A new iridoid compound extracted from patrinia scabiosaa fisch and preparation method and application thereof

Abstract

The application belongs to the technical field of medicine, and particularly relates to a new iridoid compound extracted from Patrinia scabiosaa Fisch, a preparation method and application of the iridoid compound, a structure formula of the iridoid compound is shown as formula (I), and the preparation method of the iridoid compound is provided in detail. 50 The iridoid compound has inhibiting proliferation effects on HL-60 and THP-1 cells, and IC values are 67.53 micromoles and 59.31 micromoles respectively. The iridoid compound can be used for preparing medicines for treating leukemia, and has wide application prospects.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a novel iridoid compound extracted from Patrinia scabiosifolia, its preparation method, and its application. Background Technology

[0002] Patrinia scabioscaefolia Fisch., a plant belonging to the genus Patrinia in the family Valerianaceae, is also known as Huanghua Longya, Ye Gonghua, Luchang, and Helijiqun, and is a traditional Chinese medicine. First recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), it is described as pungent, bitter, and slightly cold in nature, possessing the effects of clearing heat and detoxifying, reducing swelling and draining pus, and removing blood stasis and relieving pain. Existing literature reports the isolation and identification of over 150 active ingredients in Patrinia scabioscaefolia, including flavonoids, triterpenoid saponins, iridoids, volatile oils, sterols, and phenylpropanoids (coumarins and lignans).

[0003] Iridoids are a special class of monoterpenoid compounds that combine with sugars to form glycosides and possess a variety of physiological activities; more than 900 have been discovered to date. Currently, relatively few iridoids have been reported in *Patrinia scabra*, therefore, the extraction of iridoids with specific physiological activities from *Patrinia scabra* is of great significance.

[0004] To extract more iridoids from Patrinia scabra, those skilled in the art have made numerous efforts. For example, invention patent CN202011350445.4 discloses a method for extracting iridoids from Patrinia scabra, and obtained the iridoid compound (4β,8β)-8-methoxy-3-methyl-10-methylene-2,9-dioxane[4.3.1.03,7]decane-4-ol. However, the above-mentioned active ingredient does not have an anti-leukemia effect.

[0005] During the research process, the inventors unexpectedly extracted a new iridoid compound from Patrinia scabra. This compound had not been reported in any literature. Furthermore, the inventors conducted physiological activity experiments on the extracted iridoid compound, and the results showed that the iridoid compound also had the effect of inhibiting the growth of leukemia cells. Summary of the Invention

[0006] The primary objective of this invention is to provide a novel iridoid compound extracted from Patrinia scabra, the iridoid compound having the structural formula shown in formula (I):

[0007]

[0008] A second objective of this invention is to provide a method for preparing the aforementioned iridoid compounds, comprising the following steps:

[0009] 1) Soak dried stems of Patrinia scabiosifolia in 95% ethanol at room temperature, filter and concentrate under reduced pressure. Repeat the above operation, combine the filtrates and concentrate under reduced pressure to obtain the extract.

[0010] 2) Dissolve the extract obtained in step 1) in water, and extract it sequentially with petroleum ether, dichloromethane and ethyl acetate, and concentrate it under reduced pressure to obtain petroleum ether extract, ethyl acetate extract and dichloromethane extract of different polarities.

[0011] 3) The dichloromethane extract obtained in step 2) was separated by chromatography using an HP-20 macroporous adsorption column, and the extract was obtained by elution with four different volume ratios of ethanol-water gradient.

[0012] 4) The extract obtained in step 3) was subjected to normal phase silica gel column chromatography and eluted sequentially with seven different volume ratios of petroleum ether-ethyl acetate to obtain the extract.

[0013] 5) The extract obtained in step 4) was separated by Sephadex LH-20 dextran gel column chromatography, eluted with pure methanol, and identified by TLC to obtain 3 fractions;

[0014] 6) The fraction obtained in step 5) was separated by normal phase silica gel chromatography, using a gradient elution of chloroform:methanol in 5 different ratios to obtain the fraction;

[0015] 7) The fraction obtained in step 6) was purified by semi-preparative HPLC to obtain the new compound.

[0016] Preferably, the ethanol-water volume ratio in step 3) is 30:70, 50:50, 80:20, or 95:5.

[0017] Preferably, the ethanol-water volume ratio in step 3) is 80%.

[0018] Preferably, the volume ratio of petroleum ether to ethyl acetate in step 4) is 20:1, 10:1, 5:1, 3:1, 2:1, 1:1, or 0:1.

[0019] Preferably, the volume ratio of petroleum ether to ethyl acetate in step 4) is 0:1.

[0020] Preferably, the chloroform:methanol gradient elution ratio in step 6) is 1:0, 30:1, 15:1, 7:1, and 0:1, respectively.

[0021] A third objective of this invention is to provide the use of the aforementioned iridoid compounds in the preparation of anti-leukemia drugs.

[0022] Preferably, the leukemia cells are selected from Burrkit lymphoma leukemia cells, acute B-lymphocytic leukemia cells, acute T-lymphocytic leukemia cells, or acute myeloid leukemia cells.

[0023] Preferably, the leukemia cells are acute myeloid leukemia cells.

[0024] Preferably, the acute myeloid leukemia cells are HL-60 and / or THP-1 acute myeloid leukemia cells.

[0025] The beneficial effects of this invention are as follows: The primary objective of this invention is to provide a novel iridoid compound extracted from Patrinia scabra, and to provide a detailed method for preparing the iridoid compound. The iridoid compound exhibits inhibitory effects on the proliferation of HL-60 and THP-1 cells, with an IC50 value of [missing information]. 50 The values ​​were 67.53 μM and 59.31 μM, respectively. This material can be used to prepare drugs for treating leukemia and has broad application prospects. Attached Figure Description

[0026] Figure 1 This is a process flow diagram of the new compound of the present invention;

[0027] Figure 2 The molecular structural formula of the novel compound of this invention is shown below.

[0028] Figure 3 The main HMBC (heteronuclear multicarbon correlation spectrum) of the novel compound of this invention and 1 H- 1 HCOSY (Homonuclear Chemical Shift Correlation Spectroscopy) correlation diagram;

[0029] Figure 4 The diagram shows the NOESY (nuclear Overhaus effect spectrum) correlation of the novel compound of this invention.

[0030] Figure 5 This section describes the fitting relationship between the calculated circular dichroism chromatograms and the actual tested circular dichroism chromatograms of the novel compounds of this invention.

[0031] Figure 6 For the new compounds of this invention 1 1H-NMR (H-NMR) spectrum;

[0032] Figure 7 For the new compounds of this invention 13 C-NMR (carbon nuclear magnetic resonance) spectrum;

[0033] Figure 8 For the new compounds of this invention 1 H-1 HCOSY spectrum;

[0034] Figure 9 The HMBC spectrum of the novel compound of this invention;

[0035] Figure 10 The NOESY spectrum of the novel compound of this invention;

[0036] Figure 11 The HSQC (heteronuclear single quantum relation) spectrum of the novel compound of this invention is shown below.

[0037] Figure 12 The infrared spectrum of the novel compound of this invention;

[0038] Figure 13 This is the ultraviolet spectrum of the novel compound of this invention;

[0039] Figure 14 This is the mass spectrum of the novel compound of this invention;

[0040] Figure 15 This is a schematic diagram illustrating the cell viability of the novel compound of this invention against HL-60 and THP-1 cells.

[0041] Specific implementation methods

[0042] The present invention will be further described below with reference to the embodiments. However, the present invention is not limited to the following embodiments. It is foreseeable that various changes may occur in the implementation when combined with the prior art by those skilled in the art.

[0043] The present invention can be described in the following embodiments.

[0044] The herb *Patrinia scabiosifolia* used in this invention was collected in Qingyang, Gansu Province in September 2020, and the specimen is stored in the Natural Organics Teaching and Research Section of the State Key Laboratory of Chemistry and Chemical Engineering, Lanzhou University.

[0045] It should be noted that, unless otherwise specified, all reagents used in the following examples are commercially available.

[0046] It should be noted that, unless otherwise specified, the methods used in the following examples are all conventional methods. Example 1: Preparation and Identification of Compounds

[0047] 1. Preparation methods of compounds

[0048] A novel compound extracted and isolated from Patrinia scabra, the molecular formula of which is C0.05. 10 H 14 O4, the preparation method of this new compound is as follows:

[0049] 1) Cut the dried stems of Patrinia scabiosifolia into sections and crush them. Soak them in 95% ethanol at room temperature for 5 days. After filtration, concentrate under reduced pressure. Repeat the above operation three times. Combine the results and concentrate under reduced pressure to obtain the total extract.

[0050] 2) Dissolve the extract in water and extract it three times each with petroleum ether, dichloromethane and ethyl acetate in sequence. Concentrate under reduced pressure to obtain petroleum ether extract, ethyl acetate extract and dichloromethane extract of different polarities.

[0051] 3) The dichloromethane extract was separated by chromatography using an HP-20 macroporous adsorption column and eluted sequentially with an ethanol-water gradient of 30%, 50%, 80%, and 95% (v / v) to separate it into four fractions (Fr.1-Fr.4).

[0052] 4) The 80% ethanol extract was subjected to normal phase silica gel column chromatography and eluted sequentially with petroleum ether-ethyl acetate at volume ratios of 20:1, 10:1, 5:1, 3:1, 2:1, 1:1, and 0:1 to separate it into 7 fractions (Fr.3.1-Fr.3.7).

[0053] 5) The Fr.3.7 fraction was separated by Sephadex LH-20 dextran gel column chromatography, eluted with pure methanol, and identified by TLC to obtain three fractions (Fr.3.7.1-Fr.3.7.3).

[0054] 6) Fr. 3.7.2 was separated by normal-phase silica gel chromatography with a chloroform:methanol gradient elution (1:0, 30:1, 15:1, 7:1, 0:1) to obtain 5 fractions (Fr. 3.7.2.1-Fr. 3.7.2.5). Fr. 3.7.2.2 was further purified by semi-preparative HPLC (45% MeOH-H2O, 2.0 mL / min) to obtain 10 fractions (Fr. 3.7.2.2.1-Fr. 3.7.2.2.10). Fr. 7.2.2.2 was purified by semi-preparative HPLC (40% MeCN-H2O, 2.0 mL / min) to obtain the new compound. The specific flow chart is shown below. Figure 1 As shown.

[0055] 2. Structural identification of new compounds

[0056] The new compound is a yellow oily substance, soluble in methanol; structural identification is as follows: Figure 3-14 As shown, the optical rotation is ([α]). 23 D -40.0(c=0.1, MeOH); UV(MeOH)λ max (log ε ): 221 nm (3.98); HR-ESIMS gives the quasi-molecular ion peak m / z: 221.0804 [M+Na] +(calcd for C 10 H 14 O4Na, 221.0784), the molecular formula of this compound is presumed to be C. 10 H 14 O4 has an unsaturation degree of 4.

[0057] according to 1 HNMR and 13 12C NMR spectroscopy data show that there is an α,β-unsaturated aldehyde group [δ] in this structure. H 9.56(s), δ C 196.3; δ C 154.3, 134.4], a hydroxymethyl group [δ H 4.29, δ C 73.4] and a hydroxymethylene [δ] C 66.5] (Table 1). The one-dimensional NMR signal characteristics of this compound are similar to those of the previously reported compound Patrirscabioin M [1] The structures are very similar, the only difference being that the methoxy group at C-1 is replaced by a hydroxyl group. (This is from compound 6.) 1 H- 1 The H COSY spectrum revealed correlations between protons H-7 / H-6 / H-5 / H-9 / H-8, H-8 / H-10, and H-9 / H-1, indicating they reside in three different spin-coupled systems. In the HMBC spectrum, proton H-11 correlated with C-3 / C-4 / C-5; aldehyde protons H-3 correlated with C-5; and H-1 correlated with C-5 / C-9 / C-10, thus determining the position of the hydroxyl group at C-1.

[0058] According to the NOESY spectrum of the compound, protons H-1 and H-8 are correlated, and H-8 and H-6a are correlated, indicating that these protons are on the same side and α-oriented; protons H-9 and H-6b are correlated, indicating that these protons are on the same side and β-oriented. These NOE correlations also prove that the two five-membered rings of the compound are trans-fused. To further determine its absolute configuration, the ECD data of the compound were calculated using quantum chemical methods. Based on the relative configuration of the compound, the ECD curves of a pair of enantiomers, 6 and (-)-6, were calculated. The calculation results show that the (1S, 5S, 7S, 8S, 9S) curve matches the experimentally measured ECD curve well, indicating that the absolute configuration of the compound is (1S, 5S, 7S, 8S, 9S).

[0059] Based on the above information, the new compound was determined to be structured as (1S,5S,7S,8S,9S)-1,10-epoxy-1,7,10-trihydroxy-dolichodial, as follows. Figure 2 As shown.

[0060] Table 1 shows the composition of this new compound. 1 HNMR and 13 CNMR data.

[0061]

[0062] Example 2: Activity Test of the New Compound

[0063] The acute myeloid leukemia (AML) cell lines HL-60 and THP-1 were donated by Professor Zhao Li's research group at the First Hospital of Lanzhou University. The cytotoxicity of the novel compound BJ-6 prepared in Example 1 against HL-60 and THP-1 cells was determined using the MTT assay. HL-60 and THP-1 cells in the logarithmic growth phase were collected, centrifuged to remove the supernatant, and resuspended in RPMI 1640 medium containing 10% FBS, adjusting the cell suspension concentration to 2 × 10⁻⁶ cells / mL. 5 Cells were seeded at a concentration of 90 μL / mL in a 96-well plate. The experiment was divided into a blank group (complete culture medium), a control group (cells + complete culture medium), and experimental groups with different drug concentrations (cells + compound). The control group received 10 μL of complete culture medium, while the experimental groups received compound BJ-6 at final concentrations of 12.5, 25, 50, and 100 μM, with 5 replicates per group. After culturing at 37°C and 5% CO2 for 48 h, 10 μL of MTT (5 mg / mL) was added to each well, and the plates were incubated for another 4 h. The plates were then centrifuged at 1500 rpm for 10 min, and the culture medium was slowly aspirated from each well. 150 μL of DMSO was added to each well, and the plates were shaken until completely dissolved. The OD value was measured at 570 nm using a microplate reader.

[0064] Cell viability (%) = 1 - [(OD value of control group - OD value of experimental group) / (OD value of control group - OD value of blank group)] × 100%

[0065] Experimental results showed that the new compound inhibited the proliferation of HL-60 and THP-1 cells, with an IC50 value of [missing information]. 50 The values ​​were 67.53 μM and 59.31 μM, respectively. Figure 15 As shown.

[0066] In summary, this invention provides a novel iridoid compound extracted from Patrinia scabra, and details a method for preparing the iridoid compound. The iridoid compound exhibits inhibitory activity against the proliferation of HL-60 and THP-1 cells, with an IC50 value of [missing information]. 50 The values ​​were 67.53 μM and 59.31 μM, respectively. This material can be used to prepare drugs for treating leukemia and has broad application prospects.

[0067] References:

[0068] [1]LIU Z,NIU Y,ZHOU L,et al.Nine Unique Iridoids and IridoidGlycosides From Patrinia scabiosaefolia[J].

[0069] Frontiers in chemistry,2021,9:657028。

Claims

1. A novel iridoid compound extracted from Patrinia scabra, characterized in that, The structural formula of the iridoid ether compounds is shown in formula (I): ， （Ⅰ）。 2. The method for preparing the cycloalkenyl ether terpenoid compound according to claim 1, characterized in that, The steps include the following: 1) Soak the dried stems of Patrinia scabiosifolia in 95% ethanol at room temperature, filter and concentrate under reduced pressure. Repeat the above operation, combine the filtrates and concentrate under reduced pressure to obtain the extract. 2) Dissolve the extract obtained in step 1) in water, and extract it sequentially with petroleum ether, dichloromethane and ethyl acetate, and concentrate it under reduced pressure to obtain petroleum ether extract, ethyl acetate extract and dichloromethane extract of different polarities. 3) The dichloromethane extract obtained in step 2) was separated by chromatography using an HP-20 macroporous adsorption column, and the extract was obtained by elution with four different volume ratios of ethanol-water gradient. 4) The extract obtained in step 3) was subjected to normal phase silica gel column chromatography and eluted sequentially with seven different volume ratios of petroleum ether-ethyl acetate to obtain the extract. 5) The extract obtained in step 4) was separated by Sephadex LH-20 dextran gel column chromatography, eluted with pure methanol, and three fractions were obtained after TLC detection. 6) The fraction obtained in step 5) was separated by normal phase silica gel chromatography, using a gradient elution of chloroform:methanol in 5 different ratios to obtain the fraction; 7) The fraction obtained in step 6) was purified by semi-preparative HPLC to obtain the compound.

3. The preparation method according to claim 2, characterized in that, The ethanol-water volume ratios mentioned in step 3) are 30:70, 50:50, 80:20, and 95:5, respectively.

4. The preparation method according to claim 2, characterized in that, The volume ratio of petroleum ether to ethyl acetate mentioned in step 4) is 20:1, 10:1, 5:1, 3:1, 2:1, 1:1, or 0:

1.

5. The preparation method according to claim 2, characterized in that, In step 6), the chloroform:methanol gradient elution ratios are 1:0, 30:1, 15:1, 7:1, and 0:1, respectively.

6. The use of the iridoid compound of claim 1 in the preparation of anti-leukemia drugs.

7. The application as described in claim 6, characterized in that, The leukemia cells mentioned are selected from Burrkit lymphoma leukemia cells, acute B-lymphoblastic leukemia cells, acute T-lymphoblastic leukemia cells, or acute myeloid leukemia cells.

8. The application as described in claim 7, characterized in that, The leukemia cells mentioned are acute myeloid leukemia cells.

9. The application as described in claim 8, characterized in that, The acute myeloid leukemia cells mentioned are HL-60 and / or THP-1 acute myeloid leukemia cells.

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