Journul of Ethnopharmacology, Elsevier
Scientific
Publishers
295
30 (1990) 295 - 305 Ireland Ltd.
ANTIDIARETIC EFFECT OF A LEAF EXTRACT FROM GYMNEMA SYLVESTRE IN NON-INSULIN-DEPENDENT DIABETES MELLITUS PATIENTS
K. BASKARAN, B. KIZAR AHAMATH, E.R.B. SHANMUGASUNDARAM
of Biochemistry,
Department
K. RADHA
Postgraduute
Institute
SHANMUGASUNDARAM
of Basic Medical
Sciences
and
Madras-600
119 Ilndti (Accepted
June
26, 1990)
Summary
The effectiveness of GS,, an extract from the leaves of Gymnema syG in controlling hyperglycaemia was investigated in 22 Type 2 diabetic patients on conventional oral anti-hyperglycaemic agents. GS, (400 mglday) was administered for 18-20 months as a supplement to the conventional oral drugs. During GS, supplementation, the patients showed a significant reduction in blood glucose, glycosylated haemoglobin and glycosylated plasma proteins, and conventional drug dosage could be decreased. Five of the 22 diabetic patients were able to discontinue their conventional drug and maintain their blood glucose homeostasis with GS, alone. These data suggest that the beta cells may be regenerated/repaired in Type 2 diabetic patients on GS, supplementation. This is supported by the appearance of raised insulin levels in the serum of patients after GS, supplementation. vestre,
Introduction
Type 2 (NIDDM, non-insulin dependent) diabetes mellitus is among the most common disorders in developed and developing countries (Zimmet, 1982). Reaven (1984) has reported that abnormalities of beta cell function and secretion exist in patients with non-insulin dependent diabetes mellitus. O’Rahilly et al. (1986) observed that the inherited component of familial Type 2 diabetes may be an impaired insulin secreting response of the beta cells. While fasting serum insulin levels may be in the normal range (up to 25 rIU/ ml), glucose-induced insulin release is reduced, leaving below normal (< 55 rIU/ml) insulin levels in the post-prandial state. Correspondence
to: Prof. K. Radha Shanmugasundaram.
0378.8741/$03.50 0 1990 Elsevier Published and Printed in Ireland
Scientific
Publishers
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Ltd.
296
Control of blood glucose on a 24-h basis is the desired goal in the management of diabetes mellitus so as to prevent or delay the onset of the secondary complications of diabetes mellitus. Dieting, physical exercise and inclusion of dietary fibre have been used with limited success. Oral anti-hyperglycaemic drugs play an important role in the treatment of Type 2 diabetes mellitus (Kilo, 19821. There are two groups of oral antihyperglycaemic agents available for clinical use, i.e. the sulfonylureas and biguanides. The sulfonylureas are reported to regulate blood glucose homeostasis by stimulating pancreatic secretion of insulin (Efendic et al., 19791 and have a characteristic profile of side effects (Prout, 19741.Biguanides do not enhance the release of insulin but appear to inhibit gluconeogenesis (Altschuld and Kruger, 1968; Lloyd et al., 1975). They also aid in the peripheral utilization of glucose (Creutzfeldt et al., 19611. Many investigations of the oral anti-hyperglycaemic agents of plant origin used in traditional medicine have been conducted. One such preparation proven effective in diabetes mellitus is an extract of the leaves of Gymnema sylvestre R.Br. (Shanmugasundaram et al., 19811, a herb belonging to the Asclepiadaceae family. Sushruta (6th Century B.C.1 and practitioners of Ayurveda have recommended the use of the leaves of the ‘sala saradi’ group for the control of maturity onset diabetes in addition to dietary restriction. G. sylvestre belongs to this group and has been described in Indian Medicinal Plants (Blatter et al., 19301. GS,, an extract from G. sylvestre, has been shown to regenerate the islets of Langerhans, especially the beta cells, in both streptozotocin- and alloxantreated diabetic rats (Shanmugasundaram et al., 1988, 1990). In the present paper, GS, was subjected to clinical trial in Type 2 (non-insulin-dependent) diabetes mellitus. Materials
and Methods
GS,, the water-soluble acidic fraction of an ethanol extract of the leaves of G. sylvestre, was tested by oral administration (400 mglday, packed in a hard gelatin capsule1 to 22 Type 2 diabetic patients. The preparation of GS, has been described in an earlier paper (Shanmugasundaram et al., 19901. Patients were enlisted for GS, therapy after informed consent and were enlisted at various times. Consequently, all had been on conventional therapy by the time the present study was instituted. The 22 patients were instructed to take their conventional drugs in addition to GS, capsules. The patients were in the age group of 40 to 62 years and 3 were females. Nineteen patients were on glibenclamide therapy while 3 were on tolbutamide. The duration of diabetes varied from 1 to 12 years and the average value was 4.6 years. Blood glucose maintenance was poor, in spite of oral drugs. The dosage was adjusted to prevent hypoglycaemic episodes. The
297
details of the patients who entered therapy are given in Tables 1 and 2. For comparison, a set of 25 Type 2 diabetic patients (41- 63 years, 17 males and 8 females1 on conventional drugs alone were studied over a period of 10 - 12 months (Tables 1 and 31. The duration of diabetes in this group ranged from l-5 years and the average value was 2.7 years. Blood and urine analysis were made periodically in both groups. Venous blood samples were drawn under fasting conditions from the patients into tubes containing the disodium salt of ethylene diamine tetraacetic acid (EDTA) as anticoagulant. Plasma was separated by decantation after centrifugation of blood and used for the assay of glycosylated plasma proteins (Merelyn et al., 19811, cholesterol (Parekh and Jung, 19701, triglycerides (Rice, 19701, phospholipids (Zilversmit and Davis, 19501 and free fatty acids (Hron and Menahan, 19811. Glycosylated haemoglobin was estimated in the haemolysate by the method of Wang and Yang (19821. One aliquot of blood was collected using sodium fluoride as the anticoagulant for blood glucose estimation using the o-toluidine colour reaction (Dubowski, 1962) as modified by Sasaki and Matsui (19721, urea (Natelson et al., 19511, uric acid (Caraway, 1963) and haemoglobin (Drabkin and Austin, 19321. Serum insulin assay was conducted using 15 healthy adults, 15 cases of NIDDM on conventional therapy and 15 cases of NIDDM after 18-20 months of GS, supplementation. In all three groups, 8 volunteers provided a second sample 90 min after their usual breakfast providing 600-650 calories and this was labelled as the post-prandial sample. Insulin was assayed by the method of Yalow and Berson (19611 using the RIA kit supplied by Bhabha Atomic Energy Research Centre, Bombay. To assess the effectiveness of GS, as an instant blood glucose lowering agent, 12 adult healthy volunteers were administered 200 mg GS, under fasting state, after blood sampling in the morning. Blood samples were collected 45 min after GS, administration and blood glucose was assayed in both samples. Results Table 1 presents the general data of the patients receiving GS, supplementation and the patients on conventional drugs alone. Both groups were reasonably matched regarding age and Broca Index (%I ideal body weight). Table 2 details the raw data of the 22 NIDDM patients on GS, supplementation. Several weeks after GS, supplementation, virtually all patients developed secondary hypoglycaemic symptoms and the dose of glibenclamide or tolbutamide, as the case may be, was reduced or stopped. Most of the patients reported a sense of well-being and better alertness and less exhaustion during work, during GS, supplementation. The female patients reported that the nagging pain in their limbs disappeared within 4 weeks of GS, supplementation. The blood glucose, glycosylated haemoglobin and glycosylated plasma proteins were significantly lower after GS, supple-
S.E.M.
19
17
Conventional drugs alone
Male
Sex
the mean f
GS, supplement
Therapy
Tabular data represents
8
3
Female
CHARACTERISTICS OF THE TYPE 2 DIABETIC TIONALDRUGTHERAPYALONE
TABLE 1 PATIENTS
52.0 ?
48.5 f 1.2
1.4
Age (years)
1.7
162.0 + 1.2
163.0 f
Height (cm)
ON GS, SUPPLEMENTATION
60.0 + 3.0
61.9 & 2.3
3.0 98.9 + 2.1
99.8 f
(%I
Broca Index
TO THOSE UNDER CONVEN-
Weight (kg)
COMPARED
Age
Height
M
M
M
F
M
M
M
M
M
45
49
46
40
46
40
55
52
55
50
44
62
55
60
51
56
1
8
9
10
11
12
13
14
15
16
11
18
19
20
21
22
‘G = glibenelamide.
M
M
M
M
M
M
M
M
62
58
65
105
62
52
62
62
65
64
60
57
66
58
62
60
66
54
54
55
58
55
(kg)
Weight
T = tolbutamide.
162
165
175
182
160
155
158
145
155
160
155
165
165
162
166
167
170
160
M
56
165
M
42
180
F
5
153
161
(em)
F
M
Sex
6
46
42
4
45
2
3
42
(years)
1
Patient
DATA ON THE 22 DIABETIC
TABLE 2
5
3
4
5
12
6
4
6
6
5
1
4
3
6
5
4
4
3
4
3
4
100.0
88.0
86.6
128.0
103.3
94.6
166.9
138.0
118.2
106.6
109.1
88.0
101.5
93.6
91.1
89.6
94.3
91.6
82.0
91.1
109.4
82.0
I%)
5
(years)
Broca
Index
G
G
G
G
G
G
G
T
G
T
G
G
G
G
G
G
G
G
G
G
T
G
name’
Drug
5mg
5mg
5mg 10 mg
10 mg
7.5 mg
10 mg
1.5 g
10 mg
5mg 1.25 g
5mg
5 mg 10 mg
5mg 7.5 mg
10 mg
10 mg
10 mg
2.5 mg
II3
10 mg
GS,
Pre
Medication
18-20
2.5 mg
5 mg Nil
Nil
1.5 mg
1.5 mg
1g 1.5 mg
5mg
1g
Nil
Nil
1.5 mg
5mg 2.5 mg
5mg 2.5 mg
5mg 1.5 mg
Nil
5mg 0.5 g
months
156
120
159
206
159
213
139
220
190
187
165
138
185
108
146
162
182
218
220
186
206
119
GS,
139
110
141
205
155
176
128
186
136
183
139
108
165
110
130
126
124
181
173
131
158
152
months
8-10
120
98
132
168
136
151
108
148
118
124
126
110
143
98
118
110
121
169
148
85
129
136
months
18-20
11.3
9.3
10.0
12.5
12.3
10.6
15.0
12.7
13.3
10.9
11.2
14.2
11.2
12.3
10.8
11.9
10.3
13.1
12.1
12.6
11.6
12.8
GS,
Pre
9.2
1.9
10.1
10.7
9.8
8.9
11.0
9.3
11.6
9.1
10.3
11.8
9.6
9.4
8.7
8.3
9.1
8.9
8.6
102
9.2
9.7
months
8-10
18-20
7.9
8.1
9.6
3.9
9.3
8.4
9.6
8.0
9.2
8.1
9.2
9.5
8.4
8.2
8.6
1.9
7.8
8.1
7.9
8.6
7.8
8.8
months
3.24
3.15
4.03
3.80
3.90
4.10
3.96
4.04
3.30
4.10
4.0
3.66
3.31
3.10
3.40
1+
2.41
2+1+
2.84 3.60
1+
2+
3.14 3.40
3+ 1+
2+1+
2.40
2.93
2.15
3+
2.16 2.92
2+
3.10
2.15
3+
2.44 2.41
2+2+-
2.26 2.60
2+
2.36
2.90
3.30
3.21
3.36 3.11
3.84
2.98 3.5
3.11 3.81
3.14
1+ 1+
2+-
2.65 2.68 2.42
2+
2.28 3.24
3+ 2.53
3+ 3.35
2.81
2+
2+ 2.45
3+
-
-
2+ -
1+
2+
2+
2+
1+
1+ -
1+
2+
1+
1+
2+
2+
GS,
2.28
8-10 months
Pre
Glucosuris
2.56
months
18-20
protein
3.01
3.37
2.94
3.02
4.12
3.28
3.84
3.4
4.06
3.6
3.11
3.42
8-10 months
Pre GS,
Ijag bexoselmg
(%I
Pre
GPP
HbAlc
Blood glucose
GS, SUPPLEMENTATION
(mgldl)
BEFORE, DURING AND AFTER
duration
Disease
PATIENTS
18-20
_
-
1+ -
-
-
-
-
1+ _
-
-
-
-
_
-
1+
1+
-
1+
-
months
300
mentation. Five out of the 22 NIDDM patients on GS, supplementation were able to discontinue their conventional drugs after GS, supplementation and were able to maintain their blood glucose homeostasis with GS, alone. Table 3 presents the individual data of the patients on conventional drug therapy alone. The patients were followed up for a period of 12 months. Fasting blood glucose, glycosylated haemoglobin and glycosylated plasma proteins did not show any significant change from the initial level in patients on conventional drug therapy alone. In virtually all cases during this period, the drug requirement was increased. This is in contrast to the observations made on GS, supplemented diabetics. Table 4 summarizes the collected data on the 47 patients detailed in Tables 2 and 3. The reductions in fasting blood glucose, glycosylated haemoglobin and glycosylated plasma proteins in NIDDM patients on GS, supplementation is statistically significant (P < 0.0011, whereas no significant alterations were observed in patients on conventional drug therapy alone, suggesting that GS, may be acting on the betterment of hormone synthesis and/or its release mechanism on a 24 h basis. Blood urea, uric acid and haemoglobin levels remain in the normal range during GS, supplementation suggesting the absence of hepato- or nephrotoxicity for GS,. Plasma lipid changes during GS, supplementation and on conventional therapy alone are compared to initial values and healthy controls in Table 5. Significant reduction in plasma lipid levels are seen in patients on GS, supplementation while, with conventional drug therapy, plasma cholesterol Cp < 0.051, triglycerides (P < 0.0011 and free fatty acids Cp < 0.011 remain significantly elevated. From Table 6 it can be observed that serum insulin levels are lower than normal in NIDDM patients on conventional drugs in both fasting and in the post-prandial state, which provides data on glucose induced insulin release. However, when the group on GS, supplementation is compared with those on conventional therapy alone, there is a significant elevation of serum insulin in both the fasting and post-prandial state. In tests on the effect of oral GS, on fasting blood glucose, it was found that 45 min after GS, administration fasting blood glucose rose from 89.6 f 1.8 to 97.8 + 2.2, suggesting that GS, may not have an acute effect on blood glucose lowering. Discussion
In view of the reduction in the blood glucose, glycosylated haemoglobin and glycosylated plasma proteins, it may be postulated that GS, supplementation may lead to regeneration/repair or removal of the pancreatic damage in Type 2 diabetes mellitus, such that glucose induced secretion may be augmented. This is supported by the observation made on insulin levels (Table 6) both in the fasting and post-prandial state in GS, administered cases.
M
41
53
50
43
56
58
46
55
63
51
49
56
53
61
55
49
50
55
39
54
53
49
4
5
6
1
8
9
10
11
12
13
14
15
16
11
18
19
20
21
22
23
24
25
‘G = glibenclamide.
F
60
3
153
159
165
150
168
160
153
160
166
155
T = tolbutamide.
F
F
M
F
M
M
F
M
M
F
160
168
M
M
153
168
112
110
160
155
169
165
155
165
158
113
G G
Index (I) 104.6 101.6
duration
(years)
3
G T G G T G G G G
106.0 91.0 90.0 109.0 103.3 91.4 86.1 95.6 111.0
4
2
3
1
55
49
65
58
66
61
55
62
58
59
56
53
62
65
62
64
62
60
62
59
58
G G G
88.0 103.3 104.0 101.6 91.0 116.0 100.0
4
1
2
2
3
1
5
3
113.3
84.5
T
101.3
3
4
G
93.3
5
T
G
G
T
G
G
T
18.0
2
3
4
2
1
3
3
T
90.1
2
60
59
T
103.4
2
name’
BrCEa
Disease
Drug
10 mg
5 mg
u.5 g
1.5 mg
5mg
lg
5mg
u.15 g
10 mg
1.5 mg
1.5 g
1.5 mg
10 mg
10 mg
7.5 mg 7.5 mg
0.75 g
10 mg
0.5 g
5mg
7.5 mg
5mg
10 mg 0.15 g
10 mg
5 mg 1.5 mg 0.5 g
7.5 mg
5mg
1g 7.5 mg
1.5 mg
10 mg
10 mg
1.25 g
7.5 mg
1.25 g
1 g
0.5 g
5mg
1.5 mg
lg
5mg
lg
0.15 g
7.5 mg 1.5 mg
5w
months
10-12
7.5 mg
Initial
Medication
WHO WERE ON CONVENTIONAL
14
68
(kg1
(cml
165
Weight
PATIENTS
Height
F
M
M
M
M
M
F
M
M
M
55
2
M
Sex
54
Age (years)
1
Patient
DATA ON THE 25 DIABETIC
TABLE 3
146
168
139
155
164
153
146
114
165
118
120
124
186
115
159
132
156
124
148
134
166
124
131
186
156
Initial
12
12
lo-12
3+ 1+ 1+
3.48 2.80 3.45
3.12 2.96 3.16
1+
3.66 3.28 10.1
9.5
1+
3.41
3.26 10.3 159
186
9.6
3.18
3.11 9.9
10.1
126
2.98
3.26 9.6
9.6
164
3.04
3.16 10.3 9.4
116
1+
1+
1+
1+
2.54
2.42 9.8
9.0
1+
2.83 154
1+
3.46 2.54 12.3
11.4 159
3.14 10.9
10.6
186
1+
3.26 2.98
11.9 11.1
142
1+
3.40
3.12 10.4 109
9.8
10.6
10.1
149
1+
9.4
9.9
4.21
3.16 3.45
180
3.86
1+ 2+
3.86 3.14 9.1 10.8
9.2 11.2
182
1+
3+
3.18 2.16 10.6
9.6
116
3.11
1+
2.74
2+
3.15 2.90
2.86 2.45
10.3 10.2
10.9 11.1
9.9
11.4
139 146
10.9
159 141
2+
3.26 3.15
10.3
10.6 10.1
156
10.6
9.4
169
2+
2+
1+
2+
2+
1+
2+
2+
1c
1+
1+
1+
1+
1+
2+
1+
2+
1+
1+
2+
2+
1+
2+ 1+
3.24 3.56
11.3
10.1
135
3+ 1+
3.14 3.34
11.2
10.6
146
2+
2.95
2+
2.86
9.8 10.1
9.2
139
lo-
12 months
2+
Initial
3.08
months
Glucosuria
3.69
Initial
mg protein)
11.3
months
lo-
199
Initial
154
months
lo-
GPP (pg bexosel
HbAle (46)
Blood glucose
ALONE
(mgldl)
DRUG THERAPY
302
*Statistically
Serum insulin
LEVELS
30 f
2.3
Fasting
Normals
CONTROLS
95 f
5.3
Postprandial
(&l/ml) IN HEALTHY
13 + 2.3
Fasting
Conventional
NIDDM
AND IN CASES
significant changes (P < 0.01) compared with the group on conventional
SERUM INSULIN TION
TABLE 6
Significant changes relative to the initial value: *P < 0.05; **P < 0.01; l**P < 0.001.
21 + 2.3*
Fasting
GS, supplement
63 f
= 8.
6.7*
Postprandial
GS, SUPPLEMENTA-
therapy alone. N (fasting) = 15; N (postprandial)
50 k 5.6
Postprandial
drugs alone
OF NIDDM WITH AND WITHOUT
55.6 + 1.1 61.3 k 1.2**
197 * 2 201 f 2
148 f 2 164 + 3***
252 + 3 261 f 3*
Initial lo- 12 months
Conventional drug alone
70.9 + 1.9 62.5 + 1.9 55.4 + 1.7***
218 f 3 201 * 2 188 rt 2***
170 + 2 156 + 2 142 f 2***
260 + 4 242 f 4 231 + 3***
(mgldi)
Initial 8- 10 months 18 - 20 months
GS, supplement
UNDER
Free fatty acids (mgldl)
TO PATIENTS
Phospholipids
COMPARED
fmgldl)
UNDER GS, SUPPLEMENTATION
Triglycerides (mgldl)
S.E.M.
PATIENTS
Cholesterol
Duration
the mean f
Therapy
Tabular data represents
BLOOD LIPID PARAMETERS IN TYPE 2 DIABETIC CONVENTIONALTHERAPYALONE
TABLE 5
304
Better glycaemic control can be obtained with GS, supplementation (Tables 4 and 61 in Type 2 diabetic patients mediated through stimulation of insulin synthesis/secretion. Alternatively, GS, may be acting on the liver cells and inhibiting the process of insulin inactivation. GS, administration to healthy volunteers does not produce any acute reduction in fasting blood glucose level and it may be concluded that GS, is unlikely to stimulate insulin release as in the case of sulfonylureas. Plasma cholesterol, triglycerides and free fatty acid levels in patients on conventional drugs were elevated significantly confirming that the efficacy of the conventional drugs on lipid metabolism decreases over a period of time (Goldberg et al., 19741. In contrast, plasma lipids (cholesterol, triglycerides, phospholipids and free fatty acids) were significantly reduced in diabetics after 18-20 months of GS, supplementation and approached normal levels (Table 51. The above results confirm that GS, supplementation to Type 2 diabetic patients appears to have an advantage over conventional drug therapy alone, in reducing hyperlipaemia and may possibly delay the lipid-mediated secondary complications of atherosclerosis. Pirart (19771 and Tchobroutsky (19781 have reported that there is indirect evidence for the possible association between the duration and the severity of hyperglycaemia and the risk of developing such complications. Hyperglycaemia, hyperlipaemia and excessive glycosylation of proteins, including haemoglobin, which are the direct consequences of insulin insufficiency in Type 2 diabetes, appear to be corrected by GS, therapy, by augmenting insulin levels in circulation. Longer periods of GS, therapy and better follow up of patients are needed. Acknowledgements K.B. acknowledges the award of a fellowship by the University Grants Commission, India. Some parts of the work have been incorporated in the Ph.D. thesis submitted to the University of Madras. Insulin assays were made by Dr.M.K. Balasubramanian at Uma Clinical Laboratory, Madras. References Altschuld, R.A. and Kruger, F.A. (19681 Inhibition of hepatic gluconeogenesis in guinea pig by phenformin. Annals of the New York Academy of Sciences 148, 612-622. Blatter, E., Caius, J.F. and Mhaskar, KS. (19301 Indian Medicinal PZants, Vol. 3, 2nd Edn., Lalit Mohan Basu, Allahabad, India, pp. 1625- 1627. Caraway, W.T. (1963) Uric acid. In: D. Seligson (Ed.), Standard Methods of Clinical Chemistry, Vol. 4, Academic Press, New York, pp. 239- 247. Cruetzfeldt, W., Deuticke, U. and Soling, H.D. (19611 Potentiation of the action of exogenous insulin by N-(4-methyl benzene sulfonyll N-butyl carbamide and N-butyl biguanide in the eviserated animal. Kilinische Wochenschrift 39, 790 - 795. Drabkin, D.L. and Austin, J.H. (19321 Spectrophotometric studies, spectrophotometric constants for common haemoglobin derivatives in human, dog and rabbit blood. Journal of Biological Chemistry 98,719-733. Dubowski, K.M. (19621 An Chemistry 8, 215- 235.
o-toluidine
method
for body
fluid
glucose
determination.
Clinical
305
Efendic, S., Enzmann, F., Nylen, A., Uvnas Wallensten, K. and Luft, R. (1979) Effect of glucose/ sulfonylurea interaction on the release of insulin, glucagon and somatostatin from isolated perfused rat pancreas. Proceedings of the National Academy of Sciences U.S.A. 76, 59015904. Goldberg, M.F. (1974) In: J.R. Lynn, W.B. Snyder and A. Vaiser (Eds.1, Diabetic Retinopathy, Grune and Stratton, New York, pp. 47-63. Hron, W.T. and Menahan, L.A. (1981) A sensitive method for the determination of free fatty acids in plasma. Journal of Lipid Research 27, 377-381. Kilo, C. (1982) Current status of oral agents in diabetes. Comprehensive Therapy 8, 26-32. Lloyd, M.H., Iles, R.A., Walton, B., Hamilton, C.A. and Cohen, R.D. (1975) Effect of phenformin on gluconeogenesis from lactate and intracellular pH in the perfused guinea pig liver. Diubetes 24, 618-624. Merelyn, A.M. Naughton, A. and Cameron, D.P. (1981) Glycosylated plasma protein; A simple method for the elimination of interference by glucose in its estimation. Clinica Chimica Acta 115,111- 117. Natelson, S.. Scott, M.L. and Beffa, C. (1951) A rapid method for the estimation of urea in biological fluids by means of the reaction between biacetyl and urea. American Journal of Clinical Pathology 21, 275-278. O’Rahilly, S.P., Nugent, Z. and Rudenski, A.S. (1986) Beta-cell dysfunction, rather than insulin insensitivity, is the primary defect in familial Type-II diabetes. Lance t 2, 360 - 363. Parekh, A.C. and Jung, D.H. (1970) Cholesterol determination with ferric acetate-uranyl acetate and sulphuric acid-ferrous sulfate reagents. Analytical Chemistry 42, 1423- 1427. Pirart, J. (197’7) Diabete et complications derivatives. Presentation d’une etude prospective portant sur 4,400 cas observes entre 1947 et 1973. Diabete et Metabolisme 3, 97- 107; 173- 182; 245-256. Prout, T.E. (1974) In: W.J. Malaisse and J.Pirart (Eds.1, Proceedings, VIII Congress of the International Diabetes Federation, Excerpta Medica, Amsterdam. p. 162. Reaven, G.M. (1984) Insulin secretion and insulin action in non-insulin-dependent diabetes mellitus. Diabetes Care 7 (Suppl. l), 17-24. Rice, G.M. (1970) Triglycerides in serum. In: R.P. MacDonald (Ed.), Standard Methods of Clinical Chemistry, Vol. 6, Academic Press, New York, pp. 215-222. Sasaki, T. and Matsui, S. (1972), Effect of acetic acid concentration on the colour reaction in the o-toluidine boric acid method. Rinsho Kagaku 1, 346-353. Shanmugasundaram, K.R., Pannerselvam, C., Samudram, P. and Shanmugasundaram, E.R.B. (1981) Insulinotropic activity of Gymnema sylvestre, R. Br., An Indian medicinal herb in controlling diabetes mellitus. Pharmacological Research Communications 13, 475-586. Shanmugasundaram, E.R.B., Venkatasubramanyam, M., Vijendran, N. and Shanmugasundaram, K.R. (1988) Effect of an isolate from Gymnema sylvestre, R.Br. in the control of diabetes mellitus and the associated pathological changes. Ancient Science of Life 7, 183- 194. Shanmugasundaram, E.R.B.. Leela Gopinath, K., Shanmugasundaram, K.R. and Rajendran V.M. (1990) Possible regeneration of the islets of Langerhans in streptozotocin diabetic rats given Gymnema leaf extracts. Journal of Ethnopharmacology 30, 265-279. Sushruta (6th century B.C.), Sushruta Samhita, Vol. 2: Chikitsasthanum (Chapter XII on Prambhar Pridika, verse 9, referred from an English translation of the Sushruta Samhita by K.K. Phishgratna), S.L. Bhaduri Publisher, Calcutta, pp. 379-392. Tchobroutsky, G. (1978) Relation of diabetic control to development of microvascular complications. Diabetologia 15, 143- 152. Wang, J. and Yang, G. (1982) Calorimetric detection of glycosylated haemoglobin. Hun,un Yixylyuan Xe bao 7,325 - 328. Yalow, R.S. and Berson, S.A. (1961) Immunoassay of plasma insulin in man. Diabetes 10, 339343. Zilversmit, D.B. and Davis, A.K. (1950) Micro determination of plasma phospholipid by TCA precipitation. Journal of Laboratory and Clinical Medicine 35, 155- 160. Zimmet, P. (1982) Type II (non-insulin-dependent) diabetes: An epidemiological overview. Diabeto1ogiu 22, 399-411.
Scientific
Publishers
295
30 (1990) 295 - 305 Ireland Ltd.
ANTIDIARETIC EFFECT OF A LEAF EXTRACT FROM GYMNEMA SYLVESTRE IN NON-INSULIN-DEPENDENT DIABETES MELLITUS PATIENTS
K. BASKARAN, B. KIZAR AHAMATH, E.R.B. SHANMUGASUNDARAM
of Biochemistry,
Department
K. RADHA
Postgraduute
Institute
SHANMUGASUNDARAM
of Basic Medical
Sciences
and
Madras-600
119 Ilndti (Accepted
June
26, 1990)
Summary
The effectiveness of GS,, an extract from the leaves of Gymnema syG in controlling hyperglycaemia was investigated in 22 Type 2 diabetic patients on conventional oral anti-hyperglycaemic agents. GS, (400 mglday) was administered for 18-20 months as a supplement to the conventional oral drugs. During GS, supplementation, the patients showed a significant reduction in blood glucose, glycosylated haemoglobin and glycosylated plasma proteins, and conventional drug dosage could be decreased. Five of the 22 diabetic patients were able to discontinue their conventional drug and maintain their blood glucose homeostasis with GS, alone. These data suggest that the beta cells may be regenerated/repaired in Type 2 diabetic patients on GS, supplementation. This is supported by the appearance of raised insulin levels in the serum of patients after GS, supplementation. vestre,
Introduction
Type 2 (NIDDM, non-insulin dependent) diabetes mellitus is among the most common disorders in developed and developing countries (Zimmet, 1982). Reaven (1984) has reported that abnormalities of beta cell function and secretion exist in patients with non-insulin dependent diabetes mellitus. O’Rahilly et al. (1986) observed that the inherited component of familial Type 2 diabetes may be an impaired insulin secreting response of the beta cells. While fasting serum insulin levels may be in the normal range (up to 25 rIU/ ml), glucose-induced insulin release is reduced, leaving below normal (< 55 rIU/ml) insulin levels in the post-prandial state. Correspondence
to: Prof. K. Radha Shanmugasundaram.
0378.8741/$03.50 0 1990 Elsevier Published and Printed in Ireland
Scientific
Publishers
Ireland
Ltd.
296
Control of blood glucose on a 24-h basis is the desired goal in the management of diabetes mellitus so as to prevent or delay the onset of the secondary complications of diabetes mellitus. Dieting, physical exercise and inclusion of dietary fibre have been used with limited success. Oral anti-hyperglycaemic drugs play an important role in the treatment of Type 2 diabetes mellitus (Kilo, 19821. There are two groups of oral antihyperglycaemic agents available for clinical use, i.e. the sulfonylureas and biguanides. The sulfonylureas are reported to regulate blood glucose homeostasis by stimulating pancreatic secretion of insulin (Efendic et al., 19791 and have a characteristic profile of side effects (Prout, 19741.Biguanides do not enhance the release of insulin but appear to inhibit gluconeogenesis (Altschuld and Kruger, 1968; Lloyd et al., 1975). They also aid in the peripheral utilization of glucose (Creutzfeldt et al., 19611. Many investigations of the oral anti-hyperglycaemic agents of plant origin used in traditional medicine have been conducted. One such preparation proven effective in diabetes mellitus is an extract of the leaves of Gymnema sylvestre R.Br. (Shanmugasundaram et al., 19811, a herb belonging to the Asclepiadaceae family. Sushruta (6th Century B.C.1 and practitioners of Ayurveda have recommended the use of the leaves of the ‘sala saradi’ group for the control of maturity onset diabetes in addition to dietary restriction. G. sylvestre belongs to this group and has been described in Indian Medicinal Plants (Blatter et al., 19301. GS,, an extract from G. sylvestre, has been shown to regenerate the islets of Langerhans, especially the beta cells, in both streptozotocin- and alloxantreated diabetic rats (Shanmugasundaram et al., 1988, 1990). In the present paper, GS, was subjected to clinical trial in Type 2 (non-insulin-dependent) diabetes mellitus. Materials
and Methods
GS,, the water-soluble acidic fraction of an ethanol extract of the leaves of G. sylvestre, was tested by oral administration (400 mglday, packed in a hard gelatin capsule1 to 22 Type 2 diabetic patients. The preparation of GS, has been described in an earlier paper (Shanmugasundaram et al., 19901. Patients were enlisted for GS, therapy after informed consent and were enlisted at various times. Consequently, all had been on conventional therapy by the time the present study was instituted. The 22 patients were instructed to take their conventional drugs in addition to GS, capsules. The patients were in the age group of 40 to 62 years and 3 were females. Nineteen patients were on glibenclamide therapy while 3 were on tolbutamide. The duration of diabetes varied from 1 to 12 years and the average value was 4.6 years. Blood glucose maintenance was poor, in spite of oral drugs. The dosage was adjusted to prevent hypoglycaemic episodes. The
297
details of the patients who entered therapy are given in Tables 1 and 2. For comparison, a set of 25 Type 2 diabetic patients (41- 63 years, 17 males and 8 females1 on conventional drugs alone were studied over a period of 10 - 12 months (Tables 1 and 31. The duration of diabetes in this group ranged from l-5 years and the average value was 2.7 years. Blood and urine analysis were made periodically in both groups. Venous blood samples were drawn under fasting conditions from the patients into tubes containing the disodium salt of ethylene diamine tetraacetic acid (EDTA) as anticoagulant. Plasma was separated by decantation after centrifugation of blood and used for the assay of glycosylated plasma proteins (Merelyn et al., 19811, cholesterol (Parekh and Jung, 19701, triglycerides (Rice, 19701, phospholipids (Zilversmit and Davis, 19501 and free fatty acids (Hron and Menahan, 19811. Glycosylated haemoglobin was estimated in the haemolysate by the method of Wang and Yang (19821. One aliquot of blood was collected using sodium fluoride as the anticoagulant for blood glucose estimation using the o-toluidine colour reaction (Dubowski, 1962) as modified by Sasaki and Matsui (19721, urea (Natelson et al., 19511, uric acid (Caraway, 1963) and haemoglobin (Drabkin and Austin, 19321. Serum insulin assay was conducted using 15 healthy adults, 15 cases of NIDDM on conventional therapy and 15 cases of NIDDM after 18-20 months of GS, supplementation. In all three groups, 8 volunteers provided a second sample 90 min after their usual breakfast providing 600-650 calories and this was labelled as the post-prandial sample. Insulin was assayed by the method of Yalow and Berson (19611 using the RIA kit supplied by Bhabha Atomic Energy Research Centre, Bombay. To assess the effectiveness of GS, as an instant blood glucose lowering agent, 12 adult healthy volunteers were administered 200 mg GS, under fasting state, after blood sampling in the morning. Blood samples were collected 45 min after GS, administration and blood glucose was assayed in both samples. Results Table 1 presents the general data of the patients receiving GS, supplementation and the patients on conventional drugs alone. Both groups were reasonably matched regarding age and Broca Index (%I ideal body weight). Table 2 details the raw data of the 22 NIDDM patients on GS, supplementation. Several weeks after GS, supplementation, virtually all patients developed secondary hypoglycaemic symptoms and the dose of glibenclamide or tolbutamide, as the case may be, was reduced or stopped. Most of the patients reported a sense of well-being and better alertness and less exhaustion during work, during GS, supplementation. The female patients reported that the nagging pain in their limbs disappeared within 4 weeks of GS, supplementation. The blood glucose, glycosylated haemoglobin and glycosylated plasma proteins were significantly lower after GS, supple-
S.E.M.
19
17
Conventional drugs alone
Male
Sex
the mean f
GS, supplement
Therapy
Tabular data represents
8
3
Female
CHARACTERISTICS OF THE TYPE 2 DIABETIC TIONALDRUGTHERAPYALONE
TABLE 1 PATIENTS
52.0 ?
48.5 f 1.2
1.4
Age (years)
1.7
162.0 + 1.2
163.0 f
Height (cm)
ON GS, SUPPLEMENTATION
60.0 + 3.0
61.9 & 2.3
3.0 98.9 + 2.1
99.8 f
(%I
Broca Index
TO THOSE UNDER CONVEN-
Weight (kg)
COMPARED
Age
Height
M
M
M
F
M
M
M
M
M
45
49
46
40
46
40
55
52
55
50
44
62
55
60
51
56
1
8
9
10
11
12
13
14
15
16
11
18
19
20
21
22
‘G = glibenelamide.
M
M
M
M
M
M
M
M
62
58
65
105
62
52
62
62
65
64
60
57
66
58
62
60
66
54
54
55
58
55
(kg)
Weight
T = tolbutamide.
162
165
175
182
160
155
158
145
155
160
155
165
165
162
166
167
170
160
M
56
165
M
42
180
F
5
153
161
(em)
F
M
Sex
6
46
42
4
45
2
3
42
(years)
1
Patient
DATA ON THE 22 DIABETIC
TABLE 2
5
3
4
5
12
6
4
6
6
5
1
4
3
6
5
4
4
3
4
3
4
100.0
88.0
86.6
128.0
103.3
94.6
166.9
138.0
118.2
106.6
109.1
88.0
101.5
93.6
91.1
89.6
94.3
91.6
82.0
91.1
109.4
82.0
I%)
5
(years)
Broca
Index
G
G
G
G
G
G
G
T
G
T
G
G
G
G
G
G
G
G
G
G
T
G
name’
Drug
5mg
5mg
5mg 10 mg
10 mg
7.5 mg
10 mg
1.5 g
10 mg
5mg 1.25 g
5mg
5 mg 10 mg
5mg 7.5 mg
10 mg
10 mg
10 mg
2.5 mg
II3
10 mg
GS,
Pre
Medication
18-20
2.5 mg
5 mg Nil
Nil
1.5 mg
1.5 mg
1g 1.5 mg
5mg
1g
Nil
Nil
1.5 mg
5mg 2.5 mg
5mg 2.5 mg
5mg 1.5 mg
Nil
5mg 0.5 g
months
156
120
159
206
159
213
139
220
190
187
165
138
185
108
146
162
182
218
220
186
206
119
GS,
139
110
141
205
155
176
128
186
136
183
139
108
165
110
130
126
124
181
173
131
158
152
months
8-10
120
98
132
168
136
151
108
148
118
124
126
110
143
98
118
110
121
169
148
85
129
136
months
18-20
11.3
9.3
10.0
12.5
12.3
10.6
15.0
12.7
13.3
10.9
11.2
14.2
11.2
12.3
10.8
11.9
10.3
13.1
12.1
12.6
11.6
12.8
GS,
Pre
9.2
1.9
10.1
10.7
9.8
8.9
11.0
9.3
11.6
9.1
10.3
11.8
9.6
9.4
8.7
8.3
9.1
8.9
8.6
102
9.2
9.7
months
8-10
18-20
7.9
8.1
9.6
3.9
9.3
8.4
9.6
8.0
9.2
8.1
9.2
9.5
8.4
8.2
8.6
1.9
7.8
8.1
7.9
8.6
7.8
8.8
months
3.24
3.15
4.03
3.80
3.90
4.10
3.96
4.04
3.30
4.10
4.0
3.66
3.31
3.10
3.40
1+
2.41
2+1+
2.84 3.60
1+
2+
3.14 3.40
3+ 1+
2+1+
2.40
2.93
2.15
3+
2.16 2.92
2+
3.10
2.15
3+
2.44 2.41
2+2+-
2.26 2.60
2+
2.36
2.90
3.30
3.21
3.36 3.11
3.84
2.98 3.5
3.11 3.81
3.14
1+ 1+
2+-
2.65 2.68 2.42
2+
2.28 3.24
3+ 2.53
3+ 3.35
2.81
2+
2+ 2.45
3+
-
-
2+ -
1+
2+
2+
2+
1+
1+ -
1+
2+
1+
1+
2+
2+
GS,
2.28
8-10 months
Pre
Glucosuris
2.56
months
18-20
protein
3.01
3.37
2.94
3.02
4.12
3.28
3.84
3.4
4.06
3.6
3.11
3.42
8-10 months
Pre GS,
Ijag bexoselmg
(%I
Pre
GPP
HbAlc
Blood glucose
GS, SUPPLEMENTATION
(mgldl)
BEFORE, DURING AND AFTER
duration
Disease
PATIENTS
18-20
_
-
1+ -
-
-
-
-
1+ _
-
-
-
-
_
-
1+
1+
-
1+
-
months
300
mentation. Five out of the 22 NIDDM patients on GS, supplementation were able to discontinue their conventional drugs after GS, supplementation and were able to maintain their blood glucose homeostasis with GS, alone. Table 3 presents the individual data of the patients on conventional drug therapy alone. The patients were followed up for a period of 12 months. Fasting blood glucose, glycosylated haemoglobin and glycosylated plasma proteins did not show any significant change from the initial level in patients on conventional drug therapy alone. In virtually all cases during this period, the drug requirement was increased. This is in contrast to the observations made on GS, supplemented diabetics. Table 4 summarizes the collected data on the 47 patients detailed in Tables 2 and 3. The reductions in fasting blood glucose, glycosylated haemoglobin and glycosylated plasma proteins in NIDDM patients on GS, supplementation is statistically significant (P < 0.0011, whereas no significant alterations were observed in patients on conventional drug therapy alone, suggesting that GS, may be acting on the betterment of hormone synthesis and/or its release mechanism on a 24 h basis. Blood urea, uric acid and haemoglobin levels remain in the normal range during GS, supplementation suggesting the absence of hepato- or nephrotoxicity for GS,. Plasma lipid changes during GS, supplementation and on conventional therapy alone are compared to initial values and healthy controls in Table 5. Significant reduction in plasma lipid levels are seen in patients on GS, supplementation while, with conventional drug therapy, plasma cholesterol Cp < 0.051, triglycerides (P < 0.0011 and free fatty acids Cp < 0.011 remain significantly elevated. From Table 6 it can be observed that serum insulin levels are lower than normal in NIDDM patients on conventional drugs in both fasting and in the post-prandial state, which provides data on glucose induced insulin release. However, when the group on GS, supplementation is compared with those on conventional therapy alone, there is a significant elevation of serum insulin in both the fasting and post-prandial state. In tests on the effect of oral GS, on fasting blood glucose, it was found that 45 min after GS, administration fasting blood glucose rose from 89.6 f 1.8 to 97.8 + 2.2, suggesting that GS, may not have an acute effect on blood glucose lowering. Discussion
In view of the reduction in the blood glucose, glycosylated haemoglobin and glycosylated plasma proteins, it may be postulated that GS, supplementation may lead to regeneration/repair or removal of the pancreatic damage in Type 2 diabetes mellitus, such that glucose induced secretion may be augmented. This is supported by the observation made on insulin levels (Table 6) both in the fasting and post-prandial state in GS, administered cases.
M
41
53
50
43
56
58
46
55
63
51
49
56
53
61
55
49
50
55
39
54
53
49
4
5
6
1
8
9
10
11
12
13
14
15
16
11
18
19
20
21
22
23
24
25
‘G = glibenclamide.
F
60
3
153
159
165
150
168
160
153
160
166
155
T = tolbutamide.
F
F
M
F
M
M
F
M
M
F
160
168
M
M
153
168
112
110
160
155
169
165
155
165
158
113
G G
Index (I) 104.6 101.6
duration
(years)
3
G T G G T G G G G
106.0 91.0 90.0 109.0 103.3 91.4 86.1 95.6 111.0
4
2
3
1
55
49
65
58
66
61
55
62
58
59
56
53
62
65
62
64
62
60
62
59
58
G G G
88.0 103.3 104.0 101.6 91.0 116.0 100.0
4
1
2
2
3
1
5
3
113.3
84.5
T
101.3
3
4
G
93.3
5
T
G
G
T
G
G
T
18.0
2
3
4
2
1
3
3
T
90.1
2
60
59
T
103.4
2
name’
BrCEa
Disease
Drug
10 mg
5 mg
u.5 g
1.5 mg
5mg
lg
5mg
u.15 g
10 mg
1.5 mg
1.5 g
1.5 mg
10 mg
10 mg
7.5 mg 7.5 mg
0.75 g
10 mg
0.5 g
5mg
7.5 mg
5mg
10 mg 0.15 g
10 mg
5 mg 1.5 mg 0.5 g
7.5 mg
5mg
1g 7.5 mg
1.5 mg
10 mg
10 mg
1.25 g
7.5 mg
1.25 g
1 g
0.5 g
5mg
1.5 mg
lg
5mg
lg
0.15 g
7.5 mg 1.5 mg
5w
months
10-12
7.5 mg
Initial
Medication
WHO WERE ON CONVENTIONAL
14
68
(kg1
(cml
165
Weight
PATIENTS
Height
F
M
M
M
M
M
F
M
M
M
55
2
M
Sex
54
Age (years)
1
Patient
DATA ON THE 25 DIABETIC
TABLE 3
146
168
139
155
164
153
146
114
165
118
120
124
186
115
159
132
156
124
148
134
166
124
131
186
156
Initial
12
12
lo-12
3+ 1+ 1+
3.48 2.80 3.45
3.12 2.96 3.16
1+
3.66 3.28 10.1
9.5
1+
3.41
3.26 10.3 159
186
9.6
3.18
3.11 9.9
10.1
126
2.98
3.26 9.6
9.6
164
3.04
3.16 10.3 9.4
116
1+
1+
1+
1+
2.54
2.42 9.8
9.0
1+
2.83 154
1+
3.46 2.54 12.3
11.4 159
3.14 10.9
10.6
186
1+
3.26 2.98
11.9 11.1
142
1+
3.40
3.12 10.4 109
9.8
10.6
10.1
149
1+
9.4
9.9
4.21
3.16 3.45
180
3.86
1+ 2+
3.86 3.14 9.1 10.8
9.2 11.2
182
1+
3+
3.18 2.16 10.6
9.6
116
3.11
1+
2.74
2+
3.15 2.90
2.86 2.45
10.3 10.2
10.9 11.1
9.9
11.4
139 146
10.9
159 141
2+
3.26 3.15
10.3
10.6 10.1
156
10.6
9.4
169
2+
2+
1+
2+
2+
1+
2+
2+
1c
1+
1+
1+
1+
1+
2+
1+
2+
1+
1+
2+
2+
1+
2+ 1+
3.24 3.56
11.3
10.1
135
3+ 1+
3.14 3.34
11.2
10.6
146
2+
2.95
2+
2.86
9.8 10.1
9.2
139
lo-
12 months
2+
Initial
3.08
months
Glucosuria
3.69
Initial
mg protein)
11.3
months
lo-
199
Initial
154
months
lo-
GPP (pg bexosel
HbAle (46)
Blood glucose
ALONE
(mgldl)
DRUG THERAPY
302
*Statistically
Serum insulin
LEVELS
30 f
2.3
Fasting
Normals
CONTROLS
95 f
5.3
Postprandial
(&l/ml) IN HEALTHY
13 + 2.3
Fasting
Conventional
NIDDM
AND IN CASES
significant changes (P < 0.01) compared with the group on conventional
SERUM INSULIN TION
TABLE 6
Significant changes relative to the initial value: *P < 0.05; **P < 0.01; l**P < 0.001.
21 + 2.3*
Fasting
GS, supplement
63 f
= 8.
6.7*
Postprandial
GS, SUPPLEMENTA-
therapy alone. N (fasting) = 15; N (postprandial)
50 k 5.6
Postprandial
drugs alone
OF NIDDM WITH AND WITHOUT
55.6 + 1.1 61.3 k 1.2**
197 * 2 201 f 2
148 f 2 164 + 3***
252 + 3 261 f 3*
Initial lo- 12 months
Conventional drug alone
70.9 + 1.9 62.5 + 1.9 55.4 + 1.7***
218 f 3 201 * 2 188 rt 2***
170 + 2 156 + 2 142 f 2***
260 + 4 242 f 4 231 + 3***
(mgldi)
Initial 8- 10 months 18 - 20 months
GS, supplement
UNDER
Free fatty acids (mgldl)
TO PATIENTS
Phospholipids
COMPARED
fmgldl)
UNDER GS, SUPPLEMENTATION
Triglycerides (mgldl)
S.E.M.
PATIENTS
Cholesterol
Duration
the mean f
Therapy
Tabular data represents
BLOOD LIPID PARAMETERS IN TYPE 2 DIABETIC CONVENTIONALTHERAPYALONE
TABLE 5
304
Better glycaemic control can be obtained with GS, supplementation (Tables 4 and 61 in Type 2 diabetic patients mediated through stimulation of insulin synthesis/secretion. Alternatively, GS, may be acting on the liver cells and inhibiting the process of insulin inactivation. GS, administration to healthy volunteers does not produce any acute reduction in fasting blood glucose level and it may be concluded that GS, is unlikely to stimulate insulin release as in the case of sulfonylureas. Plasma cholesterol, triglycerides and free fatty acid levels in patients on conventional drugs were elevated significantly confirming that the efficacy of the conventional drugs on lipid metabolism decreases over a period of time (Goldberg et al., 19741. In contrast, plasma lipids (cholesterol, triglycerides, phospholipids and free fatty acids) were significantly reduced in diabetics after 18-20 months of GS, supplementation and approached normal levels (Table 51. The above results confirm that GS, supplementation to Type 2 diabetic patients appears to have an advantage over conventional drug therapy alone, in reducing hyperlipaemia and may possibly delay the lipid-mediated secondary complications of atherosclerosis. Pirart (19771 and Tchobroutsky (19781 have reported that there is indirect evidence for the possible association between the duration and the severity of hyperglycaemia and the risk of developing such complications. Hyperglycaemia, hyperlipaemia and excessive glycosylation of proteins, including haemoglobin, which are the direct consequences of insulin insufficiency in Type 2 diabetes, appear to be corrected by GS, therapy, by augmenting insulin levels in circulation. Longer periods of GS, therapy and better follow up of patients are needed. Acknowledgements K.B. acknowledges the award of a fellowship by the University Grants Commission, India. Some parts of the work have been incorporated in the Ph.D. thesis submitted to the University of Madras. Insulin assays were made by Dr.M.K. Balasubramanian at Uma Clinical Laboratory, Madras. References Altschuld, R.A. and Kruger, F.A. (19681 Inhibition of hepatic gluconeogenesis in guinea pig by phenformin. Annals of the New York Academy of Sciences 148, 612-622. Blatter, E., Caius, J.F. and Mhaskar, KS. (19301 Indian Medicinal PZants, Vol. 3, 2nd Edn., Lalit Mohan Basu, Allahabad, India, pp. 1625- 1627. Caraway, W.T. (1963) Uric acid. In: D. Seligson (Ed.), Standard Methods of Clinical Chemistry, Vol. 4, Academic Press, New York, pp. 239- 247. Cruetzfeldt, W., Deuticke, U. and Soling, H.D. (19611 Potentiation of the action of exogenous insulin by N-(4-methyl benzene sulfonyll N-butyl carbamide and N-butyl biguanide in the eviserated animal. Kilinische Wochenschrift 39, 790 - 795. Drabkin, D.L. and Austin, J.H. (19321 Spectrophotometric studies, spectrophotometric constants for common haemoglobin derivatives in human, dog and rabbit blood. Journal of Biological Chemistry 98,719-733. Dubowski, K.M. (19621 An Chemistry 8, 215- 235.
o-toluidine
method
for body
fluid
glucose
determination.
Clinical
305
Efendic, S., Enzmann, F., Nylen, A., Uvnas Wallensten, K. and Luft, R. (1979) Effect of glucose/ sulfonylurea interaction on the release of insulin, glucagon and somatostatin from isolated perfused rat pancreas. Proceedings of the National Academy of Sciences U.S.A. 76, 59015904. Goldberg, M.F. (1974) In: J.R. Lynn, W.B. Snyder and A. Vaiser (Eds.1, Diabetic Retinopathy, Grune and Stratton, New York, pp. 47-63. Hron, W.T. and Menahan, L.A. (1981) A sensitive method for the determination of free fatty acids in plasma. Journal of Lipid Research 27, 377-381. Kilo, C. (1982) Current status of oral agents in diabetes. Comprehensive Therapy 8, 26-32. Lloyd, M.H., Iles, R.A., Walton, B., Hamilton, C.A. and Cohen, R.D. (1975) Effect of phenformin on gluconeogenesis from lactate and intracellular pH in the perfused guinea pig liver. Diubetes 24, 618-624. Merelyn, A.M. Naughton, A. and Cameron, D.P. (1981) Glycosylated plasma protein; A simple method for the elimination of interference by glucose in its estimation. Clinica Chimica Acta 115,111- 117. Natelson, S.. Scott, M.L. and Beffa, C. (1951) A rapid method for the estimation of urea in biological fluids by means of the reaction between biacetyl and urea. American Journal of Clinical Pathology 21, 275-278. O’Rahilly, S.P., Nugent, Z. and Rudenski, A.S. (1986) Beta-cell dysfunction, rather than insulin insensitivity, is the primary defect in familial Type-II diabetes. Lance t 2, 360 - 363. Parekh, A.C. and Jung, D.H. (1970) Cholesterol determination with ferric acetate-uranyl acetate and sulphuric acid-ferrous sulfate reagents. Analytical Chemistry 42, 1423- 1427. Pirart, J. (197’7) Diabete et complications derivatives. Presentation d’une etude prospective portant sur 4,400 cas observes entre 1947 et 1973. Diabete et Metabolisme 3, 97- 107; 173- 182; 245-256. Prout, T.E. (1974) In: W.J. Malaisse and J.Pirart (Eds.1, Proceedings, VIII Congress of the International Diabetes Federation, Excerpta Medica, Amsterdam. p. 162. Reaven, G.M. (1984) Insulin secretion and insulin action in non-insulin-dependent diabetes mellitus. Diabetes Care 7 (Suppl. l), 17-24. Rice, G.M. (1970) Triglycerides in serum. In: R.P. MacDonald (Ed.), Standard Methods of Clinical Chemistry, Vol. 6, Academic Press, New York, pp. 215-222. Sasaki, T. and Matsui, S. (1972), Effect of acetic acid concentration on the colour reaction in the o-toluidine boric acid method. Rinsho Kagaku 1, 346-353. Shanmugasundaram, K.R., Pannerselvam, C., Samudram, P. and Shanmugasundaram, E.R.B. (1981) Insulinotropic activity of Gymnema sylvestre, R. Br., An Indian medicinal herb in controlling diabetes mellitus. Pharmacological Research Communications 13, 475-586. Shanmugasundaram, E.R.B., Venkatasubramanyam, M., Vijendran, N. and Shanmugasundaram, K.R. (1988) Effect of an isolate from Gymnema sylvestre, R.Br. in the control of diabetes mellitus and the associated pathological changes. Ancient Science of Life 7, 183- 194. Shanmugasundaram, E.R.B.. Leela Gopinath, K., Shanmugasundaram, K.R. and Rajendran V.M. (1990) Possible regeneration of the islets of Langerhans in streptozotocin diabetic rats given Gymnema leaf extracts. Journal of Ethnopharmacology 30, 265-279. Sushruta (6th century B.C.), Sushruta Samhita, Vol. 2: Chikitsasthanum (Chapter XII on Prambhar Pridika, verse 9, referred from an English translation of the Sushruta Samhita by K.K. Phishgratna), S.L. Bhaduri Publisher, Calcutta, pp. 379-392. Tchobroutsky, G. (1978) Relation of diabetic control to development of microvascular complications. Diabetologia 15, 143- 152. Wang, J. and Yang, G. (1982) Calorimetric detection of glycosylated haemoglobin. Hun,un Yixylyuan Xe bao 7,325 - 328. Yalow, R.S. and Berson, S.A. (1961) Immunoassay of plasma insulin in man. Diabetes 10, 339343. Zilversmit, D.B. and Davis, A.K. (1950) Micro determination of plasma phospholipid by TCA precipitation. Journal of Laboratory and Clinical Medicine 35, 155- 160. Zimmet, P. (1982) Type II (non-insulin-dependent) diabetes: An epidemiological overview. Diabeto1ogiu 22, 399-411.
