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Anti-diabetic potentials of Momordica charantia and Andrographis paniculata and their effects on estrous cyclicity of alloxan-induced diabetic rats
Journal of Ethnopharmacology 105 (2006) 196–200
Anti-diabetic potentials of Momordica charantia and Andrographis paniculata and their effects on estrous cyclicity of alloxan-induced diabetic rats B.A.S. Reyes a,b,∗ , N.D. Bautista c , N.C. Tanquilut b , R.V. Anunciado d , A.B. Leung c , G.C. Sanchez b , R.L. Magtoto a,c , P. Castronuevo e , H. Tsukamura f , K.-I. Maeda f a
Thomas Jefferson University, Department of Neurosurgery, Farber Institute for Neurosciences, Philadelphia, Pennsylvania 19107, USA b Institute of Veterinary Medicine and Zootechnics, Pampanga Agricultural College, Magalang 2011, Pampanga, Philippines c Institute of Animal Science, Pampanga Agricultural College, Magalang 2011, Pampanga, Philippines d College of Veterinary Medicine, University of Philippines, Los Banos, Laguna, Philippines e Department of Biology, Kutztown University, Kutztown, PA 19530, USA f Graduate School of Bioagricultural Science, Nagoya University, Nagoya 464-8601, Japan Received 10 March 2005; received in revised form 14 October 2005; accepted 18 October 2005 Available online 18 November 2005
Abstract Momordica charantia and Andrographis paniculata are the commonly used herbs by the diabetic patients in Pampanga, Philippines. While the anti-diabetic potential of Momordica charantia is well established in streptozocin- or alloxan-induced diabetic animals, the anti-diabetic potential of Andrographis paniculata in alloxan-induced diabetic rat is not known. Neither the effects of these herbs on estrous cyclicity of alloxaninduced diabetic rats are elucidated. Thus, in these experiments, Momordica charantia fruit juice or Andrographis paniculata decoction was orally administered to alloxan-induced diabetic rats. Rats that were treated with Momordica charantia and Andrographis paniculata had higher body weight (BW) compared with diabetic positive control (P < 0.01) from day 22 to day 27 (D27) but exhibited lower BW than the non-diabetic control (P < 0.05). These rats had lower feed (P < 0.05) and liquid intakes (P < 0.01) compared with diabetic positive control from day 17 to D27, but similar with the non-diabetic control. The blood glucose levels in these groups were significantly reduced from day 12 to D27 compared with diabetic positive control (P < 0.01), however, comparable with non-diabetic control. The diabetic positive control had extended mean estrous cycles (8 days) compared to Momordica charantia and Andrographis paniculata-treated diabetic rats (5 days; P < 0.05). Our results suggest that the anti-diabetic potentials of Momordica charantia and Andrographis paniculata could restore impaired estrous cycle in alloxan-induced diabetic rats. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Momordica charantia; Andrographis paniculata; Diabetes; Alloxan; Estrous cycle
1. Introduction Diabetes mellitus is the most common endocrine disease (Sexton and Jarow, 1997) and a predominant health concern affecting 16 million Americans (Yeh et al., 2003). It affected 2–3% of the total world population in 1995 (Felig et al., 1995) and had an increasing prevalence worldwide in 1998 (Alberti and Zimmet, 1998). Diabetes mellitus leads to metabolic abnormalities and is characterized by hyperglycemia resulting from
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Corresponding author. Tel.: +1 215 503 5862; fax: +1 215 955 7921. E-mail address: [email protected] (B.A.S. Reyes).
0378-8741/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2005.10.018
defects in insulin secretion, insulin action or both (Atkinson and Maclaren, 1994; Yki-Jarvinen, 1994; Teixeira et al., 2000). Insulin-dependent diabetes mellitus or Type 1 is conventionally treated with exogenous insulin while the non-insulin-dependent diabetes mellitus or Type 2 is treated with oral hypoglycemic agents such as sulphonylureas and biguanides among others (Felig et al., 1995; Rosak, 2002). Complementary and alternative medicine is widely used (Eisenberg et al., 1998; Maclennan et al., 1996; Payne, 2001) and, in the Philippines diabetes mellitus is commonly treated using medicinal plants (De Padua et al., 1997). Momordica charantia and Andrographis paniculata are commonly used herbs in the province of Pampanga, Philippines.
B.A.S. Reyes et al. / Journal of Ethnopharmacology 105 (2006) 196–200
Momordica charantia is a member of Cucurbitaceae, commonly known as ku gua, karela, bittergourd or bitter melon. It is the most popular herbal resource (Marles and Farnsworth, 1995) and is often used to treat diabetes (Arvigo and Balick, 1993). The anti-diabetic potential of Momordica charantia is well established in streptozocin- or alloxan-induced diabetic rats, mice and rabbit (Akhtar et al., 1981; Sarkar et al., 1996; Kar et al., 2003), genetically diabetic mice (Miura et al., 2001) and in humans with Type 2 diabetes (Srivastava et al., 1993). Andrographis paniculata (Burm F) Nees belongs to family Acanthaceae, commonly known as “King of Bitters” and has been used to treat various diseases (Vedavathy and Rao, 1991; Caceres et al., 1997; Kumar et al., 2004). Unlike Momordica charantia, the hypoglycemic effects of Andrographis paniculata have only been studied in streptozotocin-induced diabetic rats (Zhang and Tan, 2000) and in normal rabbits (Borhanuddin et al., 1994). Thus, using alloxan-induced diabetic rats, the anti-diabetic potential of Andrographis paniculata was investigated. Diabetes mellitus has been shown to suppress reproductive functions in humans (Griffin et al., 1994; Sexton and Jarow, 1997) and animals (Angell et al., 1996; Cagampang et al., 1997; Steger and Rabe, 1997). Specifically, diabetes mellitus suppresses luteinizing hormone secretion in streptozocin-induced diabetic rats (Cagampang et al., 1997) and disrupts estrous cycle (Cox et al., 1994). Estrous cycle is the period of reproductive cyclicity in animals that usually lasts for 4–5 days in rodents (Pineda, 2003). Though the hypoglycemic effect of Momordica charantia is well documented in diabetic animal models (Akhtar et al., 1981; Sarkar et al., 1996; Miura et al., 2001; Kar et al., 2003), it is not known whether this effect is accompanied by the restoration of reproductive functions such as estrous cyclicity. Hence, this study also aimed to elucidate the potential of Momordica charantia and Andrographis paniculata in the restoration of impaired estrous cyclicity in alloxan-induced diabetic rats. 2. Materials and methods 2.1. Plant material The elongated fruits of Momordica charantia were purchased from the local market and leaves of Andrographis paniculata were gathered from the campus of Pampanga Agricultural College, Magalang, Pampanga, Philippines. Plant materials were authenticated at the Botanical Herbarium, Museum of Natural History, University of the Philippines, Los Banos, College, Laguna, Philippines with accession numbers 67268 and 67267 for Momordica charantia and Andrographis paniculata, respectively. 2.2. Preparation of fruit juice and decoction Fruits of Momordica charantia were washed, sliced and placed in a juicer (Sanyo S6-J6, Osaka, Japan) to obtain the fruit juice. About 20 g Andrographis paniculata leaves were boiled in 100 ml of water for 5 min and decoction was filtered.
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2.3. Animals and treatment 2.3.1. Animals Female Sprague–Dawley rats weighing 140–150 g were obtained from the Research and Biotechnology Division, St. Lukes Medical Center, Quezon City, Philippines and were individually caged. Estrous cycles were recorded daily. Food and water were given ad libitum. Rats were maintained on normal laboratory chow diet containing 16% protein. 2.3.2. Induction of diabetes Only rats showing at least three consecutive estrous cycles were selected. Estrous cycle was monitored daily via vaginal cytology. Alloxan, dissolved in saline was injected intraperitoneally, at a dose of 125 mg/kg body weight (BW). The induction of alloxan-induced diabetes was confirmed by determining the urinary and glucose levels. 2.3.3. Treatments Twenty rats were used in this study at five rats per group. However, 15 were alloxan-induced diabetics and they were randomly assigned to three groups 4 days after alloxan injection. Only rats positive for urinary glucose and with a blood glucose level above 300 mg/dl including polydipsia, polyuria and polyphagia were used. The first was given Momordica charantia juice, the second with Andrographis paniculata decoction, and the third served as diabetic positive control. The fourth group served as the non-diabetic control. At 20 ml/kg BW per day, groups one and two were orally given Momordica charantia juice and Andrographis paniculata decoction, respectively. Half of the dose given at 800 h and the other half at 1600 h. Groups three and four were not given Momordica charantia juice or with Andrographis paniculata decoction. 2.3.4. Data collection The BW, daily feed and liquid intake, and blood glucose levels were measured at day 1 (D1), day 7 (D7), day 12 (D12), day 17 (D17), day 22 (D22) and day 27 (D27). For blood glucose levels, it was determined using Glucose Kit Reagent (Biosystems S.A., Barcelona, Spain) from blood collected from the tip of the tail. Samples were run in triplicate. Urinary glucose was detected from D1–D12, D17, D22 and D27 using urine test strips. 2.4. Statistical analysis All data were expressed as mean ± S.E.M. The effects of Momordica charantia juice or with Andrographis paniculata decoction on BW, feed intake, liquid intake and blood glucose levels were determined using one-way analysis of variance (Graph Pad In Stat, Graph Pad Software Inc., San Diego, CA, USA) followed by post-hoc Tukey–Kramer multiple comparisons test. The herbs’ effects on estrous cycles were analyzed using Student’s t-test.
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Fig. 1. Mean body weight (A), feed intake (B), liquid intake (C) and blood glucose levels (D) of non-diabetic control and alloxan-induced diabetic rats treated and non-treated with Momordica charantia juice and Andrographis paniculata decoction (n = 5). Values are means ± S.E.M. Values with different letters are significantly different (* P < 0.05; ** P < 0.01) from each other in each time point studied (Tukey–Kramer multiple comparisons test after ANOVA).
3. Results Fig. 1 presents BW (Fig. 1A), feed intake (Fig. 1B), and liquid intake (Fig. 1C) of the alloxan-induced diabetic rats treated with Momordica charantia juice and Andrographis paniculata decoction, the diabetic positive control, and of the non-diabetic control. Prior to alloxan treatment, all groups had comparable BW, feed intake and liquid intake. Momordica charantia juice and Andrographis paniculata decoction significantly increased the BW (P < 0.01) and lowered the feed (P < 0.05) and liquid (P < 0.01) intakes of the alloxan-induced diabetic rats compared with diabetic positive control on D17–D27. However, compared with the non-diabetic control, Momordica charantia- and Andrographis paniculata-treated rats had significantly lower BW (P < 0.01) but similar feed and liquid intakes. The diabetic positive control showed urinary glucose from D4 or D5 until D27. Conversely, the urinary glucose was not found in non-diabetic control from D1 to D27. The blood glucose levels in Momordica charantia juice- and Andrographis paniculata decoction-treated rats decreased markedly from D12 to D27 compared to diabetic positive control (Fig. 1D; P < 0.01). Compared with non-diabetic control, the blood glucose levels remained higher from D12 to D27 but no significant difference was observed. As shown in Fig. 2, rats in all groups displayed an estrous cycle of 4–5 days prior to alloxan administration (non-diabetic control: 4.4 ± 0.20 days; diabetic positive control: 4.6 ± 0.20 days; Momordica charantia juice-treated rats: 4.3 ± 0.10 days; Andrographis paniculata decoction-treated rats: 4.8 ± 0.23 days). The mean estrous cycle length of diabetic positive control was extended (P < 0.05) after alloxan administration
Fig. 2. Mean estrous cycle of non-diabetic control and alloxan-induced diabetic rats treated or non-treated with Momordica charantia juice or Andrographis paniculata decoction (n = 5). * P < 0.05 vs. before alloxan administration.
from 4.6 ± 0.20 to 7.65 ± 0.55 days. For Momordica charantia juice- and Andrographis paniculata decoction-treated rats, mean estrous cycle lengthened from 4.3 ± 0.10 to 5.24 ± 0.13 days and from 4.8 ± 0.23 to 5.36 ± 0.10 days, respectively but their differences were not significant. 4. Discussion Alloxan is cytotoxic to the pancreatic  cells thus it is an effective diabetes-induction agent. It has been widely used to induce diabetes mellitus in experimental animal models allowing investigation of hypoglycemic agents in the treatment of diabetes (Kar et al., 2003; Jayakar et al., 2004). Alloxan injection consistently produced symptoms characteristic of diabetes mellitus including hyperglycemia, decreased insulin levels, polyuria and weight loss.
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Significant reduction of blood glucose levels in alloxaninduced diabetic rats treated with Momordica charantia confirms previous reports demonstrating the anti-hypoglycemic effect of Momordica charantia in diabetic rat, mouse and rabbit models (Akhtar et al., 1981; Sarkar et al., 1996; Miura et al., 2001; Kar et al., 2003). Specifically, our results agree with the studies of Kar and colleagues (Kar et al., 2003) indicating that the blood glucose lowering effect of Momordica charantia in alloxan-induced diabetic rats occurs within 2 weeks from the onset of Momordica charantia treatment. The treatment with Andrographis paniculata led to a significant reduction of blood glucose levels indicating a hypoglycemic effect. These data support previous investigations (Zhang and Tan, 2000) in streptozocin-induced diabetic rats where Andrographis paniculata extract at 400 mg/kg BW given twice a day significantly reduced fasting serum glucose at the end of 14-day period. In our experiment, it took a shorter period to attain a significant reduction in the blood glucose level. This discrepancy could be due to the difference in the diabetes induction agents, for they used streptozocin whereas we used alloxan. Sexton and Jarow (1997) pointed that in studies using diabetic rat models, results could be influenced by diabetic induction agents such streptozocin or alloxan. There was also a difference in the sex and weight of rats, and the kind of preparation and doses. They used male 200–250 g rats while we used female 140–150 g rats. They administered Andrographis paniculata extract at 400 mg/kg while we used a decoction at 20 ml/kg. Another factor is that plant metabolites vary considerably in response to soil type, humidity and climate prevailing during growth (Evans, 2002). Nevertheless, this is the first report showing evidence that Andrographis paniculata possesses an anti-hyperglycemic effect in a rat model treated with alloxan as a diabetic induction agent. The hypoglycemic potential of Momordica charantia in the present study could be explained by the mechanisms previously described by several authors in a diabetic animal model (Welihinda and Karunanayake, 1986; Welihinda et al., 1986; Ahmed et al., 1998). As such, Momordica charantia increases the renewal of -cells in the pancreas or may permit the recovery of partially destroyed -cells (Ahmed et al., 1998) and stimulates pancreatic insulin secretion (Welihinda et al., 1982). These could likely explain the significant increase in the plasma insulin level when streptozocin-induced diabetic rats were treated with Momordica charantia (Sharma et al., 1995). Furthermore, Momordica charantia displays insulin-like properties (Ng et al., 1986), remarkably stimulates glycogen storage by the liver (Welihinda et al., 1986) and improves peripheral glucose uptake (Welihinda and Karunanayake, 1986). On the other hand, when streptozocin-induced diabetic rats were given Andrographis paniculata, the plasma insulin level did not increase (Zhang and Tan, 2000) suggesting that Andrographis paniculata does not act as an insulin secretagogue. Andrographolide, a principle with the highest content in Andrographis paniculata (Shen et al., 2002) is suggested to increase glucose utilization in peripheral tissue via an insulin-dependent mechanism (Yu et al., 2003). Taken together, the hypoglycemic effects of Andrographis paniculata may be attributed in part
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to its ability to increase glucose metabolism in a diabetic rat model. Glucose transporters mediate glucose uptake into the tissues, and, of all the glucose transporters, only glucose transporter 4 is insulin-responsive (Gorovits and Charron, 2003). A decreasing expression of glucose transporter 4 mRNA and protein caused a reduction in insulin-mediated glucose uptake in diabetes (Berger et al., 1989). Andrographolide administration in streptozocin-induced diabetic mice increased glucose transporter 4 (Yu et al., 2003). It is conceivable that Andrographis paniculata can enhance the glucose uptake through its effects on glucose transporter 4 gene expression. In the light of these data using streptozocin as a diabetic induction agent, the mechanism by which Andrographis paniculata reduces hyperglycemia in alloxan-induced diabetic model needs further studies. Impaired estrous cyclicity was evident in alloxan-induced diabetic rats. This impairment supports several reports that diabetes mellitus impairs reproductive functions (Griffin et al., 1994; Angell et al., 1996; Cagampang et al., 1997; Sexton and Jarow, 1997; Steger and Rabe, 1997). In fact, diabetes mellitus suppressed luteinizing hormone secretion in streptozocin-induced diabetic rats (Cagampang et al., 1997). In the same manner, alloxan suppressed luteinizing hormone secretion in female rats (Kinoshita et al., 2004). Additionally, diabetic gilts did not exhibit estrous or ovulation (Cox et al., 1994). Luteinizing hormone surge was also absent in immature alloxan-induced diabetic rats that led to anovulation (Kirchick et al., 1978). In the present study, the remarkable loss of body weight in diabetic rats could likely explain the impaired estrous cyclicity since it is generally accepted that the loss of BW causes a reduction in luteinizing hormone release in rats (Campbell et al., 1977). Treating alloxan-induced diabetic rats with Momordica charantia and Andrographis paniculata significantly increased BW and reduced blood glucose levels. Both of these results could be major contributing factors to the subsequent normalization of the prolonged estrous cycles. However, further investigations are required to address this issue. In summary, the results of the present study show that Andrographis paniculata possesses anti-diabetic potential in alloxaninduced diabetic rats. Moreover, this is the first physiological evidence that the anti-diabetic potentials of Momordica charantia and Andrographis paniculata restore the impaired estrous cyclicity in alloxan-induced diabetic rat model. Acknowledgements The authors are thankful to Dr. Zosimo Battad, President, Pampanga Agricultural College, Magalang, Pampanga, Philippines for providing necessary facilities and support. References Ahmed, I., Adeghate, E., Sharma, A.K., Pallot, D.J., Singh, J., 1998. Effects of Momordica charantia fruit juice on islet morphology in the pancreas of the streptozotocin-diabetic rat. Diabetes Research and Clinical Practice 40, 145–151.
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Anti-diabetic potentials of Momordica charantia and Andrographis paniculata and their effects on estrous cyclicity of alloxan-induced diabetic rats B.A.S. Reyes a,b,∗ , N.D. Bautista c , N.C. Tanquilut b , R.V. Anunciado d , A.B. Leung c , G.C. Sanchez b , R.L. Magtoto a,c , P. Castronuevo e , H. Tsukamura f , K.-I. Maeda f a
Thomas Jefferson University, Department of Neurosurgery, Farber Institute for Neurosciences, Philadelphia, Pennsylvania 19107, USA b Institute of Veterinary Medicine and Zootechnics, Pampanga Agricultural College, Magalang 2011, Pampanga, Philippines c Institute of Animal Science, Pampanga Agricultural College, Magalang 2011, Pampanga, Philippines d College of Veterinary Medicine, University of Philippines, Los Banos, Laguna, Philippines e Department of Biology, Kutztown University, Kutztown, PA 19530, USA f Graduate School of Bioagricultural Science, Nagoya University, Nagoya 464-8601, Japan Received 10 March 2005; received in revised form 14 October 2005; accepted 18 October 2005 Available online 18 November 2005
Abstract Momordica charantia and Andrographis paniculata are the commonly used herbs by the diabetic patients in Pampanga, Philippines. While the anti-diabetic potential of Momordica charantia is well established in streptozocin- or alloxan-induced diabetic animals, the anti-diabetic potential of Andrographis paniculata in alloxan-induced diabetic rat is not known. Neither the effects of these herbs on estrous cyclicity of alloxaninduced diabetic rats are elucidated. Thus, in these experiments, Momordica charantia fruit juice or Andrographis paniculata decoction was orally administered to alloxan-induced diabetic rats. Rats that were treated with Momordica charantia and Andrographis paniculata had higher body weight (BW) compared with diabetic positive control (P < 0.01) from day 22 to day 27 (D27) but exhibited lower BW than the non-diabetic control (P < 0.05). These rats had lower feed (P < 0.05) and liquid intakes (P < 0.01) compared with diabetic positive control from day 17 to D27, but similar with the non-diabetic control. The blood glucose levels in these groups were significantly reduced from day 12 to D27 compared with diabetic positive control (P < 0.01), however, comparable with non-diabetic control. The diabetic positive control had extended mean estrous cycles (8 days) compared to Momordica charantia and Andrographis paniculata-treated diabetic rats (5 days; P < 0.05). Our results suggest that the anti-diabetic potentials of Momordica charantia and Andrographis paniculata could restore impaired estrous cycle in alloxan-induced diabetic rats. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Momordica charantia; Andrographis paniculata; Diabetes; Alloxan; Estrous cycle
1. Introduction Diabetes mellitus is the most common endocrine disease (Sexton and Jarow, 1997) and a predominant health concern affecting 16 million Americans (Yeh et al., 2003). It affected 2–3% of the total world population in 1995 (Felig et al., 1995) and had an increasing prevalence worldwide in 1998 (Alberti and Zimmet, 1998). Diabetes mellitus leads to metabolic abnormalities and is characterized by hyperglycemia resulting from
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defects in insulin secretion, insulin action or both (Atkinson and Maclaren, 1994; Yki-Jarvinen, 1994; Teixeira et al., 2000). Insulin-dependent diabetes mellitus or Type 1 is conventionally treated with exogenous insulin while the non-insulin-dependent diabetes mellitus or Type 2 is treated with oral hypoglycemic agents such as sulphonylureas and biguanides among others (Felig et al., 1995; Rosak, 2002). Complementary and alternative medicine is widely used (Eisenberg et al., 1998; Maclennan et al., 1996; Payne, 2001) and, in the Philippines diabetes mellitus is commonly treated using medicinal plants (De Padua et al., 1997). Momordica charantia and Andrographis paniculata are commonly used herbs in the province of Pampanga, Philippines.
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Momordica charantia is a member of Cucurbitaceae, commonly known as ku gua, karela, bittergourd or bitter melon. It is the most popular herbal resource (Marles and Farnsworth, 1995) and is often used to treat diabetes (Arvigo and Balick, 1993). The anti-diabetic potential of Momordica charantia is well established in streptozocin- or alloxan-induced diabetic rats, mice and rabbit (Akhtar et al., 1981; Sarkar et al., 1996; Kar et al., 2003), genetically diabetic mice (Miura et al., 2001) and in humans with Type 2 diabetes (Srivastava et al., 1993). Andrographis paniculata (Burm F) Nees belongs to family Acanthaceae, commonly known as “King of Bitters” and has been used to treat various diseases (Vedavathy and Rao, 1991; Caceres et al., 1997; Kumar et al., 2004). Unlike Momordica charantia, the hypoglycemic effects of Andrographis paniculata have only been studied in streptozotocin-induced diabetic rats (Zhang and Tan, 2000) and in normal rabbits (Borhanuddin et al., 1994). Thus, using alloxan-induced diabetic rats, the anti-diabetic potential of Andrographis paniculata was investigated. Diabetes mellitus has been shown to suppress reproductive functions in humans (Griffin et al., 1994; Sexton and Jarow, 1997) and animals (Angell et al., 1996; Cagampang et al., 1997; Steger and Rabe, 1997). Specifically, diabetes mellitus suppresses luteinizing hormone secretion in streptozocin-induced diabetic rats (Cagampang et al., 1997) and disrupts estrous cycle (Cox et al., 1994). Estrous cycle is the period of reproductive cyclicity in animals that usually lasts for 4–5 days in rodents (Pineda, 2003). Though the hypoglycemic effect of Momordica charantia is well documented in diabetic animal models (Akhtar et al., 1981; Sarkar et al., 1996; Miura et al., 2001; Kar et al., 2003), it is not known whether this effect is accompanied by the restoration of reproductive functions such as estrous cyclicity. Hence, this study also aimed to elucidate the potential of Momordica charantia and Andrographis paniculata in the restoration of impaired estrous cyclicity in alloxan-induced diabetic rats. 2. Materials and methods 2.1. Plant material The elongated fruits of Momordica charantia were purchased from the local market and leaves of Andrographis paniculata were gathered from the campus of Pampanga Agricultural College, Magalang, Pampanga, Philippines. Plant materials were authenticated at the Botanical Herbarium, Museum of Natural History, University of the Philippines, Los Banos, College, Laguna, Philippines with accession numbers 67268 and 67267 for Momordica charantia and Andrographis paniculata, respectively. 2.2. Preparation of fruit juice and decoction Fruits of Momordica charantia were washed, sliced and placed in a juicer (Sanyo S6-J6, Osaka, Japan) to obtain the fruit juice. About 20 g Andrographis paniculata leaves were boiled in 100 ml of water for 5 min and decoction was filtered.
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2.3. Animals and treatment 2.3.1. Animals Female Sprague–Dawley rats weighing 140–150 g were obtained from the Research and Biotechnology Division, St. Lukes Medical Center, Quezon City, Philippines and were individually caged. Estrous cycles were recorded daily. Food and water were given ad libitum. Rats were maintained on normal laboratory chow diet containing 16% protein. 2.3.2. Induction of diabetes Only rats showing at least three consecutive estrous cycles were selected. Estrous cycle was monitored daily via vaginal cytology. Alloxan, dissolved in saline was injected intraperitoneally, at a dose of 125 mg/kg body weight (BW). The induction of alloxan-induced diabetes was confirmed by determining the urinary and glucose levels. 2.3.3. Treatments Twenty rats were used in this study at five rats per group. However, 15 were alloxan-induced diabetics and they were randomly assigned to three groups 4 days after alloxan injection. Only rats positive for urinary glucose and with a blood glucose level above 300 mg/dl including polydipsia, polyuria and polyphagia were used. The first was given Momordica charantia juice, the second with Andrographis paniculata decoction, and the third served as diabetic positive control. The fourth group served as the non-diabetic control. At 20 ml/kg BW per day, groups one and two were orally given Momordica charantia juice and Andrographis paniculata decoction, respectively. Half of the dose given at 800 h and the other half at 1600 h. Groups three and four were not given Momordica charantia juice or with Andrographis paniculata decoction. 2.3.4. Data collection The BW, daily feed and liquid intake, and blood glucose levels were measured at day 1 (D1), day 7 (D7), day 12 (D12), day 17 (D17), day 22 (D22) and day 27 (D27). For blood glucose levels, it was determined using Glucose Kit Reagent (Biosystems S.A., Barcelona, Spain) from blood collected from the tip of the tail. Samples were run in triplicate. Urinary glucose was detected from D1–D12, D17, D22 and D27 using urine test strips. 2.4. Statistical analysis All data were expressed as mean ± S.E.M. The effects of Momordica charantia juice or with Andrographis paniculata decoction on BW, feed intake, liquid intake and blood glucose levels were determined using one-way analysis of variance (Graph Pad In Stat, Graph Pad Software Inc., San Diego, CA, USA) followed by post-hoc Tukey–Kramer multiple comparisons test. The herbs’ effects on estrous cycles were analyzed using Student’s t-test.
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Fig. 1. Mean body weight (A), feed intake (B), liquid intake (C) and blood glucose levels (D) of non-diabetic control and alloxan-induced diabetic rats treated and non-treated with Momordica charantia juice and Andrographis paniculata decoction (n = 5). Values are means ± S.E.M. Values with different letters are significantly different (* P < 0.05; ** P < 0.01) from each other in each time point studied (Tukey–Kramer multiple comparisons test after ANOVA).
3. Results Fig. 1 presents BW (Fig. 1A), feed intake (Fig. 1B), and liquid intake (Fig. 1C) of the alloxan-induced diabetic rats treated with Momordica charantia juice and Andrographis paniculata decoction, the diabetic positive control, and of the non-diabetic control. Prior to alloxan treatment, all groups had comparable BW, feed intake and liquid intake. Momordica charantia juice and Andrographis paniculata decoction significantly increased the BW (P < 0.01) and lowered the feed (P < 0.05) and liquid (P < 0.01) intakes of the alloxan-induced diabetic rats compared with diabetic positive control on D17–D27. However, compared with the non-diabetic control, Momordica charantia- and Andrographis paniculata-treated rats had significantly lower BW (P < 0.01) but similar feed and liquid intakes. The diabetic positive control showed urinary glucose from D4 or D5 until D27. Conversely, the urinary glucose was not found in non-diabetic control from D1 to D27. The blood glucose levels in Momordica charantia juice- and Andrographis paniculata decoction-treated rats decreased markedly from D12 to D27 compared to diabetic positive control (Fig. 1D; P < 0.01). Compared with non-diabetic control, the blood glucose levels remained higher from D12 to D27 but no significant difference was observed. As shown in Fig. 2, rats in all groups displayed an estrous cycle of 4–5 days prior to alloxan administration (non-diabetic control: 4.4 ± 0.20 days; diabetic positive control: 4.6 ± 0.20 days; Momordica charantia juice-treated rats: 4.3 ± 0.10 days; Andrographis paniculata decoction-treated rats: 4.8 ± 0.23 days). The mean estrous cycle length of diabetic positive control was extended (P < 0.05) after alloxan administration
Fig. 2. Mean estrous cycle of non-diabetic control and alloxan-induced diabetic rats treated or non-treated with Momordica charantia juice or Andrographis paniculata decoction (n = 5). * P < 0.05 vs. before alloxan administration.
from 4.6 ± 0.20 to 7.65 ± 0.55 days. For Momordica charantia juice- and Andrographis paniculata decoction-treated rats, mean estrous cycle lengthened from 4.3 ± 0.10 to 5.24 ± 0.13 days and from 4.8 ± 0.23 to 5.36 ± 0.10 days, respectively but their differences were not significant. 4. Discussion Alloxan is cytotoxic to the pancreatic  cells thus it is an effective diabetes-induction agent. It has been widely used to induce diabetes mellitus in experimental animal models allowing investigation of hypoglycemic agents in the treatment of diabetes (Kar et al., 2003; Jayakar et al., 2004). Alloxan injection consistently produced symptoms characteristic of diabetes mellitus including hyperglycemia, decreased insulin levels, polyuria and weight loss.
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Significant reduction of blood glucose levels in alloxaninduced diabetic rats treated with Momordica charantia confirms previous reports demonstrating the anti-hypoglycemic effect of Momordica charantia in diabetic rat, mouse and rabbit models (Akhtar et al., 1981; Sarkar et al., 1996; Miura et al., 2001; Kar et al., 2003). Specifically, our results agree with the studies of Kar and colleagues (Kar et al., 2003) indicating that the blood glucose lowering effect of Momordica charantia in alloxan-induced diabetic rats occurs within 2 weeks from the onset of Momordica charantia treatment. The treatment with Andrographis paniculata led to a significant reduction of blood glucose levels indicating a hypoglycemic effect. These data support previous investigations (Zhang and Tan, 2000) in streptozocin-induced diabetic rats where Andrographis paniculata extract at 400 mg/kg BW given twice a day significantly reduced fasting serum glucose at the end of 14-day period. In our experiment, it took a shorter period to attain a significant reduction in the blood glucose level. This discrepancy could be due to the difference in the diabetes induction agents, for they used streptozocin whereas we used alloxan. Sexton and Jarow (1997) pointed that in studies using diabetic rat models, results could be influenced by diabetic induction agents such streptozocin or alloxan. There was also a difference in the sex and weight of rats, and the kind of preparation and doses. They used male 200–250 g rats while we used female 140–150 g rats. They administered Andrographis paniculata extract at 400 mg/kg while we used a decoction at 20 ml/kg. Another factor is that plant metabolites vary considerably in response to soil type, humidity and climate prevailing during growth (Evans, 2002). Nevertheless, this is the first report showing evidence that Andrographis paniculata possesses an anti-hyperglycemic effect in a rat model treated with alloxan as a diabetic induction agent. The hypoglycemic potential of Momordica charantia in the present study could be explained by the mechanisms previously described by several authors in a diabetic animal model (Welihinda and Karunanayake, 1986; Welihinda et al., 1986; Ahmed et al., 1998). As such, Momordica charantia increases the renewal of -cells in the pancreas or may permit the recovery of partially destroyed -cells (Ahmed et al., 1998) and stimulates pancreatic insulin secretion (Welihinda et al., 1982). These could likely explain the significant increase in the plasma insulin level when streptozocin-induced diabetic rats were treated with Momordica charantia (Sharma et al., 1995). Furthermore, Momordica charantia displays insulin-like properties (Ng et al., 1986), remarkably stimulates glycogen storage by the liver (Welihinda et al., 1986) and improves peripheral glucose uptake (Welihinda and Karunanayake, 1986). On the other hand, when streptozocin-induced diabetic rats were given Andrographis paniculata, the plasma insulin level did not increase (Zhang and Tan, 2000) suggesting that Andrographis paniculata does not act as an insulin secretagogue. Andrographolide, a principle with the highest content in Andrographis paniculata (Shen et al., 2002) is suggested to increase glucose utilization in peripheral tissue via an insulin-dependent mechanism (Yu et al., 2003). Taken together, the hypoglycemic effects of Andrographis paniculata may be attributed in part
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to its ability to increase glucose metabolism in a diabetic rat model. Glucose transporters mediate glucose uptake into the tissues, and, of all the glucose transporters, only glucose transporter 4 is insulin-responsive (Gorovits and Charron, 2003). A decreasing expression of glucose transporter 4 mRNA and protein caused a reduction in insulin-mediated glucose uptake in diabetes (Berger et al., 1989). Andrographolide administration in streptozocin-induced diabetic mice increased glucose transporter 4 (Yu et al., 2003). It is conceivable that Andrographis paniculata can enhance the glucose uptake through its effects on glucose transporter 4 gene expression. In the light of these data using streptozocin as a diabetic induction agent, the mechanism by which Andrographis paniculata reduces hyperglycemia in alloxan-induced diabetic model needs further studies. Impaired estrous cyclicity was evident in alloxan-induced diabetic rats. This impairment supports several reports that diabetes mellitus impairs reproductive functions (Griffin et al., 1994; Angell et al., 1996; Cagampang et al., 1997; Sexton and Jarow, 1997; Steger and Rabe, 1997). In fact, diabetes mellitus suppressed luteinizing hormone secretion in streptozocin-induced diabetic rats (Cagampang et al., 1997). In the same manner, alloxan suppressed luteinizing hormone secretion in female rats (Kinoshita et al., 2004). Additionally, diabetic gilts did not exhibit estrous or ovulation (Cox et al., 1994). Luteinizing hormone surge was also absent in immature alloxan-induced diabetic rats that led to anovulation (Kirchick et al., 1978). In the present study, the remarkable loss of body weight in diabetic rats could likely explain the impaired estrous cyclicity since it is generally accepted that the loss of BW causes a reduction in luteinizing hormone release in rats (Campbell et al., 1977). Treating alloxan-induced diabetic rats with Momordica charantia and Andrographis paniculata significantly increased BW and reduced blood glucose levels. Both of these results could be major contributing factors to the subsequent normalization of the prolonged estrous cycles. However, further investigations are required to address this issue. In summary, the results of the present study show that Andrographis paniculata possesses anti-diabetic potential in alloxaninduced diabetic rats. Moreover, this is the first physiological evidence that the anti-diabetic potentials of Momordica charantia and Andrographis paniculata restore the impaired estrous cyclicity in alloxan-induced diabetic rat model. Acknowledgements The authors are thankful to Dr. Zosimo Battad, President, Pampanga Agricultural College, Magalang, Pampanga, Philippines for providing necessary facilities and support. References Ahmed, I., Adeghate, E., Sharma, A.K., Pallot, D.J., Singh, J., 1998. Effects of Momordica charantia fruit juice on islet morphology in the pancreas of the streptozotocin-diabetic rat. Diabetes Research and Clinical Practice 40, 145–151.
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