Preparation Process Optimizing And Anti-Inflammatory Effects Of Premna Puberula Pamp. Pectin

Background:Premna puberula Pamp.belongs to the genus Premna linn.of the family Lamiaceae.The plant is rich in nutrients including pectin and proteins,and medical chemicals,flavonoids.Folk usually collect its fresh leaves to make"Fairy tofu"（Premna puberula Pamp.Pectin,PP）,which is a naturally green food with light color and strong fragrance.Its color is turquoise,has the effect of dispelling fire,clearing heat and detoxification,and has good nutritional value and health care functions.The current research proves that the key factor in the formation of the tofu is the richness of pectin in the leaves.According to the National Compendium of Chinese Herbal Medicine,Premna puberula Pamp.clears damp heat,regulates menstruation and detoxifies the body.It treats menstrual disorders,rheumatoid arthritis,and edema.Externally,it is used to treat nameless swelling and toxicity.The physicochemical properties of the same genus and its pharmacological effects have been reported in the literature,but little research has been done on Premna puberula Pamp.Objective:To prepare the Wudang functional food,Premna Puberula Pamp.Pectin（PP）,and study its quality control,preparation process,pharmacological effects and safety.Methods:The Premna Puberula Pamp.leaves were collected.The chemical composition of the alcoholic extracts of Premna Puberula Pamp.was analyzed by mass spectrometry using UPLC-Q-TOF-MS/MS,combined with the literature reports and the database of high-resolution mass spectrometry of natural products.The content of pectin in the dried leaves was determined by carbazole sulfate method.The water-soluble sugars and total flavonoids were used as quality control markers,and the water-soluble sugars and total flavonoids were determined by phenol-sulfuric acid method and sodium nitrite-aluminum nitrate method,respectively.L₉（3~4）orthogonal experimental method was used to optimize the preparation process of PP;the best preparation process was determined using ANOVA.For the pharmacological effects of PP,the swelling right hind paw of ICR mice was modeled using subcutaneous injection of carrageenan gum solution.The swelling rate was observed;and the local tissue inflammatory reactions of the model mice was investigated using vernier calipers and HE staining.The serum inflammatory factor expression was detected using ELISA.Finally,acute toxicity experiments were carried out for safety assessment of PP using maximum dosing method.Results:Fifty-three compounds were identified in the Premna Puberula Pamp.,including 19 flavonoids,16 phenolic acids,5 phenylethanol glycosides,6 fatty acids,and 7 heterocyclic compounds.The average content of pectin in dried leaves was the highest in August,at 51.349 mg/g;the content of water-soluble sugars was higher in May and October,at 262.894 mg/g and 255.798 mg/g,respectively.The total flavonoids content was highest（55.996 mg/g）in October.These results indicated that the production of PP was highest in August and the quality was best from August to October.The average content of water-soluble sugars in PP was 13.366 mg/g and the average content of total flavonoids was 4.970 mg/g.The best preparation process of PP was powder-liquid ratio 1:20,temperature 90°C,and stirring time 3 min.Data showed that PP reduced paw edema and decrease the serum level of IL-6,TNF-αand IL-1βin the model mice.There was no toxic effect of PP on mice at a total dose of 6000 mg/kg/24 h.Conclusion:The main constituents of Premna puberula Pamp.are flavonoids;Aug.～Oct.may be the best harvesting season for Premna puberula Pamp.leaves.The optimal preparation process of PP may be obtained using the orthogonal experimental method.PP had anti-inflammatory effects by reducing IL-6,TNF-αand IL-1β.Furthermore,PP was non-toxic to mice at a dose of 6000 mg/kg/24 h.