Screening For Effective Dyslipidemia-Modulating Compositions And Tablet Preparation Of Fenugreek

ObjectiveConsidering the present studies about hyperlipidemia-modulating effects of fenugreek, a plant utilized as food or medicine from ancient, we compare the lipid-modulating functions of polysaccharide(PS) and degraded polysaccharide(DPS) from this plant. Furthermore, systematic molecular mechanisms of lipid-modulating activities of each ingredient from fenugreek were analyzed. In addition, powder properties of PS and DPS were compared. Finally, fabricating crafts DPS were evaluated. This study offered fundamental studies for lipid-modulating ingredient from fenugreek and relavent new product, especially the development of health food. MethodsIn preliminary studies, aqueous extract was extracted by distilled water, and ethanol extract was prepared by ethanol-extraction from powder of fenugreek seeds. In order to obtain saponin, part of ethanol extract was purified through systematic solvents and macro reticular resins. The residue of ethanol extraction was used for preparing polysaccharide through aqueous extraction and methanol precipitation. Degraded polysaccharide was fabricated from polysaccharide by enzyme-digesting.Hyperdyslipidemia rats’ model was established. After dealed with ingredients stated above, body weigh and liver/weigh ration were calculated. Total cholesterol(TC), triglyceride(TG), low-density cholesterol(LDL), and high-density cholesterol(HDL) were tested. Liver impairing and repairing situations were observed through testing glutamic-pyruvic transaminase(ALT), Aspartate Transaminase(AST) levels in serum, and observing the liver pathological sections. Anti-peroxide activities of each ingredient were evaluated in vivo with determining serum malondialdehyde(MDA), superoxide dismutase(SOD), glutathione peroxidase(GSH-PX) levels.On the base of preliminary studies, lipid-modulating mechanisms of each ingredient were evaluated in vivo. Efflux of TC and total bile acid(TBA) contents were tested in feces. Combinative activity to cholesterol micelle of polysaccharide and degraded polysaccharide was evaluated in vitro. Protein expression of NPC1L1 and ABCG8 in small intestine was tested through western blot for evaluating influences of each ingredient to cholesterol absorption in vivo. Effects on other processes like cholesterol metabolism, reverse cholesterol transition(RCT), and synthesis of each ingredient were also detected by determining m RNA expressions of factors including CYP7A1, ABCG8, NPC1L1, ABCA1, SR-BI, HMG-Co A reductase and SREBP-1c in liver through q RT-PCR and HMG-Co A reductase in serum through ELISA method.Powder properties of polysaccharide and degraded polysaccharide were compared and analyzed. Particle size was test by sieving method. Angle of repose was tested by oscillating funnel method. Respective fluidity was evaluated by calculating Hausner index and compressibility depend on respective bulk density and tap density. Hygroscopicity was determined by standard hygroscopicity curve, which stretched by respective obtained weigh in different environment of different humidity. Viscosity-average molecular weight was tested by Ubbelohde viscometer. Fabricating crafts of degraded polysaccharide tablet was studied preliminarily, and qualities evaluations including appearance, weight variation, rigidity, friability and disintegration time were evaluated. ResultsPurified saponin and polysaccharide reached purities of 80.50% and 75.56% respectively. Degraded polysaccharide contained 27.78% reduced sugar. After fed with HFD, serum TC and TG level of rats were obviously elevated. Through the lipid-modulating activities’ studies of PS and DPS, both of them were able to reduce TC, TG, LDL and elevated HDL levels, manifesting that they could modulate normal lipid detectors in serum. Both of them normalized serum ALT level, and inhibited abnormal accumulation of lipid in liver, assuming that they can protect liver tissue under high-fat-diet condition. Improved SOD levels could be observed in both PS and DPS groups, but none of them showed obvious change of GSH-PX level. PS abnormally elevated MDA levels, implying that both of them showed antioxidant activities in vivo, but DPS is more considerable.Both polysaccharide and degraded polysaccharide showed combinative activities to cholesterol michelle, and the latter was more desired, implying that they might combine to cholesterol michelle in intestine and accelerate its efflux. Each ingredient facilitated TBA efflux, and saponin groups dose-dependently facilitated cholesterol efflux. DPS reached the effect level of high-dose PS. Saponin elevated NPC1L1 protein expression in small intestine and CYP7A1, ABCA1 m RNA expression in liver, illustrating that it can accelerate processes including cholesterol absorption, metabolism and reverse cholesterol transfer. On the other hand, both polysaccharide and degraded polysaccharide inhibited NPC1L1 but elevated ABCG8 protein expression in small intestine, assuming that they can inhibit absorption of cholesterol and bile acid. All ingredients successfully suppressed HMG-Co A reductase content in serum and m RNA expression in liver, assuming that they probably inhibit synthesis of cholesterol in vivo. However, saponin could elevate SREBP-1c m RNA expression in liver, revealed that it might accelerate synthesis of triglyceride in vivo.In aspects of Powder properties, powder size of degraded polysaccharide was similar of polysaccharide(186.54±25.99μm vs. 188.01±33.47μm), but ratio of large particle is lower. Angle of repose, Hausner index and compressibility of DPS was less than PS(P<0.05 or P<0.01), which showed desirable fluidity and glomeration. Viscosity of DPS was obviously lower than that of PS, viscometric average molecular weight of DPS is about 13% of DPS. Critical relative humidity of DPS was slightly higher than that of PS(65.72% vs. 62.84%). Optimized craft of fabricating degraded polysaccharide tablet was available and passed normal qualities tests of tablet. Appearance: the surface was clean, without plot. Theoretical tablet weight was 146 mg, and there were no sample accede the range of theoretical tablet weight±7.5%. Rigidity was 5.40±0.07 kg, friability was 0.46%, and disintegration time was 837.33 ± 9.56 s. Parameters above were accord with the tablet requirement in Chinese pharmacopoeia(2010 version). ConclusionAll ingredients showed desirable activities for ameliorating hyperlipidemia, and degraded polysaccharide showed more desirable effects. Results of mechanism studies revealed that lipid-ameliorating activities of fenugreek were due to systematic effect of influences on cholesterol transfer process in vivo including absorption, metabolism, reverse cholesterol transfer, and synthesis by different ingredients. Studies on powder properties showed that DPS was more suitable for studies on further fabricating craft. Finally, this study preliminarily studied tablet fabricating craft, for offering foundation of developing new healthy medicine for modulating dyslipidemia.