Method for separating and extracting (-)-Rabdosiin from rabdosia amethystoides

Abstract

The invention discloses a method for separating and extracting (-)-Rabdosiin from rabdosia amethystoides. The isodon amethystoides extract is dissolved in water and then is separated through a D101 macroporous adsorption resin chromatographic column, and H2O eluate is obtained. Separating the H2O eluate of the rabdosia amethystoides through an MCI chromatographic column, and carrying out pH PLC twice, so as to obtain the (-)-Rabdosiin. According to the method for separating and purifying (-)-Rabdosiin from rabdosia amethystoides provided by the invention, the structure of (-)-Rabdosiin in rabdosia amethystoides is accurately identified for the first time, and a foundation is laid for predicting possible pharmacodynamic substances and action mechanisms of the gastric rejuvenation, finding and confirming effective components of the gastric rejuvenation, researching the action mechanisms and establishing a quality control method based on the pharmacodynamic effect.

Description

Technical Field

[0001] The invention belongs to the field of natural drug chemistry, and particularly relates to a method for separating and extracting (-)-Rabdosiin from Camellia ternata. Background Art

[0002] Chinese herbal compound is the essence of traditional Chinese medicine. It is based on the principle of monarch, minister, assistant and envoy, and is the most widely used in clinical Chinese medicine. Wei Fuchun is composed of red ginseng, fragrant tea, and Citrus aurantium (fried). It is mainly used to treat chronic atrophic gastritis, precancerous lesions of gastric cancer and adjuvant treatment after gastric cancer surgery. It has been widely used in clinical practice with high safety and definite efficacy. At present, there are interpretations of the prescription. One theory is that HS is the monarch drug in the Wei Fuchun prescription, which can invigorate qi and strengthen the spleen, and XCC is the minister drug, which can regulate qi, detoxify and activate blood circulation. With ZQ regulating qi, the whole prescription can strengthen the spleen and invigorate qi, activate blood circulation and detoxify; another theory is that the treatment concept of "treatment, nourishment and protection" is the biggest feature of this medicine. Fragrant tea is for treatment, ginseng is for nourishment, and Citrus aurantium is for protection. It can be seen that the functions of each herbal material in the prescription are not yet clear.

[0003] The clarification of the effective material basis of Chinese herbal compound is the premise to ensure its safety, effectiveness and quality control. There is an expert consensus on the efficacy of WFC in treating precancerous lesions of atrophic gastritis, but there is a lack of expert consensus on its effective ingredients, pathways of action, targets and synergistic effects, which requires further in-depth and systematic research. In terms of its effective material basis, at present, studies have identified 178 chemical components in WFC tablets and 86 exposed components in rat serum, but there is a lack of accurate comparison of reference substances, and the characterization and identification of the overall components is not perfect, such as the uncertainty of the structure of high-content compounds such as Rabdosiin; pharmacokinetic studies have also been conducted on ginsenoside Rb1, naringin, ginsenoside Rb2 and oridonin, but the research on its in vivo exposed components is not comprehensive enough, especially the lack of research on the synergistic effects between the effective ingredients and the mechanism-related research, so it is very necessary to systematically and accurately identify its in vivo exposed components.

[0004] There have been relatively many studies on red ginseng and Citrus aurantium in the formula, but the research on Ampelopsis pilosula, especially Ampelopsis pubescens and Ampelopsis cyanocarpus, is not comprehensive and in-depth, and there is a lack of comprehensive characterization of their chemical components. Therefore, systematic research on their chemical components is urgently needed.

[0005] The genus Isodon belongs to the family Lamiaceae, and the whole plant or root is used as medicine. The description of Isodon plants was first recorded in the "Compendium of Materia Medica for Famine Relief". The "Flora of China" records that the whole plant is used as medicine to treat amenorrhea, mastitis, and bruises; the root is used as medicine to treat strain, muscle and bone pain, sores, and snake bites, and is an important medicine for treating snake bites. Modern pharmacological studies have shown that most Isodon plants have the effects of clearing heat and detoxifying, antibacterial and anti-inflammatory, liver protection, anti-hepatitis, and anti-cancer. They are clinically made into medicines for the treatment of hepatitis, gastroenteritis, etc. There are many varieties of Isodon, including Isodon japonica var.glaucocalyx (Maxim.) Hara, Isodon japonicus (N.Burman) H.Hara, Isodon excisus (Maxim.) There are more than 70 varieties of Amethiostoides, including I.amethystoides (Benth.) H.Hara, of which about 30 are currently used as medicinal varieties. Amethiostoides blue-calyx and Amethiostoides hairy-leaf are the main Amethiostoides varieties selected for Wei Fuchun. They have not yet been included in the 2020 edition of the Chinese Pharmacopoeia and are only used as folk Chinese herbal medicines. Amethiostoides blue-calyx and Amethiostoides hairy-leaf are very rich in chemical components, mainly containing diterpenoids, flavonoids, phenylpropanoids and organic acids. At present, most of the research on Amethiostoides focuses on diterpenoids and their pharmacological activities, but its high-content flavonoids, organic acids, and phenylpropanoids also have good pharmacological activities, such as Rabdosiin and its homologues. At present, there is a lack of systematic characterization and component identification of the overall components of Amethiostoides blue-calyx and Amethiostoides hairy-leaf, and there are few studies on the determination of their component content, and there is still uncertainty in the structural identification of some compounds.

[0006] In the previous study, the main chromatographic peaks detected in the herb were rosmarinic acid and (-)-Rabdosiin. However, only (+)-Rabdosiin was reported in the literature related to the herb, and no (-)-Rabdosiin was reported. [1] There are reports on the separation and identification of (-)-Rabdosiin, but there is no reference substance of (-)-Rabdosiin available on the market. The present invention separates and purifies Rabdosiin from the herb of Herba Cibotii, and determines the accurate configuration of several isomers of Rabdosiin by structural identification. Summary of the invention

[0007] The purpose of the present invention is to provide a method for separating and extracting (-)-Rabdosiin from Rhizoma Cibotii in view of the above-mentioned deficiencies in the prior art.

[0008] The purpose of the present invention can be achieved through the following technical solutions:

[0009] A method for separating and extracting (-)-Rabdosiin from Rhizoma Cibotii, comprising the following steps:

[0010] (1) preparing an extract of Herba Cibotii;

[0011] (2) After the extract of the herb was dissolved in water, it was separated by a polystyrene resin macroporous adsorption resin column, and then purified water, H 2 O→40%~60% EtOH→90%~95% EtOH to obtain H 2 O eluate, 40% to 60% EtOH eluate, and 90% to 95% EtOH eluate;

[0012] (3) Herba Cilantro 2 The O eluate was dissolved in water and separated by MCI column. The elution procedure was: H 2 O→8%~12% MeOH→18%~22% MeOH→45%~55% MeOH→100% MeOH, to obtain five components RAB 1, RAB 2, RAB 3, RAB 4 and RAB 5;

[0013] (4) RAB 3 was separated and prepared by pHPLC to obtain 13 components RAB 3-1 to RAB 3-13;

[0014] (5) RAB 3-10 was separated and prepared by pHPLC to obtain (-)-Rabdosiin.

[0015] As a preferred embodiment of the present invention, the method for preparing the extract of Camellia oleifera in step (1) is: heating and refluxing extraction with water or ethanol of different concentrations.

[0016] As a preferred embodiment of the present invention, the model of the polystyrene resin type macroporous adsorption resin in step (2) is D101 macroporous adsorption resin.

[0017] As a preferred embodiment of the present invention, in step (2), pure water H 2 O→50% EtOH→95% EtOH for elution.

[0018] As a preferred embodiment of the present invention, in step (2), H 2 The elution volumes of 50% EtOH, 50% EtOH and 95% EtOH were 6 times, 6 times and 6 times the column volumes respectively.

[0019] As a preferred embodiment of the present invention, the elution procedure of the MCI chromatographic column separation in step (3) is: 2 O→10%MeOH→20%MeOH→50%MeOH→100%MeOH.

[0020] As a preferred embodiment of the present invention, the pHPLC chromatographic conditions in step (4) are: acetonitrile (CH 3 CN)-1% acetic acid (HAc) was used as the mobile phase, the chromatographic column was a Venusil PrepG C18 column (250 mm×20 mm, 5 μm), and the detection wavelength was 254 nm.

[0021] As a preferred embodiment of the present invention, the pHPLC chromatographic conditions in step (5) are: methanol (MeOH)-1% acetic acid (HAc) with a volume ratio of 52:48 as the mobile phase, the chromatographic column is a Cosmosil PBr column (250mm×4.6mm, 5μm), and the retention time is 18.65min.

[0022] As a preferred embodiment of the present invention, the extract of Isodon chinensis is from Isodon calyx and / or Isodon pubescens.

[0023] As a preferred embodiment of the present invention, the extract of Isodon chinensis comes from Isodon cyanobacteria.

[0024] Beneficial effects:

[0025] The present invention provides a method for separating and purifying (-)-Rabdosiin from Rhizoma Cibotii, accurately identifies the structure of (-)-Rabdosiin in Rhizoma Cibotii for the first time, and lays a foundation for predicting the possible pharmacological substances and action mechanisms of Rhizoma Cibotii, and for the discovery, confirmation, action mechanism research of its effective ingredients and the establishment of a quality control method based on pharmacological effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 UPLC-UV-MS (negative ion) identification spectra of main identified compounds and unidentified compounds in Camellia oleifera.

[0027] Figure 2 , 4 The structural formula of the isolated and purified compound

[0028] Figure 3 、(-)-Rabdosiin 1 HNMR images

[0029] Figure 4 、(-)-Rabdosiin 13 C NMR spectra

[0030] Figure 5 、(-)-Rabdosiin 1 H 1H COSY diagram

[0031] Figure 6 、Chromatogram of (-)-Rabdosiin DETAILED DESCRIPTION

[0032] Example 1

[0033] In the previous work, it was found that the main chromatographic peaks in the herb are rosmarinic acid and (-)-Rabdosiin. In the literature related to the herb, only (+)-Rabdosiin was reported, but there was no report on (-)-Rabdosiin. [1] The separation and identification of (-)-Rabdosiin in 1994 was reported, but there is no (-)-Rabdosiin reference substance available for purchase on the market. The compounds were characterized and identified by the purchased reference substances, and the identified and unidentified compounds in 1994 were identified in the UPLC-UV-MS spectrum of 1994, see Figure 1 . Figure 1 The blue ones are compounds that have been compared with reference substances or identified relatively accurately, and have certain absorption under ultraviolet light; the red ones are compounds that have not been identified or have been preliminarily identified without reference substances. In order to more accurately identify the structure of the compounds in citrus aurantium and to quantitatively analyze the main compounds in citrus aurantium, we decided to separate, purify and identify the structure of the main compounds.

[0034] 1.1 Experimental Materials and Instruments

[0035] Thin layer chromatography silica gel pre-plate, high-efficiency silica gel GF254 (Tianjin Silida Technology Co., Ltd.); D101 macroporous adsorption resin (pure grade, Tianjin Haiguang Chemical Co., Ltd.); MCI Gel CHP 20P (Mitsubishi Chemical Corporation, Japan); deuterated reagent (Beijing Chongxi Technology Incubator Co., Ltd.); analytical column and preparative column for HPLC [Cosmosil 5C18-MS-II (250×4.6mm, 5μm) and (250×20mm, 5μm, Nacalai Tesque Co., Ltd., Japan); Cosmosil PBr (250×4.6mm, 5μm) and (250×20mm, 5μm, Nacalai Tesque Co., Ltd., Japan)].

[0036] Superconducting nuclear magnetic resonance spectrometer (Bruker 500MHz, Bruker, Germany); superconducting nuclear magnetic resonance spectrometer (Bruker 600MHz, Bruker, Germany); UHPLC-ESI-Q-OrbitrapMS (UHPLC: Ultimate 3000, MS: Q-Orbitrap, ThermoFisher Scientific, USA); analytical high performance liquid chromatograph [Waters 600E (Waters, USA), detector: Waters 2695 (254nm, 230nm dual wavelength detection); RID-10A, pump: 600Pump, controller: 600controller, online degasser: on-line Degasser AF]; preparative high performance liquid chromatograph [detectors: SPD-20A (254 nm, 230 nm dual wavelength detection), RID-10A, CBM-20A, CTO-20A, LC-8A, Shimadzu Co., Ltd., Japan]; polarimeter (AUTOPOLIV, Rudolf, Switzerland); circular dichroism spectrometer (J-815, JASCO, Japan); ultrasonic cleaner (KQ-250E, Kunshan Ultrasonic Instrument Co., Ltd.); one-hundred-thousandth balance (AX-205, Mettler Toledo, Switzerland); one-ten-thousandth balance (BP121S, Sartorius, Germany); rotary evaporator (IKARV10, IKA, Germany); ultrapure water machine (Advantage A10, Millipore, USA).

[0037] The extract of Ampelopsis pilosula comes from Ampelopsis pilosula in Deqing, Zhejiang.

[0038] 1.2 Experimental methods

[0039] With Herba Cibotii as raw material, add 10 times, 10 times and 10 times the amount of water three times in three times, heat and reflux to extract for 60 minutes, filter and combine the filtrate, concentrate the extract under reduced pressure to obtain an extract, and then freeze-dry to obtain Herba Cibotii extract powder.

[0040] The above-obtained Herba Cibotii extract (800.0 g) was dissolved in water and separated by D101 macroporous adsorption resin column chromatography, followed by H 2 O (6 times the volume), 50% EtOH (6 times the volume), 95% EtOH (6 times the volume) were used for elution, and the eluates were combined and concentrated under reduced pressure to obtain H 2 O eluate (365.4 g), 50% EtOH eluate (194.7 g), and 95% EtOH eluate (18.9 g).

[0041] Take fragrant tea 2The O eluate (150.0 g) was dissolved in water and separated by MCI column chromatography. 2 O (8 times volume), 10% MeOH (4 times volume), 20% MeOH (5 times volume), 50% MeOH (5 times volume), 100% MeOH (6 times volume) for elution, the eluates were combined, concentrated and dried under reduced pressure to obtain 5 components, namely RAB 1 (95.89g), RAB 2 (2.10g), RAB 3 (6.78g), RAB 4 (3.52g) and RAB 5 (5.55g).

[0042] RAB 3 (6.78 g) was prepared by pHPLC separation using acetonitrile (CH 3CN)-1% acetic acid (HAc) was used as the mobile phase, the chromatographic column was a Venusil PrepG C18 column (250 mm×20 mm, 5 μm), the detection wavelength was 254 nm, and 13 components [RAB 3-1 (1.95 g), RAB 3-2 (85.7 mg), RAB 3-3 (58.2 mg), RAB 3-4 (56.7 mg), RAB 3-5 (89.6 mg), RAB 3-6 (81.3 mg), RAB 3-7 (105.4 mg), RAB 3-8 (315.4 mg), RAB 3-9 (172.6 mg), RAB 3-10 (719.2 mg) (17-21 min), RAB 3-11 (113.5 mg), RAB 3-12 (62.3 mg), RAB 3-13 (7.8 mg)] were finally obtained. RAB 3-7 (105.4 mg) was separated and prepared by pHPLC, using methanol (MeOH)-1% acetic acid (HAc) with a volume ratio of 25:75 as the mobile phase, and the chromatographic column was Cosmosil 5C18-MS-II column to obtain (1S, 2S)-globoidnan B (compound 1, 19.2 mg). RAB 3-10 (719.2 mg) was separated and prepared by pHPLC, with a detection wavelength of 254 nm, using methanol (MeOH)-1% acetic acid (HAc) with a volume ratio of 52:48 as the mobile phase, and the chromatographic column was Cosmosil PBr column (250 mm×4.6 mm, 5 μm), and then lyophilized after reduced pressure concentration to obtain (-)-Rabdosiin (compound 3, 434.9 mg, retention time 18.65 min). RAB 3-11 (62.3 mg) was prepared by pHPLC separation using methanol (MeOH)-1% acetic acid (HAc) in a volume ratio of 55:45 as the mobile phase and a Cosmosil PBr column (250 mm×4.6 mm, 5 μm) to obtain (1S,2S)-Rabdosiin (compound 2, 21.6 mg) and Shimobashiric acid C (compound 4, 16.1 mg).

[0043] 1.3 Experimental Results

[0044] Compound 1:

[0045] White powder. High-resolution ESI-Q-Orbitrap MS gave its quasi-molecular ion peak m / z 537.10370 [MH] - (calcd for C 27 H 21 O 12 , 537.10275), the molecular formula is determined to be C 27 H22 O 12 .

[0046] CD (c = 0.001M, MeOH) mdeg (λ nm ): -1.27(215), -1.06(231), +2.33

[0047] (255), -0.89(291), +0.37(316), -1.79(353).

[0048] 1 H NMR (D 2 O, 500MHz) δ: [2.82 (1H, dd, J = 7.0, 13.5Hz), 2.91 (1H, dd, J = 5.0, 13.5Hz), H2-7”], 3.70 (1H, d, J = 3 .0Hz, H-2), 4.29 (1H, d, J = 3.0Hz, H-1), 5.00 (1H, dd, J = 5.0, 7.0Hz, H-8”), 6.28 (1H, br.d, ca.J = 8Hz , H-6'), 6.46 (1H, br.d, ca.J=8Hz, H-6”), 6.50 (1H, br.s, H-2’), 6.55 (1H, s, H-8), 6.56 (1H, d, J=8. 0Hz, H-5'), 6.65 (1H, d, J=8.0Hz, H-5"), 6.69 (1H, br.s, H-2"), 6.81 (1H, s, H-5), 7.53 (1H, s, H-4).

[0049] 13 C NMR (D 2 O, 125MHz) data are shown in Table 1.

[0050] No NOE correlation was observed between δ3.70 (H-2) and 6.28 (H-2') and 6.55 (H-6') in its NOESY spectrum, so it was determined that H-1 and H-2 were cis-isomers.

[0051] 1 H. 13 C NMR, [α] D 25 and CD spectrum data and literature [2,3] By comparison, the structure of the compound was identified as (1S,2S)-globoidnan B.

[0052] Compound 2:

[0053] White powder. High-resolution ESI-Q-Orbitrap MS gave its quasi-molecular ion peak m / z 717.14624 [MH]- (calcd for C 36 H 29 O 16 , 717.14501), the molecular formula is determined to be C 36 H 30 O 16 .

[0054] CD (c = 0.001M, MeOH) mdeg (λ nm ): +9.53(212), -0.57(234), +3.28

[0055] (255), -0.72(290), +0.68(314), -2.21(352).

[0056] 1 HNMR (D 2 O, 500MHz) δ: 2.71, 2.93 (1H each, both br.d, ca.J=10Hz, H 2 -7"), 2.73, 2.97 (1H each, bothbr.d, ca.J=10Hz, H 2 -7"'), 3.78(1H, br.s, H-2), 4.29(1H, br.s, H-1), 4.99(2H, m, overlapped, H-8", 8"'), 7.47(1 H, s, H-4), 6.80 (1H, s, H-5), 6.20 (1H, br.d, ca.J=8Hz, H-6'), 6.29 (1H, br.d, ca.J=8Hz, H-6”) , 6.40 (1H, br.d, ca.J=8Hz, H-6"'), 6.38 (1H, br.s, H-2'), 6.52 (1H, s, H-8), 6.57 (1H, m, overl apped, H-5'), 6.58 (2H, d, J=8.0Hz, H-5", 5"'), 6.61 (1H, br.s, H-2"), 6.62 (1H, br.s, H-2"').

[0057] 13 C NMR (D 2 O, 125MHz) data are shown in Table 1.

[0058] No NOE correlation was observed between δ3.78 (H-2) and δ6.20 (H-6') and 6.38 (H-2') in its NOESY spectrum, so it was determined that H-1 and H-2 were cis-isomers.

[0059] Its CD spectrum data and literature [2]By comparison, the structure of the compound was identified as (1S,2S)-Rabdosiin, and its NMR data was reported for the first time.

[0060] Compound 3:

[0061] White powder. High-resolution ESI-Q-Orbitrap MS gave its quasi-molecular ion peak m / z 717.14630 [MH] - (calcd for C 36 H 29 O 16 , 717.14501), the molecular formula is determined to be C 36 H 30 O 16 .

[0062] CD (c = 0.001M, MeOH) mdeg (λ nm ): +9.53(212), -0.57(234), +3.28

[0063] (255), -0.72(290), +0.68(314), -2.21(352).

[0064] 1 H NMR (D 2 O, 500MHz) δ: [2.66 (1H, dd, J=9.0, 14.0Hz), 2.88 (1H, br.d, ca.J=14Hz), H2-7”], [2.72 (1H, dd, J=9.5, 13.5Hz), 2.93 (1H, dd, J=3. 0, 13.5Hz), H2-7"'], 3.78 (1H, br.s, H-2), 4.29 (1H, br.s, H-1), 4.99 (2H, m, overlapped, H-8", 8"'), 7.47 (1H, s, H-4), 6.80 (1H, s , H-5), 6.20 (1H, br.d, ca.J=8Hz, H-6'), 6.29 (1H, br.d, ca.J=8Hz, H-6"), 6.40 (1H, br.d, ca.J=8Hz, H-6"'), 6.38 (1H, br.s, H-2') , 6.52 (1H, s, H-8), 6.57 (1H, m, overlapped, H-5'), 6.58 (2H, d, J=8.0Hz, H-5", 5"'), 6.61 (1H, br.s, H-2"), 6.62 (1H, br.s, H-2"').

[0065] 13 C NMR (D 2O, 125MHz) data are shown in Table 1.

[0066] No NOE correlation was observed between δ3.78 (H-2) and δ6.20 (H-6') and 6.38 (H-2') in its NOESY spectrum, so it was determined that H-1 and H-2 were cis-isomers.

[0067] Its CD spectrum data and literature [3] By comparison, the structure of the compound was identified as (-)-Rabdosiin, and its NMR data was reported for the first time. Figure 3-Figure 5 .

[0068] Compound 4:

[0069] White powder. High-resolution ESI-Q-Orbitrap MS gave its quasi-molecular ion peak m / z 719.16199[MH] - (calcd for C 36 H 31 O 16 , 719.16066), the molecular formula is determined to be C 36 H 32 O 16 .

[0070] CD (c = 0.001M, MeOH) mdeg (λ nm ): +2.56(203), -2.37(219), +0.04

[0071] (255).

[0072] 1 HNMR (D 2 O, 500MHz) δ: 6.72 (1H, br.s, H-2), 6.63 (1H, m, overlapped, H-5), 6.55 (1H, br.d, ca.J=8Hz, H-6), [2.63 (1H, dd, J=4.5, 14.5Hz), 2.68 (1H, dd, J=7.5, 14.5Hz), H 2 -7], 4.54 (1H, dd, J=4.5, 7.5Hz, H-8), 6.63 (1H, m, overlapped, H-2'), 6.69 (1H, m, overlapped, H-5'), 6.42 (1H, br.d, ca.J=8.0Hz, H-6'), 4.12 (1H, t, J=9.0Hz, H 2-7'), 3.81 (1H, t, J = 9.0Hz, H-8'), 6.65 (1H, m, overlapped, H-2"), 6.70 (1H, m, overlapped, H-5"), 6.43 (1H, br.d, ca.J = 8.0Hz, H-6"), 4.10 (1H, t, J = 9.0Hz, H 2 -7"), 3.70 (1H, t, J=9.0Hz, H-8"), 6.72 (1H, br.s, H-2"'), 6.61 (1H, d, J=8.0Hz, H-5"'), 6.55 (1H, br.d, ca.J=8.0Hz, H-6"'), [2.74 (1H, dd, J=9.0, 14.0Hz), 2.86 (1H, dd, J=3.5, 14.0Hz), H 2 -7"'], 4.61 (1H, dd, J=3.5, 9.0Hz, H-8"').

[0073] 13 C NMR (D 2 O, 125MHz) data are shown in Table 1.

[0074] That 1 H. 13 C. 2D NMR and CD spectral data and literature [4] By comparison, the structure of the compound was identified as Shimobashiric acid C.

[0075] Table 1 Compounds 1-4 13 C-NMR data (in D 2 O)

[0076]

[0077]

[0078] According to the above method, (-)-Rabdosiin can also be separated and purified from the hairy-leaved safflower.

[0079] References

[0080] [1]Zhaolai Hua,Rui Shen,Bin Lu,et al.Weifuchun alters tongue floraand decreases serum trefoil factor I levels in gastric intestinal metaplasia:A CONSORT-compliant article[J].Medicine,2022,101(45):e31407.

[0081] [2] Xu Fengming, Hu Huiping, Wang Zhaoquan. Study on diterpenoid components of Ipomoea alopecuroides[J]. China Journal of Traditional Chinese Medicine, 1996, (11): 38-39+64.

[0082] [3] Ding Mingming. Study on chemical components of Rhizoma Atractylodis Macrocephalae and Rhizoma Atractylodis Macrocephalae[D]. Xinxiang Medical College, 2013.

[0083] [4] Di Xuemei, Yan Fulin. Study on chemical components of Isodon pubescens[J]. Anhui Medicine, 2013, 17(09): 1470-1472.

Claims

1. A method for separating and extracting (-)-Rabdosiin from Rhizoma Cibotii, characterized in that: The following steps are involved: (1) preparing an extract of Herba Cibotii; (2) After the extract of the herb was dissolved in water, it was separated by a polystyrene resin type macroporous adsorption resin column, and eluted in sequence with pure water H2O→40%-60% EtOH→90%-95% EtOH to obtain a H2O eluate, a 40%-60% EtOH eluate, and a 90%-95% EtOH eluate; (3) The H2O eluate of R. odoratissimum was dissolved in water and separated by MCI column. The elution procedure was: H2O→8%-12% MeOH→18%-22% MeOH→45%-55% MeOH→100% MeOH, and five components RAB 1, RAB 2, RAB 3, RAB 4 and RAB 5 were obtained. (4) RAB 3 was separated and prepared by pHPLC to obtain 13 components RAB 3-1 to RAB 3-13; (5) RAB 3-10 was separated and prepared by pHPLC to obtain (-)-Rabdosiin.

2. The method according to claim 1, characterized in that The method for preparing the extract of Camellia oleifera in step (1) is: heating and refluxing extraction with water or ethanol of different concentrations.

3. The method according to claim 1, characterized in that The model of the polystyrene resin type macroporous adsorption resin described in step (2) is D101 macroporous adsorption resin.

4. The method according to claim 1, characterized in that In step (2), pure water H2O→50% EtOH→95% EtOH are used for elution in sequence.

5. The method according to claim 4, characterized in that In step (2), the elution volumes of H2O, 50% EtOH and 95% EtOH are 6 times, 6 times and 6 times the column volume respectively.

6. The method according to claim 1, characterized in that Step (3) The elution procedure of the MCI column separation is: H2O→10% MeOH→20% MeOH→50% MeOH→100% MeOH.

7. The method according to claim 1, characterized in that The pHPLC chromatographic conditions in step (4) are: acetonitrile (CH3CN)-1% acetic acid (HAc) in a volume ratio of 18:82 as the mobile phase, the chromatographic column is a Venusil PrepG C18 column (250mm×20mm, 5μm), and the detection wavelength is 254nm; the retention time of RAB 3-10 is 17-21min.

8. The method according to claim 1, characterized in that The pHPLC chromatographic conditions in step (5) are: methanol (MeOH)-1% acetic acid (HAc) in a volume ratio of 52:48 as the mobile phase, the chromatographic column is a Cosmosil PBr column (250 mm×4.6 mm, 5 μm), and the retention time is 18.65 min.

9. The method according to any one of claims 1 to 8, characterized in that: The extract of Isodon chinensis is from Isodon calyx and / or Isodon pubescens.

10. The method according to claim 9, characterized in that Isodon oleifera extract comes from Isodon oleifera.

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