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Isolation of Flavonoids from Piper delineatum Leaves by Chromatographic Techniques
采用色谱技术从胡椒属叶分离类黄酮   

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Abstract

The genus Piper (Piperaceae) is widely distributed in the tropical and subtropical regions of the world, and species belonging to this genus are included in the Ayurvedic system of medicine and in folklore medicine of Latin America. Phytochemical investigations of Piper species have led to the isolation of several classes of physiologically active compounds such as alkaloids, amides, pyrones, dihydrochalcones, flavonoids, phenylpropanoids, lignans and neolignans. In an ongoing investigation of bioactive secondary metabolites from Piper species, herein, we describe the isolation procedure of nine flavonoids, including two chalcones and two flavanones from the leaves of Piper delineatum Trel. (Piperaceae), a shrub native to tropical regions of the Americas. All compounds were elucidated by spectroscopic and spectrometric methods, and comparison with data reported in the literature.

Keywords: Piper(派珀), Flavonoids(黄酮类化合物), Isolation(隔离), Chromatography(色谱法)

Materials and Reagents

  1. Silica gel 60 (particle size 63-200 mm) (Macherey-Nagel, catalog number: 815330 )
  2. Silica gel 60 (particle size 15-40 mm) (Macherey-Nagel, catalog number: 815650 )
  3. TLC silica gel 60 F254 plates (Merck Millipore Corporation, catalog number: 105735 )
  4. SIL G/UV254 (20 x 20 cm), Macherey-Nagel, catalog number: 805023 )
  5. Pre-coated TLC-plates UV254 20 x 20 cm (Macherey-Nagel, catalog number: 821030 )
  6. Round-bottom tubes (40 ml, 200 x 20 mm) (Simax, Alamo, catalog number: 00684800 )
  7. Round-bottom tubes (8 ml, 120 x 12 mm) (Simax, Alamo, catalog number: 00684400 )
  8. Microcapillary tube 1-5 μl (Sigma-Aldrich, catalog number: P0549-1PAK )
  9. NMR simple tubes 5 mm (Wilmad® LabGlass, catalog number: 528-PP-7 )
  10. Leaves of the plant species (Piper delineatum) at the mature stage were collected in Iquitos, Maynas Province, Department of Loreto, Perú in November 2009. A voucher specimen (10484) was identified by botanist Juan Ruiz Macedo and was deposited at the Amazonense Herbarium of Universidad Nacional de la Amazonia Peruana, Iquitos, Perú.
  11. Ethanol absolute (Panreac, catalog number: 141086 )
  12. Dichloromethane (CH2Cl) (Panreac, catalog number: 131254 )
  13. Ethyl acetate (Panreac, catalog number: 141318.1212 )
  14. Hexanes (Panreac, catalog number: 121347.1612 )
  15. Chloroform (Panreac, catalog number: 161252 )
  16. Diethyl ether (Panreac, catalog number: 142770.0311 )
  17. Acetone (Panreac, catalog number: 131007.1212 )
  18. Methanol (Panreac, catalog number: 141091.1211 )
  19. Isopropanol (Panreac, catalog number: 131090 )
  20. Acetone deuterated [(CD3)2CO] (Sigma-Aldrich, catalog number: 151793-25G )
  21. Magnesium sulfate anhydrous (Panreac, catalog number: 212486 )
  22. Sulfuric acid (Panreac, catalog number: 141058 )
  23. Acetic acid glacial (Panreac, catalog number: 141008 )
  24. Sea sand washed thin grain QP (Panreac, catalog number: 211160.0416 )
  25. Sephadex LH-20 (Sigma-Aldrich, catalog number: LH20100 )
  26. Oleum: Acetic acid-water-sulfuric acid (20:4:1) (see Recipes)
  27. Coating rotor with sorbent for the CPTLC (4 mm thickness) (see Recipes)

Equipment

  1. Rotary cutter mill (Mateu and Sole, S.L., Constructores, MATSO, model: B-2, nº 258 )
  2. Soxhlet extraction apparatus (ANORSA, catalog number: 322060 )
  3. Rotary R-210 evaporator (Sigma- Aldrich, Büchi® Rotavapor®, catalog number: Z565466 )
  4. Separating funnel, 2 L (Sigma-Aldrich, catalog number: Z330663 )
  5. Erlenmeyer flaks (100 ml and 500 ml) (Duran, catalog numbers: Z232793-1EA and Z232831-1EA , respectively)
  6. Teflon funnel holder
  7. Solvent-pouring funnel (stem L, O.D. 0.65 x 22 mm) (Sigma-Aldrich, catalog number: 548804 )
  8. Evaporating flask (pear-shaped, 1L) (Sigma-Aldrich, catalog number: Z402990 )
  9. Round-bottom flask (5 L) (Sigma-Aldrich, catalog number: Z302872 )
  10. Glass Pasteur pipets 225 mm (BRAND, catalog number: 747720 )
  11. Pasteur pipette rubber bulbs, 2 ml (Sigma-Aldrich, catalog number: Z111597-12EA )
  12. Heating bath (Sigma-Aldrich, catalog number: Z563544 )
  13. Heating mantle 5 L (Selecta, catalog number: 3031450 )
  14. Hot plate (Selecta, catalog number: 1000443 )
  15. Liebig Condenser (Sigma-Aldrich, catalog number: Z531006 )
  16. Glass chromatography column (CC) for silica gel (50 x 9 cm) (Fisher Scientific, catalog number: 12058880 )
  17. Glass chromatography column (CC) for Sephadex (60 x 4.5 cm) (Fisher Scientific, catalog number: 12011550 ).
  18. Centrifugal preparative thin layer chromatography system (CPTLC) (Chromatotron, Harrison Research Inc., model: 7924T )
  19. Silica gel PF254 disks (Merck Millipore Corporation, catalog number: 107749 2500 )
  20. TLC Chamber rectangular for TLC plates (7.5 x 15.5 x 8.0 cm) (Sigma-Aldrich, catalog number: Z204226 )
  21. TLC Chamber rectangular for PTLC plates (27.0 x 26.5 x 7.0 cm) (Sigma-Aldrich, catalog number: Z126195-1EA )
  22. Glass atomizer reagent sprayer (125 ml) (Sigma-Aldrich, catalog number: Z529737-1EA )
  23. Glass vacuum filter (Fisher Scientific, catalog number: 11979659 )
  24. Micro-spatula (8 in) (Sigma-Aldrich, catalog number: Z513342-1PAK )
  25. Analytical balance, semi-micro balance (Fisher Scientific, Mettler ToledoTM, catalog number: 11142062 ; model: NewClassic MS105DU)
  26. Nuclear Magnetic Resonance (NMR) Spectrometers (Bruker, model: Bruker Avance 400 ; Bruker Avance 500 ).
  27. Optical rotations polarimeter (CHCl3 at 25 °C) (PerkinElmer, model: 241 automatic polarimeter )
  28. Ultraviolet (UV) spectrophotometer (JASCO, model: V-560 )
  29. Infrared (IR) spectrophotometer (Bruker, model: IFS 55 , IFS 28/55 )
  30. High resolution electron impact (HREI) mass spectrometer (MS) (MasSpec, model: Micromass VG Autospec magnetic sector , M series)

Procedure

Note: The whole procedure described below takes around 6 months to complete.

  1. Finely grind the air-dried leaves of Piper delineatum (252.2 g) into small pieces using a rotary cutter mill, and extract with ethanol (4 L) in a Soxhlet apparatus (Figure 1) for 24 h at around 80 °C.


    Figure 1. Plant material and extraction procedure. A. Piper spp. leaves. B. Soxhlet apparatus used for the extraction of the plant material.

  2. Evaporate the solvent under reduced pressure in a rotary evaporator (at ≤ 50 °C, around 1.30 h) provided 57.3 g (22.7%) of crude extract.
    1. Re-suspend the crude extract in 750 ml of water and slowly pour into a separating funnel (2 L) placed onto a metallic stand (Figure 2).
    2. Add 750 ml of CH2Cl2 to the separating funnel, and hand-shake gently several times. Then, allow the solvent to settle into immiscible phases and recover both phases, the organic phase (bottom layer), and the aqueous phase (top layer) into separate Erlenmeyer flasks.
    3. Place the aqueous phase inside the separating funnel and add 750 ml of CH2Cl2 and repeat the operation twice.
    4. Combine the organic phases obtained in the three extractions, and add magnesium sulfate anhydrous (10 g) to remove the residual water. Swirl the solution. If the drying agent is clumped together, add more until it looks free-flowing. Filter, through a filter funnel, the total organic phase and collect the filtrate into a 1 L evaporating flask (pear-shaped).


    Figure 2. Liquid-liquid partition of the crude extract using a separating funnel

  3. Afterwards, the organic phase is taken to dryness by removal of the CH2Cl2 under vacuum in a rotary evaporator (at ≤ 40 °C) (Figure 3), yielding 30 g (11.9%) of organic fraction (dark green solid).


    Figure 3. Rotary evaporator to remove solvent

  4. Pack a column chromatography (CC) (50 x 9 cm) by placing 1.5 cm layer of fine sand and then silica gel (particle size 63-200 mm, 430 g) to a depth of 20 cm, next cover with another 1.5 cm layer of sand to protect the adsorbent surface. Allow an appropriate volume of hexanes (4 x 1 L) to pass through the CC to encourage bubbles to rise and the silica gel to settle so that it packs tightly into the column.
    1. Dissolve the organic fraction in 40 ml CH2Cl2, mix with silica gel until it forms a slurry, and remove the solvent under the rotary evaporator until a dry powder is obtained.
    2. Spread the dried powder extract, slowly and uniformly, on the upper top of the packed column (Figure 4), and carry out the elution by gradient, using mixtures of hexanes/EtOAc of increasing polarity as eluent (10:0 to 0:10, starting with 100% hexanes, followed by hexanes/EtOAc mixtures 8:2, 6:4, 4:6, 2:8, 1:9 and finishing with 100% ethyl acetate, 1 L each solvent mixture).
    3. Collect the resulting fractions (15 fractions, 400 ml each) in round-bottomed flasks (1 L).
    4. Subsequently, remove the mobile phase from each fraction in a rotary evaporator (at ≤ 4 °C), and dissolve the residue in 20 ml CH2Cl2 and transfer to round-bottomed tubes (40 ml).


    Figure 4. Column chromatography (CC)

  5. Combine these 15 fractions into 10 subfractions (A-J) based on their similarities TLC profiles (Figure 5).
    1. To run the TLC plates, add the solvent mixtures of hexanes/ethyl acetate of increasing polarity, starting from 8:2 (8 ml hexanes plus 2 ml ethyl acetate) and finishing with 6:4, into the TLC chamber to a depth of just under 0.5 cm.
    2. Apply an aliquot of each fraction, using a microcapillary, to the baseline (marked about 0.7 cm from one end of the plate) of the TLC (around 7 x 5 cm for TLC) and place into the TLC chamber.
    3. Develop the TLC, leaving the solvent to rise up the TLC plate by capillary action, until the solvent reaches almost the top (0.5 cm of the plate).
    4. Then, visualize the developed TLC under a UV lamp using 254 nm wavelengths, and stained with oleum system and heat the plates to 80-100 °C in a hotplate until spots appear.


    Figure 5. Collected fractions from CC and analysis by TLC. A. Collected fractions in round-bottom tubes from the column chromatography showed in Figure 4. B. Visualization of thin layer chromatography (TLC) developed plates of the collected fractions that are further combined into 10 subfractions based on their TLC profiles.

  6. 1H NMR analysis reveals that fractions C (hexanes/EtOAc, 8:2), H (hexanes/EtOAc, 3:7) and J (EtOAc) are rich in flavonoids. The solvent used for the NMR experiments is deuterated acetone, (CD3)2CO.
    Note: To prepare the NMR sample: dissolve the sample in 0.6 ml of (CD3)2CO, filter through Pasteur pipette, equipped with a filter paper, and discharge into the NMR tube.
  7. Chromatograph fraction C (2.2 g) on a column chromatography (60 x 4.5 cm) packed with Sephadex LH-20 (hexanes/CHCl3/MeOH, 2:1:1 as eluent). Dissolve fraction C in the minimum volume of eluent (hexanes/CHCl3/MeOH, 2:1:1), and load to the top of the column. Develop the column using 1 L of eluent, and collect fractions of 30 ml in round-bottom tubes (40 ml), and combine by TLC profile into five fractions (C1-C5).
    Notes:
    1. Before loading the sample for column chromatography on Sephadex, pass 1.5 L of a mixture of mobile phase (hexanes/CHCl3/MeOH, 2:1:1) through the column to stabilize the Sephadex LH-20.
    2. Before loading the sample on Sephadex column chromatography, filter the sample through filter paper.
  8. Chromatograph fraction C3 (328.8 mg) by CPTLC, a preparative centrifugally accelerated radial thin-layer chromatograph (Figure 6).
    1. Apply the sample dissolved in a small volume of CH2Cl2 (3 ml), near the center of a spinning disk coated with a thin layer of sorbent (coating rotor, 4 mm thickness).
    2. The mobile phase is supplied by a teflon funnel holder about 55 cm above bench level, and the solvent flows down onto the circular plate through a capillary tube in the funnel. Elution by mixtures of hexanes/diethyl ether (8:2, 7:3, 6:4 and 1:1, 100 ml each solvent mixture) forms circular bands of the separated components which are spun off from the edge of the rotor together with solvent. 
    3. Collect 48 fractions into round-bottom tubes (8 ml).
    4. Combine fractions based on their similarities in TLC profiles, into eleven fractions (C3A-C3K).


    Figure 6. Centrifugal preparative thin layer chromatography (CPTLC, Chromatotron)

  9. Dissolve subfractions C3E (16.4 mg) and C3H (17.7 mg) in Cl2CH2 (2 ml), and further purify by PTLC (plate 20 x 20 cm, 2.5 mm SiO2 thickness) (Figure 7, steps A-E). 
    1. Load the sample as a band, as narrow as possible, at the baseline of the plate (marked about 2 cm from one end of the plate), with a Pasteur pipette (step A).
    2. Dry the plate, place in the chamber with the selected eluent (hexanes/EtOAc, 8:2 for subfraction C3E, and hexanes/CH2Cl2, 8:2 for subfraction C3H, 100 ml of eluent) and develop it (step B).
    3. Remove the plate (step C) from the chamber and visualize under UV light marking with a pencil around the band corresponding to the compound.
    4. Use a microspatula to scrape the marked band, containing the product, off the plate onto a lengthwise folder piece of clean white paper (step D).
      Note: It is very important to deposit a thin line of sample slowly and uniformly, without touching the pipet too much against the silica as this will scrape it. If the desired band is not separated enough, repeat runs with another elution.
    5. Place the scraping into a glass vacuum filter (packed with around 2 cm silica gel) and flush with EtOAc (4 x 5 ml) into a round-bottomed flask (step E). Remove the solvent under reduced pressure in a rotary evaporator.
    6. Afterwards, dissolve the sample in (CD3)2CO (0.5 ml) and with a Pasteur pipette transfer to a NMR tube to perform the NMR experiments. In this way, the following compounds can be isolated and purified: 2'-hydroxy-3,4',6'-thoxychalchone (compound I, 9.4 mg, Rf 0.42) (Boumendjel et al., 2008) from subfraction C3E, and compounds 2',4'-dihydroxy-3,6'-dimethoxychalchone (compound II, 3.6 mg, Rf 0.38) (Aponte et al., 2010) and 2', 4'-dihydroxy-6'-methoxychalchone (compound III, 8.3 mg, Rf 0.36) from subfraction C3H.
  10. Chromatograph fraction H (5.4 g) on Sephadex LH-20 (CHCl3/MeOH, 1:1, 500 ml) to afford fractions H1-H9, and collect them in round-bottom flasks (10 ml each), following the procedure described in step 7.
  11. Chromatograph fraction H6 (304.8 mg) by CPTLC (CH2Cl2/acetone of increasing polarity, 10:0 to 1:1, starting from 100% CH2Cl2 and finishing with a mixture of CH2Cl2/acetone 50%, 200 ml) to afford nine fractions (H6A-H6I), and collect in round-bottom flasks (10 ml each), using the same procedure as described in step 8. 
     

    Figure 7. Steps by step guide of preparative thin layer chromatography (PTLC) procedure. A. Load the sample as a band at the plate. B. Place the PTLC in the chamber and develop the plate. C. Remove the plate and visualize it under UV light marking the band corresponding to the compound. D. Scrape the marked band. E. Place the scraping into a glass vacuum filter and flush with a solvent.

  12. Further purify subfraction H6F (18.2 mg) by PTLC (hexanes/EtOAc, 3:2), following the same procedure described in step 9, and compounds 2',4'-dihydroxy-3,3',6'-trimethoxychalcone (compound IV, 4.7 mg, Rf 0.36) and 2',3'-dihydroxy-4',6'-dimethoxychalchone (compound V, 9.3 mg, Rf 0.40) (Chantrapromma et al., 2000) can be obtained.
  13. Chromatograph fraction H7 (593.0 mg) by CPTLC (CH2Cl2/acetone, 10:0 to 1:1) to give fractions H7A-H7G, following the same procedure as described in step 8.
  14. Subsequently, further purify subfraction H7F (23.1 mg) by PTLC (eluent: hexanes/isopropanol, 4:1) to yield compounds 2',3',5-trihydroxy-4',6',3-trimethoxychalcone (compound VI, 15.3 mg, Rf 0.47) and 2',4',4-trihydroxy-3,6'-dimethoxychalchone (compound VII, 3.8 mg, Rf 0.42) (Vogel et al., 2010,), following the same procedure described in step 9.
  15. Chromatograph fraction I (3.9 g) on Sephadex LH-20 (CHCl3/MeOH, 1:1) to afford fractions I1-I5. Further chromatograph fraction I3 (628.8 mg) by CPTLC (CH2Cl2/acetone, 10:0 to 7:3) to afford fractions I3A-I3L, following the same procedure described in step 8.
  16. Further purify subfractions I3F (16.4 mg) and I3H (21.2 mg) by PTLC (CH2Cl2/acetone, 9:1 and hexanes/isopropanol, 8:2, respectively) to afford compounds (-)-(2S)-8-hydroxy-5,7,3'-trimethoxyflavanone (compound VIII, 11.4 mg, Rf 0.44) and (-)-(2S)-7, 5'-dihydroxy-5,3'-dimethoxyflavanone (compound IX, 10.4 mg, Rf 0.39), respectively, following the same procedure described in step 9.
  17. In summary, the EtOH extract of leaves of P. delineatum was partitioned into a CH2Cl2/H2O solution. The CH2Cl2 fraction was subjected to multiple chromatographic steps to yield 9 flavonoids, whose structures were deduced by NMR and MS analysis (Figure 8).


    Figure 8. Flowchart for chromatographic steps to obtain flavonoids from leaves of Piper delineatum

Recipes

  1. Oleum: Acetic acid-water-sulfuric acid (20:4:1)
    Mix 4 ml of sulfuric acid with 16 ml of water into an Erlenmeyer (200 ml), and slowly add acetic acid glacial (80 ml). Mix well and place in a glass atomizer sprayer.
  2. Coating rotor with sorbent for the CPTLC (4 mm thickness)
    Disk coated with a thin layer of silica gel (60 PF 254) TLC standard grade (75 g) with calcium sulfate hemihydrate (30 g, CaSO4, ½ H2O) sorbent and water (187 ml).

Acknowledgments

This protocol was adapted from previously published studies, Martín-Rodríguez et al. (2015). This work was supported by the European EU, FP7-REGPOT-2012-CT2012-316137-IMBRAIN project.

References

  1. Aponte, J. C., Castillo, D., Estevez, Y., Gonzalez, G., Arevalo, J., Hammond, G. B. and Sauvain, M. (2010). In vitro and in vivo anti-Leishmania activity of polysubstituted synthetic chalcones. Bioorg Med Chem Lett 20(1): 100-103.
  2. Boumendjel, A., Boccard, J., Carrupt, P. A., Nicolle, E., Blanc, M., Geze, A., Choisnard, L., Wouessidjewe, D., Matera, E. L. and Dumontet, C. (2008). Antimitotic and antiproliferative activities of chalcones: forward structure-activity relationship. J Med Chem 51(7): 2307-2310.
  3. Chantrapromma, K., Rat, A. p. Y., Karalai, C., Lojanapiwatana, V. and Seechamnanturakit, V. (2000). A chalcone and a dihydrochalcone from Uvaria dulcis. Phytochemistry 53(4): 511-513.
  4. Harwood, L. M. and Moody, C. J. (1994). Experimental organic chemistry: principles and practice. Blackwell Science.
  5. Martín-Rodríguez, A. J., Ticona, J. C., Jiménez, I. A., Flores, N., Fernández, J. J. and Bazzocchi, I. L. (2015). Flavonoids from Piper delineatum modulate quorum-sensing-regulated phenotypes in Vibrio harveyi. Phytochemistry 117: 98-106.
  6. Vogel, S., Barbic, M., Jurgenliemk, G. and Heilmann, J. (2010). Synthesis, cytotoxicity, anti-oxidative and anti-inflammatory activity of chalcones and influence of A-ring modifications on the pharmacological effect. Eur J Med Chem 45(6): 2206-2213.

简介

蓟属(Piperaceae)广泛分布在世界的热带和亚热带地区,属于该属的种属包括在医学的阿育吠陀系统和拉丁美洲的民间传说医学中。 对Piper物质的植物化学研究已导致分离几类生理活性化合物,例如生物碱,酰胺,吡喃酮,二氢查耳酮,类黄酮,苯丙素类,木酚素和新木脂体。 在对来自Piper种的生物活性次级代谢物的进行中的研究中,我们描述了九种类黄酮的分离程序,包括来自叶脉的两种查耳酮和两种黄烷酮 。 (Pi科),灌木本地对美洲的热带地区。 所有化合物通过光谱和光谱测定法进行阐明,并与文献中报道的数据进行比较。

关键字:派珀, 黄酮类化合物, 隔离, 色谱法

材料和试剂

  1. 硅胶60(粒径63-200mm)(Macherey-Nagel,目录号:815330)
  2. 硅胶60(粒径15-40mm)(Macherey-Nagel,目录号:815650)
  3. TLC硅胶60F 254板(Merck Millipore Corporation,目录号:105735)
  4. SIL G/UV 254(20×20cm),Macherey-Nagel,目录号:805023)
  5. 预涂覆的TLC板UV sub 254×20×20cm(Macherey-Nagel,目录号:821030)
  6. 圆底管(40ml,200×20mm)(Simax,Alamo,目录号:00684800)
  7. 圆底管(8ml,120×12mm)(Simax,Alamo,目录号:00684400)
  8. 微毛细管1-5μl(Sigma-Aldrich,目录号:P0549-1PAK)
  9. NMR简单管5mm(Wilmad LabGlass,目录号:528-PP-7)
  10. 在成熟阶段的植物物种的叶子(Emperium delicatum)在2009年11月在Pero的Loreto的Maynas省的Iquitos收集。植物学家Juan Ruiz Macedo鉴定了凭证样本(10484)并保藏在亚利桑那州立大学的亚马逊植物标本室,秘鲁的伊基托斯。
  11. 乙醇绝对(Panreac,目录号:141086)
  12. 二氯甲烷(CH 2 Cl)(Panreac,目录号:131254)
  13. 乙酸乙酯(Panreac,目录号:141318.1212)
  14. 己烷(Panreac,目录号:121347.1612)
  15. 氯仿(Panreac,目录号:161252)
  16. 二乙醚(Panreac,目录号:142770.0311)
  17. 丙酮(Panreac,目录号:131007.1212)
  18. 甲醇(Panreac,目录号:141091.1211)
  19. 异丙醇(Panreac,目录号:131090)
  20. 丙酮氘代[(CD 3)2 CO](Sigma-Aldrich,目录号:151793-25G)
  21. 无水硫酸镁(Panreac,目录号:212486)
  22. 硫酸(Panreac,目录号:141058)
  23. 乙酸冰(Panreac,目录号:141008)
  24. 海砂洗细谷物QP(Panreac,目录号:211160.0416)
  25. Sephadex LH-20(Sigma-Aldrich,目录号:LH20100)
  26. 油酸:乙酸 - 水 - 硫酸(20:4:1)(参见配方)
  27. 用于CPTLC(4 mm厚)的吸附剂涂层转子(参见配方)

设备

  1. 旋转切割机(Mateu和Sole,S.L.,Constructores,MATSO,型号:B-2,n°258)
  2. 索氏萃取装置(ANORSA,目录号:322060)
  3. 旋转R-210蒸发器(Sigma-Aldrich,Büchi Rotavapor ,目录号:Z565466)
  4. 分离漏斗,2L(Sigma-Aldrich,目录号:Z330663)
  5. 锥形瓶(100ml和500ml)(Duran,目录号分别为Z232793-1EA和Z232831-1EA)
  6. 特氟龙漏斗支架
  7. 溶剂倒入漏斗(杆L,O.D. 0.65×22mm)(Sigma-Aldrich,目录号:548804)
  8. 蒸发烧瓶(梨形,1L)(Sigma-Aldrich,目录号:Z402990)
  9. 圆底烧瓶(5L)(Sigma-Aldrich,目录号:Z302872)
  10. 玻璃巴斯德移液管225mm(BRAND,目录号:747720)
  11. 巴斯德吸管橡胶球,2ml(Sigma-Aldrich,目录号:Z111597-12EA)
  12. 加热浴(Sigma-Aldrich,目录号:Z563544)
  13. 加热套5L(Selecta,目录号:3031450)
  14. 热板(Selecta,目录号:1000443)
  15. Liebig冷凝器(Sigma-Aldrich,目录号:Z531006)
  16. 硅胶(50×9cm)的玻璃色谱柱(CC)(Fisher Scientific,目录号:12058880)
  17. 用于Sephadex(60×4.5cm)(Fisher Scientific,目录号:12011550)的玻璃层析柱(CC)。
  18. 离心制备薄层色谱系统(CPTLC)(Chromatotron,Harrison Research Inc.,型号:7924T)
  19. 硅胶PF 254盘(Merck Millipore Corporation,目录号:107749 2500)
  20. 用于TLC板(7.5×15.5×8.0cm)(Sigma-Aldrich,目录号:Z204226)的TLC室矩形
  21. 用于PTLC板(27.0×26.5×7.0cm)(Sigma-Aldrich,目录号:Z126195-1EA)的TLC室矩形
  22. 玻璃雾化试剂喷雾器(125ml)(Sigma-Aldrich,目录号:Z529737-1EA)
  23. 玻璃真空过滤器(Fisher Scientific,目录号:11979659)
  24. 微刮勺(8英寸)(Sigma-Aldrich,目录号:Z513342-1PAK)
  25. 分析天平,半微量天平(Fisher Scientific,Mettler Toledo ,目录号:11142062;型号:NewClassic MS105DU)
  26. 核磁共振(NMR)光谱仪(Bruker,型号:Bruker Avance 400; Bruker Avance 500)。
  27. 旋光偏振计(25℃下的CHCl 3)(PerkinElmer,型号:241自动旋光仪)
  28. 紫外(UV)分光光度计(JASCO,型号:V-560)
  29. 红外(IR)分光光度计(Bruker,型号:IFS 55,IFS 28/55)
  30. 高分辨率电子轰击(HREI)质谱仪(MS)(MasSpec,型号:Micromass VG Autospec磁性扇区,M系列)

程序

注意:下面介绍的整个过程大约需要6个月才能完成。

  1. 使用旋转切割机将空气干燥的>子(252.2g)的叶子细磨成小块,并在索格利特装置(图1)中用乙醇(4L)提取24小时,约80℃。


    图1.植物材料和提取程序 A. Piper spp 。树叶。 B.用于提取植物材料的索格利特(Soxhlet)装置
  2. 在旋转蒸发器中(≤50℃,约1.30小时)在减压下蒸发溶剂,得到57.3g(22.7%)粗提取物。
    1. 将粗提取物重新悬浮在750ml水中,并缓慢倒入置于金属架上的分液漏斗(2L)中(图2)。
    2. 向分液漏斗中加入750ml CH 2 Cl 2,并轻轻摇动数次。然后,使溶剂沉降到不混溶相中,并将两相,有机相(底层)和水相(顶层)回收到单独的三角烧瓶中。
    3. 将水相置于分液漏斗内并加入750ml CH 2 Cl 2溶液并重复操作两次。
    4. 合并在三次萃取中获得的有机相,并加入无水硫酸镁(10g)以除去残留的水。旋转解决方案。如果干燥剂结块在一起,添加更多,直到它看起来自由流动。通过过滤漏斗过滤总有机相,并将滤液收集到1L蒸发烧瓶(梨形)中。


    图2.使用分液漏斗对粗提物进行液液分配

  3. 然后,通过在旋转蒸发器(≤40℃)下真空除去CH 2 Cl 2溶液(图3),将有机相干燥(图3),得到得到30g(11.9%)有机部分(深绿色固体)

    图3.旋转蒸发器去除溶剂

  4. 通过将1.5cm细砂层,然后硅胶(粒径63-200mm,430g)放置到20cm的深度,接下来用另一1.5cm层覆盖,封装柱色谱法(CC)(50×9cm)的砂以保护吸附剂表面。使适当体积的己烷(4×1L)通过CC,以促使气泡上升,并使硅胶沉降,使其紧密包装在柱中。
    1. 将有机级分溶解在40ml CH 2 Cl 2中,与硅胶混合,直到形成浆料,并在旋转蒸发器上除去溶剂,直到获得干燥粉末。
    2. 将干燥的粉末提取物缓慢且均匀地铺在填充柱的上部顶部(图4),并使用增加极性的己烷/EtOAc的混合物作为洗脱液(10:0至0:10 ,开始用100%己烷,随后用己烷/EtOAc混合物8:2,6:4,4:6,2:8,1:9,并用100%乙酸乙酯,1L各溶剂混合物精制)。
    3. 在圆底烧瓶(1L)中收集所得的级分(15个级分,每个400ml)。
    4. 随后,在旋转蒸发器中(≤4℃)从每个级分中除去流动相,并将残余物溶解在20ml CH 2 Cl 2溶液中,并转移至圆形(40ml)。


    图4.柱色谱(CC)

  5. 基于它们的相似性TLC谱(图5),将这15个级分合并成10个亚级分(A-J)。
    1. 为了运行TLC板,添加极性递增的己烷/乙酸乙酯的溶剂混合物,从8:2(8ml己烷加2ml乙酸乙酯)开始,并用6:4精制,进入TLC室至刚好低于0.5厘米
    2. 使用微毛细管将每个级分的等分试样施加到TLC(约7×5cm的TLC)的基线(从板的一端标记约0.7cm)并置于TLC室中。
    3. 展开TLC,通过毛细管作用使溶剂向上升至TLC板,直到溶剂几乎达到顶部(板的0.5cm)。
    4. 然后,在UV灯下使用254nm波长使显影的TLC可视化,并用发烟硫酸系统染色,并在热板中将板加热至80-100℃直到出现斑点。


    图5.来自CC的收集的级分和通过TLC分析A.来自柱层析的圆底管中收集的级分显示??在图4中。B.薄层色谱(TLC)显影显色板所收集的级分基于其TLC曲线进一步合并成10个亚级分
  6. 1 H NMR分析显示级分C(己烷/EtOAc,8:2),H(己烷/EtOAc,3:7)和J(EtOAc)富含类黄酮。用于NMR实验的溶剂是氘代丙酮,(CD 3)2 CO。
    注意:为了制备NMR样品:将样品溶解在0.6ml的(CD 3 ) 2 CO,通过装有滤纸的巴斯德吸管过滤,并排入NMR管。
  7. 在填充有Sephadex LH-20(己烷/CHCl 3/MeOH,2:1:1作为洗脱液)的柱色谱(60×4.5cm)上的色谱级分C(2.2g)。将级分C溶解在最小体积的洗脱液(己烷/CHCl 3/MeOH,2:1:1)中,并加载到柱的顶部。使用1L洗脱液展开柱,并在圆底管(40ml)中收集30ml的级分,并通过TLC图谱合并成五个级分(C1-C5)。
    注意:
    1. 在将样品加载到Sephadex上的柱层析之前,将1.5L流动相(己烷/CHCl 3/MeOH/MeOH)的混合物,2:1:1),以稳定Sephadex LH-20。
    2. 在用Sephadex柱色谱法装载样品之前,通过滤纸过滤样品。
  8. 色谱法级分C3(328.8mg)通过CPTLC,制备型离心加速径向薄层色谱(图6)。
    1. 在涂覆有薄层吸附剂的旋转盘的中心附近(涂布转子,涂布转子)施加溶解在小体积CH 2 Cl 2(3ml)中的样品, 4mm厚)。
    2. 流动相由位于台架高度上方约55cm处的特氟隆漏斗保持器提供,并且溶剂通过漏斗中的毛细管向下流到圆形板上。通过己烷/乙醚(8:2,7:3,6:4和1:1)的混合物洗脱,每种溶剂混合物为100ml)形成从转子的边缘一起旋转的分离组分的环形带溶剂。
    3. 将48个级分收集到圆底管(8ml)中。
    4. 将基于它们在TLC谱中的相似性的级分合并成十一个级分(C3A-C3K)。

        
    图6.离心制备薄层色谱(CPTLC,Chromatotron)

    注意:非常重要的是缓慢而均匀地沉积细线的样品,而不用接触移液管太多的二氧化硅,因为这会刮掉它。如果所需条带没有充分分离,请重复运行另一次洗脱。
  9. 将刮料放入玻璃真空过滤器(用约2cm硅胶填充)中,并用EtOAc(4×5ml)冲洗到圆底烧瓶中(步骤E)。在旋转蒸发器中减压除去溶剂。
  10. 然后,将样品溶解在(CD 3)2 CO(0.5ml)中,并用巴斯德移液管转移至NMR管以进行NMR实验。以这种方式,可以分离和纯化以下化合物:2'-羟基-3,4',6'-羟基香叶酮(化合物I,9.4mg,R f 0.42)(Boumendjel) et al。,2008)和化合物2',4'-二羟基-3,6'-二甲氧基香茅醛(化合物II,3.6mg,R sub 0.38)(Aponte ,2010)和来自亚级C3H的2',4'-二羟基-6'-甲氧基香茅酮(化合物III,8.3mg,R f 0.36)。
  • 在Sephadex LH-20(CHCl 3/MeOH,1:1,500ml)上的色谱级分H(5.4g)得到级分H1-H9,并将其收集在圆底烧瓶每个),按照步骤7中所述的步骤
  • 通过CPTLC(CH 2 Cl 2 /丙酮/极性增加,10:0至1:1,从100%CH 2开始)的色谱法级分H6(304.8mg) 2 CH 2 Cl 2并用CH 2 Cl 2 /丙酮50%,200ml的混合物精制),得到9个级分(H6A-H6I),并使用与步骤8中所述相同的程序收集在圆底烧瓶(各10ml)中。
     

    图7.制备薄层色谱(PTLC)步骤的逐步指南。 A.将样品作为条带加入板中。 B.将PTLC放置在室中并显影板。 C.移除板并在UV光下使其可视化,标记对应于化合物的带。 D.刮标记带。 E.将刮料放入玻璃真空过滤器中并用溶剂冲洗
  • 溶解在CH 2 CH 2(2ml)中的亚级分C3E(16.4mg)和C3H(17.7mg),并进一步通过PTLC(板20×20cm,2.5 mm SiO 2厚度)(图7,步骤AE)。
    1. 使用巴斯德移液管(步骤A),将样品作为尽可能窄的条带,在板的基线处(从板的一端标记约2cm)加载。
    2. 干燥平板,置于具有所选洗脱液的室中(己烷/EtOAc,8:2用于亚萃取C 3 E,和己烷/CH 2 Cl 2 2,8:2,用于亚级C3H,100ml洗脱液)并显影(步骤B)。
    3. 从室中取出板(步骤C),并且在UV光标记下用围绕对应于化合物的带的铅笔显现。
    4. 使用小刮刀将包含产品的标记带从板上刮下到干净的白纸的纵向折叠夹上(步骤D)。
    5. 按照与步骤9中所述相同的方法,通过PTLC(己烷/EtOAc,3:2)进一步纯化亚组分H6F(18.2mg),和化合物2',4'-二羟基-3,3',6'-三甲氧基查耳酮IV,4.7mg,R f 0.36)和2',3'-二羟基-4',6'-二甲氧基香茅酮(化合物V,9.3mg,R f 0.40) (Chantrapromma等人,2000)。
    6. 按照相同的程序,通过CPTLC(CH 2 Cl 2 /丙酮,10:0至1:1)色谱分离级分H7(593.0mg),得到级分H7A-H7G如步骤8所述。
    7. 随后,通过PTLC(洗脱液:己烷/异丙醇,4:1)进一步纯化亚组分H7F(23.1mg),得到化合物2',3',5-三羟基-4',6',3-三甲氧基查耳酮mg,R f 0.47)和2',4',4-三羟基-3,6'-二甲氧基香茅醛(化合物VII,3.8mg,R f 0.42)(Vogel 。,2010,),按照步骤9中所述的相同过程
    8. 在Sephadex LH-20(CHCl 3/MeOH/1:1)上的色谱级分I(3.9g)得到级分I1-I5。通过CPTLC(CH 2 Cl 2 /丙酮,10:0至7:3)进一步层析I3(628.8mg),得到级分I3A-I3L,步骤8中所述的步骤。
    9. 通过PTLC(CH 2 Cl 2 /丙酮,9:1和己烷/异丙醇,8:2)进一步纯化亚组分I3F(16.4mg)和I3H(21.2mg)分别为),得到化合物( - ) - (2 S)-8-羟基-5,7,3'-三甲氧基黄烷酮(化合物VIII,11.4mg,R f 0.44 )和( - ) - (2'S)-7,5'-二羟基-5,3'-二甲氧基黄烷酮(化合物IX,10.4mg,R f 0.39)分别按照步骤9中所述的相同过程进行
    10. 总之,E的叶的EtOH提取物。 delineatum 被分割成CH 2 Cl 2/H 2/H 2 O溶液。将CH 2 Cl 2部分进行多个色谱步骤,得到9种类黄酮,其结构通过NMR和MS分析推导(图8)。


      图8.从 叶子获得黄酮类化合物的色谱步骤流程图

    食谱

    1. 油:乙酸 - 水 - 硫酸(20:4:1) 将4ml硫酸与16ml水混合到锥形瓶(200ml)中,并缓慢加入冰醋酸(80ml)。混合均匀,放在玻璃雾化器喷雾器中
    2. 用于CPTLC(4mm厚)的带吸附剂的转子涂层
      用具有硫酸钙半水合物(30g,CaSO 4,1/2H 2 O)的薄层硅胶(60PF 254)TLC标准级(75g) )吸附剂和水(187ml)。

    致谢

    此协议改编自以前发表的研究,Martín-Rodríguez等人。 (2015)。这项工作得到欧洲欧盟FP7-REGPOT-2012-CT2012-316137-IMBRAIN项目的支持。

    参考文献

    1. Aponte,JC,Castillo,D。,Estevez,Y.,Gonzalez,G.,Arevalo,J.,Hammond,GB和Sauvain,M.(2010)。  体内和体内抗利什曼原虫活性的多取代合成chalcones。 20(1):100-103。
    2. Boumendjel,A.,Boccard,J.,Carrupt,PA,Nicolle,E.,Blanc,M.,Geze,A.,Choisnard,L.,Wouessidjewe,D.,Matera,ELand Dumontet, 。  Chalcones的抗恶性和抗增殖活性:前向结构活性关系。 J Med Chem 51(7):2307-2310。
    3. Chantrapromma,K.,Rat,A.p。 Y.,Karalai,C.,Lojanapiwatana,V. and Seechamnanturakit,V。(2000)。  查耳酮和来自乌拉尔草原的二氢查耳酮。 。 53(4):511-513。
    4. Harwood,LM和Moody,CJ(1994)。  实验有机化学:原理与实践 Blackwell Science 。
    5. Martín-Rodríguez,AJ,Ticona,JC,Jiménez,IA,Flores,N.,Fernández,JJ和Bazzocchi,IL(2015)。  来自 Piper delineatum的黄酮类化合物在 Vibrio harveyi 中调节群体感应调节表型。 > Phytochemistry 117:98-106。
    6. Vogel,S.,Barbic,M.,Jurgenliemk,G。和Heilmann,J.(2010)。  chalcones的合成,细胞毒性,抗氧化和抗炎活性以及A环修饰对药理作用的影响。"Eur J Med Chem < em> 45(6):2206-2213。
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    Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
    引用:Bazzocchi, I. L., Ticona, J. C., Jiménez, I. A. and Flores, N. (2016). Isolation of Flavonoids from Piper delineatum Leaves by Chromatographic Techniques. Bio-protocol 6(14): e1867. DOI: 10.21769/BioProtoc.1867.
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