阅读说明：本技术 鸡蛋花中具有抗糖尿病活性的化合物及其制备方法 (Compound with anti-diabetic activity in frangipani and preparation method thereof ) 是由 谭钦刚 张胜男 赖春华 于 2020-07-13 设计创作，主要内容包括：本发明公开了鸡蛋花中具有抗糖尿病活性的化合物及其制备方法,所述的化合物有四个,其中化合物1,2,4为新化合物,分别为三萜类化合物lup-20(29)-en-3<I>β</I>-(1-(2<I>S</I>-hydroxypropionate)-benzoic acid)(1),环烯醚萜类化合物<I>β</I>-dihydroplumericin A(2),<I>β</I>-dihydroplumericinic acid(3),吡喃酮类化合物tetrahydro-4<I>S</I>-hydroxy-6<I>β</I>-heptadecyl-2H-pyran-2-one(4),并通过光吸收检测法实验进一步研究上述鸡蛋花的化合物1-4的抗糖尿病效果。结果显示化合物1-4对<I>α</I>-葡萄糖甘酶抑制作用强于阳性对照阿卡波糖,特别是化合物1的活性尤为突出；化合物1对PTP1B抑制作用强于阳性对照齐墩果酸,具有较强的活性。本发明从鸡蛋花中发现结构新颖的、具有抗糖尿病活性的化合物,为制备抗糖尿病新药奠定基础。(The invention discloses compounds with anti-diabetic activity in plumeria and a preparation method thereof, wherein the number of the compounds is four, wherein the compounds 1, 2 and 4 are new compounds which are triterpenoids lup-20(29) -en-3 β ‑(1‑(2 S -hydroxyproprionate) -benzoic acid) (1), iridoid compounds β ‑dihydroplumericin A(2)， β -dihydroplircinic acid (3), pyrone compound tetrahydroxy-4 S ‑hydroxy‑6 β -heptadeceyl-2H-pyran-2-one (4), and the anti-diabetic effect of compounds 1-4 of the above frangipani was further investigated by photoabsorption detection assay. The results show that compounds 1-4 are paired α -grapeThe inhibition effect of the glycuronase is stronger than that of positive control acarbose, and particularly the activity of the compound 1 is particularly outstanding; the compound 1 has stronger inhibitory action on PTP1B than that of positive control oleanolic acid, and has stronger activity. The invention discovers a compound with novel structure and anti-diabetic activity from frangipani, and lays a foundation for preparing a new anti-diabetic medicine.)

1. A compound having anti-diabetic activity in plumeria, characterized in that: the number of the compounds is four, wherein the compounds 1, 2 and 4 are new compounds, namely triterpenoids lup-20(29) -en-3β-(1-(2S-hydroxypyr-opinate) -benzoic acid) (1), iridoid compoundsβ-dihydroplumericin A(2)，β-dihydro-plomerinic acid (3), pyrone compound tetrahydroxy-4S-hydroxy-6β-heptadeceyl-2H-pyran-2-one (4) having the structure shown in the following formula: ,

2. the method of claim 1, comprising the steps of:

(1) soaking flos Plumeriae Acutifoliae in 95% ethanol for 7 days for 4 times, mixing extractive solutions, distilling under reduced pressure to remove solvent, suspending the crude extract in water, extracting with ethyl acetate, and recovering solvent under reduced pressure to obtain ethyl acetate layer extract;

(2) passing the ethyl acetate part through a normal phase silica gel column, and setting a concentration gradient for elution to obtain eleven fractions: I-XI;

(3) subjecting the fraction VI to MCI chromatographic column chromatography and methanol-water gradient elution to obtain fraction VI-1 to VI-5 five fractions; VI-1 flow is subjected to ODS reverse chromatographic column chromatography, methanol-water gradient elution and recrystallization to obtainβ-dihydropliocin a compound 2;

subjecting the fraction V to MCI chromatographic column chromatography, and performing gradient elution with methanol water to obtain five fractions V-1 to V-5; passing V-4 fraction through forward silica gel column, performing ODS reverse chromatography, and gradient eluting with methanol water to obtain tetrahydroxy-4S-hydroxy-6β-heptadeceyl-2H-pyran-2-one compound 4;

v-5 decompressing and concentrating the precipitated powder, washing the powder with methanol to obtain lup-20(29) -en-3β-(1-(2S-hydroxyproprionate) -benzoic acid) compound 1;

subjecting the fraction VII to MCI chromatographic column chromatography, and performing gradient elution with methanol water to obtain four fractions VII-1 to VII-4; VII-2 separating with LH-20 gel column, eluting with methanolβDihydroplicitinic acid compound 3.

3. The method for preparing a compound having anti-diabetic activity in plumeria according to claim 2, wherein: and (3) carrying out gradient elution on the normal-phase silica gel column in the step (2) by using an eluent with the volume ratio of petroleum ether to acetone =1:0, 13 to 1, 4 to 1, 1 to chloroform to methanol =1 to 1.

4. The method for preparing a compound having anti-diabetic activity in plumeria according to claim 2, wherein: gradient elution is carried out on the methanol water in the step (3), wherein the concentration of the methanol water is gradient elution according to 70%, 75%, 80%, 85%, 90%, 95% and 100%, and the gradient elution is carried out by using acetone to obtain V-5; v-5, decompressing, concentrating and precipitating starch powder, and washing the powder by methanol; and the eluent of the normal phase silica gel column is subjected to gradient elution by adopting petroleum ether and ethyl acetate =12:1 and petroleum ether and ethyl acetate =16:1 in volume ratio respectively.

5. Use of a compound having anti-diabetic activity in a plumeria as claimed in claim 1, characterized in that: can be used for preparing medicine with antidiabetic activity.

Technical Field

The invention relates to a compound, in particular to a triterpenoid, iridoid and pyrone compound with anti-diabetic activity extracted and separated from frangipani and a preparation method thereof.

Background

The flos Plumeriae Acutifoliae is flos Plumeriae Acutifoliae of Apocynaceae (flos Plumeriae Acutifoliae)Plumeria rubraL.) dried flowers. White yellow heart, mellow smell, light and slightly bitter taste, and is a Guangxi genuine herb. The flower sun-dried tea is mainly distributed in places such as Guangdong, Guangxi, Yunnan and Fujian, and is often taken by Guangdong and Guangxi folks. The frangipani has excellent ornamental value and also has a plurality of national medicinal effects, such as treating diarrhea, dysentery, abdominal pain, toothache, ear pain, cancer and the like. According to records in the book of Chinese national Zhi Yao, it is sweet and light in flavor, cool in nature, and has the effects of treating damp-heat dysentery, detoxifying, moistening lung, etc. In Mexico, frangipani water decoction is used to treat and prevent diabetes. In recent years, studies have been made on the antidiabetic effect of the ethanolic extract of frangipani on diabetic rats induced by streptozotocin.

According to the reports in the literature, the anti-diabetic activity of the frangipani is mostly related to various crude extracts of the frangipani, and the reports on the specific active ingredients of the frangipani are few.

In order to further discover a new anti-diabetic active ingredient from the frangipani and lay a foundation for the development of a new drug, the inventor carries out deep research on chemical ingredients of the frangipani. Separating and extracting from plumeria rubra, screening by combining with anti-diabetic activity, and identifying triterpenes, iridoids and pyrones compounds with anti-diabetic activity.

Disclosure of Invention

The invention discloses triterpenes, iridoids and pyrones compounds with anti-diabetic activity extracted and separated from frangipani, and discloses an extraction and separation method, structure identification and an experiment on the anti-diabetic activity.

The technical scheme for realizing the purpose of the invention is as follows:

the frangipani contains four compounds with anti-diabetic activity, wherein the compounds 1, 2 and 4 are novel compounds,respectively triterpenoid lup-20(29) -en-3β-(1-(2S-hydroxyproprionate) -benzoic) (1), iridoid compoundsβ-dihydroplumericin A(2)，β-dihydroplircinic acid (3), pyrone compound tetrahydroxy-4S-hydroxy-6β-heptadeceyl-2H-pyran-2-one (4) having the structure shown in the following formula:

。

the preparation method of the compound with the anti-diabetic activity in the frangipani comprises the following steps:

(1) soaking 30 kg of flos Plumeriae Acutifoliae in 95% ethanol for 4 times (each for 7 days), mixing extractive solutions, distilling under reduced pressure to remove solvent, suspending the crude extract in water, extracting with ethyl acetate, and recovering solvent under reduced pressure to obtain 1.1kg of ethyl acetate layer extract;

(2) passing the ethyl acetate part through a normal phase silica gel column, and setting a concentration gradient for elution to obtain eleven fractions: i ‒ XI;

(3) performing MCI chromatographic column chromatography on the fraction VI, and performing gradient elution with methanol water to obtain five fractions VI-1 to VI-5; VI-1 flow is subjected to ODS reverse chromatographic column chromatography, methanol-water gradient elution and recrystallization to obtainβ-dihydropliocin a compound 2;

subjecting the fraction V to MCI chromatographic column chromatography, and performing gradient elution with methanol water to obtain five fractions V-1 to V-5; passing V-4 fraction through forward silica gel column, performing ODS reverse chromatography, and gradient eluting with methanol water to obtain tetrahydroxy-4S-hydroxy-6β-heptadeCyl-2H-pyran-2-one compound 4;

v-5 decompressing and concentrating the precipitated powder, washing the powder with methanol to obtain lup-20(29) -en-3β-(1-(2S-hydroxyproprionate) -benzoic acid) compound 1;

subjecting the fraction VII to MCI chromatographic column chromatography, and performing gradient elution with methanol water to obtain four fractions VII-1 to VII-4; VII-2 separating with LH-20 gel column, eluting with methanolβDihydroplicitinic acid compound 3

And (3) carrying out gradient elution on the normal-phase silica gel column in the step (2) by using an eluent with the volume ratio of petroleum ether to acetone =1:0, 13 to 1, 4 to 1, 1 to chloroform to methanol =1 to 1.

Gradient elution is carried out on the methanol water in the step (3), wherein the concentration of the methanol water is gradient elution according to 70%, 75%, 80%, 85%, 90%, 95% and 100%, and the gradient elution is carried out by using acetone to obtain V-5; v-5, decompressing, concentrating and precipitating starch powder, and washing the powder by methanol; and the eluent of the normal phase silica gel column is subjected to gradient elution by adopting petroleum ether and ethyl acetate =12:1 and petroleum ether and ethyl acetate =16:1 in volume ratio respectively.

The invention discovers triterpenes, iridoids and pyrones compounds with novel structures and anti-diabetic activity from frangipani, and lays a foundation for preparing new anti-diabetic activity medicines.

Drawings

FIG. 1 shows a method for preparing frangipani compound 1¹H NMR spectrum;

FIG. 2 shows a preparation of frangipani compound 1¹³A C NMR spectrum;

FIG. 3 is an HSQC spectrum of frangipani compound 1;

FIG. 4 is an HMBC profile of frangipani compound 1;

FIG. 5 shows a preparation of frangipani compound 1¹H-¹H COSY map;

FIG. 6 is a NEOSY spectrum of frangipani compound 1;

FIG. 7 shows Mosher's method Δ for frangipani compound 1_H ^S-R The value is obtained.

Detailed Description

Compound 1 structure identification and high resolution mass spectrum (HR-ESI-MS)m/z: 617.4229 [M – H]^‒, calcdfor C₄₀H₅₇O₅ ^‒617.4211) can be presumed to be C₄₀H₅₈O₅Having 12 unsaturations;+ 26.77(c0.43, DMSO). The IR (KBr) data show that the product contains OH (υ _max3422 cm^‒1)，COOH (υ _max1715 cm^‒1) Meta-substituted benzene ring: (υ _max964, 882, 750 cm^‒1). UV (methanol)λ _max(log) 220 (3.46), 275(1.89)。¹HNMR (500 MHz, pyridine-d ₅)_H: 0.90 (1H, m, H-1a), 1.62 (1H, m, H-1b), 1.77 (2H, m, H-2), 4.78 (1H, dd,J= 11.7, 4.8 Hz, H-3), 0.78 (1H, m, H-5), 1.35 (1H, m, H-6a), 1.45 (1H, m, H-6b), 1.35 (2H, m, H-7), 1.28 (1H, m,H-9), 1.29 (2H, m, H-11), 1.14 (2H, m, H-12), 1.62 (1H, m, H-13), 0.97 (1H,m, H-15a), 1.67 (1H, m, H-15b), 1.42 (1H, m, H-16a), 1.50 (1H, m, H-16b),1.42 (1H, m, H-18), 2.48 (1H, td,J= 11.0, 5.8 Hz, H-19), 1.41 (2H, m, H-21), 1.26 (1H, m, H-22a) 1.40 (1H, m, H-22b), 0.88 (3H, s, H-23), 0.91 (3H,s, H-24), 0.84 (3H, s, H-25), 1.00 (3H, s, H-26), 0.99 (3H, s, H-27), 0.83(3H, s, H-28), 4.75 (1H, dd,J= 2.4, 1.3 Hz, H-29a), 4.90 (1H, dd,J= 2.4,H-29b), 1.75 (3H, s, H-30), 4.96 (1H, m H-2'), 3.44 (1H, dd,J= 13.8, 5.1Hz, H-3'a), 3.51 (1H, dd,J= 13.8, 7.5 Hz, H-3'b), 8.64 (1H, s, H-5'), 8.38(1H, d,J= 7.7 Hz, H-7'), 7.49 (1H, t,J= 7.6 Hz, H-8'), 7.77 (1H, d,J=7.6 Hz, H-9');¹³CNMR (125 MHz, pyridine-d ₅) _C : 39.0 (t, C-1), 24.5 (t, C-2),82.0 (d, C-3), 38.6 (s, C-4), 56.0 (d, C-5), 18.9 (t, C-6), 34.9 (t, C-7),41.5 (s, C-8), 50.9 (d, C-9), 37.7 (s, C-10), 21.5 (t, C-11), 25.9 (t, C-12),38.7 (d, C-13), 43.5 (s, C-14), 28.2 (t, C-15), 36.2 (t, C-16), 43.7 (s, C-17), 49.0 (d, C-18), 48.7 (d, C-19), 151.5 (s, C-20), 30.6 (t, C-21), 40.7(t, C-22), 28.5 (q, C-23), 17.2 (q, C-24), 16.7 (q, C-25), 16.6 (q, C-26),15.2 (q, C-27), 18.6 (q, C-28),110.4 (t, C-29), 19.9 (q, C-30), 174.9 (s, C-1'), 72.8 (d, C-2'), 41.8 (t, C-3'), 139.3 (s, C-4'), 132.3(d, C-5'), 133.3(s, C-6'), 128.9 (d, C-7'), 129.1 (d, C-8'), 134.9 (d, C-9'), 169.8 (s, C-10'). As shown in FIG. 1, of Compound 1¹H NMR data show two terminal double bond protons (_H4.75, 4.90), and 7 corner methyl groups ((ii) ((iii))_H0.83, 0.84, 0.88, 0.91, 0.99, 1.00, 1.75). As shown in figures 2 and 3 of the drawings,¹³the carbon atom types in the C NMR and HSQC spectra and the related data give the chemical shifts of the double bond in (A)_C110.4, 151.5), suggesting that the molecular formula of compound 1 contains lupane-type triterpene skeleton.¹H NMR,¹³C NMR data also showed that the compound contained 1 meta-disubstituted benzene ring [, ]_C128.9, 129.1, 132.3, 133.3, 134.9, 139.3;_H7.49 (t, 7.6Hz), 7.77 (d, 7.6 Hz), 8.38 (d, 7.7 Hz), 8.64 (s)]Two carbonyl groups: (_C169.8, 174.9). As shown in FIG. 4, in HMBC spectrum, H-3 (b_H4.78) with C-23, 24, 1' ((C-23)_C28.5, 17.2, 174.9), it was found that the compound was substituted with an ester group at the 3-position of the lupane nucleus. This compound is reacted with the known compound lupeolβ-phenyl propionate is similar except that: according to H-2', (_H4.96) with C-1 ', 3 ', 4 ' ((C-1-C)_C174.9, 41.8, 139.3), H-3' ((II)_H3.44, 3.51) and C-1 ', 2', 4 ', 5' ((II)_C174.9,72.8, 139.3, 132.3), and as shown in figure 5,¹H-¹h-2' (in H COSY map)_H4.96) and H-3' ((II)_H3.44, 3.51), indicating that the 2' position is substituted by a hydroxyl group. And H-5' (II) according to HMBC diagram because the benzene ring is meta-disubstituted_H8.64) and H-7' ((II)_H8.38) are all mixed with C-10' ((C-10)_C169.8), it was confirmed that 6' of the benzene ring is carboxyl. As shown in fig. 6In the NOESY map, H-3: (_H4.78) and H-5 (_H0.78), H-23 (_H0.88) correlation, meaning that 3 bits areβConfiguration. The absolute configuration of the chiral carbon of the 2 'secondary alcohol was determined by the Mosher's method. (R) -MTPA and (S) The esterification of the present compounds with MTPA reagent, respectively, comparativeR) -MTPA and (S) Of the esters of MTPA¹H NMR-derived DELTA_H ^S-R According to the Δ on both sides of the hydroxyl group, as shown in FIG. 7_H ^S-R The configuration of the secondary alcohol is 2'S. In conclusion, the structure of the compound is identified as lup-20(29) -en-3β-(1-(2S-hydroxyproprionate) -benzoic acid), which is a novel compound.

Identifying the structure of compound 2 by high resolution mass spectrometry (HR-ESI-MS)m/z: 329.1001 [M + Na]^＋,calcd for C₁₆H₁₈O₆Na^＋329.0996) can be presumed to be C₁₆H₁₈O₆There are 7 unsaturations.+187.84 (c0.21, CH₃COCH₃). IR (KBr) data show OH: (υ _max3443 cm^‒1) Ester group (b)υ _max1784, 1267 cm^‒1)，C=C (υ _max1690 cm^‒1). UV (methanol)λ _max(log) 225 (3.29)。¹HNMR(400 MHz, CD₃COCD₃) _H : 5.73 (1H, d,J= 8.0 Hz, H-1), 7.45(1H, s, H-3), 3.97(1H, dt,J= 2.4, 10.0 Hz, H-5), 6.07 (1H, dd,J= 2.4, 5.6 Hz, H-6), 5.85(1H, dd,J= 6.0, 10.0 Hz, H-7), 3.52 (1H, dd,J= 2.4, 5.6 Hz, H-9), 4.45(1H, s, H-10), 2.70 (1H, m, H-11), 1.78 (2H, m, H-13), 1.11 (3H, t,J= 7.6Hz, H-14), 4.21 (2H, m, H-16), 1.28 (3H, t,J= 7.2Hz, H-17).¹³CNMR (100 MHz,CD₃COCD₃) _C : 102.4 (d, C-1), 153.1 (d, C-3), 109.7 (s, C-4), 38.8 (d, C-5),141.7 (d, C-6), 127.8 (d, C-7), 106.7 (s, C-8), 54.2 (d, C-9), 87.4 (d, C-10), 49.5 (d, C-11), 176.9 (t, C-12), 23.2 (t, C-13), 12.2 (s, C-14), 166.7(t, C-15), 60.7 (t, C-16), 14.6 (s, C-17)。¹³C NMR and DEPT data show that Compound 2 contains 16 carbon atoms, of which 2 methyl groups ((II))_C14.6, 12.2), 2 methylene groups: (_C60.7, 23.2), 8 methines (_C153.1, 141.7, 127.8, 102.4, 87.4, 54.2, 49.5, 38.8), 4 quaternary carbons ((ii)_C176.9, 166.7,109.7, 106.7), the above data indicate that compound 2 is an iridoid.¹H NMR data and¹³c NMR data and CompoundsβDihydramericin is similar except that one more(s) is included in the compound data_C60.7,_H4.21) methylene signal. From HMBC and¹H-¹on the H COSY map: in HMBC spectrum, H-17 (_H1.28) and C-16 (_C60.7) related, H-16 (_H4.21) with C-15, 17 (_C166.7, 14.6);¹H-¹h-16 (in H COSY map)_H4.21) and H-17 (_H1.28) and it was confirmed that H-16 is directly bonded to H-17 and the ethyl group is bonded to the ester group. H-5 (in NOESY map)_H3.97) with H-1(_H5.73) and H-9 (_H3.52) correlation ofβConfiguration. Taken together, the novel compounds were identifiedβ-dihydroplumericin A。

Compound (I)β-dihydroplicitinic acid (3) structural identification, ESI-MS based on low resolution mass spectrometrym/ z: 277 [M – H]^‒Presuming the formula is C₁₄H₁₄O₆，¹H NMR (400 MHz, CD₃COCD₃)_H:5.72 (1H, d,J= 5.9 Hz, H-1),7.44 (1H, s, H-3), 3.94 (1H, dt,J= 9.7, 2.3 Hz, H-5), 5.82(1H, dd,J= 5.5, 2.2 Hz, H-6), 6.05 (1H, dd,J= 5.5, 2.2 Hz, H-7), 3.50(1H, dd,J= 9.7, 5.9 Hz, H-9), 4.44 (1H, s, H-10), 2.68 (1H, ddd,J= 8.8,6.5, 1.1 Hz, H-11), 1.09 (3H, t,J= 7.4 Hz, H-14).¹³C NMR (100 MHz, CD₃COCD₃)_C102.5 (d, C-1), 153.5 (d, C-3), 106.9 (s, C-4), 39.0 (d, C-5), 127.9 (d, C-6), 141.9 (d, C-7), 109.6 (s, C-8), 54.4 (d, C-9), 87.6 (d, C-10), 49.7 (d, C-11), 177.1 (s, C-12), 23.4 (t, C-13), 12.3 (q, C-14), 167.8 (s, C-15). Identifying the compound asβ-dihidroplumericinic acid。

Compound tetrahydroxy-4S-hydroxy-6β-heptadecenyl-2H-pyran-2-one (4) structure identification according to high resolution mass spectrometry (HR-ESI-MS)m/z: 377.3034 [M + Na]⁺, calcdfor C₂₂H₄₂O₃Na⁺377.3026) can be presumed to be C₂₂H₄₂O₃There are 2 unsaturations.

+ 9.15 (c0.32,CH₃COCH₃). IR (KBr) data show OH: (υ _max3464 cm^‒1) Ester group (b)υ _max1709 cm^‒1) Long chain alkyl group(s) (iii)υ _max2922 cm^‒1). UV (methanol)λ _max(log) 214 (2.05)。¹H NMR (400 MHz, CD₃COCD₃)_H:2.61 (1H, dd,J= 17.3, 4.7 Hz, H-3a), 2.44 (1H, ddd,J= 17.3, 3.6, 1.8 Hz,H-3b), 4.28 (1H, m, H-4), 1.90 (1H, m, H-5a), 1.70 (1H, m, H-5b), 4.60 (1H,m, H-6), 1.55 (2H, m, H-7), 1.38 (1H, m, H-8a), 1.45 (1H, m, H-8b), 1.24 (1H,s, H-9~20), 1.24 (1H, s, H-21), 1.24 (1H, s, H-22), 0.83 (3H, t,J= 6.7 Hz,H-23).¹³C NMR (100 MHz, CD₃COCD₃)_C170.5 (s, C-2), 39.6 (t, C-3), 63.3 (d, C-4), 36.9 (t, C-5), 76.3 (d, C-6), 36.7 (t, C-7), 25.9 (t, C-8), 30.0-30.6(t, C-9-20), 32.8 (t, C-21), 23.5 (t, C-22), 14.5 (q, C-23). Process for preparation of Compound 4¹H NMR and¹³c NMR data is a long-chain pyrone compound, and 6-bit long side chains of the long-chain pyrone compound are all- (CH)₂)₁₆CH₃And 3 in the literatureβ-hydroxyicosan-1,5β-olide analogously, substituted in position 3 by a hydroxyl group. However, according to high resolution mass spectrometry, the compound has 2 more methylene groups than the long side chain of the compound in the literature, so the new compound is named as tetrahydro-4-hydroxy-6-heptadeceyl-2H-pyran-2-one.

The anti-diabetic effect of frangipani compounds is further illustrated by the following experiments.αGlucoronidase and protein tyrosine phosphatase 1B (PTP 1B) are important targets for treating diabetes, inhibitingαGlucoronidase and protein tyrosine phosphatase 1B (PTP 1B) are important strategies for the treatment of diabetes.

Determination of Compounds 1-4 in Plumeria rubra by light-detecting absorptionαGlucoronidase and protein tyrosine phosphatase 1B (PTP 1B) inhibitory activity.

In vitro assay protocol for evaluating monomeric compounds in frangipani using acarbose as a positive controlα-glucoronidase inhibitory activity. Paranitrobenzene is reactedα-D-glucoside (PNPG) as a substrate, usingαGlucosidase-catalyzes the hydrolysis of PNPG to yield para-nitrophenol (PNP), which is very absorbing at 405 nm. The change of the absorption intensity of the reaction system at 405 nm is monitored by a microplate reader within a certain time to calculate the sample pairα-inhibitory activity of glucosidase. Compounds 1 to 4, IC thereof₅₀Are respectively 19.45 +/-1.62μM，56.84 ± 13.98μM, 62.50 ± 23.55μM, 135.28 ±19.10μM, shows outstanding inhibitory activity, far superior to the IC of the positive control acarbose₅₀Is 363.93±95.43μM, especially compound 1, was most active.

The in vitro assay protocol using oleanolic acid as a positive control evaluated the PTP1B inhibitory activity of compounds 1-4 in frangipani. 4-nitrophenyl phosphate(s) ((s))pNPP) as substrate, using PTP1B for catalytic hydrolysispNPP produces p-Nitrophenol (NPP), which is strongly absorbing at 405 nm. The change of the absorption intensity at 405 nm of the reaction system is monitored by a microplate reader within a certain time, so that the inhibitory activity of the sample on PTP1B is calculated. Compound 1 exhibits outstanding inhibitory activity, IC thereof₅₀Is 0.21 +/-0.00μM is far superior to acarbose as positive controlIC₅₀Is 2.48 +/-0.22μM。