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A purified extract from prickly pear cactus (Opuntia fuliginosa) controls experimentally induced diabetes in rats
"~
Journalof ETHNO PHARMACOLOGY
ELSEVIER
Journal of Ethnopharmacology 55 (1996} 27 33
A purified extract from prickly pear cactus (Opuntia fuliginosa) controls experimentally induced diabetes in rats Augusto Trejo-Gonzfilez ~, Genaro Gabriel-Ortiz b, Ana Maria Puebla-Pdrez b, Maria Dolores Huizar-Contreras b, Maria del Rosario Munguia-Mazariegos ", Silvia Mejia-Arreguin b, Edmundo Calva ~,* ~'Department c4/"Biotechnology, CI1D1R-IPN, Justo Sierra 2& 59510 Jiquilpan, Michoacan, Mexico bDivision q/" Experimental Pathology, Western Branch of Biomedical Research. IMSS, Sierra Mojada 800, 44250 Guadalajara, daliseo, Mexico ~Department qf Bioehemi~trv, CINVESTA V-IPV, P.O. Box 14-740, 07000 Mexico. D.F., Mexico Received 7 June 1996: revised 22 July 1996: accepted 23 July 1996
Abstract The hypoglycemic activity of a purified extract from prickly pear cactus (Opuntiafuliginosa Griffiths) was evaluated on STZ-induced diabetic rats. Blood glucose and glycated hemoglobin levels were reduced to normal values by a combined treatment of insulin and Opuntia extract. When insulin was withdrawn from the combined treatment, the prickly pear extract alone maintained normoglycemic state in the diabetic rats. The blood glucose response to administered glucose also showed that the rats receiving the combination treatment of insulin and Opuntia extract for 7 weeks followed by Opuntia extract alone were capable of rapidly returning blood glucose to the levels of the nondiabetic rats. Although the mechanism of action is unknown, the magnitude of the glucose control by the small amount of Opuntia extract required (I mg/kg body weight per day) preclude a predominant role for dietary fiber. These very encouraging results for diabetes control by the purified extract of this Opuntia cactus make the need for clinical studies in humans evident. Keywords: Streptozotocin; Diabetes; Hypoglycemic agents: Opuntia extract; Insulin: Glycated hemoglobin: Blood glucose
1. Introduction
* Corresponding author,
T r e a t m e n t o f diabetes mellitus ( D M ) consists f u n d a m e n t a l l y o f m a n a g i n g the diet a n d / o r p h a r m a c o l o g i c a l therapies which a t t e m p t to n o r m a l i z e
0378-8741/96/$15.00 ~5 1996 Elsevier Science Ireland Ltd. All rights reserved Pll S0378-8741(96)01467-5
28
A. Trejo-Gonz{dez el a/. / Journal ~)! Ethnopharmacolog3
metabolic activities, namely glucose levels. Pharmatherapeutical treatments are based on two types of drugs: injected insulin and oral hypoglycemic agents such as biguanides and sulfonylureas. The use of the oral drugs is limited due to adverse side reactions that may cause incompatibility with other medicaments and become less effective with prolonged use (Gerich, 1985). Insulin injection decreases glycemia, but does not maintain physiologically normal blood glucose levels (Groop et al., 1985). Because of the limitations of current therapies for DM, there remains interest in alternative treatments. The prickly pear cactus (Opuntia spp.) appears to be one of the most promising sources of plant-derived DM-active suppressants. The stems of the prickly pear cactus have been used as a traditional treatment for DM, particularly among the native Nahuatl populations of Mexico (Ibafiez-Camacho and Roman-Ramos, 1979; Ibafiez-Camacho et al., 1983). Prickly pear stems have been shown to have hypoglycemic effects, both in healthy and DM-affected volunteers (lbafiez-Camacho and Roman-Ramos, 1979; Frati-Munari et al., 1987; Frati et al., 1990). It has been suggested that this effect could be related to their high soluble fiber content (Frati-Munari et al., 1987), or that a constituent(s) with hypoglycemic action increases cellular sensitivity to insulin in noninsulin dependent DM patients (Frati-Munari et al., 1988). Other documented metabolic effects of prickly pear extracts include modification of lipoprotein and cholesterol metabolism in guinea pigs (Fernandez et al., 1990; Fernandez et al., 1992; Fernandez et al., 1994). In the research reported here, we investigate the control of experimentally induced diabetes in rats by a purified extract from prickly pear cactus stems.
2. Methodology 2.1. Pur!~ed extract from prickly pear cactus stems (Opuntia extract) The Opuntia extract is an off-white amorphous powder, partially soluble in water forming a col-
55 (1996) 27 33
loidal suspension. It was prepared from Opuntia Juliginosa Griffiths (Herbarium CIMI-CIIDIR Michoacan register #4614) by a method developed by one of us (A. Trejo-Gonzfilez, patent pending on the isolation procedure).
2.2. Animals Male rats (Wistar strain, 250 350 g) were selected and housed in groups of three under standard environmental conditions (20 25°C), with 12-h light dark cycles. The rats were fed ad libitum with Purina rat chow and water. Diabetes was induced by intraperitoneal injection of streptozotocin (STZ) (Sigma X-0130) in a single dose of 50 mg/kg body weight. STZ was dissolved in a 0.01 M citrate buffer pH 4.5 at a concentration of 50 mg/ml. Diabetes in the animals was confirmed 7 days after STZ injection by the presence of blood glucose levels > 250 mg/dl. A 1-ml suspension of the purified prickly pear powder (Opuntia extract) was administered daily in an oral dose of 1 mg/kg body weight through a gastric catheter.
2.3. Treatment of animab Three groups of 7-10 nondiabetic rats were treated daily as follows: Group 1 received no treatment; Group 2 received 1 ml of physiological saline solution orally; and Group 3 received 1 ml of suspended prickly pear extract orally. Four groups of seven to 10 diabetic rats were treated daily as follows: Group 4 received no treatment; Group 5 received 1 ml of suspended pricky pear extract orally; Group 6 was treated with 3--5 units of insulin (NPH insulin of intermediate 100 activity from Lilly) injected subcutaneously according to the specific requirements of each rat; and Group 7 was treated as Group 6, but received an oral dose of 1 ml of suspended pricky pear extract 8 h after the insulin; then after the 8th week, insulin treatment was suspended and the rats received only Opuntia extract for 7 weeks more.
A. Trejo- Gonzdlez et al. / Journal Of Ethnopharmacology 55 (1996) 2 7- 33
0-0
180 170 160 .c 150 140 130 120 110 >,, 100 oo 90 80
Control Saline.Solution
t T T a
H H
Control(STZ) Opuntia Extract
-" -"
Insulin
29
ab b bc
t--
bc
Nondiabetic
70
0
I
I
I
1
r
I
I
I
2
4
6
8
10
12
14
16
0
I
I
]
]
I
I
I
I
2
4
6
8
10
12
14
16
Time (weeks)
Time (weeks)
Fig. 1. Changes in body weight of nondiabetic rats (left graph) and STZ-induced diabetic rats (right graph) treated with insulin, Opuntia extract or both. In the combined treatment insulin was discontinued at week 8 and rats received only Opumia extract thereafter. Data are means + S.D. from seven to 10 rats per treatment. Data followed by different letters at week 15 are significantly different at P = 0.05.
2.4. Measurement of hypoglycemic activity At the beginning of the experiment and every week thereafter, body weight to the nearest gram and fasting blood glucose levels were determined. Rats were held in a restraining cage to expose the tail which was held in lukewarm water, disinfected, and surgically incised to collect 1 ml of blood in a tube containing 15 units of heparin. Glucose was determined by a glucose oxidase assay (Dextrostix from Ames-Miles) and measured by a reflective glucometer (Model II, AmesMiles). At the 15th week the animals were anesthetized with ether and a 2-ml sample of blood was drawn by needle from the heart. The plasma was separated, and glucose was determined spectrophotometrically by the glucose oxidase assay (Sigma Diagnostic Kit 315). Every 7 weeks glycated hemoglobin (HbA,c) was measured by a diagnostic kit (Sigma 441-A).
2.5. Glucose tolerance test Five rats were taken at random at week 10 from each of the seven groups and fasted during 12 h before taking a blood sample. This was
followed by a subcutaneous injection of 0.5 ml of glucose (2 g/kg body weight). Blood samples were then taken at 30, 60, 120, 180 and 240 rain for measurement of glucose concentration.
2.6. Statistics For statistical purposes data were analyzed as means with standard deviations and by analysis of variance with mean separation by LSD at P = 0.05.
3. Results
Group 1 (nondiabetic and untreated rats) gained, on average, 160 g and showed a normal growth curve over the 15-week experimental period (Fig. 1). Weight gain in rats of Group 2 (nondiabetic receiving saline solution) was similar to that of Group 1. The nondiabetic rats receiving only the extract prepared from Opuntia fuliginosa (Group 3) showed less weight gain than the control Group 1. Group 4 (diabetic, STZ control) showed an average weight loss, and only four rats survived beyond week 12. Diabetic rats treated
30
A. Trejo-Gonzhlez et al. / Journal of Ethnopharmacologv 55 (1996) 27-33
Table 1 Blood glucose concentrations (mg/dl) of nondiabetic rats and STZ-induced diabetic rats receiving no treatment, Opuntia extract, insulin, or a combination of Opuntia extract and insulin Treatment groups N ondiabetic Untreated (1) Saline solution (2) Opuntia extract (3) Diabetic Untreated (4) Opuntia extract (5) lnsulin (6) Opuntia extract and insulin (7)
Initial
Week 1
Week 8
Week 15
114 _+ 9 ~ (10) 118 +_ 9 (10) 123 ± 13 (7)
119 ± 10 (10) 138 _+ 22 (10) 122 ± 11 (7)
121 _+ 7 (10) 133 _+ 25 (9) 113 + 10 (7)
107 Jr 13 (10) 105 _+ 15 (9) 123 +_ 16 (7)
113 _+ 12 114 +_ 16 120 ! 14 109 _+ 15
351 300 365 276
>400 (10) 291 + 125 (7) 267 ± 139 (4) 121 _+ 16 (7)
393 -+75 (5) 273 _+ 126 (6) 296 _+ 40 (4) 128 i 21 (7)
(10) (7) (7) (10)
_+74 + 75 ± 34 _+ 39
(10) (7) (7) (10)
~Mean -- S.D,; number in parenthesis represents the number of animals.
only with Opuntia extract (Group 5) showed an average weight gain similar to that of diabetic rats receiving insulin and Opuntia extract (Group 7), but lower than of the nondiabetic rats receiving saline solution (Group 2) or Opuntia extract (Group 3). Group 6 rats (diabetic receiving insulin) had the most variable body weights of the seven groups, but at week 15 the average was not different from that of other treated diabetic rats. Initial average blood glucose levels were similar among the seven groups (Table 1). During the course of the experiment glucose levels of the nondiabetic rats were similar to the initial values. Within 1 week, the glucose levels of diabetic untreated rats (Group 4) were three times the initial values and were maintained at these levels throughout the remainder of the experiment. Insulin-treated diabetic rats (Group 6) showed high glucose levels after 1 week, decreasing by week 2 and then stabilizing at moderately high levels. Diabetic rats receiving only Opuntia extract (Group 5) had glucose levels similar to those of the insulin-treated rats (Group 6). Diabetic rats receiving both insulin and Opuntia extract (Group 7) were the only diseased animals with normalized glucose levels. Within 2 weeks, glucose levels in this group had declined to the values found in the nondiabetic groups. Insulin treatment was suspended in rats of Group 7 due to hypoglycemia after week 8, and normal glucose levels were maintained during the following 7 weeks with administration of Opuntia extract alone.
The average glycated hemoglobin concentrations in blood of the rats at the beginning of the experiment varied from 3.2 to 5.0% (Table 2). In the nondiabetic rats these levels did not change significantly during the experiment. Untreated diabetic rats (Group 4) showed a fourfold increase in glycated hemoglobin within 1 week which was maintained until sometime after week 8, with a final average value 2.5 times the initial one. Diabetic rats receiving insulin (Group 6) showed initial changes in the levels of glycated hemoglobin similar to those of the untreated diabetic rats, but levels continued to increase gradually over the course of the experiment. Among the different groups of diabetic rats, those receiving insulin and Opuntia extract (Group 7) showed the lowest glycared hemoglobin levels at week 1. Both groups of diabetic rats receiving Opuntia extract (Groups 5 and 7) showed some initial increase in glycated hemoglobin levels which subsequently declined close to the normal range by week 15. Glucose tolerance was determined at week 10 and the results are shown in Fig. 2. In the three groups of nondiabetic rats the highest average blood glucose levels were obtained 30 min after subcutaneous glucose injection, and glucose returned to normal levels after 4 h. Diabetic untreated rats (Group 4) showed the highest blood glucose levels and these persisted during the 4-h experimental period. The average blood glucose levels of the diabetic rats receiving only Opuntia extract (Group 5) or only insulin (Group 6) were
A. Trq/o-Gonzdle: eta/. : Journal ~/ Ethnopharmaeolo~4v 55 (1996) 27 33
31
Table 2 Percent of glycated hemoglobin (HbA~) in the blood of nondiabetic and STZ-induced diabetic rats receiving no treatment, Opuntia extract, insulin, or a combination of Opuntia extract and insulin Treatment groups
Initial
Nondiabetic Untreated (1) Saline solution (2) Opuntia extract (3) Diabetic Untreated (4) Opuntia extract (5) Insulin (6) Opuntia extract and insulin (7)
Week 1
Week 8
Week 15
4.3 + 1.4'1 4.2 + 1.4 5.0 + 1. I
4.3 + 1.3 4.2 ± 1.5 4.9 ± 1.1
4.5 + 0.9 4.2 _+ 1.5 5.2 ± 1.1
3.2_+ 1.3 1.8 _+0.4 3.2 + 1.3
4.7 _+ 1.0 5.0 ± 0.1 3.2 _+0.8 3.7 ± 1.3
18.4 + 8.4 13.5 _+ 3.8 14.1 ± 4.6 9.1 ± 1.4
I7.5 ++6.3 7.7 -k 3.0 16.1 + 2.5 7.4 __+1.2
12.2 ++_4.4 6.0 _+ 3.9 20./) ± 5.7 4.8 ± 1.7
~' Mean ± S.D.: data for each treatment are based on the same number of animals cited in Table 1. c a c t u s (OpuntiaJul~ginosa G r i f f i t h s ) e x t r a c t to diabetic a n d n o n d i a b e t i c rats r e s u l t e d in significant d i f f e r e n c e s in the final a v e r a g e b o d y weights. T h e n o n d i a b e t i c rats r e c e i v i n g Opuntia e x t r a c t h a d less w e i g h t g a i n t h a n the rats o f the c o n t r o l g r o u p . Since f o o d c o n s u m p t i o n w a s n o t m e a s u r e d , it is n o t k n o w n w h e t h e r this was d u e to effects o n satiety o r d u e to d i f f e r e n c e s in efficiency o f m e t a b o l i s m . F r a t i - M u n a r i et al. (1983) r e p o r t e d t h a t in h u m a n s the c o n s u m p t i o n o f p r i c k l y p e a r c a c t u s stems r e s u l t e d in l o w e r w e i g h t gain. D i a betic rats r e c e i v i n g Opuntia e x t r a c t m a i n t a i n e d a
similar to e a c h o t h e r a n d t e n d e d to be l o w e r t h a n t h o s e o f u n t r e a t e d d i a b e t i c rats. T h e h i g h e s t a v e r age b l o o d g l u c o s e level o f t h e d i a b e t i c rats receiving insulin a n d Opuntia e x t r a c t o c c u r r e d at an i n t e r m e d i a t e high level 30 m i n a f t e r the s u g a r i n j e c t i o n , a n d d e c r e a s e d to a n o r m a l a v e r a g e v a l u e w i t h i n t h e 4-h p e r i o d .
4. Discussion and conclusions The administration
of a purified prickly pear
Diabetic
Nondiabetic 350 300
'7 -~ 250 E 200
8 15o _.= (9
~;
100 O-(D Control 50
H
Control (STZ)
H
Opuntia Extract Insulin Insulin and Opuntia Ext.
Opuntia Extract Saline Solution [
0
I
I
I
I
I
I
I
I
30 60 90 120 150 180 210 240 Time (rain)
1
0
1
I
I
I
I
]
I
I
30 60 90 120 150 180 210 240 Time (min)
Fig. 2. Glucose tolerance test in nondiabetic rats (left graph) and STZ-induced diabetic rats (right graph) treated with insulin, Opuntia extract or both. In the combined treatment insulin was discontinued at week 8 and rats received only Opuntia extract thereafter. Glucose was administered by subcutaneous injection. Data are means _+ S.D. of five rats per treatment at week 10.
32
A. Trejo-Gonz[dez et al. / Journal o.1 Ethnopharmacology 55 (1996) 27 33
steady increment in their body weights, in contrast to the decline observed in untreated diabetic rats and the wide variation observed in the insulin-treated diabetic group. It was evident that the purified Opuntia extract had a definite hypoglycemic action in diabetic rats after the first week, This is clearly shown in the rats receiving Opuntia extract, in which blood glucose levels were similar to those of insulintreated rats. In addition, the lowest blood glucose levels were obtained in the rats subjected to a combined treatment of insulin and Opuntia extract, and so low glycemic values were found alter week 8 that insulin administration was suspended and the animals received thereafter only Opuntia extract. Normoglycemia in these diabetic rats during the last 7 weeks of the experiment, therefore, was due to the hypoglycemic activity of the purified Opuntia extract alone. The glycated hemoglobin levels of the diabetic rats which received Opuntia extract or a combination of insulin and Opuntia extract returned to normal values after week 8. In diabetic rats treated with insulin alone the HbA~,. levels were not effectively controlled and did not return to normal values. It has been hypothesized that prickly pear cactus functions as a coadjutor of insulin (Meckes-Lozoya and Roman-Ramos, 1986). In the present work this could have occurred in Group 7 up to week 8, but not afterwards, since insulin administration was suspended at that time. Moreover, pancreatectomized rabbits producing no insulin but fed prickly pear stems had lower glycemia (Ibafiez-Camacho and Roman-Ramos, 1979). The subcutaneous glucose tolerance test also shows that rats administered insulin and Opuntia extract had a significant decline in the blood glucose levels with values returning near those of nondiabetic rats within 120 min. Diabetic rats treated with either insulin or Opuntia extract alone, however, showed glucose values which did not return to normal levels. Non-insulin dependent D M patients receiving fresh or cooked prickly pear stems showed lower blood glucose levels after oral administration of glucose in one study (Fernfindez-Harp et al., 1984), but no hypoglycemic effect was observed in
another (Frati-Munari et al., 1988). Researchers have concluded that for a significant hypoglycemic effect it is necessary for the patients to consume daily at least 500 g of the fresh vegetable stems or their equivalent in dried product. FratiMunari et al. (1989) experimented with commercially available dehydrated products and found them to have very low hypoglycemic activity. The control of diabetes by purified Opuntia extract can not be explained by its action as dietary fiber, since several long-term controlled studies have led to the conclusion that different soluble fiber sources do not lower or regulate blood glucose levels (Nuttall, 1993). The hypothesis proposed by Frati-Munari et al. (1988), that the prickly pear cactus improves utilization of glucose at the cellular level, is supported by the present study. Another important conclusion from the present study is that the control of diabetes by a purified Opuntia extract can be achieved with oral daily doses in the range of I mg/kg body weight. This small amount contrasts with the large quantities of fresh prickly pear cactus ( ~ 5 0 0 g) or the numerous high doses of insulin required for similar hypoglycemic effects.
Acknowledgements The authors thank Dr. Marita Cantwell for assistance in preparing this manuscript in English.
References Fernandez, M.L., Trejo, A. and McNamara, D.J. (1990) Pectin isolated from prickly pear (Opuntia sp.) modifies low density lipoprotein metabolism in cholesterol-fed guinea pigs. Journal o1' Nutrition 120, 1283-1290. Fernandez, M.L., Lin, E.C.K., Trejo, A. and McNamara, D.J. (1992) Prickly pear (Opuntia sp.) pectin reverses low density lipoprotein receptor suppression induced by a hypercholesterolemicdiet in guinea pigs. Journal q/' Nutrition 122, 2330 2340. Fernandez, M.L., Lin, E.C.K., Trejo, A. and McNamara, D.J. (1994) Prickly pear (Opuntia sp.) pectin alters hepatic cholesterol metabolism without affecting cholesterol absorption in guinea pigs fed a hypercholesterolemicdiet. Journal ~[ Nutrition 124: 817-824.
A. Trejo-Gonzdlez et al. / Journal of Ethnopharmacology 55 (1996) 27 33 Fern~ndez-Harp, J.A., Frati-Munari, A., Ch/tvez-Negrete, A., de la Riva-Pinal, H.B. and Mares-G6mez, G. (1984) Estudios hormonales en la accion del nopal sobre la prueba de tolerancia a la glucosa, lnforme prelirninar. Revista M~diea 1MSS (Mex.) 22, 387 390. Frati, A.C., Jimenez, E. and Ariza, C.R. (1990) Hypogtycemic effect of Opuntiafieus-indica in non insulin-dependent diabetes mellitus patients. Phytotherapy Researeh 4, 195 197. Frati-Munari+ A.C.+ De Leon, C., Ariza-Andraca, C.R., BatiMes-Ham, M., L6pez-Ledesma, R. and Lozoya, X. (1989) Influencia de un extracto deshidratado de nopal (Opumia fieus-indica Mill.) en la glucemia. Archivos de InvestigatiOn Mddica (Mex.) 20, 2ll-216. Frati-Munari, A.C.+ Fernandez-Harp, J.A., de la Riva, H., Ariza-Andraca, R. and Torres, M.D.C. (1983) Effects of nopal (Opuntia sp.) on serum lipids, glycemia and body weight. Arehivos de hTves't~aci6n M~dica (Mex.) 14, 117125. Frati-Munari, A.C., Gordillo, B.E., Altamirano, P. and Ariza. C.R. (1988) Hypo-glycemic effect of Opuntia streptaeantha Lemaire in NIDDM. Diabetes Care 11, 63-66. Frati-Munari, A.C., Yever-Garces, A., lslas-Andrade, S.. Ariza-Andraca, C.R. and Chavez-Negrete, A. (t987) Stud-
33
ies on the mechanism of 'hypoglycemic' effect of nopal (Opuntia sp.). Arehivos de lnrestigaei3n M&tica (Mex.) 18+ 7 12. Gerich, J.E. (1985) Sulphonylureas in the treatment ot+diabetes mellitus. Clinical Procedures 60, 439-443. Groop, L., Harno, K., Nikkila, E.A., Pelkonen. R. and Tolppanen, E.-M. (1985) Transient effect of the combination of insulin and sulfonylurea (Glibenclamide) on glycemic control in non-insulin dependent diabetics poorly controlled with insulin alone. Aeta Mediea Scandinaviea 217, 33 39. Ibafiez-Camacho, R., Meckes-Lozoya, M. and Mellado-Campos, V. (1983) The hypoglucemic effect of Opun;ia streptaeantha studied in different animal experimental models. Journal ( f Ethnopharmacolog:v 7, 175 181. lbafiez-Camacho, R. and Roman-Ramos, R. (1979) Efecto hipoglucemiante de] nopal. Arehit os de h~t,est~acibn M~;dica (Mex.) 10, 223 230. Meckes-Lozoya, M. and Roman-Ramos, R. (1986) Opuntia streptaeantha: a coadjutor in the treatment of diabetes mellitus. American Journal o! Clinical .,~Iedieine 14, 116 118. Nuttall, F.Q. (1993) Dietary fiber in the management of diabetes. Diabetes 42, 503 508.
Journalof ETHNO PHARMACOLOGY
ELSEVIER
Journal of Ethnopharmacology 55 (1996} 27 33
A purified extract from prickly pear cactus (Opuntia fuliginosa) controls experimentally induced diabetes in rats Augusto Trejo-Gonzfilez ~, Genaro Gabriel-Ortiz b, Ana Maria Puebla-Pdrez b, Maria Dolores Huizar-Contreras b, Maria del Rosario Munguia-Mazariegos ", Silvia Mejia-Arreguin b, Edmundo Calva ~,* ~'Department c4/"Biotechnology, CI1D1R-IPN, Justo Sierra 2& 59510 Jiquilpan, Michoacan, Mexico bDivision q/" Experimental Pathology, Western Branch of Biomedical Research. IMSS, Sierra Mojada 800, 44250 Guadalajara, daliseo, Mexico ~Department qf Bioehemi~trv, CINVESTA V-IPV, P.O. Box 14-740, 07000 Mexico. D.F., Mexico Received 7 June 1996: revised 22 July 1996: accepted 23 July 1996
Abstract The hypoglycemic activity of a purified extract from prickly pear cactus (Opuntiafuliginosa Griffiths) was evaluated on STZ-induced diabetic rats. Blood glucose and glycated hemoglobin levels were reduced to normal values by a combined treatment of insulin and Opuntia extract. When insulin was withdrawn from the combined treatment, the prickly pear extract alone maintained normoglycemic state in the diabetic rats. The blood glucose response to administered glucose also showed that the rats receiving the combination treatment of insulin and Opuntia extract for 7 weeks followed by Opuntia extract alone were capable of rapidly returning blood glucose to the levels of the nondiabetic rats. Although the mechanism of action is unknown, the magnitude of the glucose control by the small amount of Opuntia extract required (I mg/kg body weight per day) preclude a predominant role for dietary fiber. These very encouraging results for diabetes control by the purified extract of this Opuntia cactus make the need for clinical studies in humans evident. Keywords: Streptozotocin; Diabetes; Hypoglycemic agents: Opuntia extract; Insulin: Glycated hemoglobin: Blood glucose
1. Introduction
* Corresponding author,
T r e a t m e n t o f diabetes mellitus ( D M ) consists f u n d a m e n t a l l y o f m a n a g i n g the diet a n d / o r p h a r m a c o l o g i c a l therapies which a t t e m p t to n o r m a l i z e
0378-8741/96/$15.00 ~5 1996 Elsevier Science Ireland Ltd. All rights reserved Pll S0378-8741(96)01467-5
28
A. Trejo-Gonz{dez el a/. / Journal ~)! Ethnopharmacolog3
metabolic activities, namely glucose levels. Pharmatherapeutical treatments are based on two types of drugs: injected insulin and oral hypoglycemic agents such as biguanides and sulfonylureas. The use of the oral drugs is limited due to adverse side reactions that may cause incompatibility with other medicaments and become less effective with prolonged use (Gerich, 1985). Insulin injection decreases glycemia, but does not maintain physiologically normal blood glucose levels (Groop et al., 1985). Because of the limitations of current therapies for DM, there remains interest in alternative treatments. The prickly pear cactus (Opuntia spp.) appears to be one of the most promising sources of plant-derived DM-active suppressants. The stems of the prickly pear cactus have been used as a traditional treatment for DM, particularly among the native Nahuatl populations of Mexico (Ibafiez-Camacho and Roman-Ramos, 1979; Ibafiez-Camacho et al., 1983). Prickly pear stems have been shown to have hypoglycemic effects, both in healthy and DM-affected volunteers (lbafiez-Camacho and Roman-Ramos, 1979; Frati-Munari et al., 1987; Frati et al., 1990). It has been suggested that this effect could be related to their high soluble fiber content (Frati-Munari et al., 1987), or that a constituent(s) with hypoglycemic action increases cellular sensitivity to insulin in noninsulin dependent DM patients (Frati-Munari et al., 1988). Other documented metabolic effects of prickly pear extracts include modification of lipoprotein and cholesterol metabolism in guinea pigs (Fernandez et al., 1990; Fernandez et al., 1992; Fernandez et al., 1994). In the research reported here, we investigate the control of experimentally induced diabetes in rats by a purified extract from prickly pear cactus stems.
2. Methodology 2.1. Pur!~ed extract from prickly pear cactus stems (Opuntia extract) The Opuntia extract is an off-white amorphous powder, partially soluble in water forming a col-
55 (1996) 27 33
loidal suspension. It was prepared from Opuntia Juliginosa Griffiths (Herbarium CIMI-CIIDIR Michoacan register #4614) by a method developed by one of us (A. Trejo-Gonzfilez, patent pending on the isolation procedure).
2.2. Animals Male rats (Wistar strain, 250 350 g) were selected and housed in groups of three under standard environmental conditions (20 25°C), with 12-h light dark cycles. The rats were fed ad libitum with Purina rat chow and water. Diabetes was induced by intraperitoneal injection of streptozotocin (STZ) (Sigma X-0130) in a single dose of 50 mg/kg body weight. STZ was dissolved in a 0.01 M citrate buffer pH 4.5 at a concentration of 50 mg/ml. Diabetes in the animals was confirmed 7 days after STZ injection by the presence of blood glucose levels > 250 mg/dl. A 1-ml suspension of the purified prickly pear powder (Opuntia extract) was administered daily in an oral dose of 1 mg/kg body weight through a gastric catheter.
2.3. Treatment of animab Three groups of 7-10 nondiabetic rats were treated daily as follows: Group 1 received no treatment; Group 2 received 1 ml of physiological saline solution orally; and Group 3 received 1 ml of suspended prickly pear extract orally. Four groups of seven to 10 diabetic rats were treated daily as follows: Group 4 received no treatment; Group 5 received 1 ml of suspended pricky pear extract orally; Group 6 was treated with 3--5 units of insulin (NPH insulin of intermediate 100 activity from Lilly) injected subcutaneously according to the specific requirements of each rat; and Group 7 was treated as Group 6, but received an oral dose of 1 ml of suspended pricky pear extract 8 h after the insulin; then after the 8th week, insulin treatment was suspended and the rats received only Opuntia extract for 7 weeks more.
A. Trejo- Gonzdlez et al. / Journal Of Ethnopharmacology 55 (1996) 2 7- 33
0-0
180 170 160 .c 150 140 130 120 110 >,, 100 oo 90 80
Control Saline.Solution
t T T a
H H
Control(STZ) Opuntia Extract
-" -"
Insulin
29
ab b bc
t--
bc
Nondiabetic
70
0
I
I
I
1
r
I
I
I
2
4
6
8
10
12
14
16
0
I
I
]
]
I
I
I
I
2
4
6
8
10
12
14
16
Time (weeks)
Time (weeks)
Fig. 1. Changes in body weight of nondiabetic rats (left graph) and STZ-induced diabetic rats (right graph) treated with insulin, Opuntia extract or both. In the combined treatment insulin was discontinued at week 8 and rats received only Opumia extract thereafter. Data are means + S.D. from seven to 10 rats per treatment. Data followed by different letters at week 15 are significantly different at P = 0.05.
2.4. Measurement of hypoglycemic activity At the beginning of the experiment and every week thereafter, body weight to the nearest gram and fasting blood glucose levels were determined. Rats were held in a restraining cage to expose the tail which was held in lukewarm water, disinfected, and surgically incised to collect 1 ml of blood in a tube containing 15 units of heparin. Glucose was determined by a glucose oxidase assay (Dextrostix from Ames-Miles) and measured by a reflective glucometer (Model II, AmesMiles). At the 15th week the animals were anesthetized with ether and a 2-ml sample of blood was drawn by needle from the heart. The plasma was separated, and glucose was determined spectrophotometrically by the glucose oxidase assay (Sigma Diagnostic Kit 315). Every 7 weeks glycated hemoglobin (HbA,c) was measured by a diagnostic kit (Sigma 441-A).
2.5. Glucose tolerance test Five rats were taken at random at week 10 from each of the seven groups and fasted during 12 h before taking a blood sample. This was
followed by a subcutaneous injection of 0.5 ml of glucose (2 g/kg body weight). Blood samples were then taken at 30, 60, 120, 180 and 240 rain for measurement of glucose concentration.
2.6. Statistics For statistical purposes data were analyzed as means with standard deviations and by analysis of variance with mean separation by LSD at P = 0.05.
3. Results
Group 1 (nondiabetic and untreated rats) gained, on average, 160 g and showed a normal growth curve over the 15-week experimental period (Fig. 1). Weight gain in rats of Group 2 (nondiabetic receiving saline solution) was similar to that of Group 1. The nondiabetic rats receiving only the extract prepared from Opuntia fuliginosa (Group 3) showed less weight gain than the control Group 1. Group 4 (diabetic, STZ control) showed an average weight loss, and only four rats survived beyond week 12. Diabetic rats treated
30
A. Trejo-Gonzhlez et al. / Journal of Ethnopharmacologv 55 (1996) 27-33
Table 1 Blood glucose concentrations (mg/dl) of nondiabetic rats and STZ-induced diabetic rats receiving no treatment, Opuntia extract, insulin, or a combination of Opuntia extract and insulin Treatment groups N ondiabetic Untreated (1) Saline solution (2) Opuntia extract (3) Diabetic Untreated (4) Opuntia extract (5) lnsulin (6) Opuntia extract and insulin (7)
Initial
Week 1
Week 8
Week 15
114 _+ 9 ~ (10) 118 +_ 9 (10) 123 ± 13 (7)
119 ± 10 (10) 138 _+ 22 (10) 122 ± 11 (7)
121 _+ 7 (10) 133 _+ 25 (9) 113 + 10 (7)
107 Jr 13 (10) 105 _+ 15 (9) 123 +_ 16 (7)
113 _+ 12 114 +_ 16 120 ! 14 109 _+ 15
351 300 365 276
>400 (10) 291 + 125 (7) 267 ± 139 (4) 121 _+ 16 (7)
393 -+75 (5) 273 _+ 126 (6) 296 _+ 40 (4) 128 i 21 (7)
(10) (7) (7) (10)
_+74 + 75 ± 34 _+ 39
(10) (7) (7) (10)
~Mean -- S.D,; number in parenthesis represents the number of animals.
only with Opuntia extract (Group 5) showed an average weight gain similar to that of diabetic rats receiving insulin and Opuntia extract (Group 7), but lower than of the nondiabetic rats receiving saline solution (Group 2) or Opuntia extract (Group 3). Group 6 rats (diabetic receiving insulin) had the most variable body weights of the seven groups, but at week 15 the average was not different from that of other treated diabetic rats. Initial average blood glucose levels were similar among the seven groups (Table 1). During the course of the experiment glucose levels of the nondiabetic rats were similar to the initial values. Within 1 week, the glucose levels of diabetic untreated rats (Group 4) were three times the initial values and were maintained at these levels throughout the remainder of the experiment. Insulin-treated diabetic rats (Group 6) showed high glucose levels after 1 week, decreasing by week 2 and then stabilizing at moderately high levels. Diabetic rats receiving only Opuntia extract (Group 5) had glucose levels similar to those of the insulin-treated rats (Group 6). Diabetic rats receiving both insulin and Opuntia extract (Group 7) were the only diseased animals with normalized glucose levels. Within 2 weeks, glucose levels in this group had declined to the values found in the nondiabetic groups. Insulin treatment was suspended in rats of Group 7 due to hypoglycemia after week 8, and normal glucose levels were maintained during the following 7 weeks with administration of Opuntia extract alone.
The average glycated hemoglobin concentrations in blood of the rats at the beginning of the experiment varied from 3.2 to 5.0% (Table 2). In the nondiabetic rats these levels did not change significantly during the experiment. Untreated diabetic rats (Group 4) showed a fourfold increase in glycated hemoglobin within 1 week which was maintained until sometime after week 8, with a final average value 2.5 times the initial one. Diabetic rats receiving insulin (Group 6) showed initial changes in the levels of glycated hemoglobin similar to those of the untreated diabetic rats, but levels continued to increase gradually over the course of the experiment. Among the different groups of diabetic rats, those receiving insulin and Opuntia extract (Group 7) showed the lowest glycared hemoglobin levels at week 1. Both groups of diabetic rats receiving Opuntia extract (Groups 5 and 7) showed some initial increase in glycated hemoglobin levels which subsequently declined close to the normal range by week 15. Glucose tolerance was determined at week 10 and the results are shown in Fig. 2. In the three groups of nondiabetic rats the highest average blood glucose levels were obtained 30 min after subcutaneous glucose injection, and glucose returned to normal levels after 4 h. Diabetic untreated rats (Group 4) showed the highest blood glucose levels and these persisted during the 4-h experimental period. The average blood glucose levels of the diabetic rats receiving only Opuntia extract (Group 5) or only insulin (Group 6) were
A. Trq/o-Gonzdle: eta/. : Journal ~/ Ethnopharmaeolo~4v 55 (1996) 27 33
31
Table 2 Percent of glycated hemoglobin (HbA~) in the blood of nondiabetic and STZ-induced diabetic rats receiving no treatment, Opuntia extract, insulin, or a combination of Opuntia extract and insulin Treatment groups
Initial
Nondiabetic Untreated (1) Saline solution (2) Opuntia extract (3) Diabetic Untreated (4) Opuntia extract (5) Insulin (6) Opuntia extract and insulin (7)
Week 1
Week 8
Week 15
4.3 + 1.4'1 4.2 + 1.4 5.0 + 1. I
4.3 + 1.3 4.2 ± 1.5 4.9 ± 1.1
4.5 + 0.9 4.2 _+ 1.5 5.2 ± 1.1
3.2_+ 1.3 1.8 _+0.4 3.2 + 1.3
4.7 _+ 1.0 5.0 ± 0.1 3.2 _+0.8 3.7 ± 1.3
18.4 + 8.4 13.5 _+ 3.8 14.1 ± 4.6 9.1 ± 1.4
I7.5 ++6.3 7.7 -k 3.0 16.1 + 2.5 7.4 __+1.2
12.2 ++_4.4 6.0 _+ 3.9 20./) ± 5.7 4.8 ± 1.7
~' Mean ± S.D.: data for each treatment are based on the same number of animals cited in Table 1. c a c t u s (OpuntiaJul~ginosa G r i f f i t h s ) e x t r a c t to diabetic a n d n o n d i a b e t i c rats r e s u l t e d in significant d i f f e r e n c e s in the final a v e r a g e b o d y weights. T h e n o n d i a b e t i c rats r e c e i v i n g Opuntia e x t r a c t h a d less w e i g h t g a i n t h a n the rats o f the c o n t r o l g r o u p . Since f o o d c o n s u m p t i o n w a s n o t m e a s u r e d , it is n o t k n o w n w h e t h e r this was d u e to effects o n satiety o r d u e to d i f f e r e n c e s in efficiency o f m e t a b o l i s m . F r a t i - M u n a r i et al. (1983) r e p o r t e d t h a t in h u m a n s the c o n s u m p t i o n o f p r i c k l y p e a r c a c t u s stems r e s u l t e d in l o w e r w e i g h t gain. D i a betic rats r e c e i v i n g Opuntia e x t r a c t m a i n t a i n e d a
similar to e a c h o t h e r a n d t e n d e d to be l o w e r t h a n t h o s e o f u n t r e a t e d d i a b e t i c rats. T h e h i g h e s t a v e r age b l o o d g l u c o s e level o f t h e d i a b e t i c rats receiving insulin a n d Opuntia e x t r a c t o c c u r r e d at an i n t e r m e d i a t e high level 30 m i n a f t e r the s u g a r i n j e c t i o n , a n d d e c r e a s e d to a n o r m a l a v e r a g e v a l u e w i t h i n t h e 4-h p e r i o d .
4. Discussion and conclusions The administration
of a purified prickly pear
Diabetic
Nondiabetic 350 300
'7 -~ 250 E 200
8 15o _.= (9
~;
100 O-(D Control 50
H
Control (STZ)
H
Opuntia Extract Insulin Insulin and Opuntia Ext.
Opuntia Extract Saline Solution [
0
I
I
I
I
I
I
I
I
30 60 90 120 150 180 210 240 Time (rain)
1
0
1
I
I
I
I
]
I
I
30 60 90 120 150 180 210 240 Time (min)
Fig. 2. Glucose tolerance test in nondiabetic rats (left graph) and STZ-induced diabetic rats (right graph) treated with insulin, Opuntia extract or both. In the combined treatment insulin was discontinued at week 8 and rats received only Opuntia extract thereafter. Glucose was administered by subcutaneous injection. Data are means _+ S.D. of five rats per treatment at week 10.
32
A. Trejo-Gonz[dez et al. / Journal o.1 Ethnopharmacology 55 (1996) 27 33
steady increment in their body weights, in contrast to the decline observed in untreated diabetic rats and the wide variation observed in the insulin-treated diabetic group. It was evident that the purified Opuntia extract had a definite hypoglycemic action in diabetic rats after the first week, This is clearly shown in the rats receiving Opuntia extract, in which blood glucose levels were similar to those of insulintreated rats. In addition, the lowest blood glucose levels were obtained in the rats subjected to a combined treatment of insulin and Opuntia extract, and so low glycemic values were found alter week 8 that insulin administration was suspended and the animals received thereafter only Opuntia extract. Normoglycemia in these diabetic rats during the last 7 weeks of the experiment, therefore, was due to the hypoglycemic activity of the purified Opuntia extract alone. The glycated hemoglobin levels of the diabetic rats which received Opuntia extract or a combination of insulin and Opuntia extract returned to normal values after week 8. In diabetic rats treated with insulin alone the HbA~,. levels were not effectively controlled and did not return to normal values. It has been hypothesized that prickly pear cactus functions as a coadjutor of insulin (Meckes-Lozoya and Roman-Ramos, 1986). In the present work this could have occurred in Group 7 up to week 8, but not afterwards, since insulin administration was suspended at that time. Moreover, pancreatectomized rabbits producing no insulin but fed prickly pear stems had lower glycemia (Ibafiez-Camacho and Roman-Ramos, 1979). The subcutaneous glucose tolerance test also shows that rats administered insulin and Opuntia extract had a significant decline in the blood glucose levels with values returning near those of nondiabetic rats within 120 min. Diabetic rats treated with either insulin or Opuntia extract alone, however, showed glucose values which did not return to normal levels. Non-insulin dependent D M patients receiving fresh or cooked prickly pear stems showed lower blood glucose levels after oral administration of glucose in one study (Fernfindez-Harp et al., 1984), but no hypoglycemic effect was observed in
another (Frati-Munari et al., 1988). Researchers have concluded that for a significant hypoglycemic effect it is necessary for the patients to consume daily at least 500 g of the fresh vegetable stems or their equivalent in dried product. FratiMunari et al. (1989) experimented with commercially available dehydrated products and found them to have very low hypoglycemic activity. The control of diabetes by purified Opuntia extract can not be explained by its action as dietary fiber, since several long-term controlled studies have led to the conclusion that different soluble fiber sources do not lower or regulate blood glucose levels (Nuttall, 1993). The hypothesis proposed by Frati-Munari et al. (1988), that the prickly pear cactus improves utilization of glucose at the cellular level, is supported by the present study. Another important conclusion from the present study is that the control of diabetes by a purified Opuntia extract can be achieved with oral daily doses in the range of I mg/kg body weight. This small amount contrasts with the large quantities of fresh prickly pear cactus ( ~ 5 0 0 g) or the numerous high doses of insulin required for similar hypoglycemic effects.
Acknowledgements The authors thank Dr. Marita Cantwell for assistance in preparing this manuscript in English.
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33
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