Research On The The Reproductive Biology And Antioxidative And Antimicrobial Activities Of Polysaccharide In Vitro Of Evodia Rutaecarpa (Juss.) Beth

Evodia rutaecarpa (Juss.) Beth. is a medicanal plants belongs to Evodia Forst. Rutaceae. Fruit is medicinal, can be used for dispelling cold, reliefing pain, detoxification, insecticide; Seeds are rich in oil; aromatic oil can be extracted from leaves which could be used as yellow dye.Different developmental stages buds were used as experimental material. This paper studies on the development male and female gametophytes of flowers of E. rutaecarpa; Using histochemical techniques to observate the accumulation of polysaccharides and protein in the development of the anther and embryo sac; E. rutaecarpa polysaccharide was obtained with hot water extracting and ethanol precipitating. The scavenging activities of the polysaccharide from E. rutaecarpa on hydroxyl radical (·OH) , hydrogen peroxide (H₂O₂) and 2, 2–Diphenyl–2–picrylhydrazyl (DPPH·) were investigated by vitro Chemical Systems. Antimicrobial activity testing used the method of trace liquid dilution. And the antioxidative ability and antimicrobial activity of E. rutaecarpa polysaccharide were compared to E. lenticellata Huang Polysaccharide and Vc.The results are as follows:1. Observation of flower developmentPoly umbrella panicles with many rusty pubescent is 7–12cm in diameter, formed by many small cymes, unisexual flower, monoecism or dioecism. One small cyme has 3 floret. Partial the top one is male and the sides are female. They are all female mostly. Flower is 5 base, bract opposite, tiny, leafy or scaly. Sepals are wide oval, 1mm, pubescent. Petals are oblong, 4–6mm, petals of female flower is bigger than male flower, concave pilose. Stamens 5, they are longer than petals, filaments pubescent. Degradation ovary top 4–5 aplit; carpels usually 5, rarely 4 or 6; Style is long cylindrical, 1mm, stigma head width. Oblong ovary with thick oil–point; staminodes of female flower are scaly. Follicles are purple-red fruit, with thick oil–point, top non–beak, each Pán fruit has 1 ovoid seed, 5–6mm, black, shiny surface with small concave. Flowering is from June to August, fruiting period is from September to November. Pollinators are Apis mellifera and Calliphora.2. Microsporogenesis and development of male gametophytesThere are four microsporangiums in E. rutaecarpa. The structure of anther primodium is very simpl, its outmost layer is epidermis and the inner layers are a group of active meristem cells. Then the antherogenous occurres because the outside cells at four corners divide quickly. A group of archesporial cells are formed interior to epidermis at each corner. The archesporial cells divide periclinally to form primary sporogenous cells and primary parietal cells. Primary parietal cells divide periclinally into two layer cells , each of them divides periclinally into two layer cells. The four layer cells continue to growing and become into epidermis, endothecium (1 layer), middlelayer (2 laye, disappeared earlier) and tapetum (1 layer).The development of anther wall follows basic type, glandulartapetum. During the mitosis of microspore mother cells,the nucleus of tapetum divides into two. The primary sporogenous cells develop into microsporocyte by mitosis. The microsporocyte undergoes simultaneous meiotic division and produces tetrahedral tetrad. The tetrad is tetrahedral arrangement and surrounded by callose. Then the tetrad is separated by callose decomposition and each of them is a microspore which develops into the mature pollen which is 2–cell type. At this time, the anther wall just is consisted of epidermis and endothecium.With the spliting of the wall between neighbor anther sacs, the mature pollens are spilled out.In all developmental stages after meiosis, male gametophyte development have different degrees of degradation. So in many mature anthers very little normal mature pollen formed, pollen abortion phenomenon is obvious.3. Megasporogenesis and development of female gametophytesThe pistil was composed of 5 carpels, 5 locule, each locule has 2 ovules. The ovule is axial placentation, anatropous, double integuments and crassinucellate. Megasporogenesis is started from an archesporial cell under the nucellus epidermis. The archesportial cell divides one periclinal cell and one primary sporogenous cell. After periclinal and anticlinal division, the periclinal cell form the organization of the nucellar. The primary sporogenous cell develops into megaspore mother cell directly. The megaspore mother cell undergoes meiosis to form four megaspore in line. The three megaspore near the micropylar degradation, after three mitosis the last megaspore becomes to polygonum type embryo sac has 7 cells, 8 nucleus. There are two synergids and one egg at the micropylar end of embryo sac. The egg has large vacuole at the chalazal end and an nuclear at the micropylar end. The synergids are kept during two polar nucleus are fusing together. Two polar nucleus are located at the center part of the embryo sac. In the course of E. rutaecarpa megasporogenesis, differentiate only one archesportial cell in normal, rarely two. Abortion phenomenon appear at different times.4. Differentiation and development of secretory cavity in flowersThere is no secretory sac in flower early. Secretory sac first appeared in small inflorescence phyllaries, secondly in perianth and pedicel, secreting cells appeared in integument when ovule occurs,but no secretory cavity. A large number of initials of secretory cavity appeared in ovary when the embryo sac was mature, number of big cells in the central, who disintegrate soon, so form a big cavity. At this time, surrounding cells become flat and dissolved continuously. Then the cavity become larger and larger. The diameter of mature secretory sac is about 50μm. In the mature fruit, secretory cavity mainly present in the peel and carpopodium.5. HistochemistryIn different stages of E. rutaecarpa flower development, there is no starch grains and granular protein in tissues, but exist large number of free polysaccharides in cytoplasm. In the cells surrounding the secretory caivity, there are many starch granules that stained red, no protein particles.6. Antioxidative and antimicrobial activities of polysaccharideE. rutaecarpa polysaccharide have strong antioxidant activities on·OH,H₂O₂ and DPPH·, EC50 is 1.0mg/mL,0.1mg/mL and 0.8mg/mL respectively, the effect on elimination of H₂O₂ and·OH is obviously, and the antioxidant activity on H₂O₂ is stronger than VC at the same concentration. The antimicrobial activity of E. rutaecarpa polysaccharide is stronger than E. lenticellata Huang polysaccharide under the same conditions.