Abstract
In the current study, we investigated the effect of ethanol extract of Andrographis paniculata. (Burm.f.) Nees (Acanthaceae) (AP) on α.-glucosidase (EC 3.2.1.20) inhibition in both normal and streptozotocin-induced diabetic rats. Oral carbohydrate tolerance tests were performed in 18-h fasted rats with starch (3 g/kg), sucrose (4 g/kg), and glucose (2 g/kg) separately, in both normal and diabetic rats, 10 min after administration of 250 (D1), 500 (D2), 1000 (D3) mg/kg ethanol extract of AP, vehicle (control), and acarbose (Acar) 10 mg/kg, respectively. Blood samples were analyzed for blood glucose at 0, 30, 60, and 120 min after respective treatments and the peak blood glucose (PBG) and area under the curve (AUC) determined. Our results demonstrate that 500 mg, 1000 mg/kg ethanol extract of AP reduces and prolongs the PBG concentration, simultaneously decreasing AUC after starch and sucrose loading in normal and diabetic rats. Similarly, acarbose also reduced sucrose and starch induced blood glucose excursions, whereas it had no peak blood glucose suppressive effect after exogenous glucose load in both normal and streptozotocin-induced diabetic rats. The results suggest the possibility that the ethanol extract of AP may have PBG suppressive effect, post–carbohydrate challenge as evidenced by reduced PBG and AUC, which can be used effectively as a safer alternative to control postprandial hyperglycemia (PPH), especially in diabetic patients and borderline patients not properly controlled on diet alone.
Introduction
Plant extracts have long been used for the ethnomedical treatment of diabetes in various systems of medicine and are currently accepted as an alternative for diabetic therapy. However, for many plant extracts, there is no clear understanding of the mechanism of action. Though some preliminary screening studies on α.-glucosidase inhibitors from various plant extracts have been reported, the in vitro. α.-glucosidase inhibitory activity (Shen et al., Citation2002) may not always correlate with the in vivo. α.-glucosidase inhibitory (Ye et al., Citation2002) activity. So, it is necessary to investigate the in vivo. action after oral administration on whole live animals, which is an important step in screening plant extracts for physiological and pharmacological effects.
Andrographis paniculata. (Burm.f.) Nees (Acanthaceae) is found throughout Southeast Asia and known locally as “hempedu bumi” and “akar cerita” in Malaysia. It is a reputed herbal remedy, especially in Malaysia, for diabetes (Ahmad & Asmawi, Citation1993) and hypertension. Andrographis paniculata. (AP) finds mention in Ayurveda and is an important ingredient in about 26 Ayurvedic formulas (Tomar et al., Citation1982), several Siddha drugs, and is also official in the Indian Pharmacopoeia. (Warrier et al., Citation1980). This plant is an annual herb and has been commonly known as “king of bitter” and is reported to have antimalarial (Misra et al., Citation1992; Rahman & Furuta, Citation1999), anti-inflammatory (Shen & Chen, Citation2002; Xia et al., Citation2004), antineoplastic (Rajgopal & Kumar, Citation2003; Kumar & Sridevi, Citation2004), antiplatelet (Zhang & Tan, Citation1994), antithrombotic (Zhao & Fang, Citation1991) activities and also possesses protective activity against various liver (Kapil & Kaul, Citation1993; Visen & Shukla, Citation1993; Trivedi & Rawal, Citation2001) disorders.
It was also observed that an aqueous extract of AP could improve glucose tolerance (Borhanuddin et al., Citation1994) in normal rabbits, but failed to demonstrate any “fasting blood glucose lowering” effect after 6 weeks of administration of AP. So, perhaps the water extract of AP could have an effect on glucose absorption from gut and may prolong absorption process, suppressing the peak blood glucose levels. Similarly, unpublished data from our laboratory show that the ethanol extract of AP has an inhibitory effect on glucose absorption in the isolated modified jejunal sac preparation, suggesting an extrapancreatic mode of antihyperglycemic action.
Although there are citations of antihyperglycemic (Zhang & Tan, Citation2000b) and antidiabetic (Zhang & Tan, Citation2000a) activity of ethanol extract of AP based on free radical scavenging activity and in part to increased glucose metabolism, there are no previous reports, at least to our knowledge, on the activity of this extract on in vivo. α.-glucosidase activity. Thus the current study was designed to determine the possible effect of graded doses of ethanol extract of AP after an oral carbohydrate (starch, sucrose, glucose) load on the peak blood glucose (PBG) and area under the curve (AUC) levels in normal and diabetic rats.
Materials and Methods
Plant preparation
Leaves and aerial parts of AP from cultivated sources were supplied by Mr. Musa Yaacob from the Malaysian Agriculture Research and Development Institute (MARDI) Telong, Kota Bharu, Kelantan, Malaysia, and authenticated as Andrographis paniculata.. Voucher specimens were deposited both in MARDI, Kelantan, and Herbarium, School of Pharmaceutical Sciences, Universiti Sains Malaysia. The dried leaves were powdered using a milling machine and were extracted with 20% v/v ethanol (R & M Chemicals, Essex, UK) using the technique of cold maceration. Solvent was replenished every 24 h for 7 days. On the seventh day, the extract was filtered and separated from the marc and concentrated at 40°C by a rotary evaporator (Büchi Labortechnik, Flawil, Switzerland). Finally, the concentrated extract was loaded in a freeze-drier (Labconco Corporation, Kansas City, Missouri, USA) to obtain fine dry powder. The extract yield was 7.7%. The extract was dissolved in distilled water with ultrasonication before use.
HPLC analysis
HPLC analysis was performed on a Shimadzu LC-10AT (Shimadzu Corporation, Kyoto, Japan) system equipped with a LC-6A solvent delivery pump and SPD-10A UV/VIS detector. Data were acquired and processed by Class-VP Chromato software. The analytical column used was Nucleosil C18 (250 × 4.6 mm i.d., 5 µm; Phenomenex) along with a Guard Column C18 (10 × 4.0 mm i.d; Phenomenex) used at 35°C for the elution of analyte. The mobile phase consisting of methanol:water (65:35, v/v) was prepared and filtered through a 0.45-µm nylon-membrane filter (Millipore Corporation, Billerica, MA, USA) under vacuum before use. Methanol (JT Baker Co, Phillipsburg, NJ, USA) used as mobile phase was of HPLC grade. The analysis was performed at a flow rate of 1 mL/min with the detection set at a wavelength of 223 nm.
The commercially available standard andrographolide (AGPH) (Sigma Chemicals, St. Louis, MO, USA) stock solution was prepared at a concentration of 1 mg/mL in mobile phase. The stock solution was further diluted with the mobile phase to obtain the calibration standards of 10, 20, 30, 40, 50, and 100 µg/mL.
The sample solution of 20% v/v ethanol extract of AP was prepared by dissolving 10 mg of extract in 20 mL of mobile phase under ultrasonication for 25 min to obtain a concentration of 500 µg/mL. This solution was further diluted with the mobile phase to yield 250 µg/mL. The standard and extract samples were injected (in triplicate) in a sample volume of 20 µL. Before injection, the extract samples were filtered through a 0.2-µm, polytetrafluoroethylene (PTFE) membrane (Aervent Disposable Filters, Millipore Corporation). The HPLC profile of the standard AGPH (A) and 20% v/v ethanol extract (B) is shown in . Constituents of the drug extract were identified by comparison of HPLC retention time of authentic sample of AGPH analyzed under identical conditions.
Figure 1 (A) HPLC chromatogram of the standard andrographolide (AGPH) and (B) 20% v/v ethanol extract of AP. HPLC was performed in an UV Shimadzu LC-10AT model using Nucleosil (Phenomenex) 5-µm C18 column (250 × 4.6 mm). Mobile phase was MeOH and H2O (65:35) at a flow rate of 1 mL/min and injection volume 20 µL with SPD-10A UV detector set at 223 nm.
Animals
Female Sprague-Dawley rats weighing 200–250 g were obtained from the Central Animal House, Universiti Sains Malaysia, Penang, Malaysia, and housed in the Animal Transit Room, School of Pharmaceutical Sciences, Universiti Sains Malaysia, Penang, Malaysia, 2–3 days before the start of experiment. All the animals used were cleared by the Animal Ethics Committee, Universiti Sains Malaysia, and maintained according to international and national ethical guidelines. The animals had access to food and water ad libitum..
Diabetes was induced in the animals by a single intraperitoneal injection of streptozotocin (Sigma Aldrich Chemical Co., St. Louis, Missouri, USA) 45 mg/kg body weight in cold citrate buffer, pH 4.5. Blood glucose levels were constantly monitored using Accu-Chek Advantage-II Glucose meter (Roche Diagnostics, Manheim, Germany) and rats showing blood glucose level around 16.6 mmol/L (300 mg/dL) were included in the study. Acarbose (Bayer Pharmaceuticals, Leverkusen, Germany) was used as a positive control at a dose of 10 mg/kg body weight.
The oral carbohydrate tolerance tests were carried out in normal and diabetic groups of rats and were equally divided into various treatment groups as mentioned below.
Oral starch tolerance test
Rats were divided into five groups consisting of six rats in each group. The rats were fasted overnight for 18 h but had free access to water. Treatment group 1 rats were treated orally with 250 mg/kg body weight of 20% v/v ethanol extract of AP, treatment group 2 rats was treated orally with 500 mg/kg body weight of 20% v/v ethanol extract of AP, and treatment group 3 rats received orally 1000 mg/kg body weight of 20% v/v ethanol extract of AP. The groups were designated as D1, D2, D3, respectively. Treatment group 4 rats were treated orally with only distilled water (control) and, finally, treatment group 5 rats were treated orally with positive control acarbose (Acar) 10 mg/kg body weight. After 10 min, all rats were given starch 3 g/kg (R & M Chemicals, Essex, UK) body weight orally and the tail was snipped for blood glucose estimation before (0 min) and at 30, 60, and 120 min after starch administration.
Blood glucose concentrations were recorded and PBG and AUC determined. The maximum blood glucose concentration found during blood glucose determination was taken as the PBG. The formula for AUC determination is as follows:
Oral sucrose tolerance test
The oral sucrose tolerance test was carried out in the same way, but in this test sucrose (R & M Chemicals) at a dose of 4 g/kg body weight was used.
Oral glucose tolerance test
The oral glucose tolerance test was carried out in the same way, but in this test glucose (R & M Chemicals) at a dose of 2 g/kg body weight was used.
Statistical analysis
The values are expressed as mean±SD. Statistical difference in PBG and AUC between control and various treatment groups was determined using Statistical Package for Social Sciences (SPSS) one-way analysis of variance (ANOVA) followed by least square design (LSD) for post hoc. analysis. p < 0.05 was considered as significant.
Results
HPLC analysis
The HPLC analysis showed that the standard AGPH and extract sample eluted at the same retention time of 4.0 min and the quantity of AGPH in the extract sample calculated based on the simple linear regression curve was found to be 18.1 mg/g.
Effect of ethanol AP extract on oral starch tolerance tests in normal and diabetic rats
The results of the oral starch tolerance tests on both normal and diabetic rats demonstrated an inhibition of blood glucose increase at 30 min, brought about by ethanol extract of AP administration after an oral starch load, and further made the blood glucose curve flat. In normal rats, treatment groups D2, D3, and acarbose reduced PBG significantly at 30, 60, 120 min () while AUC levels were lowered by D3 and acarbose treatment group (). Treatment groups D2, D3, and acarbose significantly decreased PBG, while AUC level was lowered significantly ( and ) only in D3 and acarbose treatment groups in diabetic rats.
Table 1. Effect on PBG and AUC after starch loading in normal and diabetic rats treated with 250 (D1), 500 (D2), 1000 (D3) mg/kg ethanol extract of Andrographis paniculata., vehicle, and acarbose 10 mg/kg Starch was used at 3 g/kg.
Figure 2 Blood glucose response during oral starch tolerance test in normal rats treated with 250 (D1), 500 (D2), 1000 (D3) mg/kg of ethanol extract of AP, vehicle, and acarbose 10 mg/kg. Starch used at 3 g/kg. Values are the mean±SD (n = 6). *p < 0.05 compared with the control; **p < 0.001 compared with the control. (ANOVA followed by LSD post hoc. test).
Figure 3 Blood glucose response during oral starch tolerance test in diabetic rats treated with 250 (D1), 500 (D2), 1000 (D3) mg/kg of ethanol extract of AP, vehicle, and acarbose 10 mg/kg. Starch used at 3 g/kg. Values are the mean±SD (n = 6). *p < 0.05 compared with the control; **p < 0.001 compared with the control. (ANOVA followed by LSD post hoc. test).
Effect of ethanol AP extract on oral sucrose tolerance tests in normal and diabetic rats
The peak blood glucose at 30 min was suppressed on ethanol extract of AP administration followed by an oral sucrose load leading to a stunted blood glucose curve. In normal rats, D2, D3, and acarbose groups managed to suppress PBG significantly (), whereas D2, D3, and acarbose treatment groups could lower AUC concentrations significantly (). In diabetic rats, D3 and acarbose groups reduced both PBG and AUC significantly ( and ). The ethanol extract of AP shifted and delayed the blood glucose concentrations from 30 to 60 min concurrently suppressing PBG and reducing AUC.
Table 2. Effect on PBG and AUC after sucrose loading in normal and diabetic rats treated with 250 (D1), 500 (D2), 1000 (D3) mg/kg ethanol extract of Andrographis paniculata., vehicle, and acarbose 10 mg/kg Sucrose was used at 4 g/kg.
Figure 4 Blood glucose response during oral sucrose tolerance test in normal rats treated with 250 (D1), 500 (D2), 1000 (D3) mg/kg of ethanol extract of AP, vehicle, and acarbose 10 mg/kg. Sucrose used at 4 g/kg. Values are the mean±SD (n = 6). *p < 0.05 compared with the control; **p < 0.001 compared with the control. (ANOVA followed by LSD post hoc. test).
Figure 5 Blood glucose response during oral sucrose tolerance test in diabetic rats treated with 250 (D1), 500 (D2), 1000 (D3) mg/kg of ethanol extract of AP, vehicle, and acarbose 10 mg/kg. Sucrose used at 4 g/kg. Values are the mean±SD (n = 6). *p < 0.05 compared with the control; **p < 0.001 compared with the control. (ANOVA followed by LSD post hoc. test).
Effect of ethanol AP extract on oral glucose tolerance tests in normal and diabetic rats
The ethanol extracts of AP failed to effect an inhibition of blood glucose elevation after glucose loading in both normal and diabetic rats. Blood glucose level did not differ significantly at any time between groups. Similarly, acarbose failed to suppress the PBG and lower AUC after glucose loading in both normal and diabetic rats. These results reveal that neither the ethanol extract of AP nor acarbose affect absorption of glucose in the small intestine (data not shown).
Discussion
AGPH, the major biomarker and the principal component of the aerial parts of AP, is reported to have antidiabetic activity, by causing an increase in glucose utilization, simultaneously lowering plasma glucose in diabetic rats (Yu et al., Citation2003). The HPLC analysis of AGPH was carried out by following the method of Kumaran et al. (Citation2003) using a simple isocratic elution technique. This method is simple, reproducible, and can be easily applied as a measure of quality control of AP extracts for routine standardization, semi-quantitative analysis, and for optimization of extraction technique. The HPLC fingerprint showed that both the standard AGPH and extract sample eluted at the same retention time under identical conditions and the amount of AGPH in the extract sample with respect to the standard AGPH was found to be 18.1 mg/g, which was in agreement with the recent findings of Akowuah et al. (Citation2006).
The effective management of postprandial hyperglycemia (PPH) (Frantz et al., Citation2005; Yamagishi et al., Citation2005) is a key problem in diabetes mellitus because high blood glucose levels may cause the stimulation and/or progression of diabetic complications through activation of the polyol pathway (Yamagishi & Imaizumi, Citation2005) or may increase glycation of proteins (Md.Isa et al., Citation2005) and may cause the promotion of hyperinsulinemia (Freude et al., Citation2005). A prominent pathway for glucose production from food intake is the breakdown of carbohydrates and/or sucrose by amylases or glycoside hydrolases in the intestine. Thus, preventing an excessive postprandial blood glucose rise or maintaining blood glucose limit within the normal range by controlling glucose production from food sources using an oral α.-glucosidase inhibitor would be an idealistic and effective management for NIDDM patients.
Acarbose-like drugs (Laube, Citation2003), drugs that inhibit α.-glucosidase present in the epithelium of the small intestine, have been demonstrated to decrease PPH and improve impaired glucose metabolism without promoting insulin secretion in NIDMM patients (Toeller, Citation1994). Therefore, the retardation and delay of carbohydrate absorption with a plant-based α.-glucosidase inhibitor offers a prospective therapeutic approach for the management of type 2 diabetes mellitus (Reaven et al., Citation1990; Rybka et al., Citation1990; Rosenberg et al., Citation2005).
We found in the above experiments that doses D2 and D3 of the ethanol extract of AP reduced the blood glucose excursions and decreased the PBG and AUC after sucrose and starch loading in normal and diabetic rats. In normal rats also, the ethanol extract of AP demonstrated a PBG suppressive effect after sucrose and starch loading. This is in agreement with previous unpublished results from our laboratory that demonstrate the inhibitory effect of the ethanol extract of AP on glucose absorption in the isolated modified jejunal sac preparation. The tendency of ethanol extract of AP to suppress the PBG at 30 min in both normal and diabetic rats demonstrates the α. -glucosidase inhibitory activity while delaying blood glucose levels. The ethanol extract of AP seems to delay the quick digestion of starch and sucrose and lengthen the duration of carbohydrate absorption over time, thus reducing the PBG value and AUC. The above results show a striking similarity to the effects of acarbose.
In the field of phytochemistry and herbal medicines, there has been an enormous interest in the development of alternative medicines for type 2 diabetes, specifically screening for natural bioactive compounds with the ability to delay or prevent glucose absorption. This is because any control of PPH by these alternative medicines would be much safer and will improve the quality of life of persons with borderline NIDDM. α.-Glucosidase inhibitors (Watanabe & Kawabata, Citation1997), α.-amylase inhibitors (Honda & Hara, Citation1993), or glucose transport inhibitors (Kobayashi et al., Citation2000) have been screened to flatten postprandial blood glucose rise by natural compounds and are in the process of further development. Of the above-mentioned classes, only synthetic α.-glucosidase inhibitors have been clinically used for management of type 2 diabetes. It is already known that ingestion of α.-glucosidase inhibitors like acarbose or voglibiose regularly is more effective in moderating hyperglycemia in borderline NIDDM (Chiasson et al., Citation2002).
The ethanol extract of AP seems to have α.-glucosidase inhibitory activity in diabetic rats because it suppresses the PBG and reduces AUC after simultaneous starch and sucrose loading and significantly affects the absorption of starch and sucrose. It may be used as a safer alternative treatment to control PPH particularly in type 2 diabetic patients and also in borderline patients not properly controlled through diet alone. However, it is still early to suggest its use in humans, and only a thorough in-depth study can warrant its clinical use.
Acknowledgment
This research work was supported by a grant from the Ministry of Science, Technology and Environment of Malaysia (MOSTE) (grant. no. 304/PFARMASI/640043/KI05).
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