1. Introdution

Triglyceride(TG)is the most representative storage form of energy in human body. However，excessive accumulation of TG in certain tissues can induce some serious diseases such as obesity，type IIdiabetes mellitus(DM)， and metabolic syndrome(Lehner and Kuksis， 1996). TG synthesis has been assumed to occur primarily through diacylglycerol acyltransferase(DGAT)，which catalyses the final and only committed step in the glycerol phosphate pathway(Cases et al，1998). Two enzymes that display DGAT activity have been characterized as DGAT1 and DGAT2(Cases et al，2001). Mice that lacked DGAT1(Dgat1^–/^– mice)were viable and had reduced TG levels in their tissues，including white adipose tissue. These findings suggested that specific components with the inhibitory activity against DGAT1 might be a more feasible therapeutic strategy in combating human obesity and type II DM.

In the course of searching for DGAT1 inhibitors from medicines，Betula platyphylla Suk. was selected as an active natural resource. B. platyphylla，belonging to the Betucaseae family，is a monoecious deciduous tree that widely spreads in the northeast of China，the eastern of Siberia，North Korea， and Japan. The barks of B. platyphylla have been used in folk medicine for the treatment of arthritis，cancer，nephritis，dermatitis，poisoning， and chronic bronchitis. It has been reported that an extract of B. platyphylla has anti-oxidative and anticancer activities(Cho et al，2006). Triterpenoid，extracted from the barks of B. platyphylla，is an excellent drug with antiviral，antibacterial，antitumor， and anti-AIDS activities(Fan et al，2011).

The whole sample of Betula platyphylla Suk. was purchased from The Herbal Medicine Association of Taejon，Korea and was identified by Prof. Ki-hwan Bae，College of Pharmacy，Chungnam National University， and the voucher specimen(No. 06103)was deposited at the College of Pharmacy，Beihua University，Jilin，China.

NMR spectra were recorded at Bruker-Avance II 500 Instrument(¹H-NMR 500 MHz，¹³C-NMR 125 MHz)，with tetramethysilane as internal st and ard. All accurate mass experiments were performed on a Micromass QTOF2 Mass Spectrometer(Micromass，Wythenshawe，UK). Spots were visualized using UV light or 10% sulfuric acid. Positive phase silica gel was made by National Medicine Group Chemical Reagent Co.

The barks of B. platyphylla(9.0 kg)were finely cut and extracted with MeOH at 50 ^oC. After evaporation of the solvents under reduced pressure，the residue(1.8 kg)was suspended in water， and then successively partitioned with CH₂Cl₂ and n-BuOH. The CH₂Cl₂ fraction(120.0 g，IC₅₀ = 22.5 μg/mL)was subjected to silica gel column chromatography(100−200 mesh，800.0 g)using a system of n-hexane-CH₂Cl₂(1:5) and CH₂Cl₂-MeOH(200:1)to provide nine fractions(Frs. 1−9). Fr. 2(2.4 g)was purified by silica gel column chromatography and eluted with CH₂Cl₂-MeOH(100:1)to give nine fractions(Frs. 2.1−2.9). Fr. 2.2(1.1 g)was further purified by silica gel column with CH₂Cl₂-MeOH(200:1)to gain compounds 1(29.0 mg)，6(7.0 mg)， and 3(10.0 mg). Fr. 2.4(0.4 g)was subjected to silica gel column using CH₂Cl₂-MeOH(100:1)to get compounds 4(4.5 mg) and 5(1.5 mg). Fr. 2.6(0.6 g)was applied to silica gel column using the elution CH₂Cl₂-MeOH(20:1)，then compounds 2(26.0 mg)，7(39.0 mg)， and 8(10.0 mg)were obtained. Frs. 2.7(0.2 g)was subjected to silica gel column using CH₂Cl₂-MeOH(15:1)to yield compounds 9(10.0 mg) and 10(15.0 mg).

Microsomal fractions from rat liver were prepared，as described earlier(Chung et al，2004). DGAT1 activity was measured as reported previously(Coleman et al，1992). In brief，the reaction mixture was initiated by the addition of rat liver microsomal fraction，followed by gentle and brief vortexing. After incubation for 10 min at 37 ^oC，the reaction was stopped by the addition of 1.5 mL of 2-propanol- heptane-water(80:20:2)，1 mL of heptane， and 0.5 mL of water to extract the lipid. After vortexing，1.2 mL of the organic phase was transferred into a glass tube and washed once with 2.0 mL of alkaline ethanol solution. The amount of radioactivity was determined in a liquid scintillation counter(1450 Micro Beta Triux). The samples were tested for the inhibitory activity against DGAT1 in three independent experiments. The presence of DMSO in the assay medium at 2.5% concentration had no effect on the enzyme activity(Chung et al，2004). Kuraridine was the positive control in this assay(Chung et al，2004).

Compound 2: white crystal，ESI­MS m/z 426 [M⁺]，¹H-NMR(500 MHz，CDCl₃)δ: exo-methylene protons at [4.69(1H，m，H-29β)] and [4.57(1H，m，H-29α)]，a secondary alcohol group at [3.18(1H，dd，H-3)]，a vinyl methyl at [1.68(3H，s，H-30)]，six methyl signals at [1.03(3H，s，H-27)，0.97(3H，s，H-26)，0.94(3H，s，H-23)，0.83(3H，s，H-24)，0.79(3H，s，H-25)，0.76(3H，s，H-28)]. ¹³C-NMR(125 MHz，CDCl₃)δ: 38.6(C-1)，27.3(C-2)，78.9(C-3)，38.8(C-4)，55.2(C-5)，18.2(C-6)，34.2(C-7)，40.7(C-8)，50.3(C-9)，37.1(C-10)，20.9(C-11)，25.0(C-12)，38.0(C-13)，42.7(C-14)，27.4(C-15)，35.5(C-16)，42.9(C-17)，48.2(C-18)，47.9(C-19)，150.8(C-20)，29.8(C-21)，39.9(C-22)，27.9(C-23)，15.3(C-24)，16.1(C-25)，15.9(C-26)，14.5(C-27)，17.9(C-28)，109.3(C-29)，19.2(C-30). Therefore，according to the literature(Kong et al，2013)，compound 2 could unambiguously be elucidated as lupeol.

Compound 3: white crystal，ESI­MS m/z 456 [M⁺]，¹H-NMR(500 MHz，CDCl₃)δ: exo-methylene group at [4.94(1H，s，H-29α)] and [4.76(1H，s，H-29β)]，3.54(1H，td，H-19)，a secondary alcohol group at [3.46(1H，t，H-3)]，2.72(1H，t，H-15α)，2.63(1H，d，H-13)，2.23(1H，m，H-18)，1.93(1H，d，H-16α)，1.88(3H，m，H-1α，12α，22α)，1.79(3H，s，H-29)，1.75(1H，t，H-12β)，a vinyl methyl at [1.68(3H，s，H-30)]，1.59(1H，d，H-12β)，1.54(4H，m，H-2α，6α，15β，21α)，1.40(5H，m，H-6β，7β，11α，16β，22β)，methyl signal at [1.21(3H，s，H-27)]，1.20(3H，m，H-9，11β，21β)，1.07(1H，m，H-2β)，signals for four methyl at [1.06(3H，s，H-26)，1.05(3H，s，H-23)，1.00(3H，s，H-24)]，0.82(3H，s，H-25)]，0.67(1H，m，H-5). ¹³C-NMR(125 MHz，CDCl₃)δ: 40.4(C-1)，29.2(C-2)，79.0(C-3)，40.2(C-4)，56.8(C-5)，19.7(C-6)，35.8(C-7)，42.0(C-8)，51.9(C-9)，38.5(C-10)，22.1 C-11)，27.0(C-12)，39.5(C-13)，43.8(C-14)，32.1(C-15)，33.8(C-16)，57.5(C-17)，48.7(C-18)，50.9(C-19)，152.2(C-20)，31.2(C-21)，38.4(C-22)，29.6(C-23)，29.8(C-24)，17.2(C-25)，17.3(C-26)，15.8(C-27)，179.7(C-28)，110.8(C-29)，20.4(C-30). Therefore，according to the literature(Salimuzzaman et al，1988)，compound 3 could unambiguously be elucidated as betulinic acid.

Compound 9: white crystal，ESI­MS m/z 487.27 [M + H]⁺. ¹H-NMR(500 MHz，pyridine-d₅)δ: exo-methylene group at [4.90(1H，brs，H-29β) and 4.74(1H，brs，H-29α)]，a secondary alcohol group at [4.26(1H，m，H-3)]，3.54(1H，m，H-19)，2.70(1H，m，H-13)，a vinyl methyl at [1.78(3H，s，H-30)]，1.77(1H，d，H-9)，1.59-1.86(2H，m，H-11)，four methyl singlets at [1.46(3H，s，H-24)，1.12(3H，s，H-26)，0.94(3H，s，H-25)，0.93(3H，s，H-27)]. ¹³C-NMR(125 MHz，pyridine-d₅)δ: 33.0(C-1)，26.3(C-2)，73.1(C-3)，52.1(C-4)，45.1(C-5)，21.9(C-6)，34.9(C-7)，41.9(C-8)，51.2(C-9)，37.6(C-10)，21.2(C-11)，26.2(C-12)，38.2(C-13)，43.1(C-14)，30.4(C-15)，32.9(C-16)，56.7(C-17)，49.9(C-18)，47.9(C-19)，151.4(C-20)，31.3(C-21)，37.6(C-22)，179.6(C-23)，18.1(C-24)，16.9(C-25)，16.8(C-26)，14.9(C-27)，179.6(C-28)，110.0(C-29)，19.6(C-30).Therefore，according to the literature(Kiem et al，2003)，compound 9 could unambiguously be elucidated as 3α-hydroxy-lup-20(29)-en- 23，28-dioic acid.

Compound 10: white crystal，ESI­MS m/z 509.25 [M + Na]⁺. ¹H-NMR(500 MHz，pyridine-d₅)δ: aldenhyde proton at 10.00(1H，s，H-23)，exo-methylene group at [4.85(1H，brs，H-29β) and 4.63(1H，brs，H-29α)]，a secondary alcohol group at [4.26(1H，m，H-3)]，3.92(1H，m，H-11)，3.54(1H，m，H-19)，2.70(1H，m，H-13)，a vinyl methyl at [1.78(3H，s，H-30)]，1.77(1H，d，H-9)，four methyl singlets at [1.46(3H，s，H-24)，1.12(3H，s，H-26)，0.94(3H，s，H-25)，0.93(3H，s，H-27)]. ¹³C-NMR(125 MHz，pyridine-d₅)δ: 35.5(C-1)，27.3(C-2)，73.2(C-3)，53.1(C-4)，44.4(C-5)，21.5(C-6)，35.7(C-7)，43.1(C-8)，56.1(C-9)，39.2(C-10)，69.9(C-11)，38.4(C-12)，37.8(C-13)，43.4(C-14)，30.2(C-15)，33.0(C-16)，56.7(C-17)，49.5(C-18)，47.9(C-19)，151.4(C-20)，31.4(C-21)，37.7(C-22)，210.1(C-23)，17.9(C-24)，15.1(C-25)，16.8(C-26)，14.9(C-27)，178.9(C-28)，110.2(C-29)，19.7(C-30). Therefore，according to the literature(Kiem et al，2003)，compound 10 could unambiguously be elucidated as 3α，11α-dihydroxy-23-oxo-lup-20(29)-en-28-oic acid.

Bioactivity-guided fractionation of the CH₂Cl₂-soluble extract(IC₅₀ = 22.5 µg/mL)from the braks of B. platyphylla using for an in vitro DGAT1 inhibitory assay，yielded 10 lupane triterpenes: lupenone(1)，lupeol(2)，betulinic acid(3)，betulinaldehyde(4)，betulin(5)，3-deoxybetulonic acid(6)，glochidonol(7)，lup-20/29-ene-1β/3β-diol(8)，3α-hydroxy- lup-20(29)-en-23，28-dioic acid(9)， and 3α，11α-dihydroxy- 23-oxo-lup-20(29)-en-28-oic acid(10). The structures of the compounds(Figure 1)were determined by 1D and 2D NMR analysis .

Figure 1 Chemical structures of compounds 1-10 isolated from barks of B. platyphylla

All the isolates were assayed for their inhibitory activity against DGAT1， and the results were presented in Table 1. The known DGAT1 inhibitor kuraridine [IC₅₀ =(9.8 ± 0.2)µmol/L](Chung et al，2004)was used as positive control in this assay. Among the isolates，compounds 3−6，9 and 10 inhibited DGAT1 activity in a dose dependent manner with IC₅₀ values ranging from(11.2 ± 0.3)to(38.6 ± 1.2)µmol/L，while compounds 1，2，7， and 8 showed very weak inhibitory effects. Compound 2(IC₅₀ > 100 µmol/L)，with methyl substituent at C-28，exhibited a significantly lower inhibitory activity against DGAT1 than that in compounds 3−6，9， and 10 on which the same position was attached as carboxyl，aldehyde，or methylol group. Furthermore，compounds 3 and 6 showed a higher activity，indicating that as hydroxyl group substituent at C-3 may non-significantly increase the inhibitory activity against DGAT1. Moreover，compounds 3 and 9 exhibited higher activities，indicating only a positional change of groups at C-17 may not affect the inhibitory activity against DGAT1. Although structure-activity relation- ships of lupine triterpenes bearing other groups at C-28 were not thoroughly investigated，our results indicated that the substitution of electron- withdrawing group on lupine triterpenes may be important for in vitro inhibitory activity against DGAT1. Compounds 1−10 were known，but compounds 6，9， and 10 were first extracted from the barks of B. platyphylla. Therefore，further investigation and optimization of these derivatives might enable the finding of new DGAT1 inhibitors that are potentially useful in the treatment of type II DM as well as obesity.

Table 1

Table 1 Inhibitory activity of compounds 1-10 against DGAT1(±s,n=3) Compounds IC50 /(µmol·L−1) lupenone(1) >100 lupeol(2) >100 betulinic acid(3) 11.2 ± 0.3 betulinaldehyde(4) 18.6 ± 0.9 betulini(5) 38.6 ± 1.2 3-deoxybetulonic acid(6) 13.2 ± 1.1 glochidonol(7) >100 lup-20/29-ene-1β/3β-diol(8) >100 3α-hydroxy-lup-20(29)-en-23，28-dioic acid(9) 15.2 ± 0.2 3α，11α-dihydroxy-23-oxo-lup-20(29)-en-28-oic acid(10) 20.2 ± 1.5 kuraridine(positive control) 9.8 ± 0.2

Table 1 Inhibitory activity of compounds 1-10 against DGAT1(±s,n=3)