Phyllanthuse emblica polyphenols:Optimization of high-voltage pulsed electric field assisted extraction,an antioxidant and anti-inflammatory effects in vitro

Background:The polyphenols extraction of Phyllanthus emblica is primarily carried out using organic solvents,and assisted by physical fields such as ultrasound and microwave for extraction.High voltage pulsed electric field technology(PEF)is a non-thermal processing technology that has high efficiency and minimal damage to thermosensitive substances.PEF has been applied to plant extraction in many studies,however,the extraction of polyphenols from Phyllanthus emblica using the PEF has still not been reported;Objective:This study explores the optimal extraction process of polyphenols from Phyllanthus emblica using the PEF,and investigates its relaxation and anti-wrinkle based on anti-oxidation and anti-inflammatory experiment,in order to develop a Phyllanthus emblica extract with substantial efficacy;Materials and Methods:The method of Phyllanthus emblica extract using PEF is established,and compared with a traditional extraction method.The experimental conditions,such as electric field intensity(0.5–6.0 kV/cm),pulse times(20−120),extraction time(0–60 min)and material concentration(0.5%∼3%),are investigated and optimized using orthogonal experiments;Results:the polyphenols in the Phyllanthus emblica extract were highest at the electric field intensity of 5 kV/cm,120 pulses,extraction time of 30 min,and 2%material concentration.The PEFcontained more polyphenols than the conventional water extraction and ultrasound-assisted extraction.The Phyllanthus emblica extract had substantial antioxidant and anti-inflammatory effects,with a good clearance effect on DPPH(IC50 of 1.82%)and ABTS(IC50 of 1.80%)radicals.At the Phyllanthus emblica extract concentration of 1.25%,inflammatory factors(TNF-α)were reduced by 47.08%;and Conclusion:The PEF is a leading-edge and promising method for preparing Phyllanthus emblica extracts.

叶下珠黄化植原体和丛枝植原体的分子鉴定

2022年8月在海南省文昌市一个槟榔黄化病园内,分别发现叶片黄化和丛枝症状的叶下珠,前期检测均为植原体感染。为了明确叶下珠黄化植原体和丛枝植原体的分类鉴定,本研究通过克隆16S rDNA基因和核糖体蛋白(rp)基因,并进行基因序列一致性、系统发育树及虚拟RFLP等分析。结果显示:克隆获得叶下珠黄化植原体16S rDNA片段1246 bp,rp基因1212 bp;叶下珠丛枝植原体16S rDNA片段1827 bp,rp基因1240 bp。基因序列一致性显示,叶下珠黄化植原体与丛枝植原体的16S rDNA基因序列均与16SrⅠ组的植原体一致性高达98%以上,与槟榔黄化植原体海南株系的16S rDNA序列一致性达100%;而二者的rp基因序列均与rpⅠ组的植原体一致性高达99%以上。系统发育树分析显示,叶下珠黄化植原体和丛枝植原体16S rDNA基因均与翠菊黄化组(16SrⅠ)植原体聚于一个大分支,且与16SrⅠ-B亚组的槟榔黄化植原体聚于同一个小分支,亲缘关系接近;而rp基因均与翠菊黄化组(rpⅠ)植原体集聚于一个大分支,且与rpⅠ-B亚组的翠菊黄化植原体聚于同一个小分支,亲缘关系接近。虚拟RFLP分析显示,叶下珠黄化植原体和丛枝植原体的16S rDNA基因序列虚拟RFLP图谱与16SrⅠ-B的洋葱黄化植原体的参考图谱相同,且相似系数为1.00。综上表明,叶下珠黄化植原体和丛枝植原体均属于16SrⅠ-B亚组成员。研究结果可对采用铲除槟榔黄化病中间寄主的防控手段提供理论依据。

叶下珠配方颗粒、饮片汤剂药动学特性及生物等效性评价

目的评价叶下珠配方颗粒、饮片汤剂药动学特性及生物等效性。方法12只大鼠随机分为2组,分别灌胃给予叶下珠配方颗粒、饮片汤剂溶液(0.4282、2.000 g/kg),于0.083、0.167、0.25、0.5、0.75、1、2、3、4、8、12、24、36 h采血,UPLC-MS/MS法测定没食子酸、柯里拉京、鞣花酸血药浓度,计算主要药动学参数。再考察这2种制剂的生物等效性。结果2种制剂AUC_(0~∞)、AUC_(0~t)、MRT_(0~∞)、MRT_(0~t)、t_(1/2)、T_(max)、CL、C_(max)比较,差异无统计学意义(P>0.05),其C_(max)、AUC_(0~∞)比值或对数均数的差值均在90%置信度下符合生物等效标准。结论叶下珠配方颗粒、饮片汤剂药动学特性接近,具有生物等效性。

Suppressed effects of Phyllanthus urinaria L.ethyl acetate extract on hepatitis B virus both in vitro and in vivo

Background:Phyllanthus urinaria L.(P.urinaria)extract(PUE)has been used to inhibit hepatitis B virus(HBV).However,the underlying mechanism remains unclear.To investigate which PUE fractions and main components lead to against HBV and approach the relevant molecular mechanisms.Methods:P.urinaria was extracted with water,and then the decoction was extracted by petroleum ether,ethyl acetate,and n-butanol in turn.The HepG2.2.15 cell was treated with aqueous fraction,petroleum ether fraction,ethyl acetate fraction and n-butanol fraction,gallic acid(GA,C7H6O5)and corilagin(CL,C27H22O18),respectively.The medium was collected for hepatitis B surface antigen(HBsAg)and hepatitis B e antigen assays.Cell counting kit-8 method was used to identify cell proliferation.Also,the levels of cellular oxygen consumption,reactive oxygen species,and reduced glutathione were detected.The HBV modeling mice were treated with ethyl acetate fraction,entecavir and physiological saline,respectively.The serum was collected for HBsAg and inflammatory cytokines assays.Liver tissue metabolites were screened by LC-MS/MS method.Results:The ethyl acetate fraction(EAF)of P.urinaria could significantly inhibit HBV secretion in HepG2.2.15(P<0.05).Furthermore,two main constitutes in ethyl acetate fraction,GA and CL,could significantly inhibit HBV secretion and reduced cell proliferation(P<0.05).Also,GA and CL could increase cellular oxygen consumption,intracellular superoxide anions level,superoxide dismutase level and glutathione depletion.Compared with the Modeling group,EAF significantly decreased the expression levels of HBsAg,IL-1β,IFN-α(P<0.05).LC-MS/MS analysis results showed that EAF dramatically up-regulate hydroxyproline,maltotriose,betaine and down-regulate glutathione disulfide,taurocholate,taurochenodeoxycholate(P<0.05).Kyoto Encyclopedia of Genes and Genomes results show that the differential metabolites were mainly enriched in ATP-binding cassette transporters pathway.Conclusions:P.urinaria exhibits suppressed effects on HBV by modulating reactive oxygen species formation or metabolomics both in vitro and in vivo.These data indicate that P.urinaria may be an alternative therapeutic agent for the treatment of HBV-related hepatitis.

Phytochemistry and ethnomedicinal qualities of metabolites from Phyllanthus emblica L.:A review

Phyllanthus emblica or Indian gooseberry is an integrated part of Ayurvedic and Traditional Chinese Medicines.For several decades,the well-known ancient herb has been extensively utilized in traditional medicine to cure diseases like fever,diabetes,constipation,jaundice,ulcers,biliousness,anemia,anorexia,and dyspepsia.In the traditional system,Indian gooseberry has various ethnomedicinal applications.In the Ayurvedic system,different methods of administration(anupan)have shown different ethnomedicinal properties of Indian gooseberry.Seventy well-known chemical components in Indian gooseberry have been identified through phytochemical evaluation,among which the flavonoids and phenols are most prominent.From the toxicity perspective,it is considered a safe herb in India,and is taken as a food supplement in European countries.The wide-spectrum pharmacological activities of the crude extracts and isolates of Indian gooseberry are attributed to the predominance of phenols and flavonoids.Thus,it is important to study the exact mechanism of the activity of the phytochemicals in Indian gooseberry,especially in anti-cancer activities.Extract of Indian gooseberry enhances proliferation in several cancer cells in vitro,including stem cells like ovarian cancer(OC)cells,and also has been observed to possess anti-proliferative characteristics in vivo.This review intends to explore the therapeutic potential of Indian gooseberry based on scientific reports and attempts to find the gaps for future research.

叶下珠饮片质量标准优化研究

目的:优化叶下珠饮片的质量标准。方法:采用薄层色谱(TLC)法对叶下珠饮片进行定性鉴别;采用电子显微镜观察叶下珠饮片粉末及横切面特征;采用高效液相色谱法测定没食子酸、柯里拉京的含量;参照2020年版《中华人民共和国药典》测定叶下珠饮片中水分、总灰分、酸不溶灰分和醇溶性浸出物的含量。结果:TLC分析中,叶下珠饮片与对照药材在相近位置均显示紫红色斑点。显微鉴别中,饮片粉末、横切面所含各种组织结构均可作为鉴别依据。含量分析中,没食子酸的线性回归方程为Y=15068X-14509(r=0.9998),含量为0.0110%~0.2582%;柯里拉京的线性回归方程为Y=38265X-7032(r=0.9999),含量为0.1267%~0.3291%;水分、总灰分、酸不溶灰分及醇溶性浸出物含量分别为9.49%~11.29%、6.02%~9.92%、0.64%~1.67%、15.73%~30.25%。结论:该质量标准可为准确、全面控制叶下珠饮片质量提供技术支持。

Network pharmacology and experimental validation to reveal the anti-hepatitis B virus pharmacological mechanism of Phyllanthus urinaria L.

Background:To explore the pharmacological mechanism of the anti-hepatitis B virus of Phyllanthus urinaria L.through network pharmacological analysis and experimental validation.Method:The active ingredient,target of action and target of action related to hepatitis B were clarified by searching the herb group identification,GeneCards and OMIM databases,and the protein interaction relationship was obtained by using the String database,and the protein interaction network map was constructed by using Cytoscape software.We also performed gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis of key targets of the anti-hepatitis B action of Phyllanthus urinaria L.and predicted the core targets and pathways of Phyllanthus urinaria L.anti-hepatitis B.The main targets predicted by network pharmacology were then validated by HepG2.2.15 cell experiments.Results:By searching active ingredient targets and hepatitis B disease targets,a total of 19 active ingredients and 64 related targets of action were retrieved from Phyllanthus urinaria L.,and a total of 51 common targets were obtained by mapping the obtained hepatitis B disease targets and drug targets.protein protein interaction network analysis indicated that targets including TNF,JUN,AKT1,IL-10,IL-1B,CAT,HMOX1,NFE2L2,and CASP3 and other targets may be the core targets.gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed that the treatment of hepatitis B by Phyllanthus urinaria L.mainly included inflammation and oxidation-related processes,and the signaling pathways mainly included fluid shear stress and atherosclerosis,VEGF,and hepatocellular carcinoma.The results of the in vitro test showed that after the action of different concentrations of the extracts of the Phyllanthus urinaria L.in the safe concentration range on cells HepG2.2.15,HBsAg,HBeAg and hepatitis B virus DNA levels were significantly inhibited,and NFE2L2 and HMOX1 were affecting hepatitis B virus transcription and replication by regulating the oxidative stress response.Conclusion:Using an integrated network pharmacology approach,this study revealed the active components and potential targets of Phyllanthus urinaria L.for the treatment of the hepatitis B virus,providing a theoretical basis for the research and clinical application of Phyllanthus urinaria L..

Two pyrrole acids isolated from Phyllanthus emblica L.and their bioactivities

An undescribed pyrrole acid,1-(4′-methoxy-4′-oxobutyl)-1 H-pyrrole-2,5-dicarboxylic acid(1)and one known pyr-role acid(2)were isolated from the fruits of Phyllanthus emblica.The structures of these compounds were elucidated via the comprehensive analyses of IR,HRESIMS,1D and 2D spectroscopic data.A series of biological assays revealed that compounds 1 and 2 could inhibit LPS-induced over-production of nitric oxide(NO),interleukin-6(IL-6),monocyte chemotactic protein 1(MCP-1)and tumor necrosis factor-α(TNF-α)by reducing the phosphorylation of extracellular regulated protein kinases(ERK)and c-Jun N-terminal kinases(JNK)in RAW 264.7 cells.Additionally,compounds 1 and 2 were found to reduce lipid deposition and increase the mRNA expression of ATP-binding cassette transporter A1 in oxidized low-density lipoprotein-treated RAW264.7 macrophages.

叶下珠的有效部位和药理作用研究进展

叶下珠是临床常用治疗癌症的中药材,其发挥作用的有效部位包括水提物、醇提物、叶下珠生物碱、叶下珠多糖等。除显著的抗肝癌、肺癌、胃癌、乳腺癌等抗癌作用,叶下珠还具有抗病毒、抗氧化、抑菌、抗炎等药理作用。结合近5年的研究成果,文章对上述叶下珠有效部位和药理作用进行分析整理,同时提出今后关于叶下珠研究的建议,以此反映最新的叶下珠研究成果,为叶下珠临床应用提供理论依据,也为研究者下一步工作提供参考。

叶下珠水提液胃肠促动作用初探

目的对叶下珠水提液胃肠促动作用进行初步探究。方法应用在体小鼠胃排空和小肠向前推进性运动试验法,以及离体的家兔回肠肠管和豚鼠胃试验法,观察叶下珠水提液对动物胃、小肠活动情况的作用。结果相较于正常对照组,叶下珠水提液低、中、高剂量(2.5、5.0、10.0 g生药/kg)组能较明显地减少在体小鼠胃内残留率,也能较明显增加小肠向前推进运动百分比例,差异有统计学意义(P<0.05);在离体实验中,叶下珠水提液给药后明显增加离体家兔回肠肠管平滑肌收缩的幅度,相较于给药前差异有统计学意义(P<0.01),明显增加豚鼠胃底环形肌收缩的振幅和速度,相较于给药前差异也有统计学意义(P<0.01)。结论叶下珠水提液对胃肠具有明显促动作用。

叶下珠有效成分提纯工艺和质量控制研究进展

叶下珠主要成分包括黄酮类、酚酸类、木脂素类、生物碱类及多糖等,具有清热解毒、利水消肿、明目消积的功效,以及抗菌、抗氧化、抗病毒、抗血栓、提高脾细胞活性、长期使用毒副作用小的特点。临床上,叶下珠联合其他药物治疗乙型肝炎、脂肪肝效果理想。由于药效明确,开发利用前景良好,关于叶下珠各类成分提取利用和质量控制方面的研究及技术不断革新。为提供叶下珠药材开发利用的参考依据,本研究结合相关文献,综述了叶下珠部分有效成分的提纯工艺和近10年来质量控制方面的研究,发现(1)对于叶下珠中总酚类的提取,超声法适用于工业化生产,而柯里拉京的提取方面,膜过滤技术和柱色谱分离技术最有望用于工业生产;其他成分的提取工艺目前主要以传统方法为主,新方法处于实验探索阶段。(2)尽管有关叶下珠药理作用的研究较多,但是单个活性成分的构效关系研究较少,不利于进一步开发新药。(3)质量控制多集中在含量测定和指纹图谱方面,对重金属和33种农残的研究未见报道;市场上仍存在部分易混品常见的有苦味叶下珠、珠子草等,目前仍缺乏简单有效的鉴别手段。

Screening of Anti-Newcastle Disease Virus Ingredients of Phyllanthus urinaria

[ Objective ] The aim of the study was to screen the antiviral ingredients of Chinese herbal medicine Phyllanthus urinaria in order to devel- op new drugs for the treatment of viral diseases of poultry. [ Method ] The whole grass of phyllanthus was respectively decocted in 75% ethanol （E） and pure water （PF）, and then the active ingredients were separately extracted in petroleum ether （PE）, ethyl acetate （EA）, and n-butyl alcohol （BU）. The extracts were inoculated on chicken embryo fibroblast （CEF） to observe their inhibitory effect on cytopathic effect （CPE） of Newcastle disease virus （NDV）, inoculated into chicken embryos to observe the changes of the hemagglutination titer of NDV, and inoculated in chickens to determine the mortality and evaluate their effect of immune protection. [ Result] The E -PE and E -BU extracts, especially E -BU extract, inhibi- ted CPE of NDV on CEF and significantly inhibited the proliferation of NDV in chicken embryo （P〈0.05） ; the survival rate of E-PE and E-BU group was extremely significantly higher than that of ribavirin group and the normal saline control （ P 〈0.01 ）, with E - PE group more higher. [ Condusion] The water extract of phyllanthus is less effective; E -PE extract and E -BU extract should effectively inhibit the CPE and proliferation in chicken embryo of NDV and also have better immune protection against NDV.

余甘子(Phyllanthus emblica L.)Vc含量分析

采用2,6-二氯酚靛酚滴定法分析比较了余甘子各器官之间、同品种不同植株之间、不同余甘子品种之间以及与野生资源之间的Vc含量以及不同预处理方法对Vc测定的影响。结果表明,Vc在余甘子各器官中均有分布,以果实与初生幼叶中居多,茎、根中含量较低;皇帝甘与粉甘含量较多,但与其它品种的差异不大;5个野生资源单株Vc含量差异极显著,其中有3个野生单株的果肉Vc含量高于主栽品种粉甘。

余甘(Phyllanthus emblica L.)大小孢子发生及雌雄配子体发育研究

采用常规石蜡切片技术,对平丹1号余甘大小孢子发生及雌雄配子体发育进行研究。结果表明:药壁发育为双子叶型;绒毡层为腺质绒毡层;小孢子母细胞减数分裂时胞质分裂为同时型;产生四面体形四分体;成熟的花粉粒为二细胞型花粉;平丹1号余甘的雌蕊为上位子房,3心皮3室,每室含倒生胚珠2枚,为厚珠心,具珠心喙,胚囊为蓼形;平丹1号余甘部分大孢子母细胞、大孢子及胚囊发育异常,致使不能形成正常的卵细胞,不能进行受精。

苦味叶下珠药效物质及药理学研究进展

以苦味叶下珠的药效物质、临床活性、临床应用等为关键词,以Web of Science、谷歌学术、百度学术、中国知网等为数据来源,查阅了与其相关的研究文献,对苦味叶下珠的植物化学研究、药理活性、毒理学评估和禁忌证进行了分析整理,发现苦味叶下珠含有许多有价值的化学成分,且这些化学成分显示出广泛的药理活性,具有较高的抗肿瘤、抗菌、抗氧化、健胃止泻和抗溃疡活性,同时具有增强记忆力的作用,并对避孕、修复肝损伤、降血糖具有临床意义.

Anti-diabetic activity of quercetin extracted from Phyllanthus emblica L.fruit: In silico and in vivo approaches

In this study, molecular interactions of the ligands, quercetin, gallic acid, and metformin with various diabetes mellitus-related protein targets, such as glycogen phosphorylase and peroxisome proliferatoractivated receptor gamma, were assessed. It was revealed that quercetin possesses good binding affinity to both targets. Quercetin is a major constituent of methanolic extracts of Phyllanthus emblica fruit. The antihyperglycemic effect of quercetin in streptozotocin(STZ)-induced diabetic rats was examined. The isolated quercetin administered at a dose of 75 mg/kg body weight produced a maximum decrease of14.78% in blood glucose levels in the diabetic rats after 7 days of treatment. Furthermore, quercetin doses of 50 and 75 mg/kg were shown to significantly improve the profiles of triglycerides, high-density lipoprotein, very-low-density lipoprotein, low-density lipoprotein, and total cholesterol at the end of the study in STZ-induced diabetic rats. The administration of quercetin(25, 50, and 75 mg/kg body weight)daily for 28 days in STZ-induced diabetic rats resulted in a significant decrease in blood glucose and urine sugar levels, with a considerable rise in plasma insulin and hemoglobin levels. Therefore, quercetin is a potential drug with antidiabetic and antihyperglycemic action mediated by changes in the levels of glucose, cholesterol, and triglycerides as indicated by in silico and in vivo studies.

Identification and characterization of phenolics and terpenoids from ethanolic extracts of Phyllanthus species by HPLC-ESI-QTOF-MS/MS

Phyllanthus species plants are a rich source of phenolics and widely used due to their medicinal properties. A liquid chromatography–tandem mass spectrometry(LC–MS/MS) method was developed using high-pressure liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry(HPLC-ESI-QTOFMS/MS) for the identification and characterization of quercetin, kaempferol, ellagic acid and their derivatives in ethanolic extracts of Phyllanthus species. The chromatographic separation was carried out on Thermo Betasil C_8 column(250 mm×4.5 mm, 5 μm) using 0.1% formic acid in water and 0.1% formic acid in methanol as the mobile phase. The identification of diagnostic fragment ions and optimization of collision energies were carried out using 21 reference standards. Totally 51 compounds were identified which include 21 compounds identified and characterized unambiguously by comparison with their authentic standards and the remaining 30 were tentatively identified and characterized in ethanolic extracts of P. emblica, P. fraternus, P. amarus and P.niruri.

Protective effects of Phyllanthus acidus(L.) Skeels leaf extracts on acetaminophen and thioacetamide induced hepatic injuries in Wistar rats

Objective:To investigate and compare the hepatoprotective effects of crude ethanolic and aqueous extracts of Phyllanthus acidus（L.） Skeels（P.acidus） leaves on acetaminophen（APAP） and thioacetamide（TAA） induced liver toxicity in wistar rats.Silymarin was the reference hepatoprotective agent.Methods:In two different sets of experiments,the P.acidus extracts （200 and 400 mg/kg,body weight） and silymarin（100 mg/kg,body weight） were given orally for 7 days and a single dose of APAP（2 g/kg,per oral） or TAA（100 mg/kg,subcutaneous） were given to rats.The level of serum aspartate transaminase（AST）,alanine transaminase（ALT）,alkaline phosphatase（ALP）,total bilirubin and total protein were monitored to assess hepatotoxicity and hepatoprotection.Results:APAP or TAA administration caused severe hepatic damage in rats as evident from significant rise in serum AST,ALT,ALP,total bilirubin and concurrent depletion in total serum protein.The P.acidus extracts and silymarin prevented the toxic effects of APAP or TAA on the above serum parameters indicating the hepatoprotective action.The aqueous extract was found to be more potent than the corresponding ethanolic extract against both toxicants.The phenolic and flavonoid content（175.02±4.35 and 74.68±1.28,respectively） and 2,2-diphenyl-1- picrylhydrazil（DPPH）[IC50=（33.2±0.31）μg/mL]scavenging potential was found maximum with aqueous extract as compared to ethanolic extract.Conclusions:The results of present study suggests that the aqueous extract of P.acidus leaves has significant hepatoprotective activity on APAP and TAA induced hepatotoxicity,which might be associate with its high phenolic and flavonoid content and antioxidant properties.

Effect of Phyllanthus emblica Linn.on Candida adhesion to oral epithelium and denture acrylic

Objective:To investigate the effect of Phyllanthus emblica(P.emblica) Linn,ethanolic extract on the adhesion of Candida albicans(C.albicans) to human buccal epithelial cells(BECs) and denture acrylic surfaces.Methods:Human BECs and transparent acrylic strips were pretreated with ethanolic extract solution of P.emblica fruits at concentration ranged from 18.7 to 300 mg/mL.After washing BECs and the strips were inoculated with three strains of C.albicans (ATCC 10281 and two clinical isolates)(10~7 cells/mL).Normal saline solution(NSS) and 0.2% chlorhexidine gluconate were used as negative and positive controls,respectively.BECs were harvested on 12μm-polycarbonate filters(Millipore,USA).The membrane filters and the strips were stained with Gram stain.Adherent yeast cells on 100 randomly selected epithelial cells and 20 randomly selected fields on each strip were counted under microscope.The statistical significance was calculated by Kruskal-Wallis and Tukey tests at a significant level of P【0.05. Results:Significant lower numbers of all strains of yeasts adhering to BECs and acrylic strips were observed after exposure to 75-300 mg/mL of plant extract compared with NSS.Conclusions: The present study demonstrates that P.emblica ethanolic extract interferes with the adhesion of C. albicans to BECs and denture acrylic surfaces in vitro.

Development of species-specific SCAR markers for identification of three medicinal species of Phyllanthus

Phyllanthus amarus Schum. & Thonn. has been widely used in traditional medicine in Thailand as an antipyretic, a diuretic, to treat liver diseases and viral infections. Two closely related species, P. debilis L. and P. urinaria Klein ex Willd., with different and less effective medicinal properties, are less commonly used. These three species are similar in morphology and often occur in overlapping populations in nature. The latter two species can easily be mistaken for P. amarus and collected for medicinal uses, which can lead to undesirable results. DNA fingerprints of these species were obtained using RAPD-PCR techniques. RAPD markers specific for each species were identified. Primers for highly specific sequence-characterized-amplified-regions (SCAR) were then designed from nucleotide sequences of specific RAPD markers. These primers efficiently amplified SCAR markers of 408, 501 and 319 bp unique to P. amarus, P. debilis and P. urinaria respectively. This method of plant identification was rapid and highly specific when tested against DNA of several closely related species and was able to amplify specific markers from mixed DNA samples.