α-Glucosidase Inhibitory Activity of Resin From Sakhalin fir Tree ( Abies sachalinensis ) and its Bioactive Compounds

Abstract

This study investigated in vitro the α-glucosidase inhibitory activity of resin from the Sakhalin fir tree (Abies sachalinensis). The resin showed extremely high activity (IC₅₀ of 17.3 µg/mL). We isolated 8 compounds from the resin and identified them. All of the compounds isolated from A. sachalinensis resin are reported for the first time in the present study. In an α-glucosidase inhibitory assay, 6 compounds—(holophyllane C (1), (−)-(24E)-23-oxo-3,4-seco-9βH-lanosta-4(28),6,8(14),24-tetraen-3,26-dioic acid (2), abiesonic acid (3), (−)-(24E)-23-oxo-3,4-seco-9βH-lanosta-4(28),7,24-triene-3,26-dioic acid (4), 3,4-seco-4(28),6,8(14),22Z,24-mariesapentaen-26,23-olide-3-oic acid (5), and (−)-abiesonic acid 3-methyl ester (6))—showed high activity (their IC₅₀ values were 25.1, 20.9, 82.4, 20.5, 27.3, and 20.5 µg/mL, respectively). These compounds should contribute to the resin’s α-glucosidase inhibitory activity. From an industrial point of view, these findings provide new data to support that the resin of A. sachalinensis is a rich source of α-glucosidase inhibitors, which are highly valuable for their potential to be developed as functional health foods.

The genus Abies (Pinaceae) consists of 46 species, distributed mainly in temperate and boreal regions of North and Central America, Europe, Asia, and North Africa. ¹ The first phytochemical investigation of Abies plants was reported 80 years ago by Takahashi. ² More than 900 secondary metabolites of diverse chemical structures have since been identified. The resins of Pinus trees have been used for products in many fields, including food, medicine, cosmetics, paints, and coatings, as well as in the automotive industry. ^{3 -5}

Sakhalin fir (Abies sachalinensis) is a dominant tree in Hokkaido, Japan. It is used mainly as a building material. The previous studies have isolated and identified several compounds from some parts of A. sachalinensis; for example, lanostane-type triterpenoids and phenolic compounds have been isolated from the bark. ^6,7

Today’s consumer is increasingly health and safety conscious, so it is desirable to use natural products in health foods. Thus, understanding the health functions of peaches may suggest new and intensive uses for A. sachalinensis and lead to economic stimulation in Hokkaido regions.

Diabetes is a chronic metabolic disease characterized by elevated levels of blood glucose (or blood sugar), and leads over time to serious damage to the heart, blood vessels, eyes, kidneys, and nerves. More than 400 million people live with diabetes worldwide, and the prevalence is predicted to continue rising if the current trends prevail. Diabetes is a major cause of premature death, blindness, kidney failure, heart attack, stroke, and lower limb amputation. It was the seventh leading cause of death in 2016. ⁸ The leaf ethanol extract of A. sachalinensis suppressed the rise in blood glucose level in an in vivo test. ⁹ However, there has been no information about the biological activity of resin of A. sachalinensis (RAS). By evaluating the functions for their applicability to functional foods, we may find new uses for RAS in high-value-added products, which in turn may help stimulate the economy. In this paper, we try to clarify the extent of the potential health function of RAS α-glucosidase inhibitory activity, and we identify its active compound.

Materials and Methods

Plant Materials

Resin of A. sachalinensis was taken by Ms Noriko Kameyama (FUPUNOMORI Co., Ltd.) and Mr Tatsuo Kurebe (Aldebaran Co., Ltd.) from pitch-pockets of a standing tree in February 2016. The tested tree was 70 years old and located in Shimokawa-cho, Hokkaido, Japan. The resin was sealed and refrigerated in a glass vial before analyses. A voucher specimen (No. 1208) was deposited at the Department of Agro-environmental Sciences, Faculty of Agriculture, Kyushu University.

General

Optical rotations were measured with a Jasco DIP-370 polarimeter. Ultraviolet (UV) spectra were obtained by UV-visible spectrophotometer (Shimadzu 1601 PC, model TCC240, Kyoto, Japan). 1D and 2D spectra were obtained on a Bruker DRX 600 NMR spectrometer (Bruker Daltonics Inc., Billerica, MA, United States) using tetramethylsilane as an internal standard. High resolution fast-atom bombardment mass spectrometry were measured with a JEOL JMS 700 spectrometer (JEOL, Tokyo, Japan). High resolution electrospray ionisation mass spectrometry data were obtained using liquid chromatography-ion trap-time of flight tandem mass spectrometry (Shimadzu, Tokyo, Japan). Organic solvents were purchased from Wako Pure Chemical Industries (Osaka, Japan). Silica gel (75-120 mesh) was also purchased from Wako Pure Chemical Industries. Analytical thin-layer chromatography (TLC), was performed on precoated silica gel 60 GF₂₅₄ (20 × 20 cm × 0.2 mm thick) or precoated RP-C₁₈ F₂₅₄ plates (5 × 7.5 cm × 0.2 mm thick) on aluminum sheets, both purchased from Merck Co., Darmstadt, Germany. The plates were developed using the appropriate solvent systems, and the developed chromatograms were examined under UV light at 254 and 366 nm. The spots were made visible by spraying with vanillin/H₂SO₄ reagent before warming in an oven preheated to 110°C for 5 minutes. Preparative TLC was performed on precoated silica gel 60 GF₂₅₄ (20 × 20 cm × 0.2 mm thick) or precoated RP-C₁₈ F₂₅₄ glass plates (20 × 20 cm × 0.25 mm thick).

α-Glucosidase Inhibitory Activity

α-Glucosidase activity was assayed by the method described by Ueda et al ¹⁰ with minor modification. The sample solution in dimethyl sulfoxide (DMSO) (100 µL) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (100 µL) was mixed. Then, α-glucosidase (5 U/mL) and sucrose (0.1 M) in HEPES were added. After incubation at 37°C for 30 minutes, the reaction was ended by heating at 100°C for 10 minutes. The reaction mixture was centrifuged at 3000 rpm for 1 minute. The glucose formation of the supernatant was determined using a BF-5S Biosensor (Oji Scientific Instrument, Hyogo, Japan). To calculate the IC₅₀ values, each sample was dissolved in DMSO at 10, 15, 20, 25, 50, 100, and 200 µg/mL. Standard of glucose solution were prepared at 1.25, 2.5, and 5.0 mg/mL.

Extraction and Isolation Procedures

Five grams of the total MeOH extract of RAS was applied to the top of a silica gel column (45 × 2.7 cm) previously packed in n-hexane using a gradient elution of n-hexane-EtOAc (90:10→0:100) and then using EtOAc-MeOH (100:0→80:20). The effluents were collected in 50 mL fractions. Each fraction was concentrated to a small volume under reduced pressure at 40°C to obtain 122 subfractions. Subfractions having the same chromatographic pattern, as detected by TLC, were pooled together and evaporated to dryness to give finally 10 main fractions (1-10). All of these fractions were subjected to an α-glucosidase inhibition assay. The results showed that fractions 5, 8, 9, and 10 were the most active ones. Ten milligrams of Fraction 10 (eluted with n-hexane-EtOAc [30:70], initially 26.3 mg, IC₅₀ 29.4 µg/mL) was purified using preparative thin-layer chromatography (PTLC) (on precoated RP-C₁₈ F₂₅₄ plates) using MeOH to give compounds 1 (4.8 mg) and 2 (2.7 mg). Fifteen milligrams of Fraction 9 (eluted with n-hexane-EtOAc [50:50], initially 29.9 mg, IC₅₀ 38.3 µg/mL) was purified using PTLC (on precoated RP-C₁₈ F₂₅₄ plates) using MeOH to give compounds 3 (4.9 mg), 4 (2.3 mg), and 5 (1.6 mg). Twenty milligrams of Fraction 8 (eluted with n-hexane-EtOAc [60:40], initially 159.7 mg, IC₅₀ 38.3 µg/mL) was purified using PTLC (on precoated RP-C₁₈ F₂₅₄ plates) using MeOH to give compound 6 (10 mg). Twenty milligrams of Fraction 5 (eluted with n-hexane-EtOAc [70:30], initially 94.3 mg, IC₅₀ 42.6 µg/mL) was purified using PTLC (on precoated silica gel 60 GF₂₅₄) using n-hexane-EtOAc (60:40) to give compounds 7 (6.9 mg) and 8 (7.5 mg).

Results and Discussion

α-Glucosidase Inhibitory Activity of RAS

α-Glucosidase activity is related to the degradation of disaccharide, so the activity is potentially antidiabetic. The discovery of new material that inhibits α-glucosidase activity will result in the suppression of the increase of diabetes. ¹¹ The results of the assay are shown in Table 1. In the assay, we used 1-deoxynojirimycin as a positive control, and its IC₅₀ was 166.4 µg/mL. Resin of A. sachalinensis showed extremely high activity with IC₅₀ of 17.3 µg/mL, which is around 10 times higher than the positive control. A previous study reported that both the levopimaric acid and the neoabietic acid isolated from A. sachalinensis leaf suppressed the increase in blood glucose level. ⁹ In this study, we determined the activities of both acids; their IC₅₀ values were 239.7 and 73.0 µg/mL, respectively. These levels are not high compared with RAS.

Table 1

The Biological Activities of Resin of Abies sachalinensis and its Major Compounds.

		IC50 a
RAS		17.2
Compounds	Levopimaric acid	239.7 [792.5]
	Neoabietic acid	73.0 [241.4]
	Compound (1)	25.1 [51.8]
	Compound (2)	20.9 [43.3]
	Compound (3)	82.4 [171.8]
	Compound (4)	20.5 [42.3]
	Compound (5)	27.3 [58.8]
	Compound (6)	20.5 [41.3]
	Compound (7)	n.d.
	Compound (8)	n.d.

n.d., not detected; RAS, resin of Abies sachalinensis.

Positive control in the α-glucosidase inhibitory assay was 1-deoxynojirimycin, and its IC50 was 166.4 µg/mL (1019.8 µM).

^aα-Glucosidase inhibitory activity expressed as IC₅₀ µg/mL (IC₅₀ µM are also added to isolated compounds within [ ]).

Isolation and Identification of Active Compounds

Chemical studies of the active fractions using different chromatographic techniques afforded 8 compounds (1-8). They were identified by using 1D-, 2D-NMR data and mass spectrometry techniques as well as by comparing their physicochemical and spectral data with those in the literature as holophyllane C (1), ¹² (−)-(24E)-23-oxo-3,4-seco-9βH-lanosta-4(28),6,8(14),24-tetraen-3,26-dioic acid (2), ¹³ abiesonic acid (3), ^14,15 (−)-(24E)-23-oxo-3,4-seco-9βH-lanosta-4(28),7,24-triene-3,26-dioic acid (4), ¹³ 3,4-seco-4(28),6,8(14),22Z,24-mariesapentaen-26,23-olide-3-oic acid (5), ¹⁶ (−)-abiesonic acid 3-methyl ester (6), ¹³ abieta-7,17-diene-12α-methoxy-18-oic acid (7), ¹⁷ and (23R,25R)-3,4-seco-17,14-friedo-9βH-lanosta-4(28),6,8(14)-trien-26,23-olid-3-oic acid (8). ^6,13 To the best of our knowledge, the present study is the first to report all of these compounds from RAS sachalinensis. The structures of isolated compounds (1-8) are shown in Figure 1. The information of spectral data are shown in the Supplementary Material.

Figure 1

Structures of isolated compounds (1-8).

α-Glucosidase Inhibitory Activity of Isolated Compounds

As a result of the bioassay, compounds 1 to 6 showed α-glucosidase inhibitory activity (their IC₅₀ values were 25.1, 20.9, 82.4, 20.5, 27.3, and 20.5 µg/mL, respectively). The results suggest that α-glucosidase inhibitory activity of RAS should be influenced by the interactions among these isolated compounds.

Up to now, a lot of compounds have been reported as α-glucosidase inhibitors, such as triterpenoids, triterpene saponins, cyanogenic glycoside, and several flavonoids. ^{18 -20} In recent years, Rehman et al ²¹ reported several triterpenes and diterpenes from Boswellia species as α-glucosidase inhibitors. That study indicated that the carboxylic group plays a key role. Our results suggested the possibility that RAS could be used as functional food that provides antidiabetic effects. This new application may in turn activate local industry and enhance human health.

Supplemental Material

Supplementary data - Supplemental material for α-Glucosidase Inhibitory Activity of Resin From Sakhalin fir Tree (Abies sachalinensis) and its Bioactive Compounds

Supplemental material, Supplementary data, for α-Glucosidase Inhibitory Activity of Resin From Sakhalin fir Tree (Abies sachalinensis) and its Bioactive Compounds by Toshinori Nakagawa, Ahmed Ashour, Yhiya Amen, Yurie Koba, Koichiro Ohnuki, and Kuniyoshi Shimizu in Natural Product Communications