Anti-Melanogenesis Activity of Supercritical Carbon Dioxide Extract from Perilla frutescens Seeds

Perilla frutescens seed (PFS) oil is reported to inhibit skin photoaging;however, its effect on melanogenesis has not yet been investigated. Herein, we tested the anti-melanogenesis activity of an oil-based extract from PFS with supercritical carbon dioxide (scCO2). In a cell culture system, B16 mouse melanoma cells were treated with the PFS scCO2 extract and other samples. The PFS scCO2 extract decreased melanin production by approximately 90% in B16 mouse melanoma cells without cytotoxicity at 100 μg/mL. This effect was greater than that of the well-known melanogenesis inhibitor, kojic acid. Although a hexane-extracted PFS oil and a squeezed PFS oil also decreased melanin production in the B16 cells, the inhibitory effect of the PFS scCO2 extract was higher than both of these. Chemical analysis of the PFS scCO2 extract and squeezed PFS oil showed that almost 90% of the components of both oils were α-linolenic acid, linoleic acid, and oleic acid. Furthermore, the ratio of those three fatty acids across both samples was almost the same. When the three fatty acids were mixed in the same ratio as in the PFS scCO2 extract, the IC50 of the mixture for melanin production in B16 melanoma cells was identical to that of the PFS scCO2 extract. However, the IC50 of the squeezed PFS oil was approximately 6.6 times higher than that of the mixture. Although those fatty acids are the main inhibitory ingredients against melanin production in all of the extracts, some factor(s) in the squeezed PFS reduce their affinity with the cells. These results indicated that the PFS scCO2 extract could be a superior melanogenesis inhibitor. Although its main ingredients are probably the same as those of the squeezed PFS oil, it is necessary to extract with scCO2 for stronger anti-melanogenesis activity.

Studies on In Vitro Flowering and Fruiting of Perilla frutescens

Influences of PGR, sucrose, and ammonium nitrate on in vitro flowering and fruiting from cotyledon explants of P. frutescens were studied. The regenerated shoots at 2-4. cm from cotyledon explants on MS medium supplemented with 0.5 mg L^-1 BA and 1.0 mg L^-1IAA were excised and transferred to MS medium containing 30 g L^-1 sucrose, 8.25 g L^-1 ammonium nitrate, and 1.0 mg L^-1 BA. Following 40 d of culture, 86.2% of them flowered and set seeds. These seeds were germinable and developed into flowering plants in the fields. This study provides a simple system for rapid breeding of P. frutescens and studying the physiological mechanism of flowering of plants.

GC-MS analysis of volatile compounds of Perilla frutescens Britton var. Japonica accessions: morphological and seasonal variability

Objective: To investigate the composition of volatile compounds in the different accessions of Perilla frutescens(P. frutescens) collected from various habitats of China and Japan. Methods: In the present study, the essential oil from the leaves of P. frutescens cultivars from China and Japan was extracted by hydro-distillation and the chemical composition and concentration of the volatile components present in the oils were determined by gas chromatography–mass spectrometry(GC–MS) analysis. Results: Among the volatile components, the major proportion was of perilla ketone, which was followed by elemicin and beta-caryophyllene in the Chinese Perilla cultivars. The main component in the oil extracted from the Japanese accessions was myristicin, which was followed by perilla ketone and beta-caryophyllene. We could distinguish seven chemotypes, namely the perilla ketone(PK) type, perilla ketone, myristicin(PM) type, perilla ketone, unknown(PU) type, perilla ketone, beta-caryophyllene, myristicine(PB) type, perilla ketone, myristicin, unknown(PMU) type, perilla ketone, elemicine, myristicin, beta-caryophyllene(PEMB) type, and the perilla ketone, limonene, betacryophyllene, myristicin(L) type. Most of the accessions possessed higher essential oil content before the flowering time than at the flowering stage. The average plant height, leaf length, leaf width of the Chinese accessions was higher than those of the Japanese accessions. Conclusion:The results revealed that the harvest time and geographical origin caused polymorphisms in the essential oil composition and morphological traits in the Perilla accessions originating from China and Japan. Therefore, these chemotypes with desirable characters might be useful for industrial exploitation and for determining the harvest time.

紫苏(Perilla frutescens)的种籽、茎叶营养

紫苏(Perilla frutescens)原产我国,至今已有2千多年的栽培历史,历来享有食疗同源珍品之称。本研究结果表明,其种籽蛋白质、脂肪等,以及活性营养素(β-Carotene、V_(B1)、V_(B2)、V_C、V_E)、矿物元素(Ca、P、Fe、Zn……)及油脂、脂肪酸(C_(16:0)、C_(18:0)、C_(18:1)、C_(18:1)、C_(18:3)……)含量均十分显著,尤其是它的β-胡萝卜素(β-Carotene)成倍高于红胡萝卜、番茄、红苋等蔬菜。

Thai Perilla frutescens fruit oil alleviates carbon tetrachloride-induced hepatotoxicities in rats

Objective:To study the effect of perilla fruit oil against carbon tetrachloride(CCl4)-induced liver damage in rats.Methods:Perilla fruit oil was analyzed in terms of fatty acids,tocopherols and tocotrienols using chromatography.Sub-chronic toxicity of perilla fruit oil was investigated in rats for 90 d followed by a 28 d recovery period.Hematological,biochemical and pathological parameters were determined.To evaluate hepatoprotection,rats were divided into five groups and orally administered with Tween 80 for 10 d;Tween 80,silymarin,perilla fruit oil(0.1 mL/200 g)and perilla fruit oil(1 mL/200 g)for 10 d together with subcutaneous injection of CCl4(2 mL/200 g)on days 9 and 10.Liver enzymes and pathological parameters were determined.Results:Perilla fruit oil containedα-linolenic acid(56.55%of total fatty acid),β-tocopherol(49.50 mg/kg)andγ-tocotrienol(43.65 mg/kg).Rats showed significant changes in the percentage of monocytes and platelet indices following perilla fruit oil consumption for 90 d;in the percentage of neutrophils and lymphocytes,and RBC indices in the recovery period when compared with the deionized water group.Total protein and creatinine levels were increased while alkaline phosphatase and aspartate aminotransferase levels were decreased(P<0.05).Organ weight index and pathological indicators did not change significantly.The liver of CCl4-induced rats showed remarkable centrilobular fatty changes,which was ameliorated by perilla fruit oil pretreatment.Aspartate aminotransferase,alanine aminotransferase and alkaline phosphatase levels were decreased(P<0.05)in rats given perilla fruit oil.Conclusions:Perilla fruit oil is rich inα-linolenic acid,β-tocopherol andγ-tocotrienol and improves blood biomarker levels and protects against CCl4-induced hepatotoxicity.Further studies are required before supporting its use for the treatment of hepatitis.

Development and Characterization of New Microsatellite Markers for <i>Perilla frutescens</i>(L.) Britton

Based on RNA sequences using transcriptome analysis, 37 new simple sequence repeat (SSR) primer sets were developed for Perilla species. These new SSR markers were applied to analyze the genetic diversity among 15 accessions of Perilla species. A total of 182 alleles were confirmed in 37 loci, with an average of 4.9 alleles per locus and from 2 to 9 alleles per locus. The MAF (major allele frequency) per locus varied from 0.200 to 0.733, with an average of 0.463. The gene diversity (GD) ranged from 0.391 to 0.853, with an average of 0.670. The average polymorphic information content (PIC) was 0.624, ranging from 0.315 to 0.838. The new SSR markers of Perilla species reported in this study may provide potential markers to analyze the genetic diversity and genetic relationships of Perilla species. In addition, new Perilla SSR markers developed from transcriptome analysis can be useful for the identification of cultivars, conservation of Perilla germplasm resources, and genetic mapping and designating of important genes/QTLs for future Perilla crop breeding programs.

Large scale identification of SSR marker in perilla by next generation sequencing

Perilla frutescens (L.) is an edible, medicinal crop, and most popular in East Asia. Its molecular breeding and research are hampered by the paucity of molecular markers. Simple sequence repeat (SSR) markers are ubiquitous and widely used in eukaryotic genomes. EST-SSRs identification of perilla was performed in 116,387 reads generated by Illumina paired-end sequencing technology. In total 25,449 unigenes containing SSR and 33,867 SSR loci were identified, and 19,400 primer pairs were designed. Polymorphism of SSR primers was conducted by searching for insertions and deletions (INDELs), and 1,567 unique SSRs were predicted. Totally, 200 SSR primer pairs were selected for polymorphic validation among 23 perilla accessions. Results showed that 175 primer pairs produced amplicons, and 30 pairs exhibited polymorphism. Polymorphic ratio was higher by using INDEL method than using conventional primers. Phylogenetic analysis showed the 2 distinct groups: P. frutescens var. frutescens and P. frutescens var. crispa. Wrinkled leaf trait and seed trait were distinct between these 2 groups. However, no clear leaf color or geographic relationship was detected. The large scale development and identification of SSR marker in this research laid a foundation for genetic analysis and marker assisted breeding of cultivated perilla.

In vivo control of perilla rust disease by oak pyroligneous liquor

Leaves of Perilla frutescens, commonly known as perilla are widely used in Korea, due to their medicinal properties. The rust disease of perilla interrupts its wide use every year. The present study was carried out to evaluate the control of perilla rust disease by using oak pyroligneous liquor having various biological properties. The rust disease infected perilla leaves were applied with 100 μl of oak pyroligneous liquor at different concentrations such as 6.25, 12.5, 25 and 50 μg/ml. Then the leaf samples were observed for the abortion of active rust pustules (yellow color) after 48 hours of incubation at 26?C. Further, inhibition of rust pustules development by oak pyroligneous liquor was evaluated on the leaves of artificially induced perilla rust disease. The best abortion of rust pustules by oak pyroligneous liquor was noticed at the concentration of 50 μg/ml, where 74.4% of the pustules were aborted. Discoloration of rust pustules was also observed in the treated leaves from yellow to white when compared with the untreated control. In addition, oak pyroligneous liquor completely inhibited the development of rust pustules in artificially induced leaves even at low concentration (6.25 μg/ml). The results clearly indicated that oak pyroligneous liquor is a good candidate for the management of Perilla rust disease.

Effectiveness of Selected Reaction Monitoring for rapid assay of Cypermethrin Residue in Perilla Leaves

Many kinds of pesticides have been developed and used to yield a good harvest but the residues in agricul-tural products cause health problems. It is important to keep watch on these residues by using adequate methods of analysis. Pretreatment such as gel permeation chromatography (GPC) or column chromatography is often needed for the quantitative analysis of pesticide in agricultural products by conventional methods such as gas chromatography/low resolving power mass spectrometry (GC/LRMS). However, these pretreat-ments need a lot of work and take time. New methods saving the necessity of these pretreatments have been desired. We have applied selected reaction monitoring (SRM) to quantitatively determine cypermethrin residues in Perilla frutescens samples and compared the results with LRMS and HRMS in SIM mode. A background peak caused by the matrix overlapped the cypermethrin peak in the analysis using LRMS. SRM and HRMS in SIM mode provided chromatograms without matrix interference. The high selectivity of the product ion (m/z 127) produced from precursor ion (m/z 163) isolated the target peaks from the matrix peaks when using SRM. This method eliminates the pretreatment step, thus saving time and simplify ing the ana-lytical process.

Perilla frutescens:A traditional medicine and food homologous plant

Perilla frutescens,an annual herb of the Labiatae family,has been cultivated in China for more than 2000 years.P.frutescens is the one of the first medicinal and edible plant published by the Ministry of Health.Its leaves,stems and seeds can be used as medicine and edible food.Because of the abundant nutrients and bioactive components in this plant,P.frutescens has been studied extensively in medicine,food,health care and chemical fields with great prospects for development.This paper reviews the cultivation history,chemical compositions and pharmacological activities of P.frutescens,which provides a reference for the development and utilization of P.frutescens resources.

紫苏PfFUL基因调控拟南芥开花和角果发育

紫苏(FRUITFULL,FUL)基因属植物MADS-box家族中的AP1/FUL亚家族,具有调控开花时间、花分生组织分化和果实发育的功能。本研究根据紫苏转录物组注释克隆获得PfFUL基因序列,利用生物信息学方法对PfFUL蛋白的基本理化性质进行了分析,通过系统发育树分析了PfFUL与其他物种FUL的进化关系。构建35S::PfFUL植物表达载体,分别转化野生型Col-0及突变体ful-7拟南芥获得了过表达35S::PfFUL/Col-0和回补突变35S::PfFUL/ful-7植株,并对其进行表型比较分析,初步明确了PfFUL基因在植株开花及角果发育过程中的功能。PfFUL基因全长CDS为738 bp,编码245个氨基酸。系统发育分析表明,紫苏PfFUL与番茄、一串红和芡欧鼠尾草亲缘关系较近,而与拟南芥、烟草和葡萄关系较远。相比于Col-0和ful-7,转基因植株均表现出早花现象(P<0.05),35S::PfFUL/ful-7植株的角果长度可恢复到野生型表型(P>0.05),且种子皱缩的数量明显降低(P<0.01)。此外,表型观察发现,转基因植株还表现出节间生长伸长,茎生叶变窄且卷曲。本研究证实,PfFUL基因可调控植物开花和角果的发育,并参与营养生长。

干燥方式对紫苏叶品质的影响

为评价不同干燥方式对紫苏叶品质的影响,采用热泵干燥(heat pump drying,HPD)、远红外干燥(far-infrared drying,FIRD)、真空冷冻干燥(vacuum freeze drying,VFD)、热风干燥(hot air drying,HAD)4种干燥方式对紫苏叶进行处理,测定其基本营养成分含量、活性成分含量及抗氧化能力,并利用隶属函数法进行综合评价,采用顶空固相微萃取-气质联用技术鉴定其挥发性物质。结果表明,VFD处理的紫苏叶中蛋白质、粗脂肪、黄酮、多酚、迷迭香酸含量最高,色差值最小,DPPH、ABTS~+自由基清除能力最强;HPD处理的紫苏叶中还原糖含量最高;经4种干燥方式处理后,紫苏叶中挥发性物质的种类与相对含量都有不同程度的增减,烯烃类和醛类为主要挥发性物质。综合评价表明,VFD处理的紫苏叶品质优于其他干燥处理,但能耗较高;HPD处理的紫苏叶品质优于HAD和FIRD,是紫苏叶干燥处理的一种较好选择。

紫苏磷脂酸磷酸水解酶基因(PfPAH1)的鉴定及功能分析

本文基于紫苏基因组数据筛选获得两条PfPAH1基因序列,分别命名为PfPAH1-1和PfPAH1-2。应用生物信息学工具分析PfPAH1s蛋白理化性质、功能结构域及系统进化。定量PCR(qRT-PCR)检测PfPAH1s在紫苏根、茎、叶、花及不同发育时期种子的表达谱。进一步克隆到高表达的PfPAH1-1基因的编码序列并进行烟草瞬时转化以鉴定其功能。结果表明,PfPAH1-1和PfPAH1-2基因的开放阅读框(ORF)长度均为2715 bp,编码904个氨基酸残基,含有Lipin_N、Lipin_mid和属于HAD_like超家族的LNS2三个保守结构域。系统进化分析显示紫苏PfPAH1-1蛋白与唇形科植物一串红的SsPAH1亲缘关系最近,其次是葡萄和芥菜。PfPAH1-2蛋白则与唇形科的芝麻SiPAH1亲缘关系最近,其次为油橄榄,紫苏和芝麻均为油料作物,推测PfPAH1-2和SiPAH1可能具有相似功能。qRT-PCR分析表明PfPAH1-1和PfPAH1-2基因均在开花后40 d种子中表达量最高,预示着其可能参与油脂合成积累。烟草瞬时转化揭示过表达PfPAH1-1基因烟草叶片总脂肪酸及中性脂含量均高于野生型烟草,然而磷脂含量降低。

不同产地紫苏叶HPLC指纹图谱研究

为建立紫苏叶HPLC指纹图谱检测方法,采用HPLC法,以UltimateTMXB-C18(4.6mm×250mm,5μm)为色谱柱,乙腈(A)-0.1%乙酸(B)为流动相进行线性梯度洗脱,在330nm波长下以迷迭香酸为参照物测定了9个不同产地的紫苏叶的指纹图谱,并作相似度比较分析。建立了紫苏叶HPLC指纹图谱共有模式,12个共有指纹峰被标定,并对不同产地紫苏叶HPLC指纹图谱进行相似度比较,结果具有明显差异。该方法准确、简单,能较好地识别不同产地的紫苏叶样品,可为紫苏的质量评估提供有益的信息。

紫苏内生真菌Aspergillus sp.12Y03化学成分研究

采用硅胶柱层析和Sephadex LH-20凝胶柱层析等分离方法,从紫苏内生真菌Aspergillus sp.12Y03发酵产物中分离鉴定了10个化合物,经现代光谱学技术鉴定为:环-(脯氨酸-甘氨酸)(1)、环-(脯氨酸-丝氨酸)(2)、环-(丝氨酸-4-OH-脯氨酸)(3)、环-(丙氨酸-4-OH-脯氨酸)(4)、环-(苯丙氨酸-甘氨酸)(5)、环-(丙氨酸-甘氨酸)(6)、亚油酸(7)、α-亚麻酸(8)、cerevisterol(9)和22E,24R-5α,6α-环氧麦角甾-8(14),22-二烯-3β,7α-二醇(10),均为首次从该菌种中分离得到,化合物3和4具有中等强度的海虾致死活性。

紫苏内生真菌Penicillium sp.12Y25化学成分及抑菌活性研究

【目的】研究紫苏内生真菌Penicilliumsp.12Y25的化学成分及其抑菌活性,为开发新型天然药物提供参考。【方法】以分离自山西太原健康紫苏茎秆的内生真菌为研究对象,采用硅胶柱层析和Sephadex LH-20凝胶柱层析等分离手段和现代光谱学技术,对其液体发酵产物的乙酸乙酯提取物进行了分离和结构鉴定;采用菌丝速率法,测试了分离化合物对番茄灰霉病菌、西瓜枯萎病菌和小麦赤霉病菌等植物病原菌的抗菌活性。【结果】从紫苏茎秆内生真菌Penicilliumsp.12Y25发酵产物的乙酸乙酯提取物中分离鉴定了10个化合物,分别为麦角甾醇过氧化物麦角甾-4,6,8,22-四烯-3-酮、9(11)-去氢麦角甾醇过氧化物、22E,24R-7,22-麦角甾二烯-3β,5α,6β-三醇、22E,24R-7,22-麦角甾二烯-3β,5α,6β,9α-四醇、麦角甾醇、单油酸甘油酯、α-亚麻酸、对甲氧基苯乙酸和对氨基苯乙酸,均为首次从该菌种中分离得到,其中22E,24R-7,22-麦角甾二烯-3β,5α,6β-三醇和22E,24R-7,22-麦角甾二烯-3β,5α,6β,9α-四醇对番茄灰霉菌和西瓜枯萎菌有中等强度的抑制活性。【结论】紫苏内生真菌Penicilliumsp.12Y25化学成分多样,部分化合物具有植物病原菌抗菌活性。

NaCl和NaHCO_3胁迫对紫苏种子萌发的影响

通过研究不同浓度NaCl和NaHCO3胁迫对紫苏种子萌发的影响,探讨紫苏种子的耐盐碱能力。采用单盐胁迫的方法,对种子发芽率、发芽势、相对发芽率、活力指数、复萌率等指标进行了测定分析。结果表明,随NaCl和NaHCO3浓度的增加,种子发芽率、发芽势、相对发芽率、胚根长、苗鲜重呈下降趋势,之间呈显著的负相关关系。NaCl胁迫下紫苏的适宜浓度、半数抑制浓度分别是0～3.583‰、8.312‰;NaHCO3胁迫下紫苏的适宜浓度、半数抑制浓度分别是0～2.513‰、4.656‰;清水复萌试验表明,NaCl胁迫后的复萌率随浓度的增加而增加;NaHCO3胁迫后复萌率为0。综上所述,紫苏种子具有一定的抗耐盐碱能力。

HPLC法测定不同品系紫苏酚类物质的含量

采用福林酚法测定总多酚的含量,并用反相高效液相色谱法测定不同提取条件和不同紫苏品系间酚类物质组成及含量,比较不同提取方法的优劣和不同紫苏品系间的差异。结果表明:HPLC方法分离定性了8种酚类物质;以40%乙醇做提取剂的提取方法更加简便快捷。不同品系间单体酚类含量不相同,其中紫苏品系单体酚类种类比白苏品系多。在本试验所检测的单体多酚中,紫苏叶以酚酸类为主,可达80%以上,其酚酸类主要为迷迭香酸和咖啡酸,而其黄酮类则主要为槲皮素和芹菜素。本试验采用高效液相色谱法准确、重现性好,并结合对不同品系紫苏叶酚类物质含量进行比较,能够为生产原料选择和工艺提供质量控制依据。

HPLC法比较测定紫苏属植物种子维生素E含量差异

采用HPLC法测定来自中国四川和广东,以及日本和美国等地共29份紫苏属不同原(变)种种子的VE含量。结果表明,紫苏属不同原(变)种种子均含丰富的VE。所有供试材料的δ-VE、γ-VE和α-VE含量及总含量间均存在极显著差异。其中,VE总含量变幅在79.90～383.30μg/g之间,平均值为199.76μg/g;δ-VE、γ-VE和α-VE平均含量分别为5.81、190.47μg/g和3.48μg/g。以来自日本的P06-37的VE总含量最高,达383.30μg/g。比较不同原(变)种种子VE含量结果表明,不同原(变)种间δ-VE、γ-VE和α-VE含量及总含量差异未达显著水平。总体上,以白苏的δ-VE、γ-VE和α-VE平均含量及总含量均相对较高。

紫苏属植物叶中维生素的HPLC测定

[目的]研究紫苏属不同原(变)种中维生素的组成及含量。[方法]分别提取紫苏属植物叶中水溶性及脂溶性维生素,并采用高效液相色谱法进行分析。[结果]紫苏属不同原(变)种叶中维生素种类基本相同,但含量存在显著差异。[结论]紫苏属不同原(变)种苗期叶中均含有丰富的VC、VK1。