Table 13.
Separation, isolation, and purification of citrus phytochemicals from the crude extract obtained from different extraction procedures.
| Citrus Species | Fruit Part/Extraction | Separation/Isolation Method/Determination | Column/Mobile Phase | Isolated Phytochemicals | Ref. |
|---|---|---|---|---|---|
| C. sinensis L. (Florida, USA) | Peel extract | 1. Flash Chromatography-(UV spectrum = 254 nm) 2. Reverse-phase HPLC 3. Prep-HPLC (UV spectrum = 326 nm) |
1. 330 g prepacked silica gel (particle size 35–60 µm) flash column/ethyl acetate (10–40%) and hexanes (90–60%); isopropanol (15%) and hexanes (85%) 2. Semi prep-HPLC: YMC HPLC column (75 × 30 mm i.d., 5–10 µm particle size) 3. Regis Whelk-O 1–450 g column/ 35% absolute ethanol and 65% hexanes |
Nobiletin and 5,6,7,4′-tetramethoxyflavone | [215] |
|
C. sinensis
Florida, USA |
Peel extract (cold-pressed oil) |
1. Normal Phase Chromatography 2. Flash Chromatography (UV spectrum = 254 nm) 3. Reversed-phase analytical HPLC 4. Prep-HPLC 5. Semi-preparative reversed column HPLC 6. Supercritical Fluid Chromatography (UV spectrum = 220 nm) 7. Liquid chromatography (LC)–electron spray ionization mass spectrometry (ESI-MS) |
1. Silica gel (60 Å, 32–63 µm) columns (330 g) 2. Silica gel (particle size 35–60 m) flash column/ethyl acetate and hexanes; 3. Octadecyl (C18) derivatized silica gel (4.6 mm × 50 mm, 5 µm) 4. Preparative HPLC (30 mm × 75 mm, 10µm, ODS-A) columns/35% acetonitrile and 65% water 5. C18 reverse-phase column, Xterra OBDTM (19 mm × 100 mm) 6. SFC chiral column (30 mm × 250 mm, 5 µm)/CO2 7. Chromegabond WR C18, 3 µm, 120 Å; 30 mm × 3.2 mm/acetonitrile and H2O with 0.05% TFA, typical gradient of 10–90% acetonitrile |
Nobiletin, tangeretin, 3,5,6,7,8,3’,4’-heptamethoxyflavone and 5,6,7,4’-tetramethoxyflavone | [222] |
| C. sinensis | Peel oil | 1. Open column chromatography 2. Reversed-phase purification chromatography 3. analytical silica gel TLC 4. Analytical HPLC–MS |
1. Silica gel prepacked-170 g column/hexane/ethyl acetate 1:3 to 2:1 (v/v) 2. RediSep reversed-phase C18 (43 g) column/water/methanol (80/20, v/v-60/40 (v/v) |
3′,4′,3,5,6,7,8-heptamethoxyflavone | [229] |
| C. sinensis | Peel oil (Solvent extraction/Petroleum ether) |
1. Isolation 2. Crystallization 3. Hydrolysis-Reflux |
1. Carbon tetrachloride 2. Crystallization-Methanol 3. Hydrolysis-Reflux: Ethanol and Aqueous potassium hydroxide |
Nobiletin | [230] |
|
C. sinensis
(Florida, USA) |
Peel Molasses | 1. Filtration and Ultrafiltration 2.Fractionation- 3. Size exclusion chromatography (SEC) 4. Analytical HPLC (PDA = 230 and 600 nm): LCMS-ESI-MS |
1. Grade 161 glass fiber filter and Romicon model HF4 hollow fiber cartridge ultrafiltration system 2. Sepabeads SP70 column (1.5 cm × 29 cm/H2O, 50% acetone Bio-Gel P2 column (5 cm × 82 cm)/15% EthanolDE52 cellulose anion exchange column (2.5 cm × 25 cm) 3. P2 column (5 cm × 80 cm)/ 15% Ethanol 4. Alltech Alltima C8 5 μm analytical column (4.6 mm × 100 mm)/10 mM phosphoric acid/acetonitrile (90:10, v/v): (Flavonoid glycosides were analyzed 2% formic acid/water/acetonitrile |
3,5,7,10,14,16-hydroxycinnamates, Polymethoxylated flavone: Nobiletin, heptamethoxyflavone, and sinensetin, Flavonoid glycosides: Hesperidin and narirutin, narirutin 4′-glucoside, hesperetin trisaccharide, 6,8-di-C-glucosylapigenin, Phenolic glycoside: Coniferin, phlorin Phenolic acids: Ferulic acid, p-coumaric acid |
[231] |
| C. sinensis Osbeck (Local fruit Oslo, Norway) | Peel extract | 1. Open Column Chromatography; 2. Flash Chromatography; 3. Thin Layer Chromatography- UV irradiation (254 and 366 nm) |
1. MPLC column (49 × 900 mm) filled with silica gel (40–63 µm)/Chloroform, ethyl acetate, acetone, methanol 2. Silica gel, 40–63 µm/hexane-ethyl acetate (9:1 or 1:1) 3. TLC (Si gel 60 F254, 0.2 mm thickness/g hexane-ethyl acetate (1:1); toluene-dioxane-acetic acid (90:25:4) |
5-hydroxy6,7,3′,4′-tetramethoxyflavone, sinensetin (5,6,7,3′,4′-pentamethoxyflavone, 3), tetramethylscutellarein (5,6,7,4′-tetramethoxyflavone; 3,5,7,8,3′,4′-hexamethoxyflavone, tangeretin (5,6,7,8,4′-pentamethoxyflavone, 3,5,6,7,3′,4′-hexamethoxyflavone, nobiletin (5,6,7,8,3′,4′-hexamethoxyflavone, 3,5,6,7,8,3′,4′-heptamethoxyflavone; hesperidin (5,3′-dihydroxy-4′-methoxy-7-rutinosyloxyflavanone ferulic acid |
[232] |
| C. kinokuni Hort. ex Tanaka (Shizouka, Japan) | Peel extract | 1. Open Column Chromatography; 2. Preparatory-Thin Layer Chromatography |
1.Silica gel/toluene, CH2Cl2, AcOEt, acetone, and MeOH 2. Si-gel TLC/acetone–CHCl3 (1:9, 1:19 or 1:29), acetone–benzene (2:8), acetone–hexane (3:7), AcOEt–benzene (1:1), AcOEt–hexane (1:1) |
Scoparone, scopoletin, nobiletin, sinensetin, tangeretin, 5-hydroxy-6,7,8,49-tetramethoxyflavone, 5-hydroxy-6,7,39,49-tetramethoxyflavone, 5-demethylnobiletin, 6-demethoxynobiletin, 6-demethoxytangeretin, 5,6,7,49-tetramethoxyflavone, 3,5,6,7,39,49-hexamethoxyflavone, 39-hydroxy-5,6,7,8,49-pentamethoxyflavone, 3,5,6,7,8,39,49-heptamethoxyflavone, 7-hydroxy5,6,39,49-tetramethoxyflavone, 7-hydroxy-5,6,8,39,49-pentamethoxyflavone, 5,7,8,39,49-pentamethoxyflavanone, 5-O-demethylcitromitin, 3,4,39,49,59,69-hexamethoxy-29-hydroxychalcone, 29-hydroxy-4,49,59,69-tetramethoxychalcone, b-sitosterol, (2S)-5,6,7,8,49-pentamethoxyflavanone, (2S)-5,6,7,39,49-pentamethoxyflavanone, 29-hydroxy3,4,39,49,69-pentamethoxychalcone, | [233] |
| C. reticulata Blanco; (Tangerine, China) | Peel extract | 1. High speed countercurrent chromatography; 2. HPLC-PDA = 270 nm) | 1. Multilayer coil planet centrifuge- equipped with a polytetrafluoro-ethylene multilayer coil of 110 m × 1.6 mm, I.D./Two phase solvent system-n-hexane, ethyl acetate, methanol and water 2. Shim-pack VP-ODS column (250 mm × 4.6 mm, I.D.)/acetonitrile and water (50:50, v/v) |
Nobiletin, 3,5,6,7,8,3,4-heptamethoxyflavone, tangeretin and 5-hydroxy-6,7,8,3,4-pentamethoxyflavone | [218] |
| C. reticulata Blanco cv. Ponkan | Peel extract | 1. High speed countercurrent chromatography(Elite UV spectrum-200–330 nm) 2. Prep-HPLC 3. Analytical HPLC- PDA detector 4. Electrospray ionization mass spectrometry (ESI-MS) |
1. 200 mL column with six-layer coils made of 5.0 mm i.d. polytetrafluoroethylene (PTFE) tubing: Separation system composed of a K-1800 Wellchrom pump: a 150 mL sample loop made of 3 mm i.d. PTFE tubing, the high-speed countercurrent chromatograph and a B-684 collector/n-hexane–ethyl acetate–methanol–water (1:1:1:1:1.5, v/v) 2. DS-BP-30 column (250 × 30 mm I.D./methanol–water (60:40, v/v) 3. C18 column (250 × 4.6 mm i.d., 5lm)/methanol and water |
Isosinensetin, sinensetin, nobiletin and tetramethyl-o-scutellarein | [234] |
| C. sunki Hort. ex Tanaka (Jeju, S. Korea) | Peel extract (hot-water extraction) | 1. Fractionation 2. HPLC (Flavonoids) (UV spectrum = 200–400 nm) 2. Semi-prep HPLC (Polymethoxy Flavone) UV-spectrum = 200–400 nm |
1. n-hexane, chloroform, ethyl acetate, n-butanol: dissolved in EtOAc:methanol (MeOH) (1:1, v/v): filtration through 0.50-µm polytetrafluoroethylene (PTFE) 2. Sunfire™ C18 column (250 × 4.6 mm ID; 5 µm)/(A): acetonitrile (MeCN) containing 0.5% acetic acid (B) water containing 0.5% acetic acid 3. Symmetryprep™ C18 column (300 × 7.8 mm ID; 7 µm)/Methanol, Water |
Isosinensetin, sinensetin, tetra-O-methylisosutellarein, 5,7,4′-trimethoxyflavone, nobiletin, tangeretin, 5-demethylnobiletin, 5-demethyltangeretin | [235] |
| C. paradisi (White grapefruit, Florida) | Peel oil (cold-pressed oils) | 1. High speed countercurrent chromatography (UV spectrum = 310 nm) 2. HPLC with Diode Array Detection (HPLC-DAD)3. Gas Chromatography-Mass Spectrometry |
1. Three preparative coils, connected in series (diameter of tubing 2.6 mm, total volume 850 mL)/Hexane–Ethyl acetate; Methanol–Water; hexane/Ethanol–Water 2. C18-Spherisorb ODS2 column (250 × 4 mm, particle size 5 µm) (A) acetonitrile, (B) water 3. DB-5 column (15 m × 0.25 mm i.d. fused silica capillary, 0.5 µm)/Helium gas |
Meranzin hydrate, marmin, epoxybergamottin hydrate, auraptenol, Auraptenol meranzin, epoxyauraptene, and epoxybergamottin, nobiletin, tetra-omethylscutellarein (39), meranzin, isomeranzin, and heptamethoxyflavone, tangeritin, citropten, bergapten |
[236] |
| C. aurentifolia Swingle (Mexico) | Peel oil (Cold-pressed oils) | 1. High speed countercurrent chromatography (UV spectrum = 310 nm) 2. HPLC with Diode Array Detection (HPLC-DAD) 3. Gas Chromatography-Mass Spectrometry |
1. Three preparative coils, connected in series (diameter of tubing 2.6 mm, total volume 850 mL)/Hexane-Ethyl acetate; Methanol-Water; hexane/Ethanol-Water 2. C18-Spherisorb ODS2 column (250 × 4 mm, particle size 5 µm) (A) acetonitrile, (B) water 3. DB-5 column (15 m × 0.25 mm i.d. fused silica capillary, 0.5 µm)/Helium gas |
Herniarin, isopimpinellin, citropten, bergapten oxypeucedanin; bergamottin and, 5-geranyloxy-7-methoxycoumarin |
[236] |
| C. jambhiri Lush (Lemon, Nagaland, India) | Peel extract (petroleum ether) Soxhlet extraction | Open Column Chromatography | Active alumina column/Benzene Washing with petroleum ether and crystallization with Methanol |
Hesperidin, tangeretin, neohesperidin. 5-O-desmethyltangeretin | [237] |
| C. hassaku HORT. ex. TANAKA (Japan) | Peel extract (Ethanol reflux) | 1. Open Column Chromatography; 2. Preparatory-Thin Layer Chromatography; 3. Preparatory-HPLC; UV-spectrum = 313 nm |
1. Silica gel C-200 (Wako) 2. Wako-gel B-O (Wako) 3. ODS-120T column (7.8 mm i.d. × 30 cm)/Methanol–Water |
4′,5,6,7,8-pentamethoxyflavone, 3’,4′,5,6,7,8-hexamethoxyflavone, 3,3’,4′,5,6,7,8-heptamethoxyflavone, 3′, 4’,5,6,7-pentamethoxyflavone, 4′,5,7-trimethoxyflavone, 3,3’,4′,5,7,8-hexamethoxyflavone, 4’,5,7,7.8-tetramethoxyflavone, 3′,4’,5,7,8-pentamethoxyflavone |
[205] |
| Citrus Sinensis.L (sweet orange); India | Peels Solvent extraction (EtOH; Methanol–Dichloromethane–Water (MDW) (0.3:4:1, v/v); MeOH-H2O) | 1. Reversed-phase HPLC 2. Thin layer chromatography 3. HPLC–ELSD; ESI-MS (Evaporative Light Scattering Detector (ELSD) coupled to electrospray ionization mass spectrometric (ESI-MS) |
1. Adsorbosphere column–NH2, (250 × 4.6 mm column)/Mobile phase–acetonitrile–water 2. Cellulose MN 300 G/Mobile phase- N–butanol–acetone–pyridine–water (10:10:5:5, v/v/) 3. Atlantis dc-18 column (50 x 4.6mm—5μm)/ Mobile phase (A) 0.10% formic acid in HPLC grade deionized water; (B) Methanol |
Fructose, galactose, glucose, arabinose and xylose | [238] |
| Sour and sweet oranges, umbilical orange, novel orange, lime, lemon, pink and white grapefruit, aeglemarmelos, bergamot, sour and sweet tangerines and clementines (Iran) | Extraction of juice; Centrifugation | Reversed-phase chromatography UPLC–MS (λ = 254 nm) | (150 mm × 4.6 mm i.d., 5 μm particle ZORBAX Eclipse XDB-C18)/Mobile phase—2% acetonitrile and 50 mM phosphate solution (dissolve 6.8 g potassium dihydrogen phosphate in 900 mL water (pH = 2.8) | Ascorbic acid | [239] |