| Cyclocarya paliurus (Batal.) Iljinskaja (C. paliurus) is a rare and endangered plant growing in southern provinces of China and now only a few populations remained due to the great difficulty in propagation and cultivation. This plant is often used for functional foods, drugs, landscape and timber. Researches indicated that C. paliurus leaves contained many functional components such as triterpenoids, flavonoids, polysaccharides, saponins, and microelements, and therefore were valuable natural resource for functional food. Development and utilization of this plant are seriously hampered for scarce resources due to the difficulties in reproduction. In order to explore the feasibility of triterpenic acid production with hypoglycemic effect by suspension culture and provide reference for manufactures of triterpenoid functional food and the utilization of C. paliurus, cell suspension culture of C. paliurus was established, and the accumulation, extraction, separation, and identification of triterpenic acids in suspended cultured cells were studied in this paper. At the same time, the hypoglycemic effect and mechanism of triterpenic acids were explored.The crisp vigorous calli which were fit for suspension culture were obtained from the calli induced form germless immature C. paliurus leaves after sub-culturing and screening repeatedly. Good cell suspension cultures have been established by repetitious subculture and optimization of culture conditions when the suitable calli were inoculated into liquid culture medium. The optimum date for subculture was the 8th day after inoculation, and the highest biomass yield appeared at the 10th day. but the maxmum yield of TTA (total triterpenic acid) appeared at the 14th day. Higher yield of TTA could be obtained in MS medium (Murashige and Skoog mediums) when the phosphate, Ca++, and Mg++ concentration was adjusted to 1.875 mM,2 mM, and 2 mM respectively. TTA yield increased 35.70% and 22.31% respectively when the suspended cultured cells were induced by CeCl3 and salicylic acid. The yield of TTA, UA (ursolic acid), and OA (oleanolic acid) were as high as 1179.85 mg·L-1, 443.64 mg·L-1, and 159.00 mg·L-1 respectively in cell suspension culture of C. paliurus. The major triterpenic acids in the suspended cultured cells of C. paliurus were extracted, separated, and identified for the first time. High TTA extraction yield could be obtained from the suspended cultured cells by the ultrasonic extraction with acetoacetate, and the crude extraction was easy to purify for little impurities. The maximum TTA extraction yield reached 5.56% when the extraction was performed under the optimal parameters namely 40 min,50℃, and 1/10 of material/acetoacetate. Five triterpenic acids were separated from TTA by macroporous resin adsorption, silica gel column chromatography, Sephadex LH-20 column chromatography and semipreparative high performance liquid chromatography, and identified as UA, OA, BA (betulinic acid), CA ((2a,3(3)-2,3-Dihydroxy-urs-12-en-28-oic acid), and MA ((2α,3β)-2,3-Dihydroxy- olean-12-en-28-oic acid) respectively by determination of HPLC, TLC, RRLC-TOF/MS,1H NMR 13C NMR, and DEPT. This is the first report that BA and MA was separated and identified from C. paliurus.The hypoglycemic effects of triterpenic acids from suspended cultured cells of C. paliurus were investigated with in vitro model of 3T3-L1 preadipocytes, mature adipocytes, and IR mature adipocytes. The main results showed that:(1) Improvement on the proliferation of 3T3-L1 preadipocytes wasn’t observed when TTA, UA, OA, and BA were added into the culture medium, but 2.5μg·mL-1 MA did improve the proliferation. (2) TTA, UA, OA, and BA didn’t stimulate the glucose consumption of preadipocytes, but 2.5-5μg·mL-1 CA and MA increased glucose consumption with or without the stimulation of insulin. (3) 5μg·mL-1 TTA, UA, and OA significantly improved the differentiation of preadipocytes, but 5μg·mL-1 CA inhibited differentiation, and BA and MA didn’t show effects on differentiation. (4) TTA, UA, OA, CA, and MA all increased the glucose consumption of mature adipocytes with or without the stimulation of insulin, which indicated that the mechanism of triterpenic acids on the enhancement of glucose consumption differed from Ros (rosiglitazone). (5) TTA, UA, and MA could prevent mature adipocytes from formation of insilin resistance (IR). Although OA and CA couldn’t prevent IR formation, but both could alleviate IR symptom. BA had no effect on the IR formation. (6) TTA and UA could significantly alleviate IR symptom and enhance the sensitivity to insulin for IR adipocytes, MA, OA, CA showed weaker effect than that of TTA and UA, but BA had no effect on IR adipocytes. (7) All triterpenic acids significantly decreased FFA (free fatty acids) content of culture medium at suitable concentration, which might interpret the mechanism of alleviation of IR symptom and improvement of glucose consumption.IR adipocytes treated by 7.5μg·mL-1 TTA,5μg·mL-1 UA,5μg·mL-1 OA,5μg·mL-1 MA,3.6μg·mL-1 Ros and adipocytes of control, IR model were collected for experiments on hypoglycemic mechanism. This paper investigated the moleculer hypoglycemic mechanism by detecting on mRNA expression of PPARy, CAP, Glut4, Resistin, and IRS-1 which played important role in the signal transduction of insulin. The main results showed that:(1) UA might improve the sensitivity to insulin and the consumption of glucose by two pathways:firstly up-regulated PPARy and Glut4 (downstream responsive gene of PPARy) mRNA expression, secondly enhanced IRS-1 mRNA expression. (2) OA stimulated glucose consumption by down-regulation of Resistin mRNA expression. (3) MA might improve the sensitivity to insulin by increasing the expression of CAP and IRS-1 mRNA. It should be noted that MA improved CAP mRNA expression although it didn’t enhance the expression of PPARγmRNA, which suggested that there were still other pathways to regulate the expression of CAP mRNA except PPARy pathway. (4) As mixture of triterpenic acids, TTA showed comprehensive effects as a whole by two pathways:firstly increasing the expression of downstream responsive genes such as CAP and Glut4 by up-regulation of PPARγmRNA, secondly increasing the expression of IRS-1 mRNA. (5) Triterpenic acids from suspended cultured cells of C. paliurus improved the sensitivity to insulin and the consumption of glucose by the pathways different from Ros.The main results can be concluded as:(1) There were five triterpenic acids namely UA, OA, BA, CA, and MA in the suspended cultured cells of C. paliurus. (2) Cell suspension culture of C. paliurus could be used to produce triterpenic acids such as UA and OA. (3) Triterpenic acids from suspended cultured cells of C. paliurus showed good hypoglycemic effect. (4) Triterpenic acids from suspended cultured cells of C. paliurus improved glucose consumption by increasing the expression of PPARy, CAP, Glut4. and IRS-1 mRNA and decreasing the expression of Resistin mRNA. |
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