Studies On Quality Standards And Bioactivities Of Ethanol Extract From Perilla Seeds

Perilla frutescens (L.) Britt. is an annual herbaceous plant in the family Labiatae. It is also a traditional food and medicinal plant widely distributed in China. According to Chinese Pharmacopoeias (2005edition), Perilla seeds, as the ripe fruits of the plant, have many potential therapeutic properties, such as lowering adverse-rising energy, dispersing phlegm, relieving asthma, moistening intestine and so on. In this article, we systematically investigated quality classification standards, agronomic characters, oil contents, and fatty acid compositions of the Perilla seeds, and the chemical constituents and bioactivities of extracts from Perilla seed residues after extracting of the oils. Main findings are as follows:1Quality classification standards of the Perilla seedsInspection rules for Perilla seeds were established based on investigation results of purity analysis, authenticity identification, health test, weight determination, moisture determination, viability test and germination rate test. According to the results of purity,1000-seed weight, moisture content, viability, germination rate and K-clustering analysis of Perilla seeds of fifty origins from all over the China, Perilla seeds were divided into three grades:for the first grade, germination rate>84.94%, purity≥92.44%,1000-seed weight≤1.87g, moisture content<8%; for the second grade, germination rate between56.44%and84.94%, purity between92.35%and92.44%,1000-seed weight between1.87g and2.06g, and the moisture content<8%; for the third grade, germination rate between29.50%and56.44%, purity between90.95%and92.35%,1000-seed weight between2.06g and3.05g, and the moisture content<8%.2Oil contents and fatty acid compositions of the Perilla seedsBy comparing the agronomic characters, oil contents and fatty acid compositions of seeds of five Perilla variants from five different origins in China, it is shown that there was no difference in shape, color and texture. However, significant differences were indeed found in size, moisture content,1000-seed weight and oil content. In detail, average grain diameter of var. frutescens seeds was larger than others, while var. acuta and var. crispa had the smallest average grain diameter. Var. frutescens had the highest1000-seed weight and moisture content, followed by var. auriculato-dentata, var. arguta, var. acuta and var. crispa. For oil content, var. auriculato-dentata had the highest value, followed by var. arguta, var. acuta, var. crispa and var. frutescens. The USFA content in seeds of all the five variants was above90%, consisted of nine fatty acids, including a-linolenic acid, oleic acid, linoleic acid, palmitic acid and so on. However, the content and ratio of each fatty acid in seeds from different regions were significantly different. Var. auriculato-dentata possessed the highest content of a-linolenic acid, followed by var. arguta, var. crispa, var. acuta and var. frutescens. The results indicated that the quality characteristic, oil content and fatty acid compositions of Perilla seeds were substantially affected by environmental factors such as elevation, latitude, and average annual temperature. For Perilla seeds from different regions, significant differences in quality characteristics, oil contents and fatty acid compositions were found. The seed of var. auriculato-dentata from Yunnan Wenshan has the best quality among all five seeds tested.3Comparison of anti-oxidant activities of ethanol and water extracts from Perilla seed residuesPerilla seed residues after oil extraction were defatted by petroleum ether, extracted by ethanol and water successively. The yields of ethanol and water extracts were6.15%and8.20%, respectively. In ethanol extract, total polyphenols and flavonoids were83.01mg/g and30.41mg/g, respectively. Caffeic acid, rosmarinic acid and luteolin were the three major phenolic acids in the ethanol extracts, and the water extract contained38.58%of polysaccharide.Antioxidant experiments indicated that both ethanol and water extracts from the Perilla seed resiues possessed DPPH O2-and·OH free radicals scavenging activities with a dose-dependent manner. For the three kinds of free radicals, EC50were113.264,266.693and1411.183ug/mL in ethanol extract, and349.694,1076.04and1154.615μg/mL in water extract, respectively. It is clear that the antioxidant activities of ethanol extract was stronger than those of water extract. Further studies showed that addition of the ethanol extract into the Perilla seed oil could effectively reduce the lipid oxidation level and improve its oxidative stability. As antioxidant additives, the effect of0.06%ethanol extract is equivalent to0.02%of BHT in oils.4Anti-inflammatory activity and mechanism of ethanol extract of Perilla seed residuesThe anti-inflammatory activity of ethanol extract was examined using inflammatory models including both the ear edema induced by xylene and the paw edema induced by carrageenan in mice. Results show that ethanol extracts obviously inhibited ear and paw edemas of mice and a significant dose-effect relationship was observed. Thus, it is clear that ethanol extracts possessed positive an anti-inflammatory activity. Ethanol extracts was also observed to significantly inhibit the increase of proinflammatory cytokines (IL-6and TNF-α) in mice serum induced by carrageenan, reduce the content of PGE2and MDA in inflammatory exudate fluid and enhance the activity of SOD in mice serum. It can be inferred that ethanol extracts play a role in anti-inflammatory action by suppressing COX-2activity and oxidative damage in vivo.5Acute and genetic toxicities of ethanol extract of Perilla seed residuesIn acute toxicity test, no obvious symptom of poisoning and death was observed within seven days after20g/Kg BW ethanol extract of Perilla seed residues was orally administrated in mice, indicating that LD50value of ethanol extracts was far greater than20g/Kg BW. In Ames test with or without S9fractions, the numbers of revertant colonies for different doses groups of ethanol extracts were no more than twice of those for both the blank and solvent control groups, and no obvious dose-response relationship was observed. In polychromatic erythrocytes (PCE) of the mouse bone marrow test, the rate for PCE of the mice bone marrow in different doses of ethanol extracts groups was obviously lower than that of the positive control group, and there was no significant difference between different doses of ethanol extracts groups and the negative control group. In mice sperm malformation test, the rate of mice sperm malformation in different doses of ethanol extracts groups was significantly lower than that in positive control group (p<0.01), and there was also no significant difference between different doses of ethanol extracts groups and the negative control group. These results indicated that ethanol extracts has no acute toxicity and mutagenic effects, therefore, it is relatively a safe product.