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Possible mechanism of antihyperglycemic effect of Azadirachta indica leaf extract: Part V
Journal of Ethnopharmacology 67 (1999) 373 – 376 www.elsevier.com/locate/jethpharm
Short communication
Possible mechanism of antihyperglycemic effect of Azadirachta indica leaf extract: Part V R.R. Chattopadhyay Biometry Research Unit, Indian Statistical Institute, 203, Barrackpore Trunk Road, Calcutta 700 035, India Received 17 April 1998; received in revised form 11 May 1999; accepted 17 May 1999
Abstract Effect of Azadirachta indica leaf extract on serotonin inhibition in glucose mediated insulin release in rat pancreas was studied in vitro to elucidate the possible mechanism of antihyperglycemic effect of A. indica leaf extract. A. indica leaf extract blocks significantly (PB0.05) the inhibitory effect of serotonin on insulin secretion mediated by glucose. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Azadirachta indica; Antihyperglycemic activity; Insulin releasing pancreatic effect
1. Introduction Azadirachta indica A. Juss. (Meliaceae; Neem) is an indigenous plant widely available in India and Burma. Different parts of this plant have been reported to have antiseptic, wound healing, skin disease curing and antiulcer activity (Kirtikar and Basu, 1933; Chopra et al., 1956). Preliminary studies revealed that water soluble portion of alcoholic extract of A. indica leaves possessed significant antiinflammatory, antifertility, hypolipidemic and hepatoprotective activity (Chattopadhyay, 1993a, 1995; Chattopadhyay et al., 1987a, 1992). Significant antihyperglycemic effect of A. inclica leaf extract against glucose fed and adrenaline induced hyperglycemic rats and antiserotonin activity in rat uterus and fundal strip
preparations have also been reported (Chattopadhyay et al., 1986, 1987b). Possible mechanisms of antihyperglycemic effect of A. indica leaf extract on various experimental models have been studied (Chattopadhyay, 1993b,c, 1996; Chattopadhyay et al., 1993d). Results of the important findings in relation to blood sugar lowering effect of A. indica leaf extract reveals that the antihyperglycemic effect of A. indica may be due to its antiserotonin activity. Relevant literature reveals that serotonin may be present as an intracellular pool in the pancreatic islet cell which is effective in blocking insulin secretion (Feldman et al., 1972). The literature further reveals that an alpha adrenergic blocking agent like phentolamine, blocks the inhibitory effect of serotonin on glucose and dibutyl cyclic
0378-8741/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 9 9 ) 0 0 0 9 4 - X
374
R.R. Chattopadhyay / Journal of Ethnopharmacology 67 (1999) 373–376
AMP mediated insulin release. This indicates that serotonin exerts the inhibitory effect on insulin output via the alpha adrenergic system inhibiting the generation of cyclic AMP (Feldman and Libovitz, 1970). The present investigation has therefore been designed to study the effect of A. indica leaf extract on serotonin inhibition in glucose mediated insulin release in rat pancreas in vitro with a view to elucidate the possible mechanism of the antihyperglycemic effect of A. indica leaf extract.
2. Materials and methods
2.1. Collection of plant material Fresh matured leaves of A. indica were collected from the Institute’s garden and were identified by Professor S.K. Maitra (Pharmacognosy expert), School of Tropical Medicine, Calcutta. At the time of collection standard herbarium record sheets were completed with the name of the collector, collection number, specimen no., date, locality and local name.
2.2. Extraction of plant material Air-dried powder (1 kg) of A. indica leaves were extracted by percolation at room temperature with 70% EtOH. Leaf extract of A. indica was concentrated under reduced pressure (bath temperature 50°C) and finally dried in a vacuum desicator (yield=105 g of crude extract). The residue was dissolved in distilled water and filtered. The filtrate was evaporated to dryness (N1). The dried mass (yield = 50.2 g) was diluted with normal saline and used in experiments.
2.3. Pancreas incubation system Rats were anaesthetised with pentobarbital (40 mg/kg, i.p.) The pancreas was removed and cut into segments weighing 20 – 30 mg. Each piece of pancreas undergoes two sequential 15 min incubation periods. The first incubation is carried out in basal media (initial incubation) while the second incubation is carried out in media containing the
test substance (A. indica leaf extract, 25 mg/ml) added to the basal media (treatment incubation). The basal media is Kreb–Ringer bicarbonate buffer with 0.005 M pyruvate, 0.005 M fumerate, 0.005 M glutamate, 60 mg/100 ml glucose, and 400 mg/100 ml bovine serum albumin (BSA). All incubations were performed at 37°C in an atmosphere of 95% O2 –5% CO2. The pH of the buffer was 7.4. At the end of each incubation, aliquots from incubation media were placed in chilled tubes and assayed for insulin by the RIA technique (Feldman and Libovitz, 1970). The difference in insulin release between the treatment and initial incubations for each piece designated as D insulin release. The method of expressing the data (D insulin) was used because of the nonuniform distribution of the islet cells throughout the pancreas. Therefore each piece served as its own control and the data were more meaningful, since each response was relative to the initial secretion of insulin from that piece of tissue.
2.4. Isolation and identification of compounds The N1 fraction was successively extracted by Soxhlet with CHCl3 and EtOH. The alcohol extract N2 on further fractionation led to the separation of ethylacetate (N3) and normal-butanol (N4) soluble components which constituted 6.74 and 30.30 g of total fraction, respectively. Normal-butanol extract on chromotagraphic separation eluting with MeOH (purified on Sephadex LH-20) isolating compound I (75 mg) and an enriched fraction 738 mg mainly constituted compounds II (64 mg); III (330 mg); IV (90 mg) and V (22 mg) with MeOH:H2O in different ratio. Ethylacetate fraction from the crude extract purified on Sephadex LH-20 yielded the following compounds I (18 mg); IV (15 mg); V (26 mg) and VI (12 mg).
2.5. Statistical analysis The data were expressed as mean9 S.E.M. of six observations and statistically assessed by anal-
R.R. Chattopadhyay / Journal of Ethnopharmacology 67 (1999) 373–376
375
ysis of variance (ANOVA) F-test. The difference between treated and control animals were made either by Student’s t or Dunnet’s t-test (Scheff’e, 1953).
Compound IV: Kaempferol-3-O-rutinoside Compound V: Kaempferol-3-O-b-D-glucoside Compound VI: Quercetin-3-O-a-L-rhamnoside
3. Results
4. Discussion
Fig. 1 illustrates the insulin release induced by 3 mg/ml of glucose and the inhibition of this insulin release by serotonin (10 − 4 M). A. indica leaf extract (25 mg/ml) potentiates the glucose mediated insulin release and prevented the inhibitory effect of serotonin on insulin release induced by glucose. Chemical analysis revealed that the extract contains the following six compounds.
Release of insulin from beta cells of pancreas is influenced by various factors. 1. Parasympathetic stimulation 6ia vagus nerve increases insulin release (Best and Tailor, 1986). 2. Sympathetic stimulation via splanchnic nerve increases insulin release. This effect is exerted via alpha receptors. Agonists that affect primarily the beta receptors actually stimulate the release of insulin via cAMP. Under ordinary circumstances the alpha receptors mediated inhibitory effect predominates (Viswanathan et al., 1978).
Compound I: Quercetin-3-O-b-D-glucoside Compound II: Myricetin-3-O-rutinoside Compound III: Quercetin-3-O-rutinoside
Fig. 1. Effect of Azadirachta indica (25 mg/ml) on glucose mediated insulin release and the antagonistic effect on serotonin inhibition in insulin release mediated by glucose, n= 6. Group B is significantly different (P B 0.05) from control group A but group C is not. Group D is also significantly different (PB 0.05) from group B. But pairs of group AC, AD and CD are not.
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R.R. Chattopadhyay / Journal of Ethnopharmacology 67 (1999) 373–376
3. Serotonin has been reported to have an inhibitory effect on insulin release in response to adrenaline injection or glucose load (Feldman et al., 1972). From the observed findings it was noted that A. indica leaf extract blocks significantly the inhibitory effect of serotonin on insulin release mediated by glucose. Thus the blood sugar lowering activity of A. indica leaf extract may be due to its insulin releasing pancreatic effect. Chemical analysis revealed that the extract contains the six compounds which are collectively found to be responsible for blood sugar lowering activity (Chattopadhyay and Poddar, 1998). Inspite of these results indicating the insulin releasing effect in the antihyperglycemic activity of A. indica further studies are needed both on the extract and/or its chemical constituents.
Acknowledgements The author wishes to acknowledge Dr T.K. Basu, Head of the Unit for his cooperation and help.
References Best, C.H., Tailor, N.B., 1986. Physiological Basis of Medical Practice, 11th edition. Williums and Wilkins, Baltimore. Chattopadhyay, R.R., 1993a. Effect of Azaclirachta indica leaf extract on oestrous cycle of rats. Environment and Ecology 11 (4), 958 – 960. Chattopadhyay, R.R., 1993b. Possible mechanism of antihyperglycemic effect of Azadirachta indica leaf extract, Part: I. Fitoterapia (Italy) LXIV (4), 332–336. Chattopadhyay, R.R., 1993c. Possible mechanism of antihyperglycemic effect of Azadirachta indica leaf extract, Part: II. Journal of Ecobiology 4 (4), 309–312.
.
Chattopadhyay, R.R., 1995. Hypolipidemic activity of Azadirachta indica leaf extract in rats. Proceedings of Indian National Science Academy B61 (4), 281 – 284. Chattopadhyay, R.R., 1996. Possible mechanism of antibyperglycemic effect of Azadirachta indica leaf extract, Part: IV. General Pharmacology (USA) 27 (3), 431 – 434. Chattopadhyay, R.R., Poddar, G., 1998. Phytochemical screening of Azadirachta indica leaf extract as blood sugar lowering agent. Phytotherapy Research, Communicated. Chattopadhyay, R.R., Chattopadhyay, R.N., Nandy, A.K., Poddar, G., Maitra, S.K., 1986. Antiserotonin activity of a fraction of fresh leaves of Azadirachta indica (Beng. Neem). Bulletin of Calcutta School of Tropical Medicine 34, 9 – 12. Chattopadhyay, R.R., Chattopadhyay, R.N., Nandy, A.K., Poddar, G., Maitra, S.K., 1987a. Antiinflammatory activity of a fraction of fresh leaves of Azadirachta indica (Beng. Neem). Bulletin of Calcutta School of Tropical Medicine 35, 6 – 8. Chattopadhyay, R.R., Chattopadhyay, R.N., Nandy, A.K., Poddar, G., Maitra, S.K., 1987b. Preliminary report on antihyperglycemic effect of a fraction of fresh leaves of Azadirachta indica (Beng. Neem). Bulletin of Calcutta School of Tropical Medicine 35, 29 – 35. Chattopadhyay, R.R., Sarkar, S.K., Ganguly, S., Banerjee, R.N., Basu, T.K., 1992. Hepatoprotective activity of Azadirachta indica leaf extract against paracetamol-induced hepatic damage in rats. Indian Journal of Experimental Biology 30, 738 – 740. Chattopadhyay, R.R., Chattopadhyay, R.N., Maitra, S.K., 1993d. Possible mechanism of antiLyperglycemic effect of Azadirachta indica leaf extract, Part: III. Fitoterapia (Italy) LXIV (6), 535 – 538. Chopra, R.N., Nayar, S.L., Chopra, I.C., 1956. Glossary of Indian Medicinal Plants. CSIR, New Delhi, pp. 31 – 43. Feldman, J.M., Libovitz, H.E., 1970. Mechanism of epinephrine and serotonin inhibition of insulin release in golden hamster in vitro. Diabetes 19, 480 – 487. Feldman, J.M., Quickel, K.E., Libovitz, H.E., 1972. Potentiation of insulin secretion by serotonin antagonists. Diabetes 21, 779 – 785. Kirtikar, K.R., Basu, B.D., 1933. Indian Medicinal Plants. Basu, Allahabad, pp. 3 – 14. Scheff’e, H.E., 1953. A method for judging all contrasts in the analysis of variance. Biometrika 40, 87 – 113. Viswanathan, Mohan, Snehalata, C., Ramachandran, A., Viswanathan, M., 1978. Serotonin and Diabetes Mellitus. Journal of Diabetic Association (India) XVIII, 171 – 174.
Short communication
Possible mechanism of antihyperglycemic effect of Azadirachta indica leaf extract: Part V R.R. Chattopadhyay Biometry Research Unit, Indian Statistical Institute, 203, Barrackpore Trunk Road, Calcutta 700 035, India Received 17 April 1998; received in revised form 11 May 1999; accepted 17 May 1999
Abstract Effect of Azadirachta indica leaf extract on serotonin inhibition in glucose mediated insulin release in rat pancreas was studied in vitro to elucidate the possible mechanism of antihyperglycemic effect of A. indica leaf extract. A. indica leaf extract blocks significantly (PB0.05) the inhibitory effect of serotonin on insulin secretion mediated by glucose. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Azadirachta indica; Antihyperglycemic activity; Insulin releasing pancreatic effect
1. Introduction Azadirachta indica A. Juss. (Meliaceae; Neem) is an indigenous plant widely available in India and Burma. Different parts of this plant have been reported to have antiseptic, wound healing, skin disease curing and antiulcer activity (Kirtikar and Basu, 1933; Chopra et al., 1956). Preliminary studies revealed that water soluble portion of alcoholic extract of A. indica leaves possessed significant antiinflammatory, antifertility, hypolipidemic and hepatoprotective activity (Chattopadhyay, 1993a, 1995; Chattopadhyay et al., 1987a, 1992). Significant antihyperglycemic effect of A. inclica leaf extract against glucose fed and adrenaline induced hyperglycemic rats and antiserotonin activity in rat uterus and fundal strip
preparations have also been reported (Chattopadhyay et al., 1986, 1987b). Possible mechanisms of antihyperglycemic effect of A. indica leaf extract on various experimental models have been studied (Chattopadhyay, 1993b,c, 1996; Chattopadhyay et al., 1993d). Results of the important findings in relation to blood sugar lowering effect of A. indica leaf extract reveals that the antihyperglycemic effect of A. indica may be due to its antiserotonin activity. Relevant literature reveals that serotonin may be present as an intracellular pool in the pancreatic islet cell which is effective in blocking insulin secretion (Feldman et al., 1972). The literature further reveals that an alpha adrenergic blocking agent like phentolamine, blocks the inhibitory effect of serotonin on glucose and dibutyl cyclic
0378-8741/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 9 9 ) 0 0 0 9 4 - X
374
R.R. Chattopadhyay / Journal of Ethnopharmacology 67 (1999) 373–376
AMP mediated insulin release. This indicates that serotonin exerts the inhibitory effect on insulin output via the alpha adrenergic system inhibiting the generation of cyclic AMP (Feldman and Libovitz, 1970). The present investigation has therefore been designed to study the effect of A. indica leaf extract on serotonin inhibition in glucose mediated insulin release in rat pancreas in vitro with a view to elucidate the possible mechanism of the antihyperglycemic effect of A. indica leaf extract.
2. Materials and methods
2.1. Collection of plant material Fresh matured leaves of A. indica were collected from the Institute’s garden and were identified by Professor S.K. Maitra (Pharmacognosy expert), School of Tropical Medicine, Calcutta. At the time of collection standard herbarium record sheets were completed with the name of the collector, collection number, specimen no., date, locality and local name.
2.2. Extraction of plant material Air-dried powder (1 kg) of A. indica leaves were extracted by percolation at room temperature with 70% EtOH. Leaf extract of A. indica was concentrated under reduced pressure (bath temperature 50°C) and finally dried in a vacuum desicator (yield=105 g of crude extract). The residue was dissolved in distilled water and filtered. The filtrate was evaporated to dryness (N1). The dried mass (yield = 50.2 g) was diluted with normal saline and used in experiments.
2.3. Pancreas incubation system Rats were anaesthetised with pentobarbital (40 mg/kg, i.p.) The pancreas was removed and cut into segments weighing 20 – 30 mg. Each piece of pancreas undergoes two sequential 15 min incubation periods. The first incubation is carried out in basal media (initial incubation) while the second incubation is carried out in media containing the
test substance (A. indica leaf extract, 25 mg/ml) added to the basal media (treatment incubation). The basal media is Kreb–Ringer bicarbonate buffer with 0.005 M pyruvate, 0.005 M fumerate, 0.005 M glutamate, 60 mg/100 ml glucose, and 400 mg/100 ml bovine serum albumin (BSA). All incubations were performed at 37°C in an atmosphere of 95% O2 –5% CO2. The pH of the buffer was 7.4. At the end of each incubation, aliquots from incubation media were placed in chilled tubes and assayed for insulin by the RIA technique (Feldman and Libovitz, 1970). The difference in insulin release between the treatment and initial incubations for each piece designated as D insulin release. The method of expressing the data (D insulin) was used because of the nonuniform distribution of the islet cells throughout the pancreas. Therefore each piece served as its own control and the data were more meaningful, since each response was relative to the initial secretion of insulin from that piece of tissue.
2.4. Isolation and identification of compounds The N1 fraction was successively extracted by Soxhlet with CHCl3 and EtOH. The alcohol extract N2 on further fractionation led to the separation of ethylacetate (N3) and normal-butanol (N4) soluble components which constituted 6.74 and 30.30 g of total fraction, respectively. Normal-butanol extract on chromotagraphic separation eluting with MeOH (purified on Sephadex LH-20) isolating compound I (75 mg) and an enriched fraction 738 mg mainly constituted compounds II (64 mg); III (330 mg); IV (90 mg) and V (22 mg) with MeOH:H2O in different ratio. Ethylacetate fraction from the crude extract purified on Sephadex LH-20 yielded the following compounds I (18 mg); IV (15 mg); V (26 mg) and VI (12 mg).
2.5. Statistical analysis The data were expressed as mean9 S.E.M. of six observations and statistically assessed by anal-
R.R. Chattopadhyay / Journal of Ethnopharmacology 67 (1999) 373–376
375
ysis of variance (ANOVA) F-test. The difference between treated and control animals were made either by Student’s t or Dunnet’s t-test (Scheff’e, 1953).
Compound IV: Kaempferol-3-O-rutinoside Compound V: Kaempferol-3-O-b-D-glucoside Compound VI: Quercetin-3-O-a-L-rhamnoside
3. Results
4. Discussion
Fig. 1 illustrates the insulin release induced by 3 mg/ml of glucose and the inhibition of this insulin release by serotonin (10 − 4 M). A. indica leaf extract (25 mg/ml) potentiates the glucose mediated insulin release and prevented the inhibitory effect of serotonin on insulin release induced by glucose. Chemical analysis revealed that the extract contains the following six compounds.
Release of insulin from beta cells of pancreas is influenced by various factors. 1. Parasympathetic stimulation 6ia vagus nerve increases insulin release (Best and Tailor, 1986). 2. Sympathetic stimulation via splanchnic nerve increases insulin release. This effect is exerted via alpha receptors. Agonists that affect primarily the beta receptors actually stimulate the release of insulin via cAMP. Under ordinary circumstances the alpha receptors mediated inhibitory effect predominates (Viswanathan et al., 1978).
Compound I: Quercetin-3-O-b-D-glucoside Compound II: Myricetin-3-O-rutinoside Compound III: Quercetin-3-O-rutinoside
Fig. 1. Effect of Azadirachta indica (25 mg/ml) on glucose mediated insulin release and the antagonistic effect on serotonin inhibition in insulin release mediated by glucose, n= 6. Group B is significantly different (P B 0.05) from control group A but group C is not. Group D is also significantly different (PB 0.05) from group B. But pairs of group AC, AD and CD are not.
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R.R. Chattopadhyay / Journal of Ethnopharmacology 67 (1999) 373–376
3. Serotonin has been reported to have an inhibitory effect on insulin release in response to adrenaline injection or glucose load (Feldman et al., 1972). From the observed findings it was noted that A. indica leaf extract blocks significantly the inhibitory effect of serotonin on insulin release mediated by glucose. Thus the blood sugar lowering activity of A. indica leaf extract may be due to its insulin releasing pancreatic effect. Chemical analysis revealed that the extract contains the six compounds which are collectively found to be responsible for blood sugar lowering activity (Chattopadhyay and Poddar, 1998). Inspite of these results indicating the insulin releasing effect in the antihyperglycemic activity of A. indica further studies are needed both on the extract and/or its chemical constituents.
Acknowledgements The author wishes to acknowledge Dr T.K. Basu, Head of the Unit for his cooperation and help.
References Best, C.H., Tailor, N.B., 1986. Physiological Basis of Medical Practice, 11th edition. Williums and Wilkins, Baltimore. Chattopadhyay, R.R., 1993a. Effect of Azaclirachta indica leaf extract on oestrous cycle of rats. Environment and Ecology 11 (4), 958 – 960. Chattopadhyay, R.R., 1993b. Possible mechanism of antihyperglycemic effect of Azadirachta indica leaf extract, Part: I. Fitoterapia (Italy) LXIV (4), 332–336. Chattopadhyay, R.R., 1993c. Possible mechanism of antihyperglycemic effect of Azadirachta indica leaf extract, Part: II. Journal of Ecobiology 4 (4), 309–312.
.
Chattopadhyay, R.R., 1995. Hypolipidemic activity of Azadirachta indica leaf extract in rats. Proceedings of Indian National Science Academy B61 (4), 281 – 284. Chattopadhyay, R.R., 1996. Possible mechanism of antibyperglycemic effect of Azadirachta indica leaf extract, Part: IV. General Pharmacology (USA) 27 (3), 431 – 434. Chattopadhyay, R.R., Poddar, G., 1998. Phytochemical screening of Azadirachta indica leaf extract as blood sugar lowering agent. Phytotherapy Research, Communicated. Chattopadhyay, R.R., Chattopadhyay, R.N., Nandy, A.K., Poddar, G., Maitra, S.K., 1986. Antiserotonin activity of a fraction of fresh leaves of Azadirachta indica (Beng. Neem). Bulletin of Calcutta School of Tropical Medicine 34, 9 – 12. Chattopadhyay, R.R., Chattopadhyay, R.N., Nandy, A.K., Poddar, G., Maitra, S.K., 1987a. Antiinflammatory activity of a fraction of fresh leaves of Azadirachta indica (Beng. Neem). Bulletin of Calcutta School of Tropical Medicine 35, 6 – 8. Chattopadhyay, R.R., Chattopadhyay, R.N., Nandy, A.K., Poddar, G., Maitra, S.K., 1987b. Preliminary report on antihyperglycemic effect of a fraction of fresh leaves of Azadirachta indica (Beng. Neem). Bulletin of Calcutta School of Tropical Medicine 35, 29 – 35. Chattopadhyay, R.R., Sarkar, S.K., Ganguly, S., Banerjee, R.N., Basu, T.K., 1992. Hepatoprotective activity of Azadirachta indica leaf extract against paracetamol-induced hepatic damage in rats. Indian Journal of Experimental Biology 30, 738 – 740. Chattopadhyay, R.R., Chattopadhyay, R.N., Maitra, S.K., 1993d. Possible mechanism of antiLyperglycemic effect of Azadirachta indica leaf extract, Part: III. Fitoterapia (Italy) LXIV (6), 535 – 538. Chopra, R.N., Nayar, S.L., Chopra, I.C., 1956. Glossary of Indian Medicinal Plants. CSIR, New Delhi, pp. 31 – 43. Feldman, J.M., Libovitz, H.E., 1970. Mechanism of epinephrine and serotonin inhibition of insulin release in golden hamster in vitro. Diabetes 19, 480 – 487. Feldman, J.M., Quickel, K.E., Libovitz, H.E., 1972. Potentiation of insulin secretion by serotonin antagonists. Diabetes 21, 779 – 785. Kirtikar, K.R., Basu, B.D., 1933. Indian Medicinal Plants. Basu, Allahabad, pp. 3 – 14. Scheff’e, H.E., 1953. A method for judging all contrasts in the analysis of variance. Biometrika 40, 87 – 113. Viswanathan, Mohan, Snehalata, C., Ramachandran, A., Viswanathan, M., 1978. Serotonin and Diabetes Mellitus. Journal of Diabetic Association (India) XVIII, 171 – 174.