Basic Research On High-Value Processing And Utilization Of Andrographis Paniculata Active Ingredients

Andrographis paniculata(Burm.f.)Nees is a traditional Chinese medicine,its aboveground part is used for medicine.It has the effects of clearing heat-toxin,cooling blood for detumescence.Andrographolid(AD)is a lactone diterpene compound,and it is an effective ingredient of traditional Chinese andrographis medicine for its high content and easy to get.It has various effects such as anti-inflammatory,anti-viral,anti-thrombose,sedation,anti-fertility,liver protection and anticancer effects.Furthermore,it also has immune-modulating and anti-diabetic effects.In particular,its high anti-inflammatory effects and therapeutic properties for upper respiratory tract infections are widely recognized,and it is known as "Chinese anti-inflammatory drugs" and "natural antibiotics".However,its low butterfat solubility,very low water solubility and low bioavailability limit its efficiency.And andrographolide has a very bitter taste,which can lead to stomach discomfort when taken in larger doses.After it is prepared into a copolymer,it can improve the absorption and distribution of andrographolide,at the same time improve the anti-inflammatory effect of lung and colon,reduce toxic and side effects,enrich the choice of andrographolide dosage forms,and improve the stability of andrographolide.Reduce the amount of patients,save resources of Chinese medicine,and then reduce the amount of Andrographis paniculata cultivation,saving valuable land resources.In order to achieve the purpose of Andrographis ingredients high-value utilization,we selected andrographis leaves as a feedstock to extract the active ingredients of Andrographis paniculata by the ultrasonic microwave-assisted micellar extraction method,and the andrographolide was purified.The andrographolide and mannose oligosaccharides were covalently linked to form an amphiphilic copolymer,which can form stable micelles in water,and its physical and chemical characterization,in vitro and in vivo evaluation,anti-inflammatory activity were investigated.In this study,dodecyl dimethyl betaine was selected as the surfactant and the ultrasonic microwave-assisted micellar extraction method was used to extract andrographolide.On the basis of a fixed ultrasonic power of 50 W,the optimal conditions for the extraction of Andrographis paniculata were finally determined as follows:the amount of surfactant was 3%,the material-to-liquid ratio was 1:20,the microwave power was 800 W,and the microwave time was 8 min.Under these conditions,the final extraction rate of andrographolide was 2.41%,and the extraction rate of dehydrated andrographolide was 1.32%.After the Andrographis paniculata extract was settled with salt,the resulting extract was extracted with ethyl acetate,decolorized by activated carbon,and washed with methanol,chloroform,and ethanol to obtain andrographolide crystals with a purity of 97.85%,with a total yield of 70.62%.Andrographolide reacts with succinate to form dehydroandrographolide succinate(DAS),which was covalently bound to the mannosis oligosaccharide chain to form an amphiphilic copolymer structure,named dehydroandrographolide polymer succinate-mannose oligosaccharide(DAS-Man).Using HPLC,FTIR and 1H NMR to detect its chemical properties,the prepared DAS-Man was successful in linking manno-oligosaccharides and andrographolide by succinic anhydride.By comparing the peak area integrals of these two characteristic hydrogens in DAS and mannan oligosaccharides,it was judged that the drug loading of the product obtained.Taking the critical micelle concentration(CMC)of DAS-Man as the inspection standard,the optimal preparation ratio of DAS-Man was determined as the molar ratio between andrographolide and mannan oligosaccharide single sugar unit is 2:1,and the anti-solvent was ethyl acetate,the yield coefficient was 28.79%,the drug loading is 9.78%,and the cmc was 0.43 mg/mL.The results of the laser particle size analyzer showed that the mean particle size(MPS)of DAS-Man micelles was 115.1 nm,and the MPS of DAS-Man after lyophilization was 133.0 nm.The results of SEM,TEM and AFM showed that the DAS-Man powder was loose and bulky,DAS-Man micelle particles were spherical,and some particles can be observed with obvious core-shell structure.The freeze-drying made the particle size of DAS-Man micelles larger,with little effect on its dispersibility and solubility.The XRD and DSC spectra show that andrographolide is a typical crystal structure,and DAS-Man and DAS-Man freeze-dried powders exist in an amorphous form.Andrographolide is accompanied by weight loss when it absorbs heat,indicating that andrographolide was decomposed during the melting process.The weight loss curve of DAS-Man and freeze-dried DAS-Man is similar to that of mannan oligosaccharides.The reason may be that the drug loading of andrographolide in DAS-Man was low,which makes the weight loss curve similar to mannon oligosaccharides.At the same time,it showed that DAS-Man had no obvious effect on its thermal stability after being dissolved and lyophilized.The residual amounts of ethyl acetate and DMSO in DAS-Man are 0.06%and 0.09%,respectively,which are far less than the requirements for Class Ⅲ solvents in the Chinese Pharmacopoeia and can be used safely.The saturated solubility,24 h cumulative dissolution,24 h cumulative release rates and stability testes of DAS-Man showed that the DAS-Man can form stable micelles in an aqueous solution,and the dissolution rates in deionized water,simulated gastric fluid(SGF)and simulated intestinal fluid(SIF)were close to complete dissolution,and the release rate in artificial gastric juice is lower than the other two media.The chemical bonds in DAS-Man and the DAS-Man micelles were stable in these three solvent systems,then were absorbed into cells as a whole micelle,or as a whole in the colon.The in vitro simulated digestion results showed that the DAS-Man formed micelles in simulated body fluid,and the rates of free andrographolide were 0.02%,0.17%,0.63%,and 16.63%after undergoing simulated digestion in the oral cavity,stomach,small intestine,and large intestine stages,respectively.The DAS-Man micelles were stable in the oral cavity,stomach,small intestine,and destroyed in the large intestine,part of andrographolide was released,and its micelle structure was also affected with the MPS increased.Cytotoxicity experiments showed that andrographolide and DAS-Man had no obvious toxicity to Caco-2 cells when the drug concentration range was 0.31 mmol/L-0.01 mol/L.The bioavailability results showed that the AUC values of the DAS group and the DAS-Man group were 0.59 times and 1.85 times of the andrographolide group,respectively.The Tmax of DAS-Man was later than the andrographolide group with double peaks.The results of tissue distribution experiments showed that the Tmax of the spleen,kidney and small intestine in the andrographolide group was 0.5 h,the Tmax in the heart,liver,and lungs was 1 h,and the Tmax in the brain,spinal cord,and large intestine was 4 h.In the DAS-Man group,the Tmax in the heart,spleen,lung,kidney,and small intestine was 1 h,the Tmax in the liver was 2 h,and the Tmax in the brain,spinal cord,and large intestine was 4 h.The reason may be that part of DAS-Man micelles were taken into cells in a whole state,and metabolized into free andrographolide in the liver,and part of them were decomposed in the large intestine to form free andrographolide before entering blood circulation.After DAS-Man micelles were ingested and slowly decomposed in the body,the blood concentration is maintained at a high level,then the drug concentration in the kidney has also been maintained at a high level.The content of andrographolide in the lungs of DAS-Man is also higher than that of the andrographolide group.The reason was that the blood drug concentration is higher than that of the andrographolide group,and the mannose in DAS-Man was targeted to the lungs,which had a certain effect on improving the efficacy of the drug when used for lung inflammation and infection.This study investigated the anti-inflammatory effects of andrographolide and DAS-Man,and evaluated LPS-induced pneumonia in mice and OXZ-induced colitis in mice as model animals.DAS-Man and andrographolide could reduce the W/D,spleen coefficient and MPO activity of the lung tissue of inflamed mice,and could also reduce the content of TNF-α,IL-6 and IL-1β inflammatory factors in serum and tissues,and correct pathological state of lung tissue.DAS-Man and andrographolide could prolong the survival time of OXZ-induced colitis mice,reduce the DAI score of the colon tissue of the inflamed mice,reduce the shortening of the colon,and reduce the spleen coefficient.At the same time,it can also reduce the MPO activity,NO,and NO in the serum and tissues,less colon edema.The content of TNF-α,IL-4 and IL-1β inflammatory factors can improve the pathological state of the colon.DAS-Man showed better anti-inflammatory effect than andrographolide on LPS-induced acute pneumonia in mice and colon inflammation caused by OXZ.suggesting that DAS-Man has a certain protective effect on LPS-induced acute pneumonia in mice and colon inflammation caused by OXZ,and its mechanism of action may be similar to that of andrographolide can inhibit the secretion of cell inflammatory factors.