Research Of Repellent And Larvicidal Activity Of Charophytes And Product Developed

Charophyte is one of most structurally complex algae with a worldwide distribution. China has a wealth of charophyte resources in both freshwaters and estuarine systems. Up to now, there are 5 genera, 152 species,39 varieties recorded in China. The present research of charophytes mainly focused on their physiological activities, ecological charophytecters, and taxonomy, while little research has been conducted in its application. However, Zaneveld, Pal and Imahori had been reported that charophtes had some practical value in larvicide, agriculture and medicine. Therefore, more and more attention is attracted by its larvicidal effect.Subsequent research by many algologists showed varied results about whether the presence of activities played role in killing larvae in charophytes. This research was designed on the larvicidal effect of charophytes under laboratory conditions. The more sensitive techniques were used to analyze the active ingredients of charophytes to determine the specific compounds producing the larvicidal and the repellent effects, to provide theoretical basis on the exploitation of charophtes.First, the resource of charophytes in Shanxi province was investigated. Shanxi province, Located to the northern temperate region, is in the world’s largest loess plateau, rises 1000 meters above the sea level, and belongs to temperate monsoon climatic zone. There is rich in water resource in Shanxi province, include about 1000 rivers, mor than 930 lakes and reservoirs, and 257 spring water. All above provided a board natural environment for the growth of charophyte, and made it a wide distribution in Shanxi. There are 4 genera,22 species,8 varieties recorded in Shanxi. Char a inconnexa, C. calveraensis and C. aculeolata were new record in Shanxi. The distribution characteristic of charophytes in Shanxi were mosaic habitat type, mixed populations, less Nitella species, more common temperate charophyte species, less endemic species and concentrated distribution in several large basins. From culture of charophytes on simulate natural condition, the results showed that the method of charophytes culture was very simple, and possessed of popularization. From calculation, in a lake with an area of 150m2, the increased weight of charophytes was 0.1t in one month. Actually, the increased weight of charophytes is more than 0.1t in natural condition. So, the resource of charophytes is very rich and to be exploited.Second, from the larvicidal activity bioassay of the ethanol extracts of 6 charophytes in Shanxi, the result of differential larvicidal activity was measured among the various charophyte treatments to determine the specific compounds producing the larvicidal effect. In the selected 6 charophytes, only the ethanol extracts of C. inconnexa showed significant lethal effect on larvae, the LC50 was 608.29 mg·l-1.Further experiments were conducted under orthogonal experiments to determine the factors that affect the activities of the extraction. The considered effect factors with the activities were, extracting temperature > conceration time> solid-fluid ratio> extracting time. The optimum extrating conditions were shown as follows:extracting temperature 60℃, conceration time 15 d, solid-fluid ratio 1:20, extracting time 12 h.After qualitative tests, TLC, separating by silica gel column and larvicidal analysis, the consitituents 3 and 6 were extracted and separated from C. inconnexa showed good larvicidal effect. By comparing the larvicidal effect of anthraquinone compounds extracted from C. inconnexa, senna and rhubarb, better larvicidal effect was shown in C. inconnexa and senna extraction. By HPLC analysis, sennosid B was the one of main insecticidal active substance.Third, The more sensitive technique of TCT-GC/MS was used to analyze the volatile components of living charophytes to determine the specific compounds producing the repellent effect. Benzothiazole and a-Pinene were detected in the charophytes that deterred oviposition in the VOCs in the charophytes. These compounds were detected in the two species of charophytes, C. inconnexa and C. vulgaris, and these compounds have been shown to have a strong insecticidal activity. It is interesting to note that these compounds are taxon specific and did not occur in N. obtusa, which showed no repellent activity. This research also showed that charophyte has great potential in absorbing benzene, but the absorbing ability vary in species, which may have future application in wastewater treatment.At last, According to the larvicidal effect of Chara inconnexa, some products of charophte were developed, like charophyte mosquito coil and charophyte soap, and the effects of products were tested in accordance with GB. The charophyte-mosquito coil has better insecticidal effect. Half of test mosquitoes were killed within 7.1 min, which is up to the B standard. The charophyte-soap has bacteriostasis and insect repellent effects, the inhibitory rate reached more than 98%. Charophyte-soap has a significant inhibitory effect in against Gram-positive bacteria, Staphylococcus aureus and Bacillus subtil is. The effective time for mosquito repellent is more than 1.5 h.In brief, the mosquito repellent and insecticidal effects of charophyte were confirmed through this research, and the active substances were sennosid B, benzothiazole and a-Pinene. The developed products have great practical value and wide applications, and this reaearch provides theoretical basis on the exploration of charophyte.