414 Planta Med. 57(1991)

Hypoglycemic Effects of Sesquiterpene Glycosides and Polyhydroxylated Triterpenoids of Eriobo tryajaponica Nunziatina De Tommasi , Francesco De Simone , Giuseppe Cirino2, Carla Cicala2, and Cosimo Pizza"3 2

Dipartimento di Chimica delle Sostanze Naturali, Universita' di Napoli "Federico 11", Via D. Montesano 49,1-80131 Napoli, Italy Dipartimento di Farmacologia Sperimentale, Universita' di Napoli "Federico IT", Via D. Montesano 49, 1-8013 1 Napoli, Italy

Address for correspondence Received: September 9, 1990

cH3

The effects of the constituent sesquiter-

CH2

L L.-OR

pene glycosides 1—3 and polyhydroxylated triterpenoids

5—6 isolated by MeOH extraction ofEriobotryajaponica were studied in genetically diabetic mice (C57BL/KS-db/

db/Ola) and normoglycemic rats, The sesquiterpene glyco side 3 and the polyhydroxylated triterpenoids 5 and

6 produced a marked inhibition of glycosuria. Furthermore, 5 and 6 were able to reduce blood glucose levels in normoglycemic rats. While there are already some data reported on hypoglycemic activity of polyhydroxylated

triterpenoids, there are no previous data showing hypoglycemic activity of sesquiterpene glycosides.

H3C,).

H3 C

CH3

H3C

CH3

HO

H30

11']OOH

HOQ H3C 'CH3

1 Rha

6fl

5

1 R —0Lu21 'Rha—Rha

2 R —Glu-Rh—'Rha

4 R —Otu-Rha Glu

CH3

HO H3C

3 R —Gtu-'Rha'—'Rha p-D-glucopyranose

3c,1áCOOH

Rha a-L-rhamnopyranOse

CH3

Key words H3C H3ÔH

Eriobotrya japonica, hypoglycemic ef-

6

fects, sesquiterpene glycosides, polyhydroxylated triterpenoids.

HO H3C

Introduction

OOH

HO.

The leaves of Eriobotrya japonica Linde

CH3

(Rosaceae), a small tree commonly known as "loquat", are documented for use as folk medicine for the treatment of various skin diseases (1) and diabetes mellitus (2). Winter et al. (3) found that the alcoholic extract of the leaves exhibited anti-inflammatory activity in

carrageenan-induced paw edema in rat. More recently Shimizu et al. (1) reported the isolation of some known triterpenes from the Et20-soluble fraction of an EtOH ex-

tract of the leaves and, among these, maslinic acid was found to have anti-inflammatory activity. Furthermore, Noreen et al. (4) have reported a significant hypoglycemic effect in rabbits obtained using the alcoholic extract.

We have recently isolated from the MeOFI extract four new sesquiterpene glycosides 1—4 (6) and two major polyhydroxylated triterpenes, the new 3,6,19-trihydroxy-urs-12-en-28-oic acid (5) (5) and the already known 2,3-dihydroxy-urs-1 2-en-28-oic acid (6).

H3C 'CH3

7

In this work we have examined the hypoglycemic effect of the constituents of the methanol extract

obtained by sequential extraction with petroleum ether, and CHC13 followed by MeOH.

Materials and Methods General experimental procedures The following instruments were used: NMR Bruker MW-250 Spectrospin; MS, Kratos MS 902 spectrometer equipped with Kratos fab source; HPLC, Waters model 6000 A pump equipped with a U6K injector and a 401 Refractive Index Detector, GLC, Perkin Elmer sigma 115 instrument; optical rotation, Perkin Elmer 241 polarimeter; DCCC, 670 mod. Büchi apparatus

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Abstract

Planta Med. 57(1991) 415

Hypoglycemic Effects of Sesquiterpene Glycosides and Polyhydroxylated Triterpenoids ofEriobotryajaponica

Statistics

equipped with 300 tubes. The FAB-mass spectra and the t3C-DEPT NMR experiments were performed as described earlier (7).

Glycosuria was calculated as percent of the decrease in glucose in urine. fiesults were calculated as follows:

The plant was collected in April 1987 at Suzhou, Chang-Sou province. China, and was taxonomically examined by Dr. Jang Mi of the Analytic Group of the Department of Traditional Chinese Medicine, Shanghai. A voucher sample of the plant is deposited at the Herbarium of this Department.

isolation ofsesquiterpene glycosides

% decrease in glucose =

Sephadex LH-20 column (100 x 5cm). Fractions (8 ml) were eluted with MeOH and checked by TLC [silica gel plates, n-BuOH-H0Ac-

20 (750mg), 2 and 3 in fractions 22—28 (360mg), and the less

100

Results and Discussion Effects of compounds 1,2,3,5 and6 on

of residue. Part of the residue (30 g) was chromatographed on a

MeOH-H20 (7: 13 : 8) in which the stationary phase consisted of the lower phase (ascending mode, flow 12 ml/h). Fractions (4 ml) were collected to yield the more polar glycosides 1 and 3 in fractions 18—

Gx

G0 and G are respectively the values of initial

The air-dried leaves (4kg) were defatted with

Fractions 18—27 (600mg) containing the crude glycosidic mixture were further purified by DCCC with CHC13-

—

glycemia or glycosuria and the glycemia or the glycosuria at different times. The data are shown as mean + 5P.M. and statistical significance was evaluated by Student's I test.

petroleum ether and CHC13 and extracted with Me0H to give 150 g

H20 (60:15:25)].

G0

glycosuria in C57BL/KS-db/db/Ola mice

As shown in Fig. 1, a single oral administration of 200 mg/kg p.o. of 3 resulted in a marked inhibition reaching its maximal effect after 4 hours and declining to about 50% after 7 h. In the same test 1 and 2 remained inactive at the higher dose tested (300mg/kg). It is to be noted that the main difference between 3 and 1 and 2 is the lack of substitution on C-6 of the glucose unit in 3.

polar 4 in fractions 28—34 (160mg). Separation of each glycoside

isolation of triterpenes Fractions (48—80) containing a triterpene mixture (692.8mg) were fractionated by HPLC on a C18 s-Bondapak

column (30cm x 7.8mm id.) using Me0H-H20, 9: 1 (flow rate: 3.5 ml/min) as eluent to obtain pure 5 (100 mg), H1 = 3.5 mm and 6 (190 mg, H1 = 4.8 mm).

Compounds 1—6 were identified by comparison of the physical and spectral data with those reported in the litera-

txxxxxxxoee—.

mi [a]0:

—F-

8

—41° (c 1, MeOH), [MID = —277°; 4 (30mg), H1 = 12mm, [a]5: — 25.4° (c 1, MeOH), [M]0 = — 136°.

_

[a]5: —40° (c 1, MeOH), [M]D = —270°; 3(144mg),

% inhibition glicosuria

7.8mm id.) with MeOH : H20 (65 : 35) to yield 1 (288 mg), H1 = 6mm, [a]5: 50° (c 1, Me0H), [M]0 = —411°; 2(146mg), lt= 8 mm,

00000000

was achieved by HPLC on a C88 -Bondapak column (30cm x

ture(5,6).

hours

Bioassay for the hypoglycemic activity

in rats

Fig. 1 Effect on glycosuria on C578L/kS-db/db/OIa mice of () 1, () 2, and (X) 3 at a dose of 200 mg/kg p.o.; (U) tolbutamide 500 mg/kg p.o. was used as control. The results are mean of n = 4—5 experiments.

Male Wistar rats weighing 150—180g, were fasted for 20h but were given water ad libitnm. fiats were anaesthetized with urethane (1 g/kg) intraperitoneally and blood samples, for glucose determination, were collected from the jugular vein at intervals of 30 mm for 2 h. fiats were treated alternatively with the drug suspended in carboxymethylcellulose (0.5%) or with the same volume of vehicle only, 30 mm before starting the experiment. Glucose levels were determined with an enzymatic glucose oxidase method.

120

0 3 5 0

Mice C57BL/KS-db/db/Ola purchased from Olac (USA) weighing 20—25 g, were used (8). Mice were fasted one hour before the experiment and housed in groups of 4—5 in metabolic cages. Urine was collected 1 h before and 2, 4, and 7 hours after the administration of the drug or the vehicle. Tolbutamide was used as control at a dose of 500 mg/kg. Glucose levels were assessed as described above.

I

80

U 01

Bioassay for the glycosuria in mice

100

C

60

.0 C

40

0

20

0—

200

100

10

Dose (mg/kg) Fig. 2 Effect of different doses of 3 p.o. on glycosuria on C57BL/KS-db/db/ Ola mice. The results are mean of n = 4—5 experiments.

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Plant material

416 Planta Med. 57 (1991)

Nunziatina De Tommasi et al.

In Fig. 2 is shown the effect of different doses of 3 on glycosuria at the 4 hours point; 3 still remained very active even after reducing the dose by 20

a

times.

I0,

0

U

There are no previous data reported for hypoglycemic activity of sesquiterpene glycosides.

C

0 .0 -c

In another set of experiments we tested

C

4

2

7 hours

in in C578L/KS-db/db/OIa mice: () () Fig.3 3Effect Effectofofdifferent differentdoses dosesofof6po. 6 p.o. C57BL/KS-db/db/Ola mice: mg/kg; ((•) 1010 mg/kg; 1 mg/kg. The 50mg/kg; mg/kg;(U) (E]) 1 mg/kg. Theresults resultsare aremean meanofofnn4—5 4—5

compounds 5 and 6, and in Figs. 3 and 4 are reported the effects of 50, 10, and 1mg/kg p.o. on glycosuria. While 5 has the same action at any dose tested after 2 hours, 6 is active at the dose of 50mg/kg. Both compounds show a good pattern of inhibition at the 4 and 7 hours points with the three doses tested. The inhibitory effect was completely reverted only after 18—24 hours.

eriments. experiments.

Effects of 5 and 6 in normoglycemic rats

0 0,

0

necessary to have the same effect (Fig. 5). We could not test 3 because of the small amount of substance isolated from the plant.

.2

g .0 -C C

In previous studies, Noreen et al. (4) and

4

2

Villar et al. (9) have described the hypoglycemic effects respectively for Eriobotrya japonica crude alcoholic extract and tormentic acid (7) (9), a triterpene compound structurally closely related to compounds 5 and 6. In both papers

7

the mechanism of action proposed was a stimulation of

hours

Langerhans's B cells followed by an increased release of in-

Fig.4 Effectofdifferentdosesof5p.o. onglycosuria in C57BL/KS-db/db/ Ola mice: () 50mg/kg; (•) 10mg/kg; (0)1 mg/kg. The results are mean of n = 4—5 experiments.

sulin. Indeed the crude extract or the isolated compound (e.g. tormentic acid) was inactive in alloxan-treated animals.

140

In the present work three out of five compounds tested are very active as hypoglycemic agents. The

— 120

mechanism through which they act is most likely an in-

0E 0

80

00

60

.c

40

0 E

creased release of insulin from B Langerhans cells. Studies are in progress to delineate this mechanism more closely.

100

References 1 Shimizu, M., Fukumura, H., Tsuji, H., Tanaomi, S., Hayashi, T., Morita, N. (1986) Chem. Pharm. Bull. 34, 2614. 2 Chopra, R. N., Nayar, S. L. (1965) Glossary of Medicinal Plants, C.S.R.I., New Delhi, p. 168. Winter, C. A., Risley, E. A., Nuss, G. W. (1962) Proc. Soc. Exp. Biol.

20

0 30

60 minute

90

120

Fig. 5 Effect on control (0) blood glucose level of (•) 5 10mg/kg and (A) 60.1 mg/kg. Results are mean of n = 5 experiments.

111,544. Noreen, W., Wadood, A., Hidayat, H. K., Wahid, S. A. M. (1988) Planta Med. 54,196. Liang, Z. Z., Aquino, R., De Feo, V., Pizza, C. (1990) Planta Med. 56,

330.

6 De Tommasi, N., De Simone, F., Aquino, R., Pizza, C., Liang, Z. Z.

(1990) J. Nat. Prod. 53, 810. Cern, R., Aquino, R., De Simone, F., Pizza, C. (1988) J. Nat. Prod. 51, 257. Hummel, K. P., Dickie, M. H., Coleman, D. L. (1966) Science 153, 1123—112 7. Ivarra, M. D., Paya, N., Villar, A. (1988) Planta Med. 54, 282.

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We tested also the activity of 5 and 6 in nor-

moglycemic rats at a dose of 0.1, 1, and lOmg/kgp.o. administered orally 30 mm before starting the test. We monitored blood glucose levels, as described above, for 2 hours each 30 mi While 6 at a dose of 0.1 mg/kg was able to reduce blood glucose levels, a dose 100 times larger of 5 was