Abstract
Andrographis paniculata (Burm.f.) Nees (Acanthaceae) (AP), containing the major active principle, andrographolide,(AG) has been used ethnomedically in the treatment of diabetes and hypertension in Malaysia and India. Type 2 diabetes mellitus was induced by administering nicotinamide (NA) 180 mg/kg i.p., followed by streptozotocin (STZ) 45 mg/kg i.p., 15 min later and allowed for 15 days to develop. Diabetic rats treated with active principle andrographolide 10 mg/kg and ethanol extract of Andrographis paniculata at doses of 500 and 1000 mg/kg, p.o, for 21 days, caused a significant reduction in fasting serum glucose levels on all days analysed (P < 0.01). Treatment with andrographolide and extract showed an increase in glucokinase (GK; P < 0.05), hexokinase (HK; P < 0.05), and lactate dehydrogenase (LDH; P < 0.05) enzyme levels respectively. Significant reductions were observed in serum cholesterol (P < 0.05), triglycerides (P < 0.05), free fatty acids (P < 0.05), and liver glucose 6-phosphatase (G6P’Tase; P < 0.05) enzyme levels on treatment with andrographolide and extract. Glucose 6-phosphate dehydrogenase (G6PDH; P < 0.05) level also showed a significant increase on treatment with andrographolide and extract. Liver antioxidant status (P < 0.05) also improved significantly on treatment with andrographolide and extract. However, no significant increase in serum insulin was found on treatment with either andrographolide or extract, suggesting an effective extra pancreatic mechanism. Thus, the present study demonstrates that andrographolide at 10 mg/kg, and ethanol extract of Andrographis paniculata at 500 and 1000 mg/kg, p.o., on treatment for 21 days, exhibited antidiabetic, hypolipidemic, and antioxidant activities in an adult streptozotocin-nicotinamide type 2 diabetes mellitus model. The results support the traditional use of Andrographis paniculata in the treatment of type 2 diabetes mellitus, and its associated plethora of complications.
Introduction
Andrographis paniculata (Burm.f.) Nees (Acanthaceae) (AP) is an annual herb found in Southeast Asia and is reportedly used as an antidiabetic (CitationZhang & Tan, 2000) and antihypertensive (CitationZhang & Tan, 1996) in Malaysia, India and other countries. It is known as ‘akar cerita’ and ‘hempedu bumi’ in Malay dialect and is also known commonly as ‘king of bitters’. It is also cited in the literature for its antioxidant (CitationAkowuah et al., 2006), antiplatelet (CitationThisoda et al., 2006), antihyperglycemic (CitationYu et al., 2003), immunomodulatory (CitationXu et al., 2007), and anticancer properties (CitationJada et al., 2007).
The active constituents of AP are diterpene lactones including andrographolide which is considered to be the most active and important constituent in this plant (CitationAkowuah et al., 2006). AGhas been reported widely for multifarious pharmacological activities, some of them being for its antioxidative (CitationAkowuah et al., 2006), antiplatelet (CitationThisoda et al., 2006), antihyperglycemic (CitationYu et al., 2003), immunomodulatory (CitationXu et al., 2007), anticancer (CitationJada et al., 2007) and α -glucosidase inhibitory (CitationDai et al., 2006) effects.
Type 2 diabetes mellitus (T2DM) is a progressive, chronic metabolic disorder notable for the underlying defects in carbohydrate and lipid metabolism. It is typically characterized by several sequential steps involving impaired β -cell function, resulting in a relative insulin deficiency, followed by insulin resistance with decreased glucose transport into muscle and fat cells, accompanied by unrestrained hepatic glucose output, all of which contribute to an overwhelming glycemic status.
Free fatty acids (FFA) have also been implicated in the development of T2DM (CitationBoden, 2003) by causing a decreased peripheral glucose utilization leading to insulin resistance. Presently, oxidative stress due to free radical assault has also been implicated in the pathology of T2DM (CitationGovindarajan et al., 2005) and is a contributive factor in micro- and macro-vascular complications such as diabetic nephropathy, diabetic retinopathy, coronary artery disease, peripheral vascular disease, atherosclerosis, etc.
The significance of antioxidants in T2DM and low levels of tissue antioxidants have also been separately acknowledged as a risk factor for the development and progression of the disease. Chronic hyperglycaemia produces multiple, complex biochemical sequelae and diabetes-induced oxidative stress (CitationDaisuke et al., 2003) could play an important role in the quick onset and progression of T2DM. So compounds with antidiabetic, hypolipidemic and antioxidant properties could be an ideal and effective therapeutic agent for the treatment of T2DM.
We have already reported the in vivo α -glucosidase inhibitory (CitationRammohan & Asmawi, 2006) effect of the 20% v/v ethanol extract of AP in an acute model of diabetes, wherein we observed the peak blood glucose suppressive effects of the extract. The peak blood glucose suppressive effect of the ethanol AP extract may be due to the interplay among the various carbohydrate regulatory enzymes, or by enhancement of peripheral glucose metabolism or by both of the above mechanisms thus maintaining an overall glucose homeostasis and which seems to be specifically deranged in T2DM, ultimately affecting the glucose uptake, metabolism and disposal.
Furthermore, current unpublished results from our laboratory demonstrated significant in vitro α -glucosidase inhibitory effects of AG at concentrations of 10, 5, 2.5, and 1.25 mg/ml, and 20% v/v ethanol extract of AP at concentrations of 62.5, 31.25, 15.6, 7.8, 3.9, and 1.95 mg/ml, with IC50values of 11 and 17.2 mg/ml, respectively, suggesting that both AG and ethanol extract of AP may possibly have glucose lowering effects by inhibiting intestinal glucosidases.
Thus, the present study was carried out to examine the effects of AG and the ethanol extract of AP on the liver glycolytic, gluconeogenic and lipogenic enzymes in an adult STZ-NA T2DM rat model. To the best of our knowledge, this is the first report that explores the effect of andrographolide and the ethanol extract of AP on the key hepatic glycolytic, gluconeogenic and lipogenic enzymes in a chronic adult STZ-NA T2DM rat model.
Materials and Methods
Pure commercially available andrographolide (98% purity, Sigma Chemical Company, US) was first solubilised in a small volume of ethanol and then diluted with phosphate buffered saline (CitationXu et al., 2007).
Plant Preparation
Dried aerial parts including stem and leaves of AP from cultivated sources were supplied by Mr. Musa Yaacob cultivated from the nurseries of Malaysian Agriculture Research and Development Institute, (MARDI) Kelantan, Malaysia. The dried leaves were powdered and extracted with 20% v/v ethanol (R & M Chemicals, Essex, UK) by cold maceration. Extraction was carried out for 7 days, replenishing the solvent every 24 h, until complete exhaustion. Then the extract was filtered, separated from the marc, and concentrated using a rotary evaporator (Buchi Labortechnik, Switzerland) at 60°C. Finally, the concentrated extract was lyophilized (Labconco Corporation, Missouri, US) to obtain fine dry powder. The percentage yield of the extract was 10.2%.
Animals
All the rats used were cleared by the Animal Ethics Committee, Universiti Sains Malaysia, and strictly maintained according to international and national ethical guidelines. Female Sprague-Dawley rats weighing around 250–260 g were obtained from the Central Animal House, Universiti Sains Malaysia, Penang, Malaysia housed in polypropylene cages, and allowed to adjust in the Animal Transit House, 2–3 days before the start of experiment. The animals were fed standard rat pellets and given water ad libitum.
Induction of type 2 diabetic rats
T2DM was induced with adult rats (CitationMasiello et al., 1998) by administering nicotinamide (Sigma Aldrich Chemical Co., US) 180 mg/kg i.p. in cold saline, followed by streptozotocin (Sigma Aldrich Chemical Co., US) 45 mg/kg i.p in cold citrate buffer pH 4.5, 15 min later. A small drop of blood obtained from a tail snip was used to determine blood glucose levels and were constantly monitored for 15 days using a portable Accu-Chek® Advantage-II glucose meter (Roche Diagnostics, Germany). Rats showing non-fasting blood glucose level around 11–14 mmol/l (198–252 mg/dl) were included in the study. Commercially available pure andrographolide 10 mg/kg, p.o., and metformin 500 mg/kg, p.o. (Glucophage®, Lipha Pharm Ltd, United Kingdom), were used as reference marker and positive control, respectively.
Experimental Design
After 15 days (CitationKuntz et al., 2002), rats (n = 6) observed with a reading of non-fasting blood glucose of 11–14 mmol/l were designated into seven treatment groups. Each rat of a particular treatment group was orally given the respective treatment once daily for 21 days. The treatment groups were as follows:
Group I -250 mg/kg ethanol extract, p.o. (D1)
Group II -500 mg/kg ethanol extract, p.o. (D2)
Group III -1000 mg/kg ethanol extract, p.o. (D3)
Group IV - Diabetic control (DC), distilled water 4 ml/kg
Group V - Normal control (NC), phosphate buffered saline 4 ml/kg
Group VI - Andrographolide (AG), 10 mg/kg p.o.
Group VII- Metformin (MF), 500 mg/kg p.o.
Sample collection
At the end of day 21, animals were fasted overnight, sacrificed and blood collected directly from the heart into disposable Vacutainer® tubes without anticoagulant, allowed to clot, then centrifuged at 5000 rpm for 5 min. The supernatant serum was collected and separately stored in individual disposable Eppendorf® microcentrifuge tubes at 70°C until analysis. Serum was used to estimate fasting glucose levels, insulin, cholesterol, triglyceride, and free fatty acids.
Liver was immediately isolated, removed and washed with chilled isotonic 0.15% KCl-0.01 M Na+/K+ phosphate buffer, pH 7.4, a small portion was retained for estimation of glycogen and protein, and the remainder was finely minced with scissors, weighed and homogenized in a glass tube with a Teflon pestle. A 10% (w/v) crude homogenate was prepared by following the method of CitationMazel (1972) with slight modification.
In brief, 1 g of liver tissue was added to 9 volumes of isotonic 0.15% KCl-0.01 M Na+/K+ phosphate buffer, pH 7.4, and homogenized in a glass Potter-Elvehjem homogenizer for 1–2 min. The crude homogenate was centrifuged at 10000 × g for 10 min at 5°C to remove the nuclei and mitochondria. The 10,000 × g supernatant fraction contained microsomes and soluble proteins. This fraction was used to estimate liver carbohydrate metabolic enzymes (HK, GK, LDH, G6PDH and G6P’Tase), and antioxidant parameters (GSH, GST and GR).
Analytical Methods
Determination of fasting serum glucose
Fasting serum glucose was estimated by using commercially available glucose oxidase-peroxidase reactive test strips and measured on a portable blood glucose meter (Accu-Chek® Advantage-II glucose meter, Roche Diagnostics, Germany).
Determination of serum cholesterol, triglycerides, and free fatty acids
Serum cholesterol was estimated by the method of CitationKim and Goldberg (1969). Serum triglyceride and free fatty acids were analyzed by the methods of CitationSoloni (1971) and CitationSoloni and Sardina (1973), respectively.
Determination of liver carbohydrate metabolic enzymes
Liver HK was estimated according to the method of CitationDarrow and Colowick (1962). GK analysed by the method of CitationGoward (1986). LDH, G6PDH and G6P’Tase were estimated according to the methods of CitationBergmeyer and Bernt (1974), CitationBergmeyer et al. (1974), and CitationBaginsky et al. (1974), respectively. Lowry's method of protein estimation (CitationLowry et al., 1951) was used for analysis of liver proteins (data not shown here).
Determination of liver antioxidant parameters
Liver glutathione S-hydroxylase (GSH), glutathione S-transferase (GST), and glutathione reductase (GR) were estimated according to the methods of CitationJollow et al. (1974), CitationHabig et al. (1974), and CitationMavis and Stellwagen (1968), respectively.
Determination of serum insulin and liver glycogen
Serum insulin was assayed using an enzyme linked immunosorbent assay (ELISA) kit (Crystal Chem Inc, IL, US) and read on a microplate reader (Power Wave X 340®, Biotek Instruments Inc, US) and glycogen was estimated by the method of CitationMontgomery (1957).
Statistical analysis
Data were statistically evaluated using Statistical Package for Social Sciences (SPSS) one-way analysis of variance (ANOVA), followed by Tukey's Test for post-hoc analysis. Values were considered statistically significant when P < 0.05.
Results
Body Weight changes
DC group displayed a significant (P < 0.05) reduction in body weight in comparison to NC rats. shows the effect of ethanol extract of AP on body weight changes in NC, DC and treated groups. AG group showed a distinct increase (P < 0.05) in body weight which was significant. The body weights of D2, D3 treatment groups also increased significantly (P < 0.05) on treatment with 500 and 1000 mg/kg ethanol extract of AP, when compared to DC group. MF did not show any significant increase in body weight. In contrast, only D1 treatment group showed a significant (P < 0.05) fall in body weight which was similar to the DC group.
Figure 1. Effect on body weight chnges in normal and diabetic rats respective treatment for 21 days. D1-250 mg/kg ethanol extract of AP, D2-500 mg/Kg ethanol extract of AP, D3-1000 mg/Kg ethanol extract of AP, DC-Diabetic control, NC-Normal control, AG-Andrographolide, MF-Metformin. Values are mean ± S.E. (n = 6) for each group. * P < 0.05 Vs DC; one-way ANOVA followed by Tukey's Test for post hoc analysis.
Fasting serum glucose
illustrates fasting serum glucose levels in NC, DC and treated groups. DC group demonstrated a significantly (P < 0.01) increased level of fasting serum glucose when compared with NC rats on all days. AG group showed a significant (P < 0.01) fall in fasting serum glucose levels on day 7, 14, and 21. Treatment groups D1, D2, D3 showed a significant (P < 0.01) fall in fasting serum glucose on day 7, 14, and 21, respectively. MF group also displayed a potent reduction (P < 0.01) in the fasting serum glucose levels on day 7, 14, 21, which was significant.
Figure 2. Effect on fasting serum glucose levels in normal and diabetic rats after respective treatment for 21 days. D1-250 mg/kg ethanol extract of AP, D2-500 mg/kg ethanol extract of AP, D3-1000 mg/kg ethanol extract of AP, DC-Diabetic control, NC-Normal control, AG-Andrographolide, MF-Metformin. Values are mean ± S.E. (n = 6) for each group. * P < 0.05 Vs DC, **P < 0.01 Vs DC; one-way ANOVA followed by Tukey's Test for post hoc analysis.
Serum cholesterol, triglyceride, and free fatty acids
Effect of administering ethanol extract of AP on NC, DC, and treated groups on the serum lipid profile are illustrated in . Serum cholesterol (CS), triglycerides (TG), and free fatty acids (FFA) levels demonstrated a significant (P < 0.05) increase in diabetic rats as compared to NC rats. AG demonstrated a significant (P < 0.05) fall in serum cholesterol, triglycerides, and free fatty acids Treatment groups D1, D2, D3 showed a significant (P < 0.05) reduction in serum cholesterol, triglycerides, and free fatty acids in comparison to NC, and DC groups after 21 days treatment. The reduction observed in all the extract treated groups on CS and TG was lower than of NC group. Treatment with MF was also significantly (P < 0.05) effective in bringing down the elevated serum cholesterol, triglycerides, and free fatty acids.
Figure 3. Effect on serum cholesterol, triglycerides, and free fatty acids in normal and diabetic rats after respective treatment for 21 days. D1-250 mg/kg ethanol extract of AP, D2-500 mg/kg ethanol extract of AP, D3-1000 mg/kg ethanol extract of AP, DC-Diabetic control, NC-Normal control, AG-Andrographolide, MF-Metformin, CS-Cholesterol, TG- Triglycerides, FFA- Free fatty acids.Values are mean ± S.E. (n = 6) for each group.* P < 0.05 Vs DC; one-way ANOVA followed by Tukey's Test for post hoc analysis.
Liver carbohydrate metabolizing enzymes
The effect of ethanol extract of AP on NC, DC and treated rats are illustrated in . The activities of glycolytic enzymes: HK, GK, and LDH were significantly lower after 21 days of diabetes in the liver of DC group compared to NC group (P < 0.05). There was also a significant increase in turnover of gluconeogenic enzyme G6P’Tase in the DC group (P < 0.05). The lipogenic enzyme, G6PDH declined to below normal levels in diabetic rats when compared to NC (P < 0.05). AG treatment caused a statistically significant decrease in G6P’Tase levels with a significant (P < 0.05) increase in GK, HK and LDH levels. A significant (P < 0.05) increase in G6PDH enzyme levels was also observed following AG treatment. Treatment with 1000 mg/kg (D3) ethanol extract of AP caused a significant (P < 0.05) increment in the glycolytic enzymes. Though treatment with a 500 mg/kg dose of the extract demonstrated an increase in GK, HK, and LDH levels, significance (P < 0.05) was shown only in HK levels. A small but insignificant increase in levels of GK and HK levels was observed with D1 treatment after 21 days. Additionally, D1 did not cause any increase in LDH levels. G6P’Tase showed a significant (P < 0.05) sharp decline in their enzyme activities when treated with 250, 500, and 1000 mg/kg of the extract. G6PDH enzyme level showed a significant (P < 0.05) increase in activity after treatment for 21 days in D1, D2, and D3 groups. MF treatment group also showed a significant (P < 0.05) increase in turnover of glycolytic enzymes and was found to be effective in reducing the escalated G6P’Tase. G6PDH enzyme levels also restored back to near normal levels on metformin treatment which was also significant (P < 0.05).
Table 1. Effect on liver carbohydrate metabolic enzyme activities of normal and diabetic rats after espective treatment for 21 days.
Liver antioxidant parameters
DC group of rats showed a significant (P < 0.05) fall in levels of antioxidant parameters of liver: GSH, GST, and GR, respectively. illustrates the effect of ethanol extract of AP on liver antioxidant parameters in adult type 2 diabetic rats after 21 days of treatment. Treatment group D1 demonstrated a moderate increase in GSH level when treated with 250 mg/kg of the extract which was not significant, when compared to DC animals. D1 treatment also increased levels of GST and GR after 21 days of treatment but was also not statistically significant. Treatment group D2, D3 showed an increase in all the antioxidant parameters in comparison with DC group, though there was no significance for GSH and GR levels of D2 group. AG treatment also exemplified a significant (P < 0.05) increase in the levels of GSH, GST, GR, etc. MF group also illustrated a significant (P < 0.05) increase in GST and GR levels when compared to DC group but failed to cause a significant increase in GSH level.
Table 2. Effect on liver antioxidant parameters of normal and diabetic rats after respective treatment for 21 days.
Serum insulin and liver glycogen
reports the serum insulin and liver glycogen levels in NC, DC and treated groups. In DC rats, serum insulin levels showed a significant (P < 0.05) decline in comparison with NC rats. AG treatment caused a slight increase in insulin levels but was not significant. The serum insulin levels of D1, D2, D3 extract treatment groups did not cause any statistically significant increase. MF treatment demonstrated a significant (P < 0.05) increase in levels of serum insulin when compared with DC.
Table 3. Effect on serum insulin and liver glycogen levels after respective treatment for 21 days.
DC rats showed a significant (P < 0.05) fall in liver glycogen levels compared to NC group (). AG treatment also showed a significant (P < 0.05) increase in liver glycogen levels. Though administration of 250, 500, and 1000 mg/kg dose of extracts increased glycogen levels, the increase was significant (P < 0.05) only in case of the D3 group. MF administration demonstrated a potent and significant (P < 0.05) increase in liver glycogen levels, near to NC levels.
Discussion
We have already reported (CitationRammohan & Asmawi, 2006) the identification and quantification of AG in the ethanol extract of AP by performing a comparative HPLC study with the commercially available principle marker (CitationAkowuah et al., 2006), AG. The ethanol extract was found to contain 18.1 mg AG per gram of the crude extract, which is in agreement with the recent findings of CitationAkowuah et al. (2006). The earlier observed peak blood glucose suppressive effect of the 20% v/v ethanol AP extract (CitationRammohan & Asmawi, 2006) may be due to the interplay among the various carbohydrate regulatory enzymes, or by enhancement of peripheral glucose metabolism or by both of the above mechanisms maintaining an overall glucose homeostasis and specifically deranged in T2DM, ultimately affecting the glucose uptake, metabolism and disposal.
The 21 day extract treatment did not cause any increase in serum insulin levels .The serum glucose reduction seems not related to the stimulation of insulin secretion and that post prandial serum glucose reduction is probably caused without any extra load on pancreatic β -cells and/or stimulation of glucose uptake by peripheral tissues. This observed effect may in all probability be due to an extra pancreatic mechanism. There are several medicinal herbs that are known to exert significant antidiabetic activity without the stimulation of insulin secretion (CitationHannan et al., 2003; CitationShirwaikar et al., 2005).
HK is a key enzyme that catabolizes glucose, by phosphorylating it in to glucose 6-phosphate. The increase in HK caused by AG and extract treatment may be due to a direct stimulation of glycolysis in tissues by increased glucose removal, followed by an increase in glucose utilization for energy production. The fall in fasting serum glucose observed as a result of AG treatment and extract could also be due to an increase in glycolysis, by increasing turnover of glycolytic enzymes.
Phosphorylation of glucose to glucose-6-phosphate (G6P) by GK is the first step of both glycogen synthesis and glycolysis in the liver. The synthesis of the enzyme is highly sensitive to oxidative stress, with diabetes reducing the overall levels. AG treatment and extract treatment for 21 days increased the enzyme levels. The increased levels of the enzyme correlates well with the increase in liver glycogen levels and this could, to some extent, explain the observed increase in GK.
The increase in LDH levels could be attributed to an increase in peripheral glucose utilization after 21 days AG and extract treatment.
Additionally, the gluconeogenic enzyme G6P’Tase, significantly declined on once daily treatment with AG and ethanol extract of AP for 21 days. This may involve a possible extrapancreatic mode of action, by stimulating peripheral glucose utilization or enhancing glycolytic and glycogenic processes with concomitant decrease in glycogenolysis and gluconeogenesis (CitationSaxena & Vikram, 2004).
G6PDH is an important lipogenic enzyme of the pentose phosphate pathway and is usually decreased in T2DM. A decline in the enzyme activity in diabetes may result in the diminished functioning of the pentose phosphate pathway and, thereby, a subsequent decline in the production of reducing equivalents such as NADH and NADPH, thus, leading to a state of oxidative stress. Administration of AG and extract for 21 days was found to increase the liver G6PDH levels; this may possibly be due to an increase in turnover of enzymes of glycogenesis and glycolysis with a simultaneous decrease in gluconeogenic enzyme activities. It seems to cause an increase in the flux of glucose into the glycolytic pathway and pentose monophosphate shunt in an effort to reduce high blood glucose levels, evident from a reduction in serum glucose levels following 21 day AG and ethanol extract treatment. This results in increased production of the reducing equivalent, NADPH, with concomitant decrease in oxidative stress with an increase in the activity of the related liver antioxidant parameters like GSH, etc.
Another plausible mechanism is by a direct inhibition of glucose absorption from the intestine, thus reducing the fasting serum glucose level. This may be supported by our previous finding that the 20% v/v ethanol extract of AP inhibits in vivo α -glucosidase enzyme (CitationRammohan & Asmawi, 2006), involved in the digestion of carbohydrates into simple sugars in the intestine, leading to a delay, suppression, or complete inhibition of carbohydrate breakdown and the consequent glucose absorption from the intestine.
The adult STZ-NA T2DM model also illustrated some derangements in lipid metabolism, manifested by a significant increase in serum cholesterol, triglycerides, and fatty acids, results of which were consistent with earlier reported studies (CitationSaravanan & Pari, 2005). Administration of AG and parallel treatment with ethanol extract of AP (500 and 1000 mg/kg) daily for 21 days showed a significant decrease in serum cholesterol, triglycerides, and fatty acids, indicative of its hypocholesterolemic and hypotriglyceridaemic activities. The antidiabetic activity can be due to a corollary of an enhanced lipid metabolism apart from direct involvement in glucose homeostasis. The triglyceride lowering activity of AG and ethanol extract of AP could indirectly contribute to the overall antihyperglycemic activity through the interaction of glucose-fatty acid cycle or Randle's cycle (CitationFrayn, 2003). The Randle's glucose-fatty acid cycle clearly demarcates the role of increased supply of plasma triglycerides that could become a source of increased free fatty acid availability and oxidation that can impair insulin action, glucose metabolism and utilization leading to development and progression of hyperglycemia. Hence, a reduction in triglycerides by attenuation of glucose-fatty acid cycle, following oral treatment with AG and ethanol extract of AP, would also be likely to facilitate glucose oxidation and utilization (CitationMuruganandan et al., 2005) with subsequent reduction in glycemia.
Moreover, an increase in liver antioxidant parameters like GSH, GST, and GR following daily administration of 10 mg/kg AG and 500 and 1000 mg/kg ethanol AP extract for 21 days suggest probable antioxidant property, beneficial in the treatment of diabetes associated complications arising due to free radical assault.
In summary, treatment with the main active principle AG and ethanol extract for 21 days has been found to exert an antidiabetic effect. The above mentioned properties have been amply supported by the preliminary serum glucose suppressive effect of AG and the extract indicative of antidiabetic property. AG and ethanol extract also demonstrated significant reductions in serum cholesterol, triglyceride, and serum free fatty acid, which would be a valuable therapeutic option in the treatment of diabetes with associated derangements in lipid metabolism. Treatment for 21 days with AG and extract demonstrated a significant decrease in G6P’Tase and an increase in G6PDH. The positive effect of AG and extract seen on the fasting serum glucose level after 21 days of treatment, seems to be the result of gluconeogenesis and glycogenolysis (CitationShirwaikar et al., 2004), with a subsequent increase in glycolysis and glycogenesis (CitationSaraswati & Swaminathan, 2002). It may be also that intestinal glucose absorption is inhibited, resulting in peak blood glucose suppressive effect, which may be useful in abolishing a rise in blood glucose, post prandially. The active principle, AG and extract was also responsible for an increase in liver glycogen levels, possibly due to a selective promotion of glycogenesis than for glycogenolysis (CitationWu et al., 2005), thus improving glucose homeostasis. Additionally, AG and AP extract was also observed to cause a significant increase in all the liver antioxidant parameters in the present study, which is further supported by the previous work of CitationAruna et al. (1993), demonstrating that AG, in particular, caused an effective increment in GSH levels after chronic treatment of rats in a hepatotoxic model, thus proving the antioxidant effects of AG.
He and colleagues reported that AG may exert antidiabetic effect through inhibiting α -glucosidase after being metabolized to 14-deoxy-11,12-didehydroandrographolide, an α -glucosidase inhibitor in vivo. Further, α -glucosidase inhibitory activity was the cause or at least one of the mechanisms by which AP ethanol extract exerted antidiabetic effects. Stimulated glucose metabolism was found when treating diabetic rat with the ethanol extract of AP or AG. So, it can be deduced that ethanol extract of AP and AG lowers plasma glucose by inhibiting disaccharide metabolism and/or promoting glucose metabolism (CitationHe et al., 2003).
Thus, the antidiabetic activity of Andrographis paniculata is the result of an extra pancreatic mechanism and could independently be due to a distinct improvement in fasting serum glucose levels, or α -glucosidase inhibition, or by stimulating peripheral glucose utilization by facilitating oxidation and utilization or by a combination of all the mechanisms.
In conclusion, the present study demonstrates that AG at a dose of 10 mg/kg and the ethanol extract of AP, at dose levels of 500 and 1000 mg/kg, p.o., exhibited antidiabetic, hypolipidemic, and antioxidant properties in an adult STZ-NA T2DM. The beneficial combination of antidiabetic, hypolipidemic, and antioxidant properties would be of great therapeutic application in the management of T2DM and the associative abnormalities in lipid profiles. Additionally, it can also be used as a safer alternative to control post-prandial hyperglycemia (CitationRammohan & Asmawi, 2006), specifically in diabetic patients and borderline patients, not responsive to nonpharmacological treatment, exemplified by a distinct improvement in glucose disposal without inducing hypoglycemia.
Further studies in the form of bioactivity-guided fractionation of extract can be carried out to ascertain the specific activity of different fractions. A detailed investigation into its mechanism of action at molecular and cellular levels is also required to fully establish its therapeutic potential in the treatment of diabetes and diabetic complications.
Acknowledgement
This research work was supported by a grant from the Ministry of Science, Technology and Environment of Malaysia (MOSTE) (Grant.No:305/PFARMASI/6123003).
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