| Objective:On the basis of previous experiments,this study established malt germination processing technology and optimal bud length based on its delactation and digestion.Study on the characteristics of HPLC fingerprint of methanol extract from malt and the variation of alkaloids during germination;Comparison of delactation and digestion function of different bud of malt decoction;Optimization of malt germination processing technology and bud length from pharmacopositive of malt alkaloids substance and pharmacodynamics of delactation and digestion.Methods:(Ⅰ)The content of total alkaloids,hordenine in malt and amylase activity was used as index,based on the single factor experiment,the effect of soaking time,germination temperature,germination humidity and daily watering amount on the barley germination were investigated by orthogonal test.The content of total alkaloids and hordenine of malt were determined by Preliminary experimental method in our group;amylase activity of malt was determined by 3,5-dinitrosalicylic acid(DNS)colorimetry.(Ⅱ)Wonda Sil○R C18 column(4.6 mm × 250 mm,5 μm)were used with methanol(A)and 0.1 mol?L-1 potassium dihydrogen phosphate(B)in gradient elution mode at a flow rate of 1.0 m L?min-1.The detection wavelength was set at 270 nm and column temperature was 25 ℃.The fingerprints for 10 batches of malt medicinal materials were set up by HPLC,the similarity assay for 10 batches of malt medicinal materials were carried out to evaluate their quality by Chinese Materia Medica Fingerprint Similarity Evaluation System(2004A edition),while HPLC fingerprint of malt in the germination process were compared in the same condition.The dynamic changes of alkaloid substances in malt germination process were studied by HPLC and acid colorimetry.(Ⅲ)The establishment of HPRL model:the rats were injected into the back of the Metoclopramide Dihydrochloride Injection 75 mg·kg-1 weight,every morning were regular injections of 1 times,10 consecutive days.The levels of PRL,E2 and P in serum were determined by enzyme-linked immunosorbent assay(ELISA);The contents of dopamine D1 and D2 receptors in rat hypothalamus and PRL positive cells in rat pituitary were determined by immunohistochemistry.;The expression of m RNA in rat pituitary PRL cells was determined by PCR.(Ⅳ)Determination of small intestinal propelling rate and gastric emptying rate in different bud of malt decoction group by the charcoal propulsion experiment;The content of pepsin and gastrin in serum of mice in all groups were determined by ELISA.Results:(Ⅰ)The optimized malt best germination process was: soaking time for 5 h,germination temperature at 25 ℃,humidity was 70%,the weight ratio of daily watering amount and barley was 1:1.The way of sprinkling water is a small number of times,and the water content of malt is between 20% and 35%.Dry at 70 ℃ for 12 h.(Ⅱ)There were nineteen common peaks in the fingerprints of ten batches of samples,of which the 7th peak was hordenine.The 5th,7th,8th,10 th,12th,15 th,16th,17 th peak were newly added after the germination of barley;the peak area of 1th,6th,9th,13 th peak gradually increased and then decreased,to reach the maximum in the bud length of 0.75 cm and the peak area of 3th,5th,8th,17 th,18th,19 th peak gradually increased and then leveling in malt germination process.The content of total alkaloid was the largest in malt bud length of 0.75 cm.(Ⅲ)Influence on the content of PRL in serum: compared with the normal group(3.91±1.49 ng·ml-1),the content of PRL in serum of rats in model group(30.404±5.516 pg·ml-1,P<0.01)were significantly increased;compared with the model group,the content of PRL in serum of rats in Bromocriptine positive group and all different bud of malt decoction group were significantly decreased(P<0.01),of which Bromocriptine positive group and 0.75cm(4.41±2.49 ng·ml-1),1.00cm(4.72±2.45 ng·ml-1),1.25cm(4.69±3.61 ng·ml-1)bud of malt decoction group were closed to normal group.The results showed that different bud of malt decoction had certain therapeutic effect on HPLC,and malt length was the best from 0.75 cm to 1.25 cm.Compared with the normal group(553.09±48.74),the number of PRL positive cells in rat pituitary in model group(27415.48±1072.01,P<0.01)were significantly increased;Compared with the model group,the number of PRL positive cells in rat pituitary in Bromocriptine positive group and all different bud of malt decoction group were significantly decreased(P<0.01),of which 0.50 cm to 1.25 cm bud of malt decoction group were closed to normal group,especially in 0.75 cm(1794.35 ±347.62)bud of malt group.Compared with the normal group(4.23±5.27),The expression of m RNA in rat pituitary PRL cells in model group(22.78±4.12,P<0.01)were significantly increased;compared with the model group,The expression of m RNA in rat pituitary PRL cells in Bromocriptine positive group and 0.50cm(8.41±6.90),0.75cm(6.43±3.74),1.00cm(7.83±4.21)bud of malt decoction group were significantly decreased,especially in 0.75 cm(P<0.01)bud of malt group.Compared with the normal group(674.52 ±72.90),the number of dopamine D1 receptors in rat hypothalamus in model group(9.55 ±6.07),bromocriptine positive group(170.66 ±12.00)and all malt group were significantly decreased(P < 0.01);Compared with the model group,the number of dopamine D1 receptors in rat hypothalamus in bromocriptine positive group and all different bud of malt decoction group were significantly increased(P<0.01 or P<0.05),But it were still significantly lower than the normal group.Compared with the normal group(222.41±33.28),the number of dopamine D2 receptors in rat hypothalamus in model group(41.89±14.01),bromocriptine positive group and 0.25 cm,0.50 cm,1.50 cm,1.75 cm,2.00 cm bud of malt decoction were significantly decreased(P<0.01 or P<0.05);Compared with the model group,the number of dopamine D2 receptors in rat hypothalamus in all different bud of malt decoction group were significantly increased(P<0.01 or P<0.05),of which 0.75cm(180.04±37.72),1.00cm(179.29±22.99),1.25cm(179.28±30.59)were closed to normal group.The results indicated that these three groups could restore the number of dopamine D2 receptors in rat hypothalamus to normal,and suggested that D2 receptor should be the main target for malt delactation.(Ⅳ)Compared with the normal group,different bud of malt decoction group can increased small intestinal propelling rate and gastric emptying rate(P < 0.01 or P < 0.05),of which 0.75 cm to 2.00 cm bud of malt decoction group can significantly increased small intestinal propelling rate(P<0.01);0.75 cm to 1.25 cm bud of malt decoction group can significantly increased gastric emptying rate(P<0.01).Combined with the effect of malt on the intestinal propulsion rate and gastric emptying rate in mice,the malt length ranged from 0.75 cm to 1.25 cm.Conclusion:(Ⅰ)The quality of malt medicinal materials sold on the market were different,and there is no uniform processing standard for germination.The malt germination processing technology of this paper established is stable and feasible,which can ensure the uniformity of malt length and the stable content of effective substances in malt.(Ⅱ)From the point of view of chemical composition and the content of alkaloids,it’s suggested that the bud length of malt germinating should be from 0.75 to 1.00 cm.Based on the effect of delactation and digestion of malt,it’s suggested that the bud length of malt germinating should be from 0.75 cm to 1.25 cm.(Ⅲ)The mechanism of malt delactation is not only direct secretion of PRL cells in rat pituitary gland,but also regulation of PRL secretion through hypothalamus dopamine D2 receptor.(Ⅳ)It is suggested that the new edition of Chinese Pharmacopoeia should increase the quality control standard of malt medicinal materials.(Unification the malt germination processing technology and the bud length;Limiting the malt germination temperature and humidity;increase the malt effective substances as the index.)... |
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