Isolation, Purification And Structural Identification Of Polysaccharides From Prunella Vulgaris Linn And Their Biological Activities

Prunella vulgaris L., which belongs to the family of Lamiaceae, is an edible and medicinal perennial plant widely cultured in China, Korea, Japan, and Europe. It is widely consumed as a healthy food and traditional Chinese medicine(TCM) for treatments of jaundice, hepatitis, gonorrhea, tuberculosis, diabetes mellitus and etc. It is also popularly consumed as a soup ingredient and herbal tea in Southern China. Phytochemical studies have demonstrated that P. vulgaris contains various active ingredients, of which polysaccharide is one of the main active ingredients. However, to date, no detailed studies on purification,structural characteristics and their biological activities of P. vulgaris polysaccharides have been reported. In the present study, systematic studies about the extraction, isolation and purification, physicochemical property and structure-activity relationship of P. vulgaris polysaccharides were carried out. In addition, the preparation, antiproliferative activity and its mechanism of P. vulgaris polysaccharide-zinc(P1-Zn) complex were investigated. Main results are listed as follows:(1) The basic composition of P. vulgaris included moisture(22.4%), ash(12.13%),protein(10.63%), fat(4.53%), fiber(24.75%) and carbohydrate(9.81%). Response surface methodology(RSM) was used to optimize the ultrasonic extraction of P. vulgaris polysaccharides(PVP) according to the response of polysaccharide yield. The optimal extraction conditions were ultrasonic power 210 W, extraction time 50 min and extraction temperature 70 °C. Under the optimal extraction conditions, the maximum yield of polysaccharide was 2.11%, which was agreed closely with the predicted value(2.03%). The content of total carbohydrate, protein, galacturonic acid, glucuronic acid, and total phenols of PVP were 87.5%, 11.4%, 6.26%, 0.37%, and 1.95%, respectively. The molecular weight distribution consisted of four fractions: 4720 kDa(56.1%), 31.8 kDa(3.02%), 4.87 kDa(11.6%) and 0.69 kDa(29.2%). Monosaccharide composition analysis suggested that PVP comprised of rhamnose, arabinose, xylose, mannose, glucose, and galactose with molar percentages of 2.8%, 28.2%, 38.5%, 11.0%, 3.0% and 16.5%, respectively. PVP showed good antioxidant activities evaluated by the methods of DPPH and ABTS radical scavenging capacity and ferric-reducing antioxidant power(FRAP) assays. In addition, PVP showedstrong inhibitory effects on the growth of human hepatoma HepG2, gastric carcinoma SGC-7901, and human breast cancer MCF-7 cells.(2) P. vulgaris polysaccharides(PV-P) were extracted using hot water and fractionated using a DEAE-Sepharose fast-flow column to obtain three fractions of water(PV-P1), 0.1 M NaCl(PV-P2) and 0.2 M NaCl(PV-P3). Three fractions had different chemical composition,monosaccharide composition and types of glycosidic linkages. Structural analyses showed that PV-P1 had a higher molecular weight and degree of branching as compared to PV-P2 and PV-P3. Tertiary structure analysis indicated that PV-P1, PV-P2 and PV-P3 did not have triple-helical conformation. PV-P2 and PV-P3 exhibited stronger antioxidant activities than PV-P1, and might be potential candidates for antioxidant agents. However, PV-P1 showed stronger immunomodulatory activities than PV-P2 and PV-P3 in term of stimulation of the production of immune cytokines, including nitric oxide(NO), tumor necrosis factor-α(TNF-α), and interleukin-6(IL-6) in murine macrophage RAW264.7 cells.(3) A heterpolysaccharide, here called P1, was obtained from PV-P1 by purification using a Sephadex G-100 column. Chemical and physical analyses indicated that P1 had a spherical conformation with an average molecular weight of 1,750 kDa, and consisted of arabinose(28.37%), xylose(54.67%), mannose(5.61%), glucose(5.46%), and galactose(5.89%). The main linkage types of P1 were proved to be(1â†'5)-linked α-L-Ara,(1â†')-linked α-L-Ara,(1â†'3)-linked α-D-xyl,(1â†'3)-linked β-D-Gal,(1â†'3,6)-linkedβ-D-Gal,(1â†'3,6)-linked α-D-Man and(1â†'6)-linked α-D-Glc according to periodate oxidation-Smith degradation and NMR analyses. P1 could significantly enhance the secretion of NO, TNF-α, and IL-6 in murine RAW264.7 cells involving the toll-like receptor 2(TLR2),TLR4 and complement receptor 3(CR3). Further studies showed that P1 exhibited stable immune activities in the pH range of 4.0-10.0 and below 121 °C.(4) P1-zinc complex(P1-Zn) was firstly prepared by the reaction of P1 and zinc acetate to achieve its enhanced antiproliferative activity. The content of zinc in P1-Zn was 27 mg/g as determined by atomic absorption spectrophotometer(AAS). The formation of P1-Zn was confirmed by the results of infrared(IR) spectroscopy, conductivity measurement, and scanning electron microscope(SEM). Furthermore, P1-Zn exhibited a significantantiproliferative effect(98.4% inhibition rate at 500 μg/m L) on HepG2 cells through induction of apoptosis, which was characterized by the G0/G1 phase cell cycle arrest,disruption of mitochondrial membrane potential(ΔΨm), the activation of caspase-3 and-9,and reactive oxygen species(ROS) overproduction. These results suggested that P1-Zn inhibited the proliferation of HepG2 cells via cell cycle arrest at the G0/G1 phase and mitochondrial pathway.(5) Oral administration of P. vulgaris polysaccharides could enhance the activities of antioxidant enzymes, including superoxide dismutase(SOD) and glutathione peroxidase(GSH-Px) in the serum of rats as compared to the untreated control, especially at a high dose of 300 mg·kg-1·d-1. In addition, P. vulgaris polysaccharides could improve the indexes of the spleen and thymus of rats and enhance the production of serum IL-6 and IFN-γ. These results suggested that P. vulgaris polysaccharides had antioxidant and immunomodulatory activities in vivo.