for the treatment of diabetes involving the decrease in postprandial hyperglycemia by inhibiting conversion of carbohydrate into glucose and then its absorption from the intestine. This inhibition reduces glucose absorption through delayed carbohydrate digestion and extended digestion time [25].
The genus Calophyllum comprises of 180 to 200 species of which C. inophyllum Linn is the most abundant species. It is widespread in tropical areas, which tolerates varied kinds of soil such as coastal sand, clay or even degraded soil [26]. The plant possesses a wide variety of uses ranging from traditional, medicinal and industrial applications; the wood has been used in general construction and boat building, as well as for flooring, furniture, musical instruments, handicrafts, and a variety of other purposes [27]. Several species of this genus are known to be used in folk medicine [28]. The extracted oil from the fruit is used as a remedy for sciatica, shingles, neuritis, rheumatism, ulcers, and skin diseases; while seed oil is reported to have medicinal and healing properties [29]. Decoction from dried leaves is widely used in curing rheumatism, skin infections, cuts and sores [30]. Extracts from leaves and stem bark expressed antihyperglycemic and antihyperlipidemic activities [31], while leaf extract was identified to inhibit oxidative stress [32]. Its fruits are effectively utilized in the treatment of dermatitis bark is locally utilized for treating vaginal disorders after childbirth, the passing of blood, gonorrhea, and internal haemorrhages [33]. The broad spectrum of biological activities expressed by C. inophyllum has been associated with the chemical composition of its different parts. The root is furnished with xanthones such as brasilixanthone, 1,3,5-trihydroxy-2-methoxy-xanthone, caloxanthone A, pyranojacareubin, caloxanthone B and tovopyrifolin [34]. The genus Calophyllum has been reported to be rich in coumarins [35], triterpenoids [36], and flavonoids [37]. Several coumarins isolated from two Calophyllum species were found to inhibit HIV-1 replication and cytophaticity activities. Xanthone derivative obtained from the root bark of C. inophyllum Linn has been identified as antimicrobial and cytotoxic agent [38]. Five bioactive compounds isolated from C. inophyllum Linn leaves namely; mixture of calophyllic and isocalophyllic acids, 3-oxo-friedelin-28-oic acid, canophyllic acid, amentoflavone, and shikimic acid showed dose-dependent lipid-lowering activity in in-vivo experiments [39]. Calophyllolide a complex coumarin from C. inophyllum Linn was reported as an anticoagulant and anti-inflammatory agent [40]. The plant has also been identified as a good anticancer agent [41]. Ojah et al, identified the chemical constituents of ten essential oils extracted from C. inophyllum Linn by GC-MS analysis. The study revealed that the plant is furnished with monoterpenes, sesquiterpenes and their oxygenated analogs [42]. Although some phytochemical constituents from the plant have been reported and volatile chemical constituents in the plant have been characterized in previous studies, no study has been performed on the α-amylase and α-glucosidase inhibitory potential of essential oils from different parts of the plant.
The aim of this study is to evaluate the α-amylase and α-glucosidase inhibitory activities of ten essential oils from C. inophyllum Linn.
MATERIALS AND METHODS
PLANT MATERIAL
Fresh samples of C. inophyllum Linn were collected from the Botanical garden, University of Ibadan, Ibadan, Oyo State, Nigeria. The samples were authenticated in the Herbarium, Department of Botany, University of Ibadan, Nigeria, where voucher samples were deposited with specimen voucher number UIH - 22659. The collection of the samples was done during the daytime. The plant was sorted into ten parts: leaf, stalk, flower, seed, pod, peel, stem wood, stem bark, root wood, and root bark.
EXTRACTION OF ESSENTIAL OILS BY HYDRODISTILLATION
Each separated part (leaf, stalk, flower, seed, pod, peel, stem wood, stem bark, root wood, and root bark) of C. inophyllum Linn was air-dried, pulverized and hydro-distilled for 3 hours in an all-glass Clevenger-type apparatus designed to British Pharmacopeia (BP) specifications. Essential oils were procured in 0.219 to 0.560% yields. Each of the oils had a distinct characteristic pleasant smell. The essential oils were refrigerated until the assay was carried out.
EXTRACTION OF WHEAT ALPHA-AMYLASE
500 g of malted whole wheat flour was added slowly with mild stirring to 1 L of 0.2 % calcium acetate solution at room temperature and continuously stirred for 2 hours on a stirrer. The suspension was then centrifuged at 40 °C at 12000 g for 10 minutes. The resultant clear brown supernatant was stored at 2 °C to 3 °C prior to heat treatment. Since β-amylase interferes with the enzymatic determination of α-amylase, it was inactivated by heating the extract at 70 °C for 15 minutes. α-amylase is resistant to inactivation by this treatment at pH between 6.5 and 8.0. The pH of the extract was first adjusted to 6.6 with cold ammonium hydroxide (4%). Heat treatment was carried out at 85 °C to 90 °C and other at 72 °C to 74 °C using a water bath with continuous stirring. The extract was then cooled to 2 °C to 3 °C until use [43].
DETERMINATION OF ALPHA-AMYLASE INHIBITION ACTIVITY
The assay mixture containing 200 µL of sodium phosphate buffer (0.02 M), 20 µL of enzyme (0.25 mg/mL) and the essential oils over a concentration range 20-100 µg/mL were incubated for 10 minutes at room temperature followed by addition of 200 µL of starch (5 mg/mL) in all test tubes. The reaction was terminated with the addition of 400 µL DNS reagent and placed in boiling water bath for 5 minutes, cooled and diluted with 15 mL of distilled water and absorbance was measured at 540 nm using a Shimadzu UV-2000i double beam spectrophotometer. The control samples were prepared without the essential oils. The inhibition (%) was calculated according to the following formula (Eqn. 1)
Where:
Abs540 (control) is the absorbance of the control at wavelength 540 nm.
Abs540 (EO) is the absorbance of essential oils at wavelength 540 nm.
The IC50 values were determined from plots of percent inhibition versus log inhibitor concentration and were calculated by non-linear regression analysis from the mean inhibitory values. Acarbose was used as the reference α-amylase inhibitor. All tests were performed in triplicate [44].
DETERMINATION OF YEAST ALPHA-GLUCOSIDASE INHIBITION ACTIVITY
p-Nitrophenyl-α-D-glucopyranoside, acarbose, baker’s yeast alpha glucosidase was dissolved in 100 mM phosphate buffer (pH 6.8) and used as the enzyme extract. p-Nitrophenyl-α-D-glucopyranoside was used as the substrate. Essential oils were used in the concentration ranging from 0.02 to 0.1 mg/mL. Different concentrations of plant extracts were mixed with 320 µL of 100 mM phosphate buffer (pH = 6.8) at 30 °C for 5 minutes. 3 mL of 50 mM sodium hydroxide was added to the mixture and the absorbance was read at 410 nm using a Shimadzu UV-2000i double beam spectrophotometer. The control samples were prepared without any essential oil. The inhibition (%) was calculated according to the formula below (Eqn. 2):
bs410 (control) is the absorbance of the control at wavelength 410 nm.
Abs
Abs410 (control) is the absorbance of the control at wavelength 410 nm.
Abs410 (EO) is the absorbance of essential oils at wavelength 410 nm.
The IC50 values were determined from plots of percent inhibition versus log inhibitor concentration and were calculated by non-linear regression analysis from the mean inhibitory values. Acarbose was used as the positive control and all tests were performed in triplicate [45].
RESULTS AND DISCUSSION
Several medicinal plants have been reported to possess anti-diabetic activity and hence, the use of herbal drugs as complementary and alternative therapy to existing medications for the treatment of diabetes mellitus. Development of α-amylase and α-glucosidase inhibitor from natural products such as medicinal plants has been considered as a unique opportunity for a more economic management of diabetes. In recent years the popularity of alternative medicine has increased geometrically for various reasons ranging from potency to affordability [46]. Pterocarpus soyauxii a unique Nigerian medicinal plant has been identified as a potent anti-diabetic agent [47]. Aqueous extract of Salacia oblonga has been also identified as a good α-amylase and α-glucosidase inhibitor [48]. Hence the need to explore more plants with good anti-diabetic activity.
PERCENTAGE YIELD OF ESSENTIAL OILS FROM C. INOPHYLLUM LINN
Essential oils obtained from ten (10) parts of C. inophyllum Linn gave characteristic odours and colours. The oils were procured in 0.219 to 0.506 % yields (Table 1), with the highest yield from the peel, which gave 0.560 %, while the root had the lowest yield (0.219%), which may be due to its high fiber content.
ALPHA-AMYLASE INHIBITION OF C. INOPHYLLUM LINN
The percentage inhibition obtained for the standard anti-diabetic drug acarbose was relatively high for the concentration range used (0.1-0.02 mg/mL) as indicated on Table 2. Maximum percentage inhibition of 95.7 % was obtained for acarbose at 0.1 mg/mL and decreased slightly to 59.65 % at 0.02 mg/mL. The ten oils (Leaf, Leaf-stalk, Flower, Seed, Pod, Peel, Stem wood, Stem bark, Root wood, and Root bark) exhibited concentration-dependent inhibition similar to acarbose the standard anti-diabetic drug used. Percentage inhibition of the standard drug (95.68 %) was in close range with pod essential oil with inhibition efficiency of 80.32% at 0.1 mg/mL (Table 2). A graph of percentage inhibition versus concentration (mg/mL) of essential oils was plotted from which the IC50 values were obtained for each fraction using linear regression analysis in reference to the central standard. An inverse relationship exists between the percentage inhibition efficiency and the IC50 values. The higher the IC50 value the lower the activity of the essential oils and vice versa. The following IC50 values were obtained in the determination of α-amylase inhibition: [(Leaf, 0.043±0.05 mg/mL); (Leaf-stalk, 0.044±0.02 mg/mL); (Flower, 0.045±0.05 mg/mL); (Seed, 0.042±0.03 mg/mL); (Pod, 0.040±0.051 mg/mL); (Peel, 0.047±0.04 mg/mL); (Stem wood 0.047±0.02 mg/mL); (Stem bark, 0.049±0.05 mg/mL); (Root wood, 0,048±0.05 mg/mL) and (Root bark, 0.046±0.04 mg/mL)] compared to the standar anti-diabetic acarbose (0.034±0.02 mg/mL). The standard anti-diabetic drug with the lowest IC50 value of 0.034±0.05 mg/mL exhibited the highest α-amylase inhibition activity followed closely by Pod essential oil (0.040±0.05 mg/mL) as indicated on the bar chart in Figure 1. The least activity was expressed by the highly fibrous stem bark essential ol 0.049±0.01 mg/mL. The high α-amylase inhibition of the pod essential oil in comparison with the standard anti-diabetic drug must be due to the presence of some important bioactive phyto-contituents.
ALPHA-GLUCOSIDASE INHIBITION OF C. INOPHYLLUM LINN
The percentage inhibition obtained for the standard anti-diabetic drug acarbose was relatively high for the concentration range used (0.1-0.02 mg/mL). Optimum percentage inhibition of 96.34% was obtained for acarbose at 0.1 mg/mL and decreased slightly to 60.28 % at 0.02 mg/mL. This trend indicates that the percentage inhibition of the standard used in the study was concentration-dependent. The ten oils (Leaf, Leaf-stalk, Flower, Seed, Pod, Peel, Stem wood, Stem bark, Root wood, and Root bark) exhibited concentration-dependent inhibition similar to acarbose the standard anti-diabetic drug used. Percentage inhibition of the standard was in close range with the pod essential oil with inhibition efficiency of 80.32 % at 0.1 mg/mL as indicated in Table 3. A graph of percentage inhibition versus concentration (mg/mL) of essential oils was plotted from which the IC50 values were obtained for each essential oil using linear regression analysis in reference to the central standard. An inverse relationship exists between the percentage inhibition efficiency and the IC50 values. The higher the IC50 value the lower the activity of the essential oils and vice versa. The following IC50 values were obtained in the determination of α-amylase inhibition: [(Leaf, 0.044±0.02 mg/mL); (Leaf-stalk, 0.043±0.03 mg/mL); (Flower, 0.044±0.04 mg/mL); (Seed, 0.048±0.02 mg/mL); (pod, 0.038±0.04 mg/mL); (Peel, 0.048±0.03 mg/mL); (Stem wood, 0.048±0.04 mg/mL); (Stem bark, 0.048±0.02 mg/mL); (Root wood, 0.047±0.04 mg/mL) and (Root bark, 0.045±0.04 mg/mL)] compared to the standard anti-diabetic drug acarbose (0.032±0.04 mg/mL). The standard anti-diabetic drug with the lowest IC50 value of 0.032±0.04 mg/mL exhibited the highest α-amylase inhibition activity followed closely by the pod essential oil (0.038±0.01 mg/mL) as indicated on Figure 2. The least activity was expressed by the Seed oil (0.048±0.02 mg/mL).
ALPHA-AMYLASE AND GLUCOSIDASE INHIBITION OF C. INOPHYLLUM LINN
The pod essential oils showed peak α-amylase and α-alpha glucosidase activity while essential oils from other parts showed appreciable inhibition activity. The high inhibition efficiency expressed by pod essential oils in both the α-amylase and α-glucosidase inhibition studies must be triggered by the presence of bioactive constituents. Generally, the alpha amylase and alpha glucosidase inhibition of all parts of C. inophyllum Linn were concentration dependent. The high α-amylase and α-glucosidase inhibition of the pod essential oils in comparison with the standard anti-diabetic drug must be due to the presence of some impact bioactive phyto-contituents in the plant. Results obtained for α-amylase and α-glucosidase inhibition are consistent with values reported earlier in literature [49, 50].
CONCLUDING REMARKS
The α-amylase and α-glucosidase inhibitory activities of C. inophyllum Linn revealed that the plant could be an interesting component against postprandial hyperglycemia and other diseases associated with diabetes mellitus. Future researches are necessary to confirm this observational study.
RECOMMENDATIONS
It is highly recommended that bio-assay guided isolation and characterization of non-volatile components from various parts of C. inophyllum Linn should be carried out to identify specific compounds responsible for the activities observed in this study.
In vivo study should be carried out on volatile and non-volatile components of this plant prior to clinical study.
