# α-Amylase and α-Glucosidase Inhibitory Saponins fromPolyscias fruticosa Leaves

Authors: Tran Thi Hong Hanh; Nguyen Hai Dang; Nguyen Tien Dat
Journal: Journal of Chemistry
 (2016)

---

## Abstract

Three bisdesmosidic saponins 3-O-[β-D-glucopyranosyl-(1→4)-β-D-glucuronopyranosyl] oleanolic acid 28-O-β-D-glucopyranosyl ester (1), polyscioside D (2), and 3-O-{
β-D-glucopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→4)]-β-D-g
l
u
c
u
r
o
n
o
p
y
r
a
n
o
s
y
l
} oleanolic acid 28-O-β-D-glucopyranosyl-(1→2)-β-D-galactopyranosyl ester (3) were isolated from a methanol extract ofPolyscias fruticosa (L.) Harms leaves. Compound1 was obtained as a main constituent and compound3 was reported for the first time and named as polyscioside I. Saponin1 inhibited porcine pancreas α-amylase and yeast α-glucosidase activities while2 and3 were inactive. Synergistic inhibitory effect on α-amylase was observed from the combination of low concentrations of1 and acarbose. The findings suggest the use ofP. fruticosa and its major saponin1 for the prevention and treatment of diabetes and its complications.


---

## Body

## 1. Introduction

Polyscias fruticosa (L.) Harms (synonymsNothopanax fruticosus (L.) Miq. andPanax fruticosus L.), belonging to the family Araliaceae, is widely used in Vietnam as a tonic agent for the treatment of ischemia and inflammation and to increase blood flow in the brain. The leaves of this plant are also eaten as a salad [1]. Previous studies showed thatP. fruticosa leaf extracts exhibited antipyretic, anti-inflammatory, analgesic, and molluscicidal properties [2, 3] and antidiabetic activity [4]. A few reports on the chemical composition of this plant indicated the presence of polyacetylene [5] and saponins [6]. Diabetes is a group of metabolic diseases characterized by chronic hyperglycemia resulting from deficiency in insulin secretion or action. Recent reports revealed that a high postprandial plasma glucose level is more harmful than fasting blood glucose. Therefore, it is important to control the postprandial blood glucose level to reduce complications and mortality. One therapeutic approach for treating diabetes is to decrease postprandial glycemia by inhibiting enzymes responsible for carbohydrate hydrolysis, such as α-glucosidase and α-amylase [7].Our search for antidiabetic agents of natural origin identified a methanol extract ofP. fruticosa leaves that showed significant inhibitory activity against α-glucosidase and α-amylase. A phytochemical investigation of the methanol extract ofP. fruticosa leaves led to the isolation of three bisdesmosidic saponins (Figure 1). Their structures were elucidated as 3-O-[β-D-glucopyranosyl-(1→4)-β-D-glucuronopyranosyl] oleanolic acid 28-O-β-D-glucopyranosyl ester (1) [6, 8], 3-O-{
β-D-glucopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→4)]-β-D-g
l
u
c
u
r
o
n
o
p
y
r
a
n
o
s
y
l
} oleanolic acid 28-O-β-D-glucopyranosyl ester or polyscioside D (2) [6], and 3-O-{
β-D-glucopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→4)]-β-D-g
l
u
c
u
r
o
n
o
p
y
r
a
n
o
s
y
l
} oleanolic acid 28-O-β-D-glucopyranosyl-(1→2)-β-D-galactopyranosyl ester (3), newly named polyscioside I. All compounds were evaluated for their inhibitory activity against porcine pancreas α-amylase and yeast α-glucosidase.Figure 1
Structure of compounds1–3 and key HMBC correlations of3.


## 2. Experimental

### 2.1. General Experimental Procedures

Optical rotation values were recorded a JASCO P-2000 digital polarimeter (JASCO, Tokyo, Japan). NMR experiments were carried out on a Bruker AM500 FT-NMR spectrometer (Bruker, Rheinstetten, Germany) using tetramethylsilane (TMS) as internal standard. The HR-ESI-MS was recorded on API Q-STAR PULSAR I of Applied Biosystem. The bioassay absorbance was read by xMark Microplate Spectrophotometer reader (Bio-Rad Laboratories, USA).


### 2.2. Plant Material

The leaves ofPolyscias fruticosa were collected in Tien Hai, Thai Binh, in July 2011 and identified by Professor Tran Huy Thai, Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology. The voucher specimens were deposited at the herbarium of the Institute of Ecology and Biological Resources.


### 2.3. Extraction and Isolation

The air-dried and powderedP. fruticosa leaves (2.0 kg) were extracted with methanol (5 L × 3 times) at room temperature. The combined extracts were concentrated to give 300 g of crude extract, which was then resuspended in water (1.5 L) and successively partitioned with hexane and ethyl acetate (each 0.5 L × 3 times) to obtain 75 and 92 g of hexane and ethyl acetate residues, respectively. The water residue was filtered through a Diaion HP-20 column eluted by 0%, 25%, and 100% methanol in water. The 100% methanol-eluted fraction was diluted with water (1 : 1 v/v) and kept overnight at 4°C. Compound1 as white crystals (500 mg) was obtained by filtering. The filtrate was concentrated and chromatographed on a silica gel column eluted with a gradient of 0%, 50%, and 100% methanol in ethyl acetate to afford three fractions, F1–F3. Fraction F2 was fractionated on a silica gel column eluted with chloroform-methanol-water (50 : 10 : 1 v/v) to afford compound2 (10 mg). Compound3 (20.5 mg) was purified from F3 by a RP-18 column (methanol-water 1 : 1 v/v).Polyscioside I (
3
). White powder, mp. >300°C. α
D
2
4
=
+
12.4 (c 0.1, MeOH). 1H NMR (500 MHz, DMSO-d
6): δ 0.67 (3H, br s, H-26), 0.73 (3H, br s, H-24), 0.85 (6H, br s, H-25, 29), 0.87 (3H, br s, H-30), 0.97 (3H, br s, H-23), 1.06 (3H, br s, H-27), 3.01 (1H, m, H-3), 3.60 (1H, m, H-2′′′), 4.25 (1H, d,J = 7.0 Hz, H-1′′), 4.29 (1H, d,J = 6.5 Hz, H-1′), 4.62 (1H, d,J = 7.5 Hz, H-1′′′′′), 4.48 (1H, d,J = 7.0 Hz, H-1′′′′), 5.14 (1H, m, H-11), 5.30 (1H, d,J = 8.0 Hz, H-1′′′). 13C NMR (125 MHz, DMSO-d
6): see Table 1. HR-ESI-MS (positive): m
/
z 1281.6127 [M + H]+, (calcd. 1281.6116 for C60H97O29).Table 1
13C-NMR data of compounds 1–3 measured in DMSO-d
6.
C
1
2
3
C
1
2
3
1
38.1
38.1
38.1
3-O-GluA
2
25.4
22.9
25.4
1′
105.0
103.6
103.6
3
87.9
88.4
88.3
2′
73.5
79.3
79.5
4
38.7
38.6
38.6
3′
75.4
75.4
75.3
5
55.0
55.0
55.0
4′
82.9
82.3
82.3
6
17.7
17.7
17.7
5′
75.3
75.1
75.1
7
32.2
32.3
32.7
6′
172.0
171.8
171.5
8
40.0
40.7
40.0
4′-O-Glu
9
47.0
47.0
47.0
1′′
103.8
103.3
103.3
10
36.2
36.2
36.2
2′′
73.4
73.5
73.6
11
22.9
22.9
22.9
3′′
76.3
76.2
76.3
12
121.6
121.7
121.5
4′′
70.0
69.9
69.9
13
143.4
143.4
143.5
5′′
77.0
77.0
77.0
14
41.2
41.2
41.1
6′′
61.1
61.1
61.0
15
27.1
27.7
27.9
2′-O-Glu
16
22.5
22.5
21.9
1′′′′
—
103.1
103.2
17
45.9
45.9
45.8
2′′′′
—
73.5
73.6
18
40.7
40.7
40.7
3′′′′
—
76.1
76.0
19
45.5
45.5
45.5
4′′′′
—
70.1
70.0
20
30.2
30.3
30.2
5′′′′
—
76.8
76.7
21
33.2
33.2
33.2
6′′′′
—
60.9
61.0
22
31.6
31.6
32.3
28-O-Glc/Gal
23
27.6
27.7
27.7
1′′′
94.1
94.1
91.9
24
16.6
16.1
16.7
2′′′
72.3
72.3
76.9
25
15.1
15.1
15.1
3′′′
76.6
76.6
74.1
26
16.4
16.9
16.7
4′′′
69.5
69.5
70.7
27
25.4
25.5
25.5
5′′′
77.6
77.7
76.8
28
175.2
175.2
175.2
6′′′
60.7
60.7
61.8
29
32.7
32.7
32.7
2′′′-O-Glu
30
23.3
23.3
23.4
1′′′′′
—
—
102.3
2′′′′′
—
—
75.0
3′′′′′
—
—
77.0
4′′′′′
—
—
69.4
5′′′′′
—
—
77.5
6′′′′′
—
—
60.5Acid Hydrolysis of
3. Compound3 (10 mg) was heated in 2 N HCl (5 mL) at 80°C for 2 h, and then the solution was extracted with ethyl acetate (2 mL × 3). The sugar products in the aqueous layer were identified as glucose (Rf 0.65), galactose (Rf 0.60), and glucuronic acid (Rf 0.12) in comparison with standards by silica gel thin layer chromatography (developed with acetone-methanol-water 10 : 9 : 1 and sprayed by 10% sulfuric acid solution containing 2% vanillin). After preparative thin layer chromatography, the optical rotation of each sugar fraction was checked. The positive optical rotation values led to the assignment of D-glucuronic acid, D-glucose, and D-galactose [9].


### 2.4. Assay forα-Glucosidase Inhibition

The yeastα-glucosidase (G0660, Sigma-Aldrich) enzyme inhibition assay was performed by the digestion ofp-nitrophenyl-α-D-glucopyranoside [10]. The sample solution (2 μL dissolved in DMSO) and 0.5 U/mL α-glucosidase (40 μL) were mixed in 120 μL of 0.1 M phosphate buffer (pH 7.0). After 5 min preincubation, 5 mMp-nitrophenyl-α-D-glucopyranoside solution (40 μL) was added and the solution was incubated at 37°C for 30 min. The absorbance of released 4-nitrophenol was measured at 405 nm by using a microplate reader (Molecular Devices, CA). Acarbose was used as positive control (IC50 650.4 μg/mL).


### 2.5. Assay forα-Amylase Inhibition

The porcine pancreasα-amylase (A3176, Sigma-Aldrich) enzyme inhibitory activity was measured using the method reported by Ashok Kumar et al. [11] with slight modifications. Substrate was prepared by dissolving 2-chloro-4-nitrophenyl-α-D-maltotrioside (93834, Sigma-Aldrich) in phosphate buffer (pH 7.0). The sample (2 μL dissolved in DMSO) and 0.5 unit/mL α-amylase (50 μL) were mixed in 100 μL of 0.1 M phosphate buffer (pH 7.0). After 5 min preincubation, substrate solution (50 μL) was added and the solution was incubated at 37°C for 15 min. The absorbances were measured at 405 nm. Acarbose was used as positive control (IC50 57.6 μg/mL).


## 2.1. General Experimental Procedures

Optical rotation values were recorded a JASCO P-2000 digital polarimeter (JASCO, Tokyo, Japan). NMR experiments were carried out on a Bruker AM500 FT-NMR spectrometer (Bruker, Rheinstetten, Germany) using tetramethylsilane (TMS) as internal standard. The HR-ESI-MS was recorded on API Q-STAR PULSAR I of Applied Biosystem. The bioassay absorbance was read by xMark Microplate Spectrophotometer reader (Bio-Rad Laboratories, USA).


## 2.2. Plant Material

The leaves ofPolyscias fruticosa were collected in Tien Hai, Thai Binh, in July 2011 and identified by Professor Tran Huy Thai, Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology. The voucher specimens were deposited at the herbarium of the Institute of Ecology and Biological Resources.


## 2.3. Extraction and Isolation

The air-dried and powderedP. fruticosa leaves (2.0 kg) were extracted with methanol (5 L × 3 times) at room temperature. The combined extracts were concentrated to give 300 g of crude extract, which was then resuspended in water (1.5 L) and successively partitioned with hexane and ethyl acetate (each 0.5 L × 3 times) to obtain 75 and 92 g of hexane and ethyl acetate residues, respectively. The water residue was filtered through a Diaion HP-20 column eluted by 0%, 25%, and 100% methanol in water. The 100% methanol-eluted fraction was diluted with water (1 : 1 v/v) and kept overnight at 4°C. Compound1 as white crystals (500 mg) was obtained by filtering. The filtrate was concentrated and chromatographed on a silica gel column eluted with a gradient of 0%, 50%, and 100% methanol in ethyl acetate to afford three fractions, F1–F3. Fraction F2 was fractionated on a silica gel column eluted with chloroform-methanol-water (50 : 10 : 1 v/v) to afford compound2 (10 mg). Compound3 (20.5 mg) was purified from F3 by a RP-18 column (methanol-water 1 : 1 v/v).Polyscioside I (
3
). White powder, mp. >300°C. α
D
2
4
=
+
12.4 (c 0.1, MeOH). 1H NMR (500 MHz, DMSO-d
6): δ 0.67 (3H, br s, H-26), 0.73 (3H, br s, H-24), 0.85 (6H, br s, H-25, 29), 0.87 (3H, br s, H-30), 0.97 (3H, br s, H-23), 1.06 (3H, br s, H-27), 3.01 (1H, m, H-3), 3.60 (1H, m, H-2′′′), 4.25 (1H, d,J = 7.0 Hz, H-1′′), 4.29 (1H, d,J = 6.5 Hz, H-1′), 4.62 (1H, d,J = 7.5 Hz, H-1′′′′′), 4.48 (1H, d,J = 7.0 Hz, H-1′′′′), 5.14 (1H, m, H-11), 5.30 (1H, d,J = 8.0 Hz, H-1′′′). 13C NMR (125 MHz, DMSO-d
6): see Table 1. HR-ESI-MS (positive): m
/
z 1281.6127 [M + H]+, (calcd. 1281.6116 for C60H97O29).Table 1
13C-NMR data of compounds 1–3 measured in DMSO-d
6.
C
1
2
3
C
1
2
3
1
38.1
38.1
38.1
3-O-GluA
2
25.4
22.9
25.4
1′
105.0
103.6
103.6
3
87.9
88.4
88.3
2′
73.5
79.3
79.5
4
38.7
38.6
38.6
3′
75.4
75.4
75.3
5
55.0
55.0
55.0
4′
82.9
82.3
82.3
6
17.7
17.7
17.7
5′
75.3
75.1
75.1
7
32.2
32.3
32.7
6′
172.0
171.8
171.5
8
40.0
40.7
40.0
4′-O-Glu
9
47.0
47.0
47.0
1′′
103.8
103.3
103.3
10
36.2
36.2
36.2
2′′
73.4
73.5
73.6
11
22.9
22.9
22.9
3′′
76.3
76.2
76.3
12
121.6
121.7
121.5
4′′
70.0
69.9
69.9
13
143.4
143.4
143.5
5′′
77.0
77.0
77.0
14
41.2
41.2
41.1
6′′
61.1
61.1
61.0
15
27.1
27.7
27.9
2′-O-Glu
16
22.5
22.5
21.9
1′′′′
—
103.1
103.2
17
45.9
45.9
45.8
2′′′′
—
73.5
73.6
18
40.7
40.7
40.7
3′′′′
—
76.1
76.0
19
45.5
45.5
45.5
4′′′′
—
70.1
70.0
20
30.2
30.3
30.2
5′′′′
—
76.8
76.7
21
33.2
33.2
33.2
6′′′′
—
60.9
61.0
22
31.6
31.6
32.3
28-O-Glc/Gal
23
27.6
27.7
27.7
1′′′
94.1
94.1
91.9
24
16.6
16.1
16.7
2′′′
72.3
72.3
76.9
25
15.1
15.1
15.1
3′′′
76.6
76.6
74.1
26
16.4
16.9
16.7
4′′′
69.5
69.5
70.7
27
25.4
25.5
25.5
5′′′
77.6
77.7
76.8
28
175.2
175.2
175.2
6′′′
60.7
60.7
61.8
29
32.7
32.7
32.7
2′′′-O-Glu
30
23.3
23.3
23.4
1′′′′′
—
—
102.3
2′′′′′
—
—
75.0
3′′′′′
—
—
77.0
4′′′′′
—
—
69.4
5′′′′′
—
—
77.5
6′′′′′
—
—
60.5Acid Hydrolysis of
3. Compound3 (10 mg) was heated in 2 N HCl (5 mL) at 80°C for 2 h, and then the solution was extracted with ethyl acetate (2 mL × 3). The sugar products in the aqueous layer were identified as glucose (Rf 0.65), galactose (Rf 0.60), and glucuronic acid (Rf 0.12) in comparison with standards by silica gel thin layer chromatography (developed with acetone-methanol-water 10 : 9 : 1 and sprayed by 10% sulfuric acid solution containing 2% vanillin). After preparative thin layer chromatography, the optical rotation of each sugar fraction was checked. The positive optical rotation values led to the assignment of D-glucuronic acid, D-glucose, and D-galactose [9].


## 2.4. Assay forα-Glucosidase Inhibition

The yeastα-glucosidase (G0660, Sigma-Aldrich) enzyme inhibition assay was performed by the digestion ofp-nitrophenyl-α-D-glucopyranoside [10]. The sample solution (2 μL dissolved in DMSO) and 0.5 U/mL α-glucosidase (40 μL) were mixed in 120 μL of 0.1 M phosphate buffer (pH 7.0). After 5 min preincubation, 5 mMp-nitrophenyl-α-D-glucopyranoside solution (40 μL) was added and the solution was incubated at 37°C for 30 min. The absorbance of released 4-nitrophenol was measured at 405 nm by using a microplate reader (Molecular Devices, CA). Acarbose was used as positive control (IC50 650.4 μg/mL).


## 2.5. Assay forα-Amylase Inhibition

The porcine pancreasα-amylase (A3176, Sigma-Aldrich) enzyme inhibitory activity was measured using the method reported by Ashok Kumar et al. [11] with slight modifications. Substrate was prepared by dissolving 2-chloro-4-nitrophenyl-α-D-maltotrioside (93834, Sigma-Aldrich) in phosphate buffer (pH 7.0). The sample (2 μL dissolved in DMSO) and 0.5 unit/mL α-amylase (50 μL) were mixed in 100 μL of 0.1 M phosphate buffer (pH 7.0). After 5 min preincubation, substrate solution (50 μL) was added and the solution was incubated at 37°C for 15 min. The absorbances were measured at 405 nm. Acarbose was used as positive control (IC50 57.6 μg/mL).


## 3. Results and Discussion

Compound3 was obtained as a white crystal and its high-resolution electrospray ionization mass spectrometry spectrum revealed an ion peak at m
/
z 1281.6127 [M + H]+, corresponding to the molecular formula C60H96O29. The 1H-NMR spectrum of1 showed seven methyl groups at δ
H 0.67 (3H, br s, H-26), 0.73 (3H, br s, H-24), 0.85 (6H, br s, H-25, 29), 0.87 (3H, br s, H-30), 0.97 (3H, br s, H-23), and 1.06 (3H, br s, H-27) and five glycosidic anomeric protons at δ
H 4.25 (1H, d,J = 7.0 Hz, H-1′′), 4.29 (1H, d,J = 6.5 Hz, H-1′), 4.48 (1H, d,J = 7.0 Hz, H-1′′′′), 5.30 (1H, d,J = 8.0 Hz, H-1′′′), and 4.62 (1H, d,J = 7.5 Hz, H-1′′′′′). The large coupling constants of all anomeric protons indicated the β-configuration of the sugars. 13C-NMR and DEPT spectra indicated the presence of an olean triterpene skeleton with seven CH3, ten CH2, five CH, and 8 C signals (Table 1). The aglycone was identified as oleanolic acid, a common triterpene of saponins in the genusPolyscias [6]. The δ
C values of C-3 at δ
C 88.3 and C-28 at δ
C 175.2 suggested that compound3 was a bisdesmosidic saponin with sugar units attached to these positions. In addition, five sugar moieties were recognized by five anomeric methine groups at δ
C 103.6 (C-1′), 103.3 (C-1′′), 91.9 (C-1′′′), 103.2 (C-1′′′′), and 102.3 (C-1′′′′′). These data are similar to those of compound2, except for the addition of a sugar moiety and the upfield shift of the anomeric signal at δ
C from 94.1 to 91.9. Acid hydrolysis of compound3 allowed the identification of D-glucuronic acid, D-glucose, and D-galactose. Extensive analysis of the HMBC spectrum of compound3 allowed the position of the sugar units to be determined. The couplings from H-1′ (δ
H 4.29) to C-3 (δ
C 88.3) indicated that the glucuronic and glucosyl moieties attached to C-3. The correlations from H-1′′ (δ
H 4.25) to C-4′ (δ
C 82.3) and between H-4′′′′ (δ
H 4.48) and C-2′ (δ
C 79.5) confirmed that the two glucosyl units were attached to C-2′ and C-4′, respectively. Indeed, the 13C-NMR data of this fragment coincided well with the β-D-glucopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→4)]-β-D-glucuronopyranosyl of compound2 (Table 1). The HMBC correlations from H-1′′′ (δ
H 5.31) to C-28 (δ
C 175.2) and the anomeric C-1′′′ resonance shifted upfield at δ
C 91.9 suggesting the galactose unit attached to C-28. The HMBC couplings from H-1′′′′′ (δ
H 4.62) to C-2′′′ (δ
C 76.9) and from H-2′′′ (δ
H 3.60) to C-1′′′′′ (δ
C 102.3) and the COSY crosspeak between H-1′′′ and H-2′′′ indicated that the glucose unit attached to C-2′′′. Thus, compound3 was elucidated as 3-O-{
β-D-glucopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→4)]-β-D-g
l
u
c
u
r
o
n
o
p
y
r
a
n
o
s
y
l
} oleanolic acid 28-O-β-D-glucopyranosyl-(1→2)-β-D-galactopyranosyl ester, named polyscioside I.The inhibitory effects of the isolated compounds against porcine pancreasα-amylase and yeast α-glucosidase were evaluated in comparison with the antidiabetic acarbose (Table 2). Compound1 potently inhibited α-amylase and α-glucosidase with IC50 values of 27.1 and 440.5 μg/mL, respectively. In contrast, compounds2 and3 did not show any effect on both enzymes. Dou et al. reported that the glucuronic acid unit at C-3 and glucosidic moiety at C-28 of the aglycone were the functional groups essential for α-amylase and α-glucosidase inhibition; the addition or replacement of glucose by another sugar unit might thus abolish the inhibitory activity [12]. In oral sucrose tolerance tests, several oleanolic acid glycosides showed significant antihyperglycemic activity in the oral sucrose tolerance in rats [13, 14]. However, the 2′-O-β-D-glucopyranosyl unit attached to the glucuronic acid moiety was assumed to markedly reduce the hypoglycemic effect [15]. Consistent with this, in our study, compounds2 and3 possessing a 2′-O-β-D-glucopyranosyl unit were inactive regarding α-amylase and α-glucosidase inhibition. Previous studies showed thatP. fruticosa leaf extracts strongly inhibited rat intestinal α-glucosidase [16] and exhibited hypoglycemic and antihyperglycemic activity in normal and streptozotocin-diabetic albino rats [4]. Our results suggest that the observed hypoglycemic activity might be related to the inhibition of α-amylase and α-glucosidase enzymes.Table 2
Inhibitory effects (IC50  ± SD, μg/mL) of 1–3 for α-amylase and α-glucosidase.
Compounds
α-Amylase
α-Glucosidase
1
27.1 ± 2.05
440.5 ± 12.7
2
—
—
3
—
—
Acarbose
57.6 ± 4.12
650.4 ± 33.4It was of interest to establish whether compound1 and acarbose interact synergistically or additively in the inhibition of α-glucosidase and α-amylase. Therefore, the assay was performed in solutions containing these agents alone or in a mixture at different concentrations. The results showed that the combination of compound1 and acarbose produced a synergistic inhibitory effect on porcine pancreas α-amylase (Figure 2) but had no effect on yeast α-glucosidase (data not shown). Interestingly, higher synergistic inhibition was found from the combination of low concentrations of individual agents. At 1.0 μg/mL, compound1 and acarbose alone exhibited only 1.5% and 4.2% inhibition, respectively. However, their combination increased the inhibition up to 32.8%. At a higher concentration of 10 μg/mL, the percentage of α-amylase inhibition of the mixture was equal to the sum of compound1 and acarbose, suggesting that they produced additive inhibition. A slight increase in inhibition was obtained from the mixture in comparison with that achieved by the individual agents at 100 μg/mL. These findings indicate that saponin1 produced synergistic effects on porcine pancreas α-amylase inhibition when combined with a low concentration of acarbose.Figure 2
The percentage inhibition by combination of1 and acarbose on porcine pancreatic α-amylase. Results are expressed as means ± SD; n
=
3. p
∗
<
0.05 compared with acarbose alone.Acarbose is a potentα-glucosidase inhibitor and has been used in the treatment of type 2 diabetes. It can also be used in combination with other agents, such as sulfonylurea and metformin, in patients with diabetes. However, the long-term use and high-dose administration of this drug cause severe side effects, such as diarrhea, flatulence, and abdominal or stomach pain [17–19]. Our study showed that the combination ofP. fruticosa saponin1 with acarbose at a low concentration produced more synergistic inhibition than either drug alone, suggesting that they might provide a significant clinical benefit in delaying postprandial hyperglycemia and diminishing the adverse effects of acarbose.


## 4. Conclusion

Three bisdesmosidic saponins, including two known 3-O-[β-D-glucopyranosyl-(1→4)-β-D-glucuronopyranosyl] oleanolic acid 28-O-β-D-glucopyranosyl ester (1) and 3-O-{
β-D-glucopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→4)]-β-D-g
l
u
c
u
r
o
n
o
p
y
r
a
n
o
s
y
l
} oleanolic acid 28-O-β-D-glucopyranosyl ester (or polyscioside D,2) and one new 3-O-{
β-D-glucopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→4)]-β-D-g
l
u
c
u
r
o
n
o
p
y
r
a
n
o
s
y
l
} oleanolic acid 28-O-β-D-glucopyranosyl-(1→2)-β-D-galactopyranosyl ester (named polyscioside I,3), were isolated from the leaves ofP. fruticosa. Compound1 showed inhibitory effects against porcine pancreas α-amylase and yeast α-glucosidase activities and produced a synergistic effect with acarbose in α-amylase inhibition. Further study is needed to clarify the mechanism of enzyme inhibition as well as the hypoglycemic properties of saponin1.



---

*Source: 2082946-2016-04-30.xml*