Studies On The Chemical Constituents Of Phlomis Younghusbandii

Objective:1. To study the chemical constituents of Phlomis younghusbandii.2. To establish RP-HPLC methods for the determination of iridoid glycosides and phenylethanoid glycosides, respectively in Phlomis younghusbandii.3. To study on the compositions and the contents of iridoid glycosides and phenylethanoid glycosides among Phlomis younghusbandii,Lamioplomis rotata,Phlomis umbrosa and Phlomis medicinalis Diels, which can provide reference for their further development.4. To study the analgesic and anti-inflammatory effects of Phlomis younghusbandii.Methods:1. Chemical constituents were isolated and purified by macroporous resin, TLC, silica gel column chromatography and Pre-HPLC. Their structures were identified by physical and chemical properties and MS, NMR spectra date.2. Five iridoid glycosides (sesamoside, shanzhiside methyl ester,7,8-dehydropenstemoside, penstemoside,8-O-acetylshanzhiside methyl ester) and three phenylethanoid glycosides (acteoside, alyssonoside, isoacteoside) were determined by HPLC. HPLC analysis was performed on Symmetry C18 (4.6 mm×150 mm,5μm, Waters) column, for the iridoid glycosides the mobile phase was gradient elution of acetonitrile-water,0-5 min:7% acetonitrile; 5-10 min:7%-12% acetonitrile; 10-40 min:12% acetonitrile. The flow rate was 1.0 mL·min-1. The column temperature was 20℃and the detection wavelength was 235 nm; for the phenylethanoid glycosides the mobile phase was the mixture of methanol-water (36:64, pH=4.5, HAc). The flow rate was 1.0 mL·min-1. The column temperature was 20℃and the detection wavelength was 330 nm.3. HPLC was used to identify and determine the content of iridoid glycosides and phenylethanoid glycosides in the four medicinal plants:Phlomis younghusbandii Lamioplomis rotata,Phlomis umbrosa and Phlomis medicinalis Diels.4. Administered the water extract of Phlomis younghusbandii by intraperitoneal injection, the acetic-acid induced writhing times, the hot plate threshold, carrageenan-induced hind paw edema, xylene-induced ear swelling, and acetic acid induced the peritoneal capillary permeability in mice were observed and determined to evaluate the analgesic and anti-inflammatory activities of Phlomis younghusbandii.Results:1. Fourteen compounds were isolated and identified respectively as shanzhiside methyl ester (1), sesamoside (2),7,8-dehydropenstemoside (3), penstemoside (4),8-O-acetylshanzhiside methyl ester (5), phlomiol (6), acteoside (7), alyssonoside (8), isoacteoside (9), triacontanoic acid (10), triacontanoic acid,2-4(4-hydroxyphenyl)ethyl ester (11),β-sitosterol (12),β-sitosterol heteroside (13) and sucrose (14).2. The linear ranges of sesamoside, shanzhiside methyl ester,7,8-dehydropenstemoside, penstemoside,8-O-acetylshanzhiside methyl ester acteoside, alyssonoside, isoacteoside in HPLC were 48-648(r=0.999 3),48-336 (r=0.999 5),40-280(r=0.999 4),10-70 (r=0.999 6), 40-280(r=0.999 7),20-50(r=0.999 6),10-130(r=0.999 5),10-130 (r=0.999 7)μg·mL-1, respectively. The average recoveries (n=6) of them were all between 96% and 104%, RSD<5%.3. Phlomis younghusbandii,Lamioplomis rotata,Phlomis umbrosa and Phlomis medicinalis Diels have the similar chromatograms and contain plenty of iridoid glycosides and phenylethanoid glycosides. In particular, the larger content sesamoside, shanzhiside methyl ester, acteoside are widely included in the four medicinal plants.4. The water extract of Phlomis younghusbandii could remarkably reduce the number of writhing reaction induced by acetic acid in mice and the high dose could prolong the leg-licking time in mice provoked by hot plate. It could significantly inhibit mice auricula swelling and paw edema induced by xylene and carrageenan respectively, it also reduced permeability of abdominal capillaries and inflammatory peritoneal exudation induced by acetic acid.Conclusions:1. Compounds 3,7,8,9,10,11,13,14 were all isolated from the plant for the first time, especially the phenylethanoid glycoside compounds were isolated for the first time.2. The methods are simple, accurate and good reproducibility, and can be used for quality control of Phlomis younghusbandii.3. Phlomis younghusbandii,Lamioplomis rotata,Phlomis umbrosa and Phlomis medicinalis Diels have the similar iridoid glycosides and phenylethanoid glycosides. 4. The water extract of Phlomis younghusbandii showed a certain analgesic and anti-inflammatory activities.