Study On Compound Lipid-lowering Soft Capsule And Its Mechanism

Objective: To develop pure natural health food, compound lipid-lowering softcapsule with grape seed procyanidins and lycopene as main efficacy components; at thesame time, to study on its quality standard, acute oral toxicity, lipid-lowering effect andmechanisms. Methods: To research the optimum process conditions of procyanidinsextracting in grape seed by supercritical CO2extraction (SCF-CO2). By single factor andorthogonal experiment design, the optimum process conditions were studied. Theprescription of capsule consisted of grape seed procyanidins, lycopene and sunflowerseed oil. The quality standard of the capsule was established according to the relevantspecifications of governmental health food standards. The acute oral toxicity andlipid-lowering effect of the capsule were studied according to health food inspection andevaluation of technical specifications (2003), and its mechanisms of lipid-lowering effectwere studied. Results:1. The development of compound lipid-lowering soft capsule:(1)The technical study of extracting procyanidins from grape seed by supercritical CO2:1)The single factor experiment results showed when the extraction pressure was15MPa,the extraction temperature was50℃, the extraction time was30min, and the volume ofethylalcohol was400mL respectively, the extraction yield of procyanidins was thehighest.2) The orthogonal experiment results showed that the optimum processconditions: the extraction pressure was18MPa，the extraction temperature was50℃，the extraction time was40min, and the volume of ethylalcohol was500mL.3) Themethod for determination of procyanidins content in supercritical CO2extraction fluid ofgrape seed by rapid resolution liquid chromatogruphy (RRLC) was established. ZorbaxXDB-C18Column (4.6mm×50mm,1.8μm) was used, and1.5%acetic acidmethanol-1.5%acetic acid water (90:10, V/V) was used as mobile phase. The flow ratewas0.9mL·min-1, the detection wavelength was280nm and the column temperature was at 40℃. The line correlation of procyanidins content was observed from0.60~3.60mg/mL,y=639.0818x-28.961, R2=0.9997.(2) To study the prescription screening and preparingtechnology of capsule:1) Prescription of capsule: sunflower seed oil63.5%,grape seedprocyanidins8.0%, lycopene oleoresin28.5%, the flowability, cut off and suspensionstability corresponded with requirement of pharmaceutics. On the basis of the basicprescription of soft capsule shell,0.5%TiO2,0.2%amaranth,0.06%grape purple wereadded, the effect of shield and appearance were satisfying. The results of laboratory scaletest and pre-production showed that the preparation technology was scientific andfeasible.2) The quality standard for capsule was established, the content of lycopene incapsule was detected by high-performance liquid chromatography (HPLC) withKromasilC18Column (4.6mm×150mm,5μm), moving phase: acetonitrile:methanol(50:50, V/V), flow rate:1mL·min-1, wavelength:472nm and the column temperaturewas30℃. The line correlation of lycopene content was observed from0.2000～20.0000μg/mL, y=37743676x+893291.2, r=0.9996. The average recovery was101.20%,RSD was1.09%. The content of procyanidins in capsule was detected by catalyticcolorimetry with ferric ions. The line correlation of procyanidins content was observedfrom0.00~1.50mg/mL, y=0.33969x-0.00889, r=0.99909. The average recovery was102.507%, the RSD was5.565%. The uniformity, disintegration time limited of capsuleconformed to the requirements of pharmacopoeia of the People’s Republic of China(2010); the content of functional components and peroxide value of capsule were up tothe standard, and the quality guarantee period was at least2years.2. Toxicology andfunction evaluation:(1) The acute toxicity test was carried out with mice, at the dose of10g/kg·BW, the capsule had no effect on body weight of normal mice (P＞0.05). Theacute oral toxicity test showed that the maximum tolerated dose (MTD) of the capsulewas higher than10g/kg·BW.(2) The influence of GSPE, lycopene and capsuleprescription on normal and hyperlipidemia mice:1) The GSPE, lycopene and capsuleprescription were dosed respectively, which had no effect on body weight, serum TC, TG,HDL and LDL of normal mice (P＞0.05).2) In middle and high dose groups of capsuleprescription, serum TC and LDL levels were decreased, serum HDL level was enhanced(P＜0.05); at the same dose,GSPE or lycopene had no same effect (P＞0.05).(3)Assessment of assisting blood lipids reduction function of capsule.1) From the0week,in comparison with control group, the body weight of other groups was markedlyincreased (P＜0.01); from the1st week, the increase of body weight in model group wasfaster than that of high dose capsule group (P＜0.01); in sixth week, the body weight of all other groups was lighter compared with model group (P＜0.05);2) Compared withmodel group, capsule and positive control Zhibituo groups could decrease the sreum TG,TC levels in the4th and the6th week (P＜0.05).3) In the4th and the6th week,compared with model group, the sreum HDL level was enhanced in high dose capsulegroup (P＜0.05), but the sreum LDL level was decreased in capsule and positive controlZhibituo groups (P＜0.01).4) Compared with model group, the sreum ApoB, LPa andApoB/ApoAI levels decreased in middle, high capsule groups and positive controlZhibituo group (P＜0.01), the sreum ApoAI level enhanced lightly (P＞0.05).5) The allcapsule groups and positive control Zhibituo group could decrease the liver MDA leveland enhance the liver SOD and GSH-Px activities (P＜0.01).6) The organ coefficientwas no difference among groups (P＞0.05).7）The capsule could alleviate the liverpathologic changes induced by high fat diet distinctly.3. Study on the mechanisms ofcompound lipid-lowering soft capsule:(1) Effect of capsule on lipid-metabolizedenzyme level in hyperlipidemia rats.1) The liver HMG-CoA level in middle, high dosegroup and positive control Zhibituo group was decreased，HL level was increased (P＜0.05);2) Compared with the model group, the serum LCAT and LPL levels of high dosegroup were increased significantly (P＜0.01);(2) Study on the molecular mechanisms oflipid-lowing effect in capsule.1) In high dose group treated with capsule, the relativeexpression of LDL-R mRNA in liver was higher than that of mode lgroup (P＜0.05);2)Compared with the model group, the middle, high capsule groups treated with capsuleupregulated the relative expressions of PPARγ mRNA in liver (P＜0.01);3) The relativeexpressions of liver LXRa mRNA in all capsule groups were higher than those of modelgroup, but the difference was no statistical significance (P＞0.05);4) In high dosecapsule group, the relative expression of liver SR-BI mRNA was increased significantly(P＜0.01).5）Compared with the model group, the relative expression of liver ABCA1mRNA was increased in high dose capsule group (P＜0.01). Conclusions:1.Theoptimum process conditions of procyanidins extracting in grape seed: the extractionpressure is18MPa; the extraction temperature is50℃; the extraction time is40min; theethylalcohol addition is500mL. The determination of grape seed proanthocyanidins inthe extract liquor with rapid resolution liquid chromatogruphy (RRLC) is accuracy andprecision, it can provide a new reference method for determination of procyanidinscontent. The prescription of capsule is reasonable, the preparation technology of capsuleis scientific and operable; quality control is simple, quick and accurate. All of relatedindexes conform to the requirements of pharmacopoeia of the People’s Republic of China (2010) and the related standards. The quality guarantee period is2years at least. Thedetection methods of functional components in capsule are accurate and reliable. In softcapsule, the average content of procyanidins is87.9000mg/g, RSD is1.32%, the averagecontent of lycopene in soft capsule is15.2418mg/g, RSD is1.27%.2. The maximumtolerated dose (MTD) of capsule is higher than10g/kg·BW. The prescription of capsulecan decrease serum TC and LDL levels and enhance serum HDL level; lipid-loweringeffect of capsule is better than that of grape seed procyanidins or lycopene usedrespectively. So the lycopene and grape seed procyanidins may have synergistic effect inlipid-lowering. The capsule can reduce the serum TG, TC, LDL, ApoB, LPa levels andthe value of ApoB/ApoA1, increase liver SOD and GSH-Px activities, decrease MDAcontent. The capsule can alleviate the liver pathologic changes induced by high fat dietdistinctly.3. The lipid-lowering mechanisms of capsule: elevating the HL, LPL andLCAT levels, inhibitting the enzyme activity of HMG-CoA in hyperlipidemia rats,accelerating the degradation of TG and TC, and reducing cholesterol synthesis. Capsulecan enchance the relative expressions of LDL-R mRNA, PPARγ mRNA, SR-BI andABCA1mRNA, but the expression of LXRa mRNA does not change significantly. Thereverse cholesterol transport (RCT) is major lipid-lowering mechanism.