Study On The Dynamic Accumulation Of Catalpol In Fresh Leaves Of Rehmannia Glutinosa And Its Anti-psoriasis Effect

The leaf of rehmannia is an important vegetative organ of the plant. At present, the leaves have not yet been "Pharmacopoeia" containing,it is usually been used privately, or included in the prescription of the private doctors. The leaves are mainly used for the treatment of skin disorders,with the exact effect and high cure rate. But its active ingredient is still unknown, which is worth to be explored further.Catalpol is one of the main component in leaves and root tuber of rehmannia glutinosa libosch. It belongs to the iridoid glycosides compounds, the catalpol content is low in plant and the chemical property is unstable. Research and development of catalpol is a hotspot recently, including the following two main aspects: (1) To discover the new activity of catalpol. (2) The method for catalpol preparation. The purpose of this paper is to reveal the changes regularity of catalpol in fresh leaves, which would guide the development of resources scientifically and reasonably; To research the purification process of catalpol in leaves of Rehmannia glutinosa libosch., which will provide more samples for the further test; To explore the catalpol role in preventive and treatment of psoriasis, to reveal the material basis for the treatment of psoriasis; To explore the biological activity of the leaves and identify the structure of the active ingredient, so as to provide new ideas for comprehensive utilization of the leaves.Firstly, I determined the catalpol content in leaves and root tuber of Rehmannia glutinosa libosch. at different developmental stages by HPLC, explored distribution and accumulation regularity of catalpol in leaves. The results showed that the catalpol content in leaves increased rapidly at first, then it declined gradually. It reached the highest value in August or September, which was twice than root tuber in the same period. The water content of leaves also reached the peak value at the same stage. Experiments showed that the changes of the catalpol content is closely related to the state of plant development: when the leaves grow prosperously, it product a mount of catalpol to protect the plant, so the catalpol content be detected become high; Along with the root tuber grow and strengthen, the catalpol content of root tuber improves gradually till it reaches highest value at harvest time. Comparing the catalpol content between leaves and root tuber at the harvest time, we found that the latter was a bit higher than the former, but the difference was little. These results suggest that we should collect the leaves, which used to be abandoned. The leaves of Rehmannia glutinosa libosch. is a new drug sources.Secondly, by using the macroporous resin, we explored the separation and purification process of catalpol in leaves of Rehmannia glutinosa libosch. Recently, catalpol is chiefly purified from root tuber, with higher costs and complex process. We tried to utilize the catalpol in the leaves. Screening 12 different types of resin, we identified that H103 resin has good adsorption and desorption capacity of catalpol. Then we studied the process parameters for purification catalpol with H103, determined that the maximum capacity was 2BV, eluate concentration was 10%EtOH, adsorption rate was 1-2BV/h, Solution concentration was 1mg/ml, end-eluting at 8BV, elution flow rate was 1-3BV/h. Under the conditions of the process, the transfer rate of catalpol was more than 90%, the purity improved 2.6 times. The study laid the foundation for developing catalpol in leaves of Rehmannia glutinosa libosch.Thirdly, we investigated the influence of catalpol on animal models with psoriasis, evaluated catalpol by selecting four animal tests. The results showed that the catalpol had the preventive and therapeutic effect on psoriasis.(1) Catalpol inhibited the epithelial cell proliferation. We injected estrostilben in peritoneal of female mice, which made vaginal epithelial in proliferative phase of estrogen. Then, by oral drug delivery, we investigated the influence of catalpol on mitosis of vaginal epithelial cells. The results showed that in doses 50-200mg/kg, catalpol effectively inhibited the mitosis of mouse vaginal epithelial cells, which hint that catalpol probably inhibit the epidermal hyperplasia of psoriasis.(2) Preventive and therapeutic effect on pathological changes of guinea pigs skin induced by propranolol. 5% propranolol was used on guinea pig ear to make psoriasis-like skin rash. Meanwhile, oral drug delivery for 3 weeks. The results showed that psoriasis-like organizational change had been improved obviously, catalpol could promote the restoration of psoriasis-like organizational change, it had the preventive and therapeutic effect on psoriasis.(3) Catalpol affected the formation of granular layer in mouse tail. Mouse tail is lack of granular layer naturaly, which can simulate parakeratosis of psoriasis. If the medicine could promote the formation of granular layer in mouse tail, maybe it has the ability to anti- psoriasis. The results showed that in doses 50-200mg/kg, although the cell number had a certain degree of increase, the statistical comparison was of no difference, which showed that catapol was weak to resume normal keratinization.(4) The antipruritic effect of catalpol. After 21 days of oral administration, we shaved the guinea pig right hind foot. Histamine phosphate was dropwised and the concentration was increased gradually, till guinea pig licked back up to the right hind foot, calculated the threshold to itch. The results showed that in the 100-200 mg/kg dose, threshold to itch increased significantly. The statistical comparison differed significantly. Catalpol significantly againsted the itching induced by histamine phosphate.Eventually, we made a preliminary activity screening of leaves and carry out the separation and purification on the active site. Using 95% EtOH to extract the leaves, dissolved ethanol extract N1 in water, and then used EtOAC to extract N1and obtain ed N3, the part of the remaining water was named N2. We studied the effects of Three parts on normal glucose-induced hyperglycemia in mice. Three days of oral administration, and then oral glucose 2g/10ml/kg at the end of 60 minutes after the dose. The mice tail was cut off for blood collection at the end of 30 minutes after glucose, the test strips was used to detect blood sugar level. The results showed that N1, N2 did not show hypoglycemic effect, N3 in the 800mg/kg showed hypoglycemic effect, when the dose increased to 1600mg/kg, the hypoglycemic effect was more marked, which appeared dose-dependent. N3 was the fat-soluble parts, we carried out isolation and purification of the site, and gained two plant sterols—β-sitosterol and daucosterol from it. Through literature research, we know thatβ-sitosterol and daucosterol not only have the effect on promoting insulin secretion, but more importantly they have a wide range of immunomodulatory activity. This discovery will provide a new way of thinking on comprehensive development of leaves of Rehmannia glutinosa libosch.The study made some foundations for prevention and treatment of psoriasis with catalpol, and provide a new method for catalpol preparation, and also provide a new thread for comprehensive utilization of leaves of Rehmannia glutinosa libosch.