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Isolation of Triterpenes from Propolis (Bee Glue)
从蜂胶中分离三萜   

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Abstract

Propolis (bee glue) is a natural substance produced by bees upon collection of mainly plant resins. Bees use it as antiseptic sealing agent between honeycombs and to preserve the hive from external contamination. Numerous scientific studies have been published on the biological properties of propolis including its anti-inflammatory, anti-oxidant, immunostimulant, antitumour and antimicrobial activity. Different propolis chemotypes have been characterised based on the nature of the plant-derived substances present and the geographical origin of collection. Here, we describe the isolation of nine triterpenes from a sample of propolis originating from North-Western Cameroon. All compounds were identified following analysis of their spectroscopic data and comparison with previously published reports.

Materials and Reagents

  1. TLC silica gel 60 F254 plates (VWR International, catalog number: 1.05554.0001 )
  2. Wilmad® NMR tubes 5 mm diam., precision (Sigma-Aldrich, catalog number: Z274275 )
  3. Snap-cap vials (VWR International, catalog number: 548-0555 )
  4. Raw propolis (Nature’s Laboratory Ltd, catalog number: P5 )
  5. Ethanol 96% v/v (extra pure, Specified Laboratory Reagent) (Thermo Fisher Scientific, catalog number: 10162252 )
  6. Hexanes (for HPLC, 95% n-hexane approx) (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10703611 )
  7. Ethyl acetate, extra pure, Specified Laboratory Reagent (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10080130 )
  8. Sodium sulfate anhydrous, extra pure (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10606082 )
  9. Silica gel 60 (0.063–0.200 mm) (VWR International, catalog number: 1.07734.1000 )
  10. Silica gel 60H (VWR International, catalog number: 1.07736.1000 )
  11. 98% p-Anisaldehyde (Sigma-Aldrich, catalog number: A88107 )
  12. Acide sulfurique fumant (pure) (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10222282 )
  13. Acetic acid glacial (pure) (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10375020 )
  14. Methanol (extra pure, Specified Laboratory Reagent) (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10214490 )
  15. Sephadex® LH-20 (Sigma-Aldrich, catalog number: LH20100 )
  16. α-Amyrin (Sigma-Aldrich, catalog number: 53017 )
  17. Cycloartenol (Sigma-Aldrich, catalog number: 08172 )
  18. Lupeol (Sigma-Aldrich, catalog number: S957712 )
  19. Dichloromethane, extra pure, stabilised with amylene, Specified Laboratory Reagent (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10458210 )
  20. Chloroform-d (CDCl3) (Sigma-Aldrich, catalog number: 10080130 )
  21. Anisaldehyde-sulphuric acid reagent (see Recipes)

Equipment

  1. Heated ultrasonic water bath (340 W) (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10215332 )
  2. Büchi rotary evaporator, Rotavapor® R-210 (VWR International, catalog number: 531-0850 )
  3. UV viewing cabinet CC10 (Thermo Fisher Scientific, Fisher Scientific, catalog number: 11778201 )
  4. UV lamp UVG-11 230 V 50/60 Hz 4 W hand held short wave (Thermo Fisher Scientific, Fisher Scientific, catalog number: 11718241 )
  5. UV lamp UVL-21 230 V 50/60 Hz 4 W hand held long wave (Thermo Fisher Scientific, Fisher Scientific, catalog number: 11728241 )
  6. Analytical balance (VWR International, catalog number: 611-2267 )
  7. Precision balance (VWR International, catalog number: 611-2695 )
  8. Vacuum water jet pump (VWR International, catalog number: 181-9100 )
  9. Pear-shaped (separating) funnel (1,000 ml) (Scientific Glass Laboratories, catalog number: SFP1L )
  10. Round bottom flasks, 250 ml (Total number of 12) (Scientific Glass Laboratories, catalog number: FRS250/B19 or FRS250/B24 )
  11. Glass foot measuring cylinder with hexagonal base, borosilicate glass, class “B” (25 ml and 250 ml) (Scientific Glass Laboratories, catalog number: MCB/25 and MCB/250 )
  12. Conical Erlenmeyer flasks (500 ml) (Scientific Glass Laboratories, catalog number: FC500/B24 )
  13. Filter funnels, borosilicate (Scientific Glass Laboratories, catalog number: BFF75 and BFF100 )
  14. Rubber vacuum tubing NW8 (VWR international, catalog number: 189.3111 )
  15. Glass Pasteur pipettes 150 mm (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10006021 )
  16. Glass Pasteur pipettes 230 mm (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10209381 )
  17. Glass micropipettes (homemade from glass Pasteur pipettes 230 mm using a Bunsen burner)
  18. Büchner filter with cone and thread, GL14, for vacuum liquid chromatography (VLC) (Scientific Glass Laboratories, catalog number: TBF/4 / B24 / POR3 )
  19. Twin trough chambers for TLC plates (VWR International, catalog number: 552-0011 )
  20. Glass atomiser reagent sprayer (VWR International, catalog number: 552-0031 )
  21. Glass chromatography columns (CC) with sintered discs (Scientific Glass Laboratories, catalog number: R2/40 )
  22. Duratool DO1600 hot air heat gun (Amazon)
  23. Whatman qualitative filter paper, grade 1 (Sigma-Aldrich, catalog number: WHA1001125 )
  24. Exactive Orbitrap mass spectrometer (MS) operating in a positive and negative electrospray ionisation (ESI) switching mode (Thermo Fisher Scientific)
  25. JEOL 505HA high resolution electron impact (HREI) mass spectrometer (MS) using direct probe at elevated temperature (110~160 ºC) at 70 eV (JEOL USA)
  26. JEOL Lambda Delta 400 NMR spectrometer (JEOL USA, model: JEOL Nuclear Magnetic Resonance Spectrometers )

Software

  1. Xcalibur software (version 2.2) for MS data processing (Thermo Fisher Scientific, model: Xcalibur software)
  2. Mestre Nova (MNova) software (version 8.0.0) for NMR processing (Mestrelab Research SL, model: version 8.0.0)

Procedure

  1. The whole procedure described below takes around 6 weeks to complete. All steps are done at room temperature (20-25 °C) unless otherwise stated. Weigh propolis (ca. 50 g) in a conical Erlenmeyer flask then add 70% ethanol in water (500 ml) to the flask.
  2. Place the flask in the ultrasonic water bath on high for 1 h at 60 °C.
  3. Recover the supernatant into a second conical Erlenmeyer flask and re-extract the raw material left-over in the original flask using fresh 70% ethanol (500 ml) as in step 2. Repeat this process 10 times and combine all supernatants.
  4. Filter the pooled supernatants into a round-bottom flask using a glass filter funnel overlaid with filter paper. Discard the insoluble material (brown solid) remaining on the filter paper. The filtered extract is a clear amber solution. Keep this filtered extract in a fridge at 4 °C.
  5. Concentrate the filtered extract in the round-bottom flask to dryness under reduced pressure at ≤40 °C using a rotary evaporator. To achieve this, adjust the water bath to 60 °C and apply a vacuum of 175 mbar. Complete evaporation will occur in around 30 min.
  6. Add distilled water (250 ml) and pure (96%) ethanol (25 ml) to the dried extract to obtain a suspension.
  7. Place a separating funnel onto a metallic stand (Figure 1), close its stopcock and slowly pour the suspension into the separating funnel.
  8. Add hexane (250 ml) to the separating funnel.
  9. Hand-shake the funnel gently several times. Remove the top stopper, then allow for the solvents to settle until into two immiscible phases. Recover the aqueous phase (bottom layer) and the hexane phase (top layer) in separate Erlenmeyer flasks. Do this by slowly opening the stopcock and closing it just before the curved meniscus between the two liquids reaches the stopcock.
  10. Place the aqueous phase back inside the separating funnel.
  11. Repeat steps 8 and 10 twice using fresh hexane.
  12. Add ethyl acetate (250 ml) to the funnel and repeat steps 9 to 10 (the aqueous phase is the bottom layer and the ethyl acetate phase is the top layer).
  13. Repeat step 12 twice using fresh ethyl acetate.
  14. Sprinkle some anhydrous sodium sulfate into the recovered hexane and the ethyl acetate phases and gently manually rotate the Erlenmeyer flasks to observe if the cloudiness (i.e. residual water droplets) disappears. Continue sprinkling sodium sulphate and rotating the flasks until the liquids are completely clear.
  15. Filter each organic phase into a round-bottom flask using a glass filter funnel overlaid with filter paper. Discard the insoluble material (sodium sulfate) remaining on the filter paper. The filtered extracts are clear solutions; concentrate them to dryness under reduced pressure at ≤40 °C using a rotary evaporator. To achieve this, adjust the water bath to 60 °C and apply a vacuum of 335 mbar (hexane) or 240 mbar (ethyl acetate). Complete evaporation will occur in around 20 min.
  16. Store the hexane and the ethyl acetate extracts at −20 °C.
  17. Dissolve a portion (5 to 10 g) of the hexane extract in 10 ml of a suitable solvent (e.g. dichloromethane, ethyl acetate and/or hexane) and mix with silica gel 60 (3 g). Leave the mixture to dry on a bench at 20-25 °C. A free-flowing and completely dried powder is usually obtained overnight.
  18. Connect the Büchner filter for VLC to a vacuum water jet pump and pack it with silica gel 60 H. Leave a space (ca. 3 cm) above the compacted layer of silica layer so as to accommodate the sample and an appropriate volume of solvent. Allow hexane to pass through the column under vacuum to check the uniformity of the column.
  19. Slowly and uniformly spread the powder prepared in step 17 as a thin layer (ca. 1 cm) directly on to the top of the packed VLC column. Carry out the elution with hexane-ethyl acetate mixtures of increasing polarity (starting with 100% hexane, hexane/ethyl acetate 95:5, 9:1, 85:15, 8:2, 7:3, 6:4, 1:1, 4:6, 25:75, 3:7, 2:8, 1:9 and lastly 100% ethyl acetate). Add a defined volume of solvent (ca. 300 ml) to the top of the VLC column each time and apply vacuum until the column dries up (Figure 2) (Coll and Bowden, 1986; Pelletier et al., 1986). Collect each fraction in a round-bottomed flask and concentrate to dryness under reduced pressure at ≤ 40 °C using a rotary evaporator. To achieve this, adjust the water bath to 60 °C and apply a vacuum of 240 mbar. Complete evaporation will occur in around 20 min.
  20. Dissolve an aliquot of each fraction (ca. 5-10 mg) in dichloromethane (ca. 5 ml). Use a glass micropipette to apply 5-10 spots of each fraction as bands ca. 1 cm above the bottom edge of a thin layer chromatography (TLC) silica gel plate (Figure 3A).
  21. Place a filter paper inside a TLC chamber and pour a mixture (20 ml) of hexane/ethyl acetate (8:2, v/v) into the chamber. Leave the solvent in the chamber for 30 min at room temperature (20 °C-25 °C) to help reach solvent saturation. Pour a mixture (20 ml) of hexane/ethyl acetate (7:3, v/v) into second TLC chamber. Place the spotted TLC plates in the chambers and leave them to develop in an ascending direction until the solvent reaches ca. 1 cm below the top (Figure 3B).
  22. Take the developed TLC plates out of the chambers and observe under UV light using short (λ= 254 nm) and long (λ= 366 nm) wavelengths. Spray anisaldehyde-sulphuric acid reagent (see Recipes) over the plates and heat the plates to 105-110 °C with a heat gun for ca. 1 min until coloured spots appear (Figure 3C) (Waldi, 1965). Calculate Rf values for individual spots as the ratio of the distance (in cm) from the centre of the spot to the baseline over the distance (in cm) from the solvent line to the baseline (Stahl and Mangold, 1975; Stock and Rice, 1974). Triterpenes typically appear as bright pink spots on TLC plates following spraying with anisaldehyde-sulfuric acid reagent and heating (Figure 3D).
  23. The first pink coloured spot appears in the VLC fraction eluted with 15% ethyl acetate in hexane. Mix this fraction with silica gel 60 (3 g) in dichloromethane. Leave the mixture to dry on a bench at 20-25 °C. A free-flowing and completely dried powder is usually obtained overnight.
  24. In a glass, beaker, mix enough silica gel 60 in hexane to obtain a slurry which has the right consistency so as to be poured to fill a CC glass column. Leave the column stopcock open when filling with the slurry to let the latter settle inside the column. Continue pouring the slurry inside the column until the silica particles settle and form a horizontal layer about 5 cm from the top of the column. Ensure that there is a sufficient volume (around 5 ml) of solvent on top of the silica layer so the packing material does not dry out. Take care to prevent the inclusion of any air bubbles (Note 1) (Braithwaite and Smith, 1996; Ravindranath, 1989). Apply the powdered fraction to the top of the packed column.
  25. Carry out the elution using hexane and ethyl acetate mixtures of increasing polarity (from 100% hexane, hexane/ethyl acetate 9:1, 8:2, 7:3, 6:4, 1:1, 4:6, 3:7, 2:8, 1:9 and lastly 100% ethyl acetate) (Figure 4). Collect all eluates in vials, and analyse their contents by TLC (steps 20-22).
  26. One of the eluates appears as a single pink spot following spraying with anisaldehyde sulphuric acid reagent (Rf = 0.40 in hexane/ethyl acetate 8:2). Concentrate this eluate to dryness under reduced pressure at ≤ 40 °C, dissolve it in chloroform-d (2.5 ml) and transfer it into an NMR tube. Carry out 1H, 13C NMR and MS analysis to reveal the presence of four triterpenes, namely α-amyrin (Hernández Vázquez et al., 2012; Basyuni et al., 2006), β-amyrin (Basyuni et al., 2006; Mahato and Kundu, 1994), lupeol (Basyuni et al., 2006; Thanakijcharoenpath and Theanphong, 2007) and cycloartenol (Kamisako et al., 1987; Zhu et al., 2012).
  27. Combine the VLC fractions eluted with 25% and 30% ethyl acetate in hexane, respectively. Concentrate them to dryness under reduced pressure at ≤ 40 °C using the rotary evaporator.
  28. In a glass, beaker, mix enough Sephadex® LH-20 (Note 2) in 5% hexane in dichloromethane to obtain a slurry which has the right consistency so as to be poured to fill a CC glass column. Leave the column stopcock open when filling with the slurry to let the latter settle inside the column. Continue pouring the slurry inside the column until the Sephadex® LH-20 particles settle and form a horizontal layer about 5 cm from the top of the column. Ensure there is only the minimal volume of solvent remaining on the surface of the packing material so it does not dry out. Take care to prevent the inclusion of any air bubbles (Note 1). (Determann and Brewer, 1975; Kremmer and Boross, 1979).
  29. Apply the combined fractions, re-dissolved in 5% hexane in dichloromethane, onto the top of the column.
  30. Elute with 5% hexane in dichloromethane to afford an eluate which appears as a single pink spot on TLC following spraying with anisaldehyde-sulfuric acid reagent and heating (Rf = 0.23 in hexane/ethyl acetate 8:2). Concentrate this eluate to dryness under reduced pressure at ≤ 40 °C, dissolve it in chloroform-d (2.5 ml) and transfer it into an NMR tube. Carry out 1H, 13C NMR and MS analysis to reveal the presence of two triterpenes, namely mangiferonic acid (Escobedo-Martinez et al., 2012), and ambonic acid (Da Silva et al., 2005).
  31. Combine the VLC fractions eluted with 40% and 50% ethyl acetate in hexane, respectively. Concentrate them to dryness under reduced pressure at ≤ 40 °C using a rotary evaporator and subject them to gel filtration (see step 28).
  32. Elute with 5% hexane in dichloromethane to afford one eluate containing a bright pink spot.
  33. Fractionate this eluate further by CC (see step 24) using hexane/ethyl acetate mixtures of increasing polarity (from 100% hexane, hexane/ethyl acetate 9:1, 8:2, 7:3, 6:4, 1:1, 4:6, 3:7, 2:8, 1:9 and lastly 100% ethyl acetate). One of the eluates appears as a single pink spot on TLC following spraying with anisaldehyde sulphuric acid reagent (Rf = 0.40 in hexane/ethyl acetate 7:3). Concentrate this eluate to dryness under reduced pressure at ≤40 °C using a rotary evaporator, dissolve it in chloroform-d (2.5 ml) and transfer it into an NMR tube. Carry out 1H, 13C NMR and MS analysis to reveal the presence of two triterpenes, namely mangiferolic acid (Escobedo-Martinez et al., 2012) and ambolic acid (Escobedo-Martinez et al., 2012).
  34. Subject a portion (5 to10 g) of the ethyl acetate extract to VLC eluting with hexane: ethyl acetate mixtures of increasing polarity (starting with 100% hexane, hexane/ethyl acetate 95:5, 9:1, 85:15, 8:2, 7:3, 6:4, 1:1, 4:6, 3:7, 25:75, 2:8, 1:9 and lastly 100% ethyl acetate) (steps 13-15).
  35. Fractionate the VLC fraction eluted with 40% ethyl acetate in hexane, by gel filtration (step 28) eluting with 5% hexane in dichloromethane, followed by dichloromethane, to yield a mixture of ambolic acid and isomangiferolic acid (Escobedo-Martinez et al., 2012) along with mangiferonic and mangiferolic acids as pure compounds.

Representative data


Figure 1. Typical set up for liquid-liquid partition using a separating funnel


Figure 2. Typical set up for vacuum liquid chromatography (VLC)

A

B

C

D

Figure 3. Thin layer chromatography (TLC) analysis. A. Spotting of sample on TLC plate; B. Spotted plate placed in the TLC tank for elution; C. Heating of plate; D. Calculation of Rf values for triterpene (pink) spots.


Figure 4. Typical set up for column chromatography

Notes

  1. To prevent the formation of air bubbles, the slurry should be poured slowly onto the inside walls of the column and the packed column should not be allowed to dry out. Any air bubbles can be removed by gently finger tapping the column.
  2. It is also possible to use some recycled Sephadex® LH-20. The latter is obtained by soaking any used Sephadex® LH-20 slurry in a mixture of dichloromethane and methanol in a Büchner filter connected to a vacuum water jet pump. Once the vacuum has been applied, the Sephadex® LH-20 is left to air-dry on the bench at 20-25 °C.

Recipes

  1. Anisaldehyde-sulphuric acid reagent (Waldi, 1965)
    Mix 0.5 ml of p-anisaldehyde with 10 mL glacial acetic acid and 85 mL methanol
    Slowly add sulphuric acid (5 mL) to that mixture
    Mix well, place in a glass atomiser (amber) bottle and store at 2-8 °C

Acknowledgments

This protocol was adapted from the previously published study, Kardar et al. (2014). This work was supported by the Leverhulme Trust, UK (Research Project Grant RPG-150). We thank T. Zhang for technical assistance and Nature’s Laboratory Ltd for the supply of propolis.

References

  1. Basyuni, M., Oku, H., Inafuku, M., Baba, S., Iwasaki, H., Oshiro, K., Okabe, T., Shibuya, M. and Ebizuka, Y. (2006). Molecular cloning and functional expression of a multifunctional triterpene synthase cDNA from a mangrove species Kandelia candel (L.) Druce. Phytochemistry 67(23): 2517-2524.
  2. Braithwaite, A. and Smith, F.J. (1996). Chromatographic methods. 5th edition. Blackie Academic and professional 117–64.
  3. Coll, J. C. and Bowden, B. F. (1986). The application of vacuum liquid chromatography to the separation of terpene mixtures. J Nat Prod 49, 934–6.
  4. Da Silva, M. S. S., Cito, A. M. G. L., Chaves, M. H., Lopes, J. A. D. (2005). Cycloartane triterpenoids of propolis from Teresina-PI. Quim Nova 28, 801–804.
  5. Determann, H. and Brewer, J. E. (1975). Gel chromatography. In: Heftmann, E. (ed). Chromatography- A laboratory handbook of chromatographic and electrophoretic methods. Springer, 164-88.
  6. Escobedo-Martinez, C., Concepcion Lozada, M., Hernandez-Ortega, S., Villarreal, M. L., Gnecco, D., Enriquez, R. G. and Reynolds, W. (2012). (1) H and (13) C NMR characterization of new cycloartane triterpenes from Mangifera indica. Magn Reson Chem 50(1): 52-57.
  7. Hernández Vázquez, L., Palazon, J., Navarro-Ocaña, A. (2012). The pentacyclic triterpenes -amyrins: a review of sources and biological activities. In: Rao, Venketeshwer (ed). Phytochemicals-a global perspective of their role in nutrition and health. In Tech Publisher.
  8. Kamisako, W., Honda, C., Suwa, K. and Isoi, K. (1987). Studies of 13C NMR spectra of13C-enriched cycloartenol biosynthesized from [1-13C]-, [2-13C]- and [1,2,-13C2]-acetate. Revised 13C NMR spectral assignments of cycloartenol and cycloartanol and 13C NMR spectral support for the generally accepted skeleton formation mechanism of cycloartenol. Magn Res Chem 25, 683–687.
  9. Kremmer, T. and Boross, L. (1979). Gel chromatography. Translated by Gabor, M. John Wiley and Sons. Budapest 17-31.
  10. Mahato, S. B.and Kundu, A. P. (1994). 13C NMR spectra of pentacyclic triterpenoids – acompilation and some salient features. Phytochemistry 37, 1517-1575.
  11. Pelletier, S. W., Choksi, H. P. and Desai, H. K. (1986). Separation of diterpenoid alkaloid mixtures using vacuum liquid chromatography. J Nat Prod 49, 892–900.
  12. Ravindranath, B. (1989). Principles and practice of chromatography. Ellis Harwood Ltd. 193-292.
  13. Stahl, E. and Mangold, H. K. (1975). Techniques of thin layer chromatography. In: Heftmann, E. (ed). Chromatography- A laboratory handbook of chromatographic and electrophoretic methods. Springer-Verlag, 164-88.
  14. Stock, R. and Rice, C. B. (1974). Chromatographic methods. Chapman and Hall 279-317.
  15. Thanakijcharoenpath, W. and Theanphong, O. (2007). Triterpenoids from the stem of Diospyros glandulosa. Thai J Pharm Sci 31, 1-8.
  16. Waldi, D. (1965). Spray reagents for thin layer chromatography. In: Stahl, E. (ed). Thin layer chromatography A laboratory handbook. Springer, 483-502.
  17. Zhu, Z. H., Liu, W. H., Ge, L. G., Yu, Q., Zhao, W. C., Yang, J. H. and Wan, H. T. (2012). Molecular cloning and characterization of a cDNA encoding cycloartenol synthase from Fritillaria thunbergii Miq. Afr J Biotechnol 11, 6896–6903.

简介

蜂胶(蜂胶)是在收集主要植物树脂时由蜜产生的天然物质。 蜂用作蜂窝之间的防腐密封剂,并保护蜂巢免受外部污染。 已经发表了关于蜂胶的生物学性质的许多科学研究,包括其抗炎,抗氧化,免疫刺激,抗肿瘤和抗微生物活性。 已经基于存在的植物衍生物质的性质和收集的地理起源来表征不同的蜂胶化学型。 在这里,我们描述了从源自喀麦隆西北部的蜂胶样品中分离出九种三萜。 在分析其光谱数据并与先前发表的报告比较后鉴定所有化合物。

材料和试剂

  1. TLC硅胶60F254板(VWR International,目录号:1.05554.0001)
  2. 精密(直径5毫米,精密(Sigma-Aldrich,目录号:Z274275))的Wilmad?
  3. Snap-cap小瓶(VWR International,目录号:548-0555)
  4. 原料蜂胶(Nature's Laboratory Ltd,目录号:P5)
  5. 乙醇96%v/v(超纯,指定实验室试剂)(Thermo Fisher Scientific,目录号:10162252)
  6. 己烷(用于HPLC,约95%正己烷)(Thermo Fisher Scientific,Fisher Scientific,目录号:10703611)
  7. 乙酸乙酯,超纯,指定实验室试剂(Thermo Fisher Scientific,Fisher Scientific,目录号:10080130)
  8. 无水硫酸钠,超纯(Thermo Fisher Scientific,Fisher Scientific,目录号:10606082)
  9. 硅胶60(0.063-0.200mm)(VWR International,目录号:1.07734.1000)
  10. 硅胶60H(VWR International,目录号:1.07736.1000)
  11. 98%p - 茴香醛(Sigma-Aldrich,目录号:A88107)
  12. 酰化硫磺消烟剂(纯)(Thermo Fisher Scientific,Fisher Scientific,目录号:10222282)
  13. 乙酸冰(纯)(Thermo Fisher Scientific,Fisher Scientific,目录号:10375020)
  14. 甲醇(超纯,指定实验室试剂)(Thermo Fisher Scientific,Fisher Scientific,目录号:10214490)
  15. Sephadex LH-20(Sigma-Aldrich,目录号:LH20100)
  16. α-淀粉酶(Sigma-Aldrich,目录号:53017)
  17. Cycloartenol(Sigma-Aldrich,目录号:08172)
  18. Lupeol(Sigma-Aldrich,目录号:S957712)
  19. 二氯甲烷,超纯,用戊烯稳定,指定实验室试剂(Thermo Fisher Scientific,Fisher Scientific,目录号:10458210)
  20. 氯仿-d(CDCl 3)(Sigma-Aldrich,目录号:10080130)
  21. 苯甲醛 - 硫酸试剂(参见配方)

设备

  1. 加热超声水浴(340W)(Thermo Fisher Scientific,Fisher Scientific,目录号:10215332)
  2. Büchi旋转蒸发器,Rotavapor R-210(VWR International,目录号:531-0850)
  3. UV观察箱CC10(Thermo Fisher Scientific,Fisher Scientific,目录号:11778201)
  4. UV灯UVG-11 230V 50/60Hz 4W手持短波(Thermo Fisher Scientific,Fisher Scientific,目录号:11718241)
  5. UV灯UVL-21 230V 50/60Hz 4W手持式长波(Thermo Fisher Scientific,Fisher Scientific,目录号:11728241)
  6. 分析天平(VWR International,目录号:611-2267)
  7. 精密天平(VWR International,目录号:611-2695)
  8. 真空水喷射泵(VWR International,目录号:181-9100)
  9. 梨形(分离)漏斗(1,000ml)(Scientific Glass Laboratories,目录号:SFP1L)
  10. 圆底烧瓶,250ml(总数12)(Scientific Glass Laboratories,目录号:FRS250/B19或FRS250/B24)
  11. 具有六边形底部,硼硅酸盐玻璃,"B"级(25ml和250ml)(Scientific Glass Laboratories,目录号:MCB/25和MCB/250)的玻璃足量筒
  12. 锥形锥形烧瓶(500ml)(Scientific Glass Laboratories,目录号:FC500/B24)
  13. 过滤漏斗,硼硅酸盐(Scientific Glass Laboratories,目录号:BFF75和BFF100)
  14. 橡胶真空管NW8(VWR international,目录号:189.3111)
  15. 玻璃巴斯德移液管150mm(Thermo Fisher Scientific,Fisher Scientific,目录号:10006021)
  16. 玻璃巴斯德移液管230mm(Thermo Fisher Scientific,Fisher Scientific,目录号:10209381)
  17. 玻璃微量移液管(使用Bunsen燃烧器从玻璃巴斯德移液管自行制备)
  18. 用于真空液相色谱(VLC)(Scientific Glass Laboratories,目录号:TBF/4/B24/POR3)的具有锥体和螺纹GL14的Büchner过滤器
  19. 用于TLC板的双槽室(VWR International,目录号:552-0011)
  20. 玻璃雾化试剂喷雾器(VWR International,目录号:552-0031)
  21. 具有烧结盘(Scientific Glass Laboratories,目录号:R2/40)的玻璃色谱柱(CC)
  22. Duratool DO1600热风加热枪(亚马逊)
  23. Whatman定性滤纸,1级(Sigma-Aldrich,目录号:WHA1001125)
  24. 以正负电喷雾电离(ESI)切换模式(Thermo Fisher Scientific)操作的Exactive Orbitrap质谱仪(MS)
  25. JEOL 505HA高分辨率电子轰击(HREI)质谱仪(MS)在70 eV(JEOL USA)的高温(110?160℃)下使用直接探头
  26. JEOL Lambda Delta 400 NMR光谱仪(JEOL USA,型号:JEOL核磁共振光谱仪)

软件

  1. 用于MS数据处理的Xcalibur软件(版本2.2)(Thermo Fisher Scientific,型号:Xcalibur软件)
  2. 用于NMR处理的Mestre Nova(MNova)软件(版本8.0.0)(Mestrelab Research SL,型号:8.0.0版)

程序

  1. 以下描述的整个过程大约需要6周才能完成。除非另有说明,所有步骤在室温(20-25℃)下进行。将蜂胶(约50g)在锥形锥形烧瓶中称重,然后向烧瓶中加入70%乙醇的水溶液(500ml)。
  2. 将烧瓶置于高温超声水浴中1小时,60℃
  3. 将上清液回收到第二个锥形烧瓶中,如步骤2所述,使用新鲜的70%乙醇(500ml)再次提取原始烧瓶中剩余的原料。重复该过程10次,并合并所有上清液。 >
  4. 使用覆盖有滤纸的玻璃过滤漏斗将合并的上清液过滤到圆底烧瓶中。弃去滤纸上残留的不溶物(棕色固体)。过滤的提取物是澄清的琥珀色溶液。将过滤的提取物放在4°C的冰箱中。
  5. 在≤40℃下使用旋转蒸发器在减压下将过滤的提取物浓缩在圆底烧瓶中至干。为此,将水浴调节至60℃并施加175毫巴的真空。约30分钟内完全蒸发。
  6. 向干燥的提取物中加入蒸馏水(250ml)和纯(96%)乙醇(25ml)得到悬浮液。
  7. 将分液漏斗放在金属支架上(图1),关闭其活栓,并将悬浮液缓慢倒入分液漏斗中。
  8. 向分液漏斗中加入己烷(250ml),
  9. 轻轻摇动漏斗几次。取出顶部塞子,然后让溶剂沉降,直到两个不混溶相。在单独的锥形瓶中回收水相(底层)和己烷相(顶层)。通过缓慢打开活栓并在两种液体之间的弯曲的弯月面到达活栓之前关闭活塞。
  10. 将水相放回分液漏斗中。
  11. 使用新鲜己烷重复步骤8和10两次
  12. 向漏斗中加入乙酸乙酯(250ml),重复步骤9至10(水相为底层,乙酸乙酯相为顶层)。
  13. 用新鲜乙酸乙酯重复步骤12两次
  14. 将一些无水硫酸钠撒入回收的己烷和乙酸乙酯相中,轻轻地手动旋转锥形瓶以观察浊度(即残余水滴)是否消失。继续喷洒硫酸钠并旋转烧瓶直到液体完全清除
  15. 使用覆盖有滤纸的玻璃过滤漏斗将每个有机相过滤到圆底烧瓶中。丢弃残留在滤纸上的不溶物(硫酸钠)。过滤的提取物是澄清溶液;在≤40℃下使用旋转蒸发器在减压下浓缩至干。为此,将水浴调节至60℃,并施加335毫巴(己烷)或240毫巴(乙酸乙酯)的真空。约20分钟内完全蒸发。
  16. 在-20℃下储存己烷和乙酸乙酯萃取液
  17. 将一部分(5至10g)己烷提取物溶解在10ml合适的溶剂(例如二氯甲烷,乙酸乙酯和/或己烷)中,并与硅胶60(3g)混合。将混合物在20-25℃的工作台上干燥。通常在一夜之间获得自由流动且完全干燥的粉末
  18. 将VLC的Büchner过滤器连接到真空水喷射泵,并用硅胶60 H包装。在压实的二氧化硅层上方留出一个空间(约3厘米),以便容纳样品和适当体积的溶剂。使己烷在真空下通过柱以检查柱的均匀性。
  19. 将在步骤17中制备的粉末作为薄层(约1cm)缓慢且均匀地铺展到填充的VLC柱的顶部。用极性递增的己烷 - 乙酸乙酯混合物(用100%己烷,己烷/乙酸乙酯95:5,9:1,85:15,8:2,7:3,6: 1,4:6,25:75,3:7,2:8,1:9,最后是100%乙酸乙酯)。每次向VLC柱的顶部加入限定体积的溶剂(约300ml),并施加真空直到柱干燥(图2)(Coll和Bowden,1986; Pelletier等人 >,1986)。在圆底烧瓶中收集各级分,并在≤40℃下使用旋转蒸发器在减压下浓缩至干。为此,将水浴调节至60℃,并施加240毫巴的真空。约20分钟内完全蒸发。
  20. 将每个级分(约5-10mg)的等分试样溶解在二氯甲烷(约5ml)中。使用玻璃微量移液器将每个部分的5-10个点作为条带。在薄层色谱(TLC)硅胶板的底部边缘上方1cm处(图3A)。
  21. 将滤纸放置在TLC室内,并在室内倒入己烷/乙酸乙酯(8:2,v/v)的混合物(20ml)。在室温(20°C -25°C)下将溶剂留在室中30分钟,以帮助达到溶剂饱和。在第二TLC室中倒入己烷/乙酸乙酯(7:3,v/v)的混合物(20ml)。将点样的TLC板放置在室中,让它们以上升的方向发展,直到溶剂达到约。距离顶部1厘米(图3B)。
  22. 将展开的TLC板从室中取出,并使用短波长(λ= 254nm)和长波长(λ= 366nm)在UV光下观察。在板上喷洒茴香醛 - 硫酸试剂(参见配方),并用热风枪将板加热至105-110℃。 1分钟,直至出现色斑(图3C)(Waldi,1965)。计算单个斑点的Rf值,作为从溶剂线到基线的距离(cm)上从斑点中心到基线的距离(以cm计)的比值(Stahl和Mangold,1975; Stock and Rice, 1974)。在用茴香醛 - 硫酸试剂喷雾和加热后,三萜通常在TLC板上显示为亮粉色斑点(图3D)。
  23. 第一个粉红色的斑点出现在用己烷中的15%乙酸乙酯洗脱的VLC级分中。将该级分与硅胶60(3g)在二氯甲烷中混合。将混合物在20-25℃的工作台上干燥。通常在一夜之间获得自由流动且完全干燥的粉末
  24. 在玻璃烧杯中,在己烷中混合足够的硅胶60以获得具有适当稠度的浆料,以便倾倒以填充CC玻璃柱。在填充浆料时让柱塞旋转打开,以使浆料在柱内沉降。继续将浆料倾倒在柱内,直到二氧化硅颗粒沉降并从柱顶部形成约5cm的水平层。确保在二氧化硅层顶部有足够体积(约5ml)的溶剂,因此包装材料不会变干。注意防止包含任何气泡(注1)(Braithwaite和Smith,1996; Ravindranath,1989)。将粉末部分加到填充柱的顶部。
  25. 使用极性增加的己烷和乙酸乙酯混合物进行洗脱(从100%己烷,己烷/乙酸乙酯9:1,8:2,7:3,6:4,1:1,4:6,3:7 ,2:8,1:9,最后是100%乙酸乙酯)(图4)。收集小瓶中的所有洗脱液,并通过TLC分析其内容物(步骤20-22)。
  26. 在用茴香醛硫酸试剂(Rf = 0.40,在己烷/乙酸乙酯8:2中)喷雾后,洗脱液之一表现为单个粉红色斑点。在≤40℃下将该洗脱液在减压下浓缩至干,将其溶于氯仿-d(2.5ml)中,并转移至NMR管中。进行1 H,13 C NMR和MS分析以揭示存在四种三萜烯,即α-淀粉酶(HernándezVázquezet al。 ,2012; Basyuni等人,2006),β-淀粉酶(Basyuni等人,2006; Mahato和Kundu,1994),lupeol(Basyuni等人, et al。,2006; Thanakijcharoenpath和Theanphong,2007)和环木菠萝烯醇(Kamisako等人,1987; Zhu等人,2012)。 >
  27. 合并分别用25%和30%乙酸乙酯的己烷溶液洗脱的VLC级分。使用旋转蒸发器在≤40°C下减压浓缩至干
  28. 在玻璃烧杯中,在5%己烷/二氯甲烷中混合足够的Sephadex LH-20(注释2),得到具有适当稠度的浆料,以便倒入填充CC玻璃柱。在填充浆料时让柱塞旋转打开,以使浆料在柱内沉降。继续将浆料倾倒在柱内,直到Sephadex LH-20颗粒沉淀并且从柱顶部形成约5cm的水平层。确保在包装材料表面上只剩下最小量的溶剂,以便不会变干。注意防止含有气泡(注1)。 (Determann和Brewer,1975; Kremmer和Boross,1979)。
  29. 将合并的级分重新溶解于在二氯甲烷中的5%己烷中,在柱的顶部。
  30. 用5%己烷/二氯甲烷洗脱,得到洗脱液,在用茴香醛 - 硫酸试剂喷雾并加热(Rf = 0.23,在己烷/乙酸乙酯8:2中)后,其在TLC上表现为单个粉红色点。在≤40℃下将该洗脱液在减压下浓缩至干,将其溶于氯仿-d(2.5ml)中,并转移至NMR管中。进行1 H,13 C NMR和MS分析以揭示存在两种三萜烯,即甘草酸(Escobedo-Martinez et al。 ,2012)和氨基酸(Da Silva等人,2005)。
  31. 合并分别用40%和50%乙酸乙酯的己烷溶液洗脱的VLC级分。使用旋转蒸发器在≤40℃下减压浓缩至干,并进行凝胶过滤(参见步骤28)。
  32. 用5%己烷/二氯甲烷洗脱,得到含亮粉色斑点的洗脱液。
  33. 通过CC(参见步骤24)进一步分离该洗脱液,使用增加极性的己烷/乙酸乙酯混合物(从100%己烷,己烷/乙酸乙酯9:1,8:2,7:3,6:4,1:1, 4:6,3:7,2:8,1:9,最后是100%乙酸乙酯)。在用茴香醛硫酸试剂(Rf = 0.40,在己烷/乙酸乙酯7:3中)喷雾后,洗脱液之一在TLC上表现为单个粉红点。使用旋转蒸发器在≤40℃下减压浓缩该洗脱液至干,将其溶解在氯仿-d(2.5ml)中,并转移至NMR管中。进行1 H,13 C NMR和MS分析,显示存在两种三萜,即泛酸酸(Escobedo-Martinez et al。 ,2012)和双氢酸(Escobedo-Martinez等人,2012)。
  34. 将乙酸乙酯萃取物的一部分(5至10g)用极性递增的己烷:乙酸乙酯混合物洗脱(用100%己烷,己烷/2,7:3,6:4,1:1,4:6,3:7,25:75,2:8,1:9,最后是100%乙酸乙酯)(步骤13-15)。 >
  35. 分馏用40%乙酸乙酯的己烷溶液洗脱的VLC级分,通过凝胶过滤(步骤28),用5%己烷的二氯甲烷溶液洗脱,然后用二氯甲烷洗脱,得到双环酸和异富勒酸的混合物(Escobedo-Martinez等al。,2012)以及作为纯化合物的甘草酸和芒果叶酸

代表数据


图1.使用分液漏斗的液 - 液分区的典型设置


图2.真空液相色谱法(VLC)的典型设置

A

B

C

D

图3.薄层色谱(TLC)分析 A.在TLC板上点样样品; B.点样板置于TLC罐中用于洗脱; C.板的加热; D.三萜(粉红色)斑点的Rf值的计算。


图4.柱层析的典型设置

笔记

  1. 为了防止气泡的形成,将浆料缓慢倒入塔的内壁上,并且不应该使填充塔干燥。任何气泡都可以轻轻地用手指点击柱子来除去。
  2. 也可以使用一些再生的Sephadex LH-20。后者通过将任何使用的Sephadex LH-20浆液浸入与真空水喷射泵连接的Büchner过滤器中的二氯甲烷和甲醇的混合物中而获得。一旦施加真空,将Sephadex?sup-LH-20在20-25℃下在工作台上风干。

食谱

  1. 苯甲醛 - 硫酸试剂(Waldi,1965)
    将0.5ml的对 - 茴香醛与10ml冰乙酸和85ml甲醇混合
    向该混合物中缓慢加入硫酸(5mL) 混合均匀,放入玻璃雾化器(琥珀色)瓶中,并在2-8℃下保存

致谢

该方案改编自以前发表的研究,Kardar等人(2014)。这项工作得到了英国Leverhulme信托基金(研究项目赠款RPG-150)的支持。我们感谢T. Zhang的技术援助和Nature's Laboratory Ltd供应蜂胶。

参考文献

  1. Basyuni,M.,Oku,H.,Inafuku,M.,Baba,S.,Iwasaki,H.,Oshiro,K.,Okabe,T.,Shibuya,M.and Ebizuka,Y。 来自红树树种的多功能三萜合酶cDNA的分子克隆和功能性表达<坎布里亚candel (L.)Druce。 Phytochemistry 67(23):2517-2524。
  2. Braithwaite,A。和Smith,F.J。(1996)。色谱方法。第5版。
  3. Coll,J.C。和Bowden,B.F。(1986)。 真空液相色谱法在分离萜烯混合物中的应用 J Nat Prod 49,934-6。
  4. Da Silva,M.S.S.,Cito,A.M.G.L.,Chaves,M.H.,Lopes,J.A.D。(2005)。 来自Teresina-PI的蜂胶环萜三萜类化合物 Quim Nova 28 ,801-804。
  5. Determann,H。和Brewer,J.E。(1975)。凝胶色谱法。在:Heftmann,E。(ed)。 色谱 - 色谱和电泳方法的实验室手册。 Springer,164-88。
  6. Escobedo-Martinez,C.,Concepcion Lozada,M.,Hernandez-Ortega,S.,Villarreal,M.L.,Gnecco,D.,Enriquez,R.G.and Reynolds,W。来自印度猕猴桃的新环三甲基甘油三萜类化合物的(1)H和(13) 50(1):52-57
  7. HernándezVázquez,L.,Palazon,J.,Navarro-Oca?a,A.(2012)。五环三萜类化合物:来源和生物活性的综述。在:Rao,Venketeshwer(ed)。 植物化学 - 它们在营养和健康中的作用的全球视角。在Tech Publisher。
  8. Kamisako,W.,Honda,C.,Suwa,K。和Isoi,K。(1987)。 从[1-13C] - 生物合成的13 C富集的环木菠萝烯醇的13 C NMR谱, 2-13C] - 和[1,2 -13C2] - 乙酸乙酯。修订的13 C NMR光谱分配的环木菠萝烯醇和环木菠萝烷醇和13 C NMR光谱支持普遍接受的环木菠萝烯醇的骨架形成机制。 Magn Res Chem 25,683-687。
  9. Kremmer,T。和Boross,L。(1979)。凝胶色谱法。由Gabor,M.John Wiley和Sons编写。 布达佩斯 17-31。
  10. Mahato,S.B.and Kundu,A.P。(1994)。 五环三萜类化合物的13 C NMR光谱 - 合成和一些显着特征。 Phytochemistry 37,1517-1575。
  11. Pelletier,S.W.,Choksi,H.P.and Desai,H.K。(1986)。 使用真空液相色谱法分离二萜类生物碱混合物。 /em> 49,892-900。
  12. Ravindranath,B。(1989)。 色谱法的原理和实践 em> Ellis Harwood Ltd。 193-292。
  13. Stahl,E.and Mangold,H.K。(1975)。薄层色谱技术。在:Heftmann,E。(ed)。 色谱 - 色谱和电泳方法的实验室手册。 Springer-Verlag,164-88。
  14. Stock,R.and Rice,C.B。(1974)。色谱方法。 279-317。
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  17. Zhu,Z.H.,Liu,W.H.,Ge,L.G.,Yu,Q.,Zhao,W.C.,Yang,J.H.and Wan,H.T。(2012)。 分子克隆和表征编码来自贝母贝类的环木菠萝脑醇合酶的cDNA Miq 11,6896-6903。
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引用:Kardar, M. N. and Seidel, V. (2015). Isolation of Triterpenes from Propolis (Bee Glue). Bio-protocol 5(20): e1630. DOI: 10.21769/BioProtoc.1630.
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