Studies On Chemical Constituents Of Cistanche Tubulosa

Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com

Intro

Cistanche tubulosa is a kind of parasitic plant parasitic on the roots of multi-branched Tamarix vamosissma or other desert plants. It is distributed in the southern region of Xinjiang, China. Its medicinal part is its dry fleshy stem. Its medicinal materials are Cistanche, Grand-Rue, or Inch-Rue. It is commonly used for kidney tonifying and the Yang strengthening medicine in traditional Chinese medicine. Modern pharmacological studies believe that it has anti-aging functions such as prolonging life, so it is also called "desert ginseng". There are few studies on its chemical components, and some phenylethanoid glycosides were extracted by Kobayashi Hiromi et al. Six monomeric compounds I, IL, III, IV, V, and VI were isolated from the extract and identified as D-Mannitol by chemical and spectral methods, β- Sitosterol, daucosterol, 8-epibrucine, echinacoside, and Cistanoside A. The latter two glycosides are active components for strengthening the Yang and anti-aging. Compounds I, II, III, IV, and VI have not been reported in Cistanche deserticola yet.

Experimental

1. materials and instruments

The experimental Cistanche deserticola was purchased from Hotan pharmaceutical company, Xinjiang. The macroporous resin was type AB-B, produced by Nankai University. Dextran gel LH-20 was the product of the Shanghai Long March pharmaceutical factory. Type X4 micro melting point instrument was used for melting point measurement, and the thermometer was not calibrated. The IR instrument used was Shimadzu-440, and the models of H-NMR and C-NMR instruments were Varian FT-80A.

2. extraction and separation

Take 8kg of Cistanche deserticola, reflux and extract with 95% EtOH, place the extract overnight, precipitate Compound I, concentrate the solution under reduced pressure to the extract situation, add an appropriate amount of water for suspension, and extract successively with EtOAc and n-BuOH to obtain 3 parts: EtOAc, n-BuOH and water layers. Compounds II and III were obtained by the silica gel column chromatography and eluted by CHCl3 MeOH (100: l → 10: l); The aqueous layer was eluted with water and 50% EtOH through an activated carbon column, and the 50% EtOH eluted part was eluted with silica gel column chromatography and CHCl3 EtOH (10:4) to obtain compound IV; The part of n-BuOH eluted with water and methanol respectively through the macroporous resin column after decompression and concentration, collect the methanol elution part, and then obtain phenolic glycosides through the polyamide column layer. The phenolic glycosides were then separated and depurated by the silica gel column (CHCl3:MeOH: H2O 6:4:l elution) and Dextran gel LH-20 (MeOH: H20 was 1:1 eluted), and the yellowish amorphous powder V and VI. were obtained.

3. Components identification

Compound I: colorless acicular crystal (EtOH), mp166 ~ 168 ℃, [a] 15D + 21.3 ° (measured in borax, 1g of this component, and 1.28g of borax, add water to 10ml), IR (KBr) υ 3400, 3250, 1460, 1350, 1080, 1020cm-1. H-NMR(D2O) δ 3.4~4.0ppm. Colorless square crystals were obtained by acetylation with the conventional method, mp 121 ~ 122 ℃, IR (KBr) υ 1735, 1450, 1370, 1370, 1215, 1085, 1030cm-1, the above data are compared with mannitol standard, and the results are consistent.

Compound II: colorless acicular crystal (EtOH), mp140 ~ 142 ℃, IR (KBr) υ 3400, 2950, 1630, 1460, 1380, 1060cm-1. And the melting point of the mixture with β- sitosterol standard does not decrease. Thin-layer chromatography Rf values are consistent, so Compound II is determined as β- Sitosterol.

Compound III: white amorphous powder, α- Naphthol test was positive. mp282～284℃, IR(KBr) υ3400, 2950, 1630, 1465, 1380, 1075, 1020cm-1. Compared with the standard directly, III was determined as daucosterol.

Compound IV: colorless acicular crystal (EtOH), mp147 ~ 150 ℃, IR (KBr) υ 3400, 2800~2400, 1680, 1630, 1430cm-1. H-NMR(C5D5N) δ 1.16 (3H, D, j = 7Hz, CH3), 2.1 ~ 2.6 (2h, m, H-6), 2.7 (1H, m, H-8), 3.25 (1H, m, H-9), 3.5 (1H, m, H-5), 4.3 ~ 5.2 (H-7, masked by glycosyl proton signal), 5.4 (1H, D, j = 7Hz, terminal proton), 5.82 (1H, D, j = 4Hz, h-1), 7.85 (1H, s, H-3) ppm. C-NMR(C5D5N) δ 15.8 (C-10), 29.6 (C-5), 43.2 (C-8), 44.2 (C-6), 47.3 (C-9), 63.0 (C-6’,), 71.7 (C-4’), 74.3 (C-2’), 78.1 (C-7, C-3’), 78.5 (C-5’), 95.0(C-l), 100.5(C-1’), 114.7(C-4), 150.4(C-3), 170.3 (C-11) ppm. Penta acetylate (IV A) of IV: 70mg crystal IV was dissolved in 0.7ml pyridine and 0.7ml acetic anhydride and placed at room temperature for 2h. The reaction product was eluted by silica gel column chromatography and CHCl3 MeOH (20: l) to obtain 40mg white penta acetylate. IR(KBr) 3350, 1750, 1675, 1630, 1365cm-1. H-NMR(CDCl3) δ 0.99 (3H, d, j = 7Hz, CH3), 1.96 (3H, s, OCH3), 2.02 (9H, s, OCOCH3 * 3), 2.10 (3H, s, OCOCH3), 2.15 ~ 2.50 (2H, m, H-6), 2.7 (1H, m, H-8), 2.95 (1H, m, H-9), 3.75 (1H, m, H-5), 4.2 ~ 5.3 (H-7 masked by glycosyl proton signal), 5.35 (1H, D, J=2.5Hz, H-1), 7.43 (1H, s, H-3) ppm. According to the above spectral data of IV and IV a, combined with the literature values, IV was identified as 8-epibrucine.

Compound V: amorphous powder, silica gel thin layer chromatography Rf value of 0.3 (CHCl3: MeOH: H2O for 6: 4: l expansion, 1%FeCl3 spray detection), UV (H2O) λ 222, 320nm. IR(KBr) υ 3350, 1690, 1625, 1596, 1515cm-1. H-NMR (D2O) δ 1.07 (3H, d, CH3), 2.86 (2H, t, AR-CH2-CH2 -), 4.3, 4.37, 5.l6 (1H each, glucose and rhamnosyl end group protons), 6.6 ~ 7.1 (6H, aryl H), 6.4 and 7.7 (1H each, d, J=16Hz, caffeoylene protons) ppm. C-NMR (D2O+DSS) δ 133.8, 119.0, 145.0, 146.5, 119.4, 123.8, 73.0, 37.0 (phenylethanol group above), 129.3, 116.0, 150.7, 147.1, 117.9, 125.3, 170.7, 116.0, 149.6 (caffeic acid group above), 104.6, 76.8, 83.6, 71.8, 75.2, 70,3 (intermediate glucose group above), 103.0, 73.5, 73.5, 74.5, 72.2, 19.6 (rhamnose group above), 105.0, 75.8, 78.3, 72.5, 78.3, 63.1 (terminal glucose group above) ppm. Compared with the literature, V is identified as echinacoside.

Compound VI: amorphous powder, silica gel thin-layer chromatography RF 0.35 (the development and detection conditions are the same as above), UV and IR spectra are very similar to those of echinacoside, and there is a δ 3.80 peak in the H-NMR spectra, there is a δ 57.1 peak in the C-NMR spectrum, proved there is an OCH3 group on it. C-NMR(D2O+DSS) δ 134.0, 116.0, 149.5, 146.4, 119.3, 123.9, 73.0, 37.2 (the above is phenyl ethanol group, δ 149.5 indicated the substitution position of OCH3), 129.5, 116.0, 150.7, 147.2, 117.9, 125.4, 170.9, 116.5, 149.6 (caffeoyl above), 104.6, 76.7, 83.2, 71.8, 75.3, 70.3 (intermediate glucosyl above), 104.0, 73.5, 73.5, 74.3, 72.3, 19.7 (rhamnosyl above), 105.1, 75.9, 78.3, 72.5, 78.3, 63.0 (terminal glucosyl above), 57.l (methoxy) ppm. The above data are consistent with the spectral data of Cistanoside A isolated from the same genus Cistanche salsa.