Anti-complementary Constituents Of Pogostemon Cablin

The complement system, a vital innate immune response, which composed of more than30different protein that widespread in the serum, tissue fluid and the cell membrane, was found that complement the action of antibody directed against bacteria and erythrocytes. And its activation process is characterized by a series of serine protease cascade enzymatic hydrolysis reactions. Complement system is an important part of specific and nonspecific immune mechanism, characterized by antimicrobial defense reaction, immune regulation and mediating immune pathological injury. However, the excessive activation of complement may cause abnormal reaction of the immune system, resulting in damage to normal tissue and the pathological process involving in many diseases, such as Rheumatoid arthritis, alzheimer’s disease, systemic lupus erythematosus, ischemic in perfusion, acute muscle infarction and acute respiratory distress syndrome. Preliminary experiments showed that the methon extract of Pogostemon cablin possessed potent anti-complementary activity on the classical pathway and the alternative pathway(CH5o=0.017mg-mL-1, APso=0.46mg-mL"1). However, the chemical ingredient with anti-complementary activity from Pogostemon cablin have never been reported. This research mainly focused on anti-complement compounds isolated and purified from Pogostemon cablin (Blanco) Benth., which is used in traditional Chinese medicine for the treatment of upset stomach, vomiting, diarrhea, headache, and fever. Twenty-four compounds were separated from the petroleum ether, ethyl acetate and n-BuOH soluble fractions. All isolates were evaluated for their in vitro anti-complementary activity both on the classical pathway (CP) and alternative pathway(AP). Mechanism study indicated that7interacted with Clq, C2, C5and C9components, while22interacted with Clq, C2, C4, C5and C9components.1. Activity Identification of the Bioactive ExtractsThe optimal dilutions of sera (resources of complements) that gave submaximal lysis in the absence of complement inhibitors were determined to perfect the hemolysis assay to determine different extracts of Pogostemon cablin. The results showed that the petroleum ether extract(CHso=0.025mg·mL-1), EtOAc extract (CH50=0.10mg-mL-1) and n-BuOH extract(CH50=0.26mg·mL-1) were the active fractions, while the volatile oil of P.cablin showed little activity. After further research, the fraction which was extracted volatile oil showed potent anti-complementary activity.2. Isolation and Identification of Anti-complementary ConstituentsTwenty-four compounds were isolated from the ethyl acetate, n-BuOH and petroleum ether soluble fractions including15flavonoids:5-hydroxy-3,7,3’,4’-tetramethoxyflavone (1),5-hydroxy-7,3’,4’-trimethoxyflavanone (2),5,4’-dihydroxy-3,7,3’-trimethoxyflavone (3),5-hydroxy-3,7,4’-trimethoxyflavone (4),5,4’-dihydroxy-7,3’-dimethoxyflavone (5), luteolin (6), quercetin-7,3’,4’-trimethyl ether (7), ermanine (8),3,5,7-trihydroxy-3’,4’-dimethoxyflavone (9), quercetin (10), apigenin (11), kaempferol(12),5-hydroxy-7,3’,4’-trimethoxyflavone (13), kaempferol-7-O-β-D-glucopyranoside(14) and kaempferol-3-O-β-D-glucopyranosid e-7-O-α-L-rhamnoside (15); three terpenoids:oleanic acid (16),1β-hydroxy-10βH-guaia-4,11-dien-3-one (22),(5R)-5-hydroxypatchol(23); two steroidals:β-sitosterol (19), β-daucosterol (20); two phenolic acids:vanillic acid (17), benzylalcohol (18); one adenosine:2’-deoxythymidine (21); one pyrone:4-methyl-6-isopenthyl-pyrone (24).3. Anti-complementary Activity Test of Constituents of RcablinComopunds3,7,10,12,16and22exhibited anti-complementary activities through the classical pathway and alternative pathway (CHso:0.072-1.08mg·mL-1; AP50:0.39-0.49mg·mL-1), while5,6,23and24showed inhibitory effect on the classical pathway only(CH50:0.054-0.54mg·mL-1). Mechanism study demonstrated that7interacted with C1q, C2, C5and C9components, while22interacted with C1q, C2, C4, C5and C9components.4. GC-MS Analysis of Petroleum Ether Extract10samples of Pogostemon cablin were collected and GC-MS method was used to analyze chemical composition. And10more constituents were identified from active fraction of petroleum ether extract, including alkane and derivatives, sesquiterpene and monoterpene. The results substantiated that these compounds might be the possible fundation for its potent anti-complementary activity.By using GC-MS,3compounds including1β-hydroxy-10βH-guaia-4,11-dien-3-one (22),(5R)-5-hydroxypatchol (23),4-methyl-6-isopenthyl-pyrones (24) were measured to study the relation between the components and the activity of P.cablin collected from different regions. The results showed that there’s relationship between the activity of petroleum ether extract and compounds22,23and24.