Studies On The Extraction Process,Immunomodulatory And Hematopoietic Effects Of Caulis Spatholobi

Hematopoietic dysfunction is due to the significant reduce of the bone marrow hematopoietic tissue or partial failure of haemopoiesis and then making hematopoietic disorders caused by disease or physical factors,which is a clinical sequelae of diseases such as chemotherapy,radiation therapy,bone marrow transplantation,chemical toxic injury and some autoimmune diseases including immune dysfunction,aplastic anemia and the like.At present,the treatment programs for hematopoietic disorders at home and abroad are very limited,the treatment costs are extremely high,and the treatment effect is not satisfactory.The key challenge in treating hematopoietic disorders is how to reconstruct the hematopoietic and immune functions in the patient.Caulis Spatholobi extraction?CSE?is a multi-component extraction with a multi-target and low toxicity compared with the single chemical,but the current reports about CSE mostly and directly utilized ethanol and acetic acid Ethyl ester or other organic reagents to extract which process is lack of pertinence to the extraction of active ingredients,while the water-soluble extractions of Caulis Spatholobi is inadequate,and the lack of systematic studies of its immunomodulatory and promote hematopoietic activity and its underlying mechanisms limited the development of Caulis Spatholobi.This paper mainly studies the exploitation and optimization of the extraction process of CSE applying Supercritical carbon dioxide extraction?SCO₂?and systematically evaluates the immunomodulatory activity and hematopoietic activity of CSE.This research mainly includes the following aspects:1.Studies on the extractive process of Caulis SpatholobiSupercritical carbon dioxide extraction technology was performed to extract the effective substances with the extraction rate of flavonoids as an indicator.First of all,liquid ratio,extraction temperature,pressure and time on the total flavonoids extraction rate were investigated through single-factor experiments.Then,we optimize the parameters and its range through orthogonal to support Caulis Spatholobi as the raw materials for the production of traditional Chinese medicine.The results of the single-factor experiment showed that the extraction rate of flavonoids was in the range of 1:3¹:5 mg/mL,the extraction temperature range was 30⁵0?,the extraction time range was 3⁵ h,the separation tank carbon dioxide pressure range was 25⁴5 Mpa.According to the results of single-factor experiments,the orthogonal experiment table of four factors and three levels was designed.The results showed that the order of the four factors such as the ratio of material to liquid,extraction temperature,time and pressure were as follows:temperature>solid/liquid ratio>time>pressure.Parameters which lead to the highest extraction rate of flavonoids are as follows:the ratio of solid to liquid?g/mL?was 1:7,the extraction temperature was 50?,the extraction time was 4 h and the pressure of carbon dioxide was 25 Mpa.The results of the validation experiments showed that the best extraction technology in orthogonal experiment could ensure the extraction rate of total flavonoids in the extraction to reach 1.7%or more,so this process is the best technology for extracting Caulis Spatholobi.2.Studies on the of immunomodulatory activity and its underlying mechanisms of CSEIn order to determine the role of CSE in immunomodulation,Hydrocortisone?HC?was applied to establish immunosuppressive model mouse,and after 21 days of treatment,CSE could enhance the acitvity of immune cells and promoted the release of immunomodulatory factors that enhanced the IgG and IgM levels in serum as well as the immunomodulatory cytokines including IL-2,IL-6,IL-10,IL-12,IFN-IFN-?.By NK killing test and lymphocyte proliferation assay,delayed type hypersensitivity experiments and serum hemolysin experiments,CSE was provedto enhance the body's innate immune system,cellular immune system and NK cell function.In addition,hydrocortisone caused a significant increase of phosphorylation of NF?B and a significant decrease of phosphorylation of I?B in mice lymphocytes,while CSE could significantly change the abnormal expression of NF?B and I?B.Thus,CSE possesses the hematopoietic capacity and immune function effects.3.Studies on the of hematopoietic activity and its underlying mechanisms of CSEIn order to study the hematopoietic activity and its underlying mechanisms of CSE,the model of hematopoietic suppression in mice was established by 60Co?-ray irradiation.Hematopoietic disorders caused by radiation were alleviated by the treatment of CSE and it could increase the body weights,spleen indics,thymus indics and increase the number of white blood cells in mice in a time-dependent manner,which indicats that CSE also has immunomodulatory ability in the 60Co?-ray induced mouse model.In addition,colony formation experiments demonstrated that CSE promoted the proliferation and differentiation of hematopoietic progenitor cells in mouse bone marrow,which indicates that CSE can enhance the hematopoietic-related cells such as erythrocytes and platelets.CSE enhanced the hematopoietic ability of mice by regulating the levels of IL-3 and GM-CSF.At the same time,the phosphorylation levels of MEK and Erk in the spinal cord of mice induced by 60Co?-rays decreased significantly,while CSE significantly increased the activation of these two proteins.Based on the above results,it is concluded that CSE can improve the hematopoietic disorder caused by 60Co?-rays through the MEK/Erk signaling pathway by increasing the number of leukocytes,regulating the level of hematopoietic factors,increasing the immune capacity and proliferation and differentiation of bone marrow hematopoietic progenitor cells.In summary,CSE extracted by supercritical carbon dioxide could regulate the immune function and hematopoietic function and respectively achieved through the NF?B signaling pathway and the MEK/Erk signaling pathway.This article provides a pharmacodynamic and pharmacological basis for the application of CSE in the treatment of hematopoietic disorders and the application as immunomodulators after radiotherapy and chemotherapy of tumors.