An iridoid ether glycoside compound, a preparation method thereof, and use thereof in the preparation of antidepressant drugs

Abstract

The present invention belongs to the field of natural medicines and chemical medicines for treating and preventing depression, and specifically discloses an iridoid glycoside compound, a preparation method thereof, and use of the iridoid glycoside compound in preparing antidepressant drugs. The iridoid glycoside compound extracted from the soft-branched yellow cicada has a structure selected from allamanoid C, allamanoid E, and allamanoid H, has a novel chemical structure, and has low toxicity to normal cells. Allamanoid C, allamanoid E, and allamanoid H exhibit significant neuroprotective effects on HT22 cell damage induced by corticosterone at low concentrations, and their activity is stronger than that of the positive control drug fluoxetine hydrochloride. Allamanoid C, allamanoid E, and allamanoid H have significant antidepressant effects on mouse despair depression models (tail suspension model and forced swim model), and therefore allamanoid C, allamanoid E, and allamanoid H can be used to prepare antidepressant drugs.

Description

Technical Field

[0001] The present invention relates to the field of natural medicines and chemical medicines for treating and preventing depression, in particular to an iridoid ether terpenoid glycoside compound, a preparation method thereof and application thereof in preparing antidepressant drugs. Background Art

[0002] Depression is a mental disorder characterized by low mood and loss of interest, for which there is no effective treatment. Statistics show that over 350 million people suffer from depression nationwide, with 95 million in China, nearly 50% of whom are students. This number is expected to continue to rise, imposing a significant economic and social burden. Treatments for depression primarily include medication and psychotherapy, with medication being an important component. Currently, the main medications used to treat depression include monoamine oxidase inhibitors, selective serotonin reuptake inhibitors, and tricyclics. However, these medications generally suffer from slow onset of action, low efficacy, and are associated with numerous adverse reactions.

[0003] Allamanda cathartica Linn. is a vine-like shrub of the genus Allamanda in the Apocynaceae family. Also known as yellow warbler, small yellow warbler, and soft-branched flower warbler, it is native to Brazil and is now widely distributed in tropical regions. It is cultivated in southern provinces of my country, including Guangxi, Guangdong, Fujian, and Taiwan. In traditional medicine, the roots of the plant are used to treat snake bites, the stem bark is used as a diuretic, and the leaves are used as a laxative. The chemical composition of the plant is still understudied. Previous studies have revealed that the plant contains iridoids and their glycosides, sterols, alkaloids, fatty acids, and volatile oils. Iridoid glycosides are a class of active compounds found widely in the plant kingdom, exhibiting diverse biological activities, including anti-inflammatory, anti-tumor, antidiabetic, hepatoprotective, neuroprotective, and cardiovascular effects. These compounds have complex and diverse structures, and their biological activities and mechanisms of action are closely related to their chemical structure.

[0004] However, there are no reports on the antidepressant effects of iridoid glycosides. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides an iridoid glycoside compound, a preparation method thereof, and an application thereof in the preparation of antidepressant drugs.

[0006] To achieve the above object, the present invention is implemented according to the following technical solutions:

[0007] One of the objects of the present invention is to provide an iridoid glycoside compound, wherein the structure of the iridoid glycoside compound is one of allamanoid C, allamanoid E, and allamanoid H as shown in Formula I:

[0008]

[0009] A second object of the present invention is to provide a method for preparing an iridoid glycoside compound, comprising the following steps:

[0010] S1. Crush the dried branches and leaves of the soft-branched yellow cicada, extract them multiple times by immersion in an organic solvent, and combine the multiple extracts;

[0011] S2. The combined extracts are concentrated and then separated and purified by macroporous resin column chromatography, Sephadex LH-20 column chromatography, octadecyl bonded phase silica gel column chromatography and preparative high performance liquid chromatography to obtain allamanoid C, allamanoid E and allamanoid H, respectively.

[0012] Furthermore, the organic solvent is an ethanol solution with a volume concentration of 95%.

[0013] A third object of the present invention is to provide an application of an iridoid glycoside compound in the preparation of antidepressant drugs.

[0014] A fourth object of the present invention is to provide an antidepressant comprising one or more of allamanoid C, allamanoid E, and allamanoid H as shown in Formula I. Of course, the antidepressant may further comprise a pharmaceutically acceptable carrier, diluent, or excipient.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] 1. The present invention discovered a new class of iridoid glycoside compounds, allamanoid C, allamanoid E, and allamanoid H, derived from the branches and leaves of the soft-branched yellow cicada. These compounds are composed of two monosaccharide molecules linked to the C-1 position of a frangipani-type iridoid glycoside. They have a novel chemical structure and are less toxic to normal cells.

[0017] 2. The present invention found that allamanoid C, allamanoid E and allamanoid H showed significant neuroprotective effects on HT22 cell damage induced by corticosterone at lower concentrations, and their activity was stronger than that of the positive control drug fluoxetine hydrochloride.

[0018] 3. The present invention found that allamanoid C, allamanoid E and allamanoid H have significant antidepressant effects on mouse despair depression models (tail suspension model, forced swim model), so allamanoid C, allamanoid E and allamanoid H can be used to prepare antidepressant drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is the iridoid glycoside compound allamanoid C 1 H NMR spectrum.

[0020] Figure 2 It is the iridoid glycoside compound allamanoid C 13 C NMR spectrum.

[0021] Figure 3 This is the HSQC spectrum of the iridoid glycoside compound allamanoid C.

[0022] Figure 4 It is the iridoid glycoside compound allamanoid C 1 H– 1 H COSY spectrum.

[0023] Figure 5 This is the HMBC spectrum of the iridoid glycoside compound allamanoid C.

[0024] Figure 6 This is the NOESY spectrum of the iridoid glycoside compound allamanoid C.

[0025] Figure 7 It is the iridoid glycoside compound allamanoid E 1 HNMR spectrum.

[0026] Figure 8 It is the iridoid glycoside compound allamanoid E 13 C NMR spectrum.

[0027] Figure 9 This is the HSQC spectrum of the iridoid glycoside compound allamanoid E.

[0028] Figure 10 It is the iridoid glycoside compound allamanoid E 1 H– 1 H COSY spectrum.

[0029] Figure 11This is the HMBC spectrum of the iridoid glycoside compound allamanoid E.

[0030] Figure 12 This is the NOESY spectrum of the iridoid glycoside compound allamanoid E.

[0031] Figure 13 It is the iridoid glycoside compound allamanoid H 1 H NMR spectrum.

[0032] Figure 14 It is the iridoid glycoside compound allamanoid H 13 C NMR spectrum.

[0033] Figure 15 This is the HSQC spectrum of the iridoid glycoside compound allamanoid H.

[0034] Figure 16 It is the iridoid glycoside compound allamanoid H 1 H– 1 H COSY spectrum.

[0035] Figure 17 This is the HMBC spectrum of the iridoid glycoside compound allamanoid H.

[0036] Figure 18 This is the NOESY spectrum of the iridoid glycoside compound allamanoid H.

[0037] Figure 19 This is a graph showing the toxicity of iridoid glycosides allamanoid C, allamanoid E and allamanoid H to HT-22 cells.

[0038] Figure 20 This figure shows the protective effects of iridoid glycosides allamanoid C, allamanoid E and allamanoid H on the corticosterone-induced HT-22 injury model.

[0039] Figure 21 This figure shows the antidepressant effects of the iridoid glycoside compounds allamanoid C, allamanoid E and allamanoid H on the mouse tail suspension model.

[0040] Figure 22 This figure shows the antidepressant effects of the iridoid glycoside compounds allamanoid C, allamanoid E and allamanoid H on the forced swimming model of mice. DETAILED DESCRIPTION

[0041] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0042] The mice used in the following examples were female Kunming and C57BL / 6J mice purchased from the Guangdong Provincial Laboratory Animal Center. All other related reagents used, unless otherwise specified, were commercially available, and the methods involved, unless otherwise specified, were all well-known methods.

[0043] Example 1. Preparation of compounds allamanoid C, allamanoid E and allamanoid H

[0044] (1) 15 kg of naturally dried branches and leaves of Allamanda cathartica Linn. var. cathartica were ground into a coarse powder and extracted three times with 95% ethanol at room temperature. The extracts were combined and concentrated under reduced pressure to obtain 2.3 kg of total extract. The total extract was suspended in water and extracted with petroleum ether, ethyl acetate, and n-butanol, respectively, to obtain 190 g of the petroleum ether extract, 295 g of the ethyl acetate extract, and 410 g of the n-butanol extract.

[0045] (2) The obtained 410 g n-butanol extraction fraction was subjected to macroporous adsorption resin column chromatography, and gradient elution was performed using an ethanol-water binary mixed solvent as the mobile phase (volume ratios were: 0:100, 10:90, 40:60, 80:20 and 95:5, respectively). After thin layer chromatography (TLC) analysis, similar fractions were combined to obtain five main fractions, namely Fr.A, Fr.B, Fr.C, Fr.D, and Fr.E. 94 g of the fraction Fr.B was loaded onto a Sephadex LH-20 column and eluted using a methanol-water binary mixed solvent (volume ratio of 1:1) as the mobile phase at a flow rate of 1 ml / min. The eluate was collected and similar fractions were combined by TLC analysis to obtain six fractions, namely Fr.Ba, Fr.Bb, Fr.Bc, Fr.Bd, Fr.Be, and Fr.Bf.

[0046] (3) 15.3 g of the fraction Fr.Bc was subjected to reverse-phase ODS column chromatography, and gradient elution was performed using a methanol-water binary mixed solvent as the mobile phase (volume ratios were 10:90, 20:80, 30:70, 40:60, 60:40, 80:20, and 100:0, respectively). Similar fractions were combined by TLC to obtain 8 fractions, namely Fr.Bca, Fr.Bcb, Fr.Bcc, Fr.Bcd, Fr.Bce, Fr.Bcf, Fr.Bcg, and Fr.Bch.

[0047] (4) 2.1 g of the fraction Fr.Bcc was loaded onto a Sephadex LH-20 column and eluted with a methanol-water binary mixed solvent as the mobile phase (volume ratio of 1:1) at a flow rate of 0.5 ml / min. The eluate was collected and similar fractions were combined by TLC analysis to obtain five fractions, namely Fr.Bcca, Fr.Bccb, Fr.Bccc, Fr.Bccd, and Fr.Bcce.

[0048] (5) The obtained fraction Fr.Bccb was concentrated and dissolved in methanol, and then further separated and purified by reverse phase preparative high performance liquid chromatography (PHPLC), using a methanol-water binary mixed solvent with a volume ratio of 14:86 as the mobile phase and a flow rate of 5 mL / min for elution, to obtain 3.6 mg of compound allamanoid C; the obtained fraction Fr.Bccc was concentrated and dissolved in chromatographic grade methanol, and then further separated and purified by reverse phase preparative high performance liquid chromatography (PHPLC), using a methanol-water binary mixed solvent with a volume ratio of 12:88 as the mobile phase and a flow rate of 5 mL / min for elution, to obtain 4.1 mg of compound allamanoid E; the obtained fraction Fr.Bcce was concentrated and dissolved in chromatographic grade methanol, and then further separated and purified by reverse phase preparative high performance liquid chromatography (PHPLC), using a methanol-water binary mixed solvent with a volume ratio of 11:89 as the mobile phase and a flow rate of 5 mL / min for elution, to obtain 2.0 mg of compound allamanoid H.

[0049] Example 2 Structural Identification of Compounds Allamanoid C, Allamanoid E, and Allamanoid H

[0050] The NMR data were measured using a Bruker AVANCE-400 NMR spectrometer. The solvent was deuterated methanol (CD3OD), the internal standard was tetramethylsilane (TMS), and the NMR shift (δ) was 10 -6 The unit of ppm is given, the unit of coupling constant (J) is Hz, and the peak shape of overlapping signals is not marked. HR-ESI-MS data were measured using an Agilent 6210LC / MSD TOF-MS mass spectrometer; ultraviolet spectra (UV) were measured by a JASCO V-550 UV / visible spectrometer; infrared spectra (IR) were measured using a JASCO FT / IR-480 Fourier transform infrared spectrometer (KBr pellet); optical rotation values ​​(OR) were measured using a JASCO P-1020 polarimeter; macroporous adsorption resin HP-20 was purchased from Shanghai Kaiyin Chemical Co., Ltd.; preparative HPLC was performed using an Agilent 1260 high performance liquid chromatograph with an MWD detector. TLC analysis of GF 254Thin-layer silica gel precast plates were purchased from Yantai Institute of Chemical Industry; column chromatography silica gel (200–300 mesh) was provided by Qingdao Ocean Chemical Co., Ltd.; Sephadex LH-20 gel chromatography packing was produced by Amershanm Biosciences; and ODS column chromatography packing was produced by Merck.

[0051] The compound allamanoid C is a light yellow powder, [α]25D = -137.0 (c 0.30, methanol); UV (MeOH) λ max :208nm; IRvKBrmax3180,2832,1752,1750,1299,1067cm -1 ; HR-ESI-MS m / z 655.1859 [M+Na] + (Molecular formula C 27 H 36 NaO 17 The calculated value is 655.1845). 1 HNMR, 13 CNMR, HSQC, 1 H– 1 HCOSY, HMBC and NOESY NMR spectra are shown in Figures 1 to 6 . 1H Spectrum ( 1 HNMR) and carbon spectroscopy ( 13 The data attribution of CNMR is shown in Table 1.

[0052] Table 1. allamanoid C 1 H (400MHz) and 13 C (100 MHz) NMR data

[0053]

[0054] Based on the above physicochemical constants and spectral data, the structure of allamanoid C was identified as:

[0055]

[0056] The compound allamanoid E is a light yellow powder, [α]25D = -3.3 (c 0.60, methanol); UV (MeOH) λ max :213nm; IRvKBrmax3453,2872,1748,1294,1076cm -1 ; HR-ESI-MS m / z 655.1860 [M+Na] + (Molecular formula C 27 H 36 NaO 17The calculated value is 655.1845). 1 HNMR, 13 CNMR, HSQC, 1 H– 1 HCOSY, HMBC and NOESY NMR spectra are shown in Figures 7 to 12 . 1H Spectrum ( 1 HNMR) and carbon spectroscopy ( 13 The data attribution of CNMR is detailed in Table 2.

[0057] Table 2. allamanoid E 1 H (400MHz) and 13 C (100 MHz) NMR data

[0058]

[0059] Based on the above physicochemical constants and spectral data, the structure of allamanoidE was identified as:

[0060]

[0061] The compound allamanoid H is a light yellow powder, [α]25D = -13.0 (c1.00, methanol); UV (MeOH) λ max :207nm; IRvKBrmax3370,2870,1748,1300,1076cm -1 ; HR-ESI-MS m / z 625.1758 [M+Na] + (Molecular formula C 27 H 36 NaO 17 The calculated value is 625.1739). 1 HNMR, 13 CNMR, HSQC, 1 H– 1 HCOSY, HMBC and NOESY NMR spectra are shown in Figures 13 to 18 . 1H Spectrum ( 1 HNMR) and carbon spectroscopy ( 13 The data attribution of CNMR is shown in Table 3.

[0062] Table 3. allamanoid H 1 H (400MHz) and 13 C (100 MHz) NMR data

[0063]

[0064] Based on the above physicochemical constants and spectral data, the structure of allamanoid H was identified as:

[0065]

[0066] Example 3. In vitro antidepressant activity of allamanoid C, allamanoid E, and allamanoid H

[0067] In this example, the antidepressant activity of the compound in vitro was evaluated using the classic corticosterone-induced HT22 cell injury model.

[0068] 1. Methods:

[0069] 1.1. Determination of cytotoxicity of compounds:

[0070] Mouse hippocampal neuron HT-22 cells (purchased from Shanghai Saibaikang Biotechnology Co., Ltd.) were cultured in DMEM culture medium containing 10% FBS and 1% P / S. The HT-22 cells were seeded in a 96-well cell culture plate and cultured overnight in a 37°C, 5% CO2 incubator; the original culture medium was discarded, and 100 μL of the above-mentioned culture medium containing the test sample was added to each well. At the same time, a cell control well was set up and the cells were placed in an incubator for further culture for 48 hours. The culture medium was discarded, and 10 μL of CCK-8 solution and 90 μL of MEM culture medium were added to each well. The cells were placed in a cell incubator and incubated in the dark for 1 hour; the absorbance value (OD value) was detected at a wavelength of 465 nm using a multifunctional microplate reader to calculate the cell survival rate.

[0071] 1.2. Protective effects of compounds on the corticosterone-induced HT-22 injury model:

[0072] Mouse hippocampal neuronal HT-22 cells (purchased from Shanghai Saibaikang Biotechnology Co., Ltd.) were seeded into 96-well cell culture plates and cultured overnight in a 37°C, 5% CO2 incubator. The original culture medium was discarded, and 100 μL of the above-mentioned culture medium containing the test sample was added to each well. At the same time, 200 μM corticosterone (MedChemExpress, HY-B1618) was added and cultured for 24 hours. A normal cell control group and a corticosterone model group were set up. After 24 hours of culture, the cell survival rate was calculated according to the operating instructions attached to the CCK-8 detection kit.

[0073] 2. Results:

[0074] 2.1. Cytotoxicity results are shown in Figure 19Allamanoid C, allamanoid E, and allamanoid H showed no significant toxic effects at doses below 50 μM. Results are presented as mean ± SEM, and statistical analysis was performed using one-way ANOVA (*P < 0.05, **P < 0.01).

[0075] 2.2. Protective effects of compounds on corticosterone-induced HT-22 cell damage Figure 20 Allamanoid C, allamanoid E, and allamanoid H exhibited significant neuroprotective effects against corticosterone-induced HT22 cell damage at lower concentrations, indicating that this class of compounds possesses excellent antidepressant activity, which is stronger than the positive control drug fluoxetine hydrochloride. The results are presented as mean ± SEM, and the statistical method was one-way ANOVA (compared with the control group, **P < 0.01; compared with the corticosterone group (COR), ## P<0.01).

[0076] In conclusion, allamanoid C, allamanoid E and allamanoid H all have significant antidepressant activity.

[0077] Example 4: In vivo antidepressant effect experiments of allamanoid C, allamanoid E, and allamanoid H

[0078] In this example, the antidepressant efficacy of the compound in vivo was evaluated using classic mouse behavioral despair depression models (tail suspension model, forced swim model).

[0079] 1. Methods:

[0080] C57BL / 6J mice (18-22 g, purchased from Spefox (Beijing) Biotechnology Co., Ltd.) were housed at (20 ± 2)°C, 60% humidity, and on a 12-h light / 12-h dark light cycle with free access to food and water. Animal experiments adhered to international animal experimentation ethics guidelines and were conducted after one week of maintenance. Normal mice were randomly divided into eight groups, each containing 10 mice: a blank control group; the active drug fluoxetine (10 mg / kg); allamanoid C (low-dose group: 5 mg / kg, high-dose group: 10 mg / kg); allamanoid E (low-dose group: 5 mg / kg, high-dose group: 10 mg / kg); and allamanoid H (low-dose group: 5 mg / kg, high-dose group: 10 mg / kg). Each group was intraperitoneally injected once daily for three days. Behavioral testing was performed 1 hour after the last dose. Mice in the normal control and model groups were injected with saline containing 1% DMSO and 5% Tween as a solvent control.

[0081] 1.2. Mouse Tail Suspension Test: One hour after the last dose, secure the mouse's tail at one-third of its length to a horizontal metal bar in a tail suspension box with tape, hanging upside down with the head 15-20 cm above the table. Observe for 6 minutes. The cumulative immobility time within the last 4 minutes is recorded as depression (despair) duration.

[0082] 1.3. Forced Swim Test in Mice: One hour after the last drug administration, mice were placed in a 15 cm diameter, 25 cm height, and 13 cm deep (the bottom of the water was too deep for the mice's hind paws to reach). The water temperature was (25 ± 1)°C. The entire process was videotaped for 6 minutes and analyzed using ForcedSwim Version 2.0 (Clever Sys Inc.) software. The duration of immobility during the last 4 minutes indicated the mice's level of depression (or despair). The glass tank was cleaned and the water changed before each experiment.

[0083] 2. Results:

[0084] 2.1. Tail suspension test results Figure 21 The results showed that compared with the blank control group, the immobility time of mice in the fluoxetine group, allamanoid C, allamanoid E and allamanoid H group was reduced after administration. The results are mean ± SEM, and the statistical method is one-way ANOVA (*P < 0.05, **P < 0.01).

[0085] 2.2. Results of the forced swim test are shown in Figure 22 The results showed that compared with the blank control group, the immobility time of mice in the fluoxetine group, allamanoid C, allamanoid E and allamanoid H group was reduced after administration. The results are mean ± SEM, and the statistical method is one-way ANOVA (*P < 0.05, **P < 0.01).

[0086] In conclusion, allamanoid C, allamanoid E and allamanoid H all have good antidepressant effects in vivo.

[0087] In summary, antidepressants can be prepared using one or more combinations of allamanoid C, allamanoid E, and allamanoid H. Of course, the antidepressants may further contain a pharmaceutically acceptable carrier, diluent, or excipient.

[0088] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. Any technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. An iridoid glycoside compound, characterized in that: The structure of the iridoid glycoside compound is one of allamanoid C, allamanoid E, and allamanoid H as shown in Formula I:

2. A method for preparing the iridoid glycoside compound according to claim 1, characterized in that: The following steps are involved: S1. Crush the dried branches and leaves of the soft-branched yellow cicada, extract them multiple times by immersion in an organic solvent, and combine the multiple extracts; S2. The combined extracts are concentrated and then separated and purified by macroporous resin column chromatography, Sephadex LH-20 column chromatography, octadecyl bonded phase silica gel column chromatography and preparative high performance liquid chromatography to obtain allamanoid C, allamanoid E and allamanoid H, respectively.

3. The method for preparing an iridoid glycoside compound according to claim 2, wherein: The organic solvent is an ethanol solution with a volume concentration of 95%.

4. Use of the iridoid glycoside compound according to claim 1 in the preparation of antidepressants.

5. An antidepressant drug, characterized in that: The method comprises one or more of allamanoid C, allamanoid E and allamanoid H as shown in formula I as claimed in claim 1.

6. The antidepressant drug according to claim 5, characterized in that It also contains a pharmaceutically acceptable carrier, diluent or excipient.

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