Assessment of anti-diabetic effect of Vietnamese herbal drugs

The prevalence of type 2 diabetes mellitus is increasing in Vietnam as well as in other developing countries (China, Indian subcontinent, and Africa). Searching for hypoglycemic agents with origin from domestic herbals was considered as a useful way to find novel therapy of the disease.

After treatment i.p. or orally of normal mice with extract of Anemarrhena asphodeloides (A.a.), Angiopteris evecta (A.e.) and Gynostemma pentaphyllum (G.p.), blood glucose levels of the mice were decreased. All of those 3 extracts also suppressed the rise in blood glucose in normal mice during a glucose tolerance test.

At both 3.3 and 16.7 mM glucose, 2, 4 and 8 mg/ml Anemarrhena asphodeloides (A.a. or TH2J increased the insulin release of Wistar (W) and Goto- Kakizaki (GK) rat islets. In perifusions of islets, A.a. also increased insulin secretion that returned to basal levels when A.a. was omitted from the perifusate. Thus, ethanol extract of the roots of A.a. contains a substance, TH2, that stimulates insulin secretion from islets of normal W and GK rats. The mechanism behind TH2-stimulated insulin secretion involves an effect on the exocytotic machinery of the B-cell, mediated via pertussis toxin-sensitive Gc-proteins.

G.p. extract had a hypoglycemic effect in rats and mice. We have isolated the active compound, phanoside, a gypenoside with molecular mass of 914.5 Da. When given orally to rats, phanoside (40 and 80 mg/kg) improved glucose tolerance and enhanced plasma insulin levels. Phanoside stimulated insulin release at 3.3 and 16.7 mM glucose from isolated rat pancreatic islets of both W and GK rats. Interestingly, B- cell sensitivity to phanoside is higher at 16.7 mM than at 3.3 mM glucose, since significant insulin responses were observed with phanoside between 31.25 and 125 pM only at the high glucose levels. When W rat islets were incubated at 3.3 mM glucose with 150 pM phanoside and 0.25 mM diazoxide to keep K-ATP channels open, insulin secretion was similar to that in islets incubated in 150 pM phanoside alone. At 16.7 mM glucose, phanoside-stimulated insulin secretion was reduced in the presence of 0.25 mM diazoxide. In W islets depolarized by 50 mM KC1 and with diazoxide, phanoside stimulated insulin release 2-fold at 3.3 mM glucose but did not further increase the release at 16.7 mM glucose. When using nimodipine to block L-type Ca2+ channels in B-cells, phanoside-induced insulin secretion was unaffected at 3.3 mM glucose but decreased at 16.7 mM glucose. In perifusion of islets, phanoside (75 and 150 |iM) dose-dependently increased insulin secretion that returned to basal levels when phanoside was omitted. Thus, the effect of phanoside seems to be exerted distal to K-ATP channels and L-type Ca2+ channels that is on the exocytotic machinery of the B-cells.

In conclusion, from 8 Vietnamese herbal drugs, we. have found 3 extracts which decreased blood glucose of the mice. Two of them (A. a and G.p. extract) stimulated insulin secretion from rat islets. Ethanol extract of A. a. (TH2) stimulated insulin secretion by an effect on the exocytotic machinery of the B cell mediated via pertussis toxin sensitive Ge-protein. From G.p. we islolated a novel substance, phanoside, that directly stimulates the exocytosis of insulin.

Keywords: G-protein, type 2 diabetes, phanoside, antidiabetic plant, hypoglycemic agent, insulin secretion, pancreatic islets, Gynostemma pentaphyllum, Anemarrhena asphodeloides, Angiopteris evecta.

Contents

Abstract 2

List of original papers 4

Abbreviations 5

Background 7

1. General 7

2. Treatment of diabetes in traditional medicine 8

3. Gynnostemma pentaphyllum 9

4. Anemarrhena asphodeloides 9

5. Modulation of insulin secretion in pancreatic B-cells 10

6. Goto-Kakizaki (GK) rats 13

Aims 15

Materials and Methods 16

1. Methods of extraction 16

2. Purification and characterization of compound structures 17

3. Screening study 19

4. Glucose tolerance test 19

5. Isolation of pancreatic islets 20

6. Batch incubation of islets 20

7. Perifusion of islets 21

8. Effect of pharmacological agents on insulin secretion stimulated by active substances 21

9. Measurement of insulin secretion from islets 22

10. Cell viability 22

11. Data analysis 23

Results and discussion 24

1. Screening of the hypoglycemic effect of eight herbs (Paper I) 24

2. Insulin secretion is stimulated by ethanol extract of Anemarrhena asphodeloides (TH2) in isolated islet of healthy Wistar and diabetic Goto-Kakizaki rats (Paper

II) 27

3. Phanoside, a novel hypoglycemic substance from Gynostemma pentaphyllum (Paper III-IV) 30

4. The mechanism by which phanoside stimulates insulin secretion from rat islets (Paper IV) 36

Conclusions 40

Acknowledgements 41

Financial supports 42

References 43

Paper I-IV 51