Validated TLC method for simultaneous quantitation of kutkoside and picroside‐I from Kutki extract

Introduction – The two iridoid glycosides kutkoside and picroside‐I are the active hepatoprotective principles of Picrorhiza kurroa Royle ex Benth (Scrophulariaceae), commonly known as Kutki. Quantitation of these phytoconstituents is important for the routine quality control of Kutki extract. Objective – To develop and validate a simple, precise and rapid thin‐layer chromatography (TLC) method for the simultaneous quantitation of kutkoside and picroside‐I in Kutki extract. Methodology – The analysis was performed on a TLC precoated silica gel 60 F₂₅₄ plate with ethyl acetate:methanol:glacial acetic acid:formic acid (25:5:1:1, v/v/v/v) as mobile phase. Densitometric evaluation of kutkoside and picroside‐I was carried out at 265 nm and the mobile phase showed good resolution with R_f values 0.42 ± 0.03 and 0.61 ± 0.03 for kutkoside and picroside‐I, respectively. The method was validated in terms of specificity, linearity, accuracy and precision. Results – The content of kutkoside and picroside‐I was found to be 2.18 and 1.90%, respectively, and was comparable with those obtained by HPLC. The linearity was found to be in the range of 80–480 ng/spot for both kutkoside and picroside‐I. The average recovery values were found to be 96.5 and 96.0% for kutkoside and picroside‐I, respectively. Conclusion – The developed method was found to be relatively simple, precise and reproducible for the simultaneous quantitation of kutkoside and picroside‐I. The method does not employ any derivatisation procedure and can be used as a quality control tool for the routine analysis of commercial Kutki extracts. Copyright © 2010 John Wiley & Sons, Ltd.     

Chemical composition,antioxidant and macromolecule damage protective effects of Picrorhiza kurroa Royle ex Benth

In the present study, we identified the chemical constituents of 70% hydroalcoholic fraction of Picrorhiza kurroa by LC–ESI–MS/MS which showed the presence of iridoid glucosides such as picroside I, picroside II, picroside III, picroside IV, kutkoside, pikuroside and flavonoids like apocynin and vanillic acid. P. kurroa exhibited DPPH radical scavenging and metal chelating activities with IC₅₀ of 75.16 ± 3.2 and 55.5 ± 4.8 μg/mL and also showed potent reducing power and total antioxidant activities. The extract inhibited macromolecule damage such as H₂O₂ induced plasmid DNA damage and AAPH induced oxidation of bovine serum albumin and lipid peroxidation of rat hepatic tissues.     

The detection of radical scavenging compounds in crude extract of borage (Borago officinalis L.) by using an on-line HPLC-DPPH method

The rapid evaluation of antioxidant activity of crude borage (Borago officinalis L.) extract was determined by using DPPH free radical method. This borage extract resulted in a rapid decrease of the absorbance and showed very high hydrogen-donating capacity towards the 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radical. A new HPLC-DPPH on-line method was applied for a screening of several radical scavenging components in this borage extract as well as for quantitative analysis. This on-line HPLC-DPPH method was developed using a methanolic solution of DPPH-stable radical. The HPLC-separated analytes reacted post-column with the DPPH solution in methanol. The induced bleaching was detected as a negative peak photometrically at 515 nm. The separation of antioxidative components was carried out by gradient HPLC with mobile-phase composition ranging from 2% to 80% acetonitrile with 2% acetic acid in water, UV detection was carried out at 280 nm. The HPLC analysis of borage extract revealed the presence of several radical scavenging components in the borage extract. The results obtained from the chromatograms suggest that some compounds present in the extract possess high radical quenching ability. The dominant antioxidative compound in the crude extract of borage leaves was identified as rosmarinic acid.     

大豆肽体外抗氧化活性研究

研究大豆肽的体外抗氧化的作用。采用邻二氮菲-Fe^2＋检测大豆肽对羟自由基（·OH）的清除作用,邻苯三酚检测大豆肽对超氧阴离子（·O2^-）的清除作用,用硫代巴比妥酸法测定小鼠肝匀浆丙二醛（MDA）的含量,用比色法测定小鼠红细胞溶血度,来研究大豆肽的抗氧化效果。结果表明：大豆肽可以清除·OH和·O2^-,抑制·OH所致的丙二醛的产生,减少H2O2所致的红细胞溶血,在2～15g／L内均具有明显的量效关系。表明大豆肽在体外具有明显的抗氧化效果。     

Free radical scavenging potential of Picrorhiza kurrooa Royle ex Benth

For assessing free radical scavenging potential of P. kurrooa, the antioxidant activity of P. kurrooa extract was studied by lipid peroxidation assay using rat liver homogenate. The extract (1 mg/ml) showed marked protection (up to 66.68%) against peroxidation of liver phospholipids. Besides, reduced glutathione showed very encouraging activity. The extract also exhibited significant scavenging activity. Thus augmenting the wide use of plant in the indigenous system of medicine, which may partly be due to antioxidant and free radical scavening activity of the extract.     

Development of informative genic SSR markers for genetic diversity analysis of Picrorhiza kurroa

Picrorhiza kurroa is a medicinal perennial herb of economic importance due to its hepatoprotective properties mainly accounted by picroside I and picroside II. To fulfill the current demand of the market indiscriminate collection from its natural habitat pose a great threat to this endangered species. To strategize the conservation of natural populations, a set of 20 highly informative novel genic SSR markers were identified. The utility of these makers was successfully tested for the genetic diversity characterization of P. kurroa populations (n = 28) from three geographical locations. These markers produced 136 alleles (average of 6.8) with mean observed heterozygosity, expected heterozygosity, Shannon’s information index, and PIC value of 0.971, 0.798, 1.681, and 0.737 respectively, revealing a higher extent of genetic diversity in P. kurroa. Further, clustering of all the individuals according to their geographical locations indicates at a spatial population structure in P. kurroa. The current study suggests that informative SSR makers identified here can be potentially used for diversity characterization targeting wider geographical locations for selection of elite/quality genotypes for commercial cultivation and genetic rescue of this endangered species.     

In vitro free radical scavenging activity of hepatic metallothionein induced in an Indian freshwater fish, Channa punctata Bloch

Mammalian metallothioneins (MT) have been reported to scavenge free radicals. There is no experimental evidence to show that fish MT has a similar property. In the present study cadmium-induced MT (Cd-MT) from the liver of an Indian freshwater fish Channa punctata Bloch was investigated for its free radical scavenging activity using three different in vitro assays. Exposure to cadmium chloride (0.2 mg/kg body weight; three doses on alternate days) resulted in a marked induction of Cd-MT in liver. Only a single isoform of Cd-MT was found to be induced. Molecular weight of Cd-MT was found to be 14 kDa as deduced by SDS-PAGE analysis. The purified Cd-MT effectively scavenged the following free radicals: superoxide radical (O2*-), 2,2'-azinobis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS*+) and 1,1-diphenyl-picrylhydrazyl radical (DPPH*). The radical scavenging effect was found to be concentration-dependent. Also, the purified MT exhibited an inhibitory effect on ferric nitrilotriacetate (Fe-NTA) induced oxidative DNA damage in vitro. The cysteine residues of MT are proposed to be the main candidate for its radical scavenging activity. Findings of the present study strongly suggest a free radical scavenging role for fish MT. Present study adds to the little existing knowledge about fish MT and its possible biological functions.     

Evaluation of antioxidant properties of root bark of Hemidesmus indicus R. Br. (Anantmul).

Hemidesmus indicus R. Br. (Asclepiadaceae) is a well known drug in Ayurveda system of medicine. In the present study, antioxidant activity of methanolic extract of H. indicus root bark was evaluated in several in vitro and ex vivo models. Further, preliminary phytochemical analysis and TLC fingerprint profile of the extract was established to characterize the extract which showed antioxidant properties. The in vitro and ex vivo antioxidant potential of root bark of H. indicus was evaluated in different systems viz. radical scavenging activity by DPPH reduction, superoxide radical scavenging activity in riboflavin/light/NBT system, nitric oxide (NO) radical scavenging activity in sodium nitroprusside/Greiss reagent system and inhibition of lipid peroxidation induced by iron-ADP-ascorbate in liver homogenate and phenylhydrazine induced haemolysis in erythrocyte membrane stabilization study. The extract was found to have different levels of antioxidant properties in the models tested. In scavenging DPPH and superoxide radicals, its activity was intense (EC50 = 18.87 and 19.9 microg/ml respectively) while in scavenging NO radical, it was moderate. It also inhibited lipid peroxidation of liver homogenate (EC50 = 43.8 microg/ml) and the haemolysis induced by phenylhydrazine (EC50 = 9.74 microg/ml) confirming the membrane stabilization activity. The free radical scavenging property may be one of the mechanisms by which this drug is effective in several free radical mediated disease conditions.     

The antioxidative activity of traditional Japanese herbs

The objective of this research was to examine the radical scavenging activity of traditional Japanese herbs. Samples used in the experiments were gennoshoko (Geranium nepalense var. thunbergii), yomogi (Artemisia vulgaris var.indica), senburi (Swertia japonica), iwa-tobacco (Conandron ramondioides), sarunokoshikake (Elfvingia applanata), kanzo (Glycyrrhiza uralensis Fisch) and matatabi (Actinidia polygama). The water-soluble components of the herbs were extracted in boiling water, and the volatile oil was extracted by a distillation apparatus or steeping in some organic solvents such as petroleum ether and ethyl ether. The radical scavenging activity was determined by the decrease of free radicals of DPPH detected by both colorimetric assay and HPLC method at 517 nm. The extracts of gennoshoko, yomogi and iwa-tobacco showed remarkable radical scavenging activity. The volatile oil of yomogi obtained by distillation or steeping in organic solvents had especially strong antioxidative activity.     

有柄石韦醇提物抗氧化活性研究

通过DPPH自由基清除测定、还原能力、总抗氧化能力、羟自由基清除测定,评价有柄石韦Pyrrosia petiolosa醇提物的抗氧化活性。结果显示,有柄石韦醇提物清除DPPH·的IC50为75.82 μg·mL-1,清除·OH的IC50为46.30 μg·mL-1。有柄石韦醇提物清除DPPH· 的能力强于抗坏血酸,清除·OH的能力弱于抗坏血酸;同时,其还原能力和总抗氧化能力均强于抗坏血酸。该结果说明有柄石韦醇提物具有较强的抗氧化活性。     

冬葵果多糖的抗氧化作用研究

研究冬葵果多糖(Fructus Malvae polysaccharides,FMP)对氧自由基的清除作用及对脂质过氧化的抑制作用。采用分光光度法测定FMP清除Fenton体系产生的羟自由基(.OH)、邻苯三酚自氧化体系产生的超氧阴离子自由基(O2-.)的能力及评价对Fe(Ⅱ)-H2O2体系(.OH)诱导的脂质过氧化终产物丙二醛(MDA)的抑制作用。结果显示,在化学模拟体系中,冬葵果多糖对.OH具有很强的清除作用,与VC比较达到显著水平(P<0.01),对O2-.的清除能力与VC相当。在体外,多糖浓度达到1.72 mg/mL可明显降低MDA的含量,与空白液比较达到显著水平(P<0.01)。以上结果表明,FMP具有明显的抗氧化活性。     

蛹虫草MF27高抗氧化活性提取物筛选及保肝作用研究

本研究对一株优质蛹虫草菌株MF27不同提取物进行体外抗氧化活性比较,筛选得到高抗氧化活性提取物,并进一步探究该提取物对CCl₄诱导的小鼠肝损伤的修复作用。以DPPH自由基和羟自由基的清除率为抗氧化评价指标,从菌丝体发酵液、菌丝体水提物/醇提物、以及子实体水提物/醇提物中筛选菌株MF27的高抗氧化活性提取物;以CCl₄致小鼠急性肝损伤为模型,通过检测血清生化指标、肝功指标的变化,来评价该高活性提取物的体内抗氧化保肝效果。体外抗氧化实验结果表明,MF27的不同提取物均具有较好的体外抗氧化活性,但对清除DPPH和OH自由基能力最好的提取物是子实体水提物,其对DPPH自由基的半数有效浓度（EC₅₀）为0.096mg/mL,对羟自由基的半数有效浓度（EC₅₀）为0.196mg/mL,当在1mg/mL 时对DPPH自由基的清除率为94.94%,对羟自由基的清除率为70.17%;体内抗氧化保肝结果显示,给药组（子实体水提物）相比模型组而言,小鼠血清中ALT、AST以及肝脏中MDA水平极显著降低（P<0.01）、SOD水平极显著升高（P<0.01）,表明子实体水提物能有效改善氧化性肝损伤,同时与阳性对照（联苯双酯）对比,给药组在肝脏指数上表现出相当的作用（P>0.05）。本研究表明菌株MF27的最有效抗氧化活性提取物是子实体水提物,它对体内氧化性肝损伤有一定的修复能力,揭示MF27子实体水提物具有成为抗氧化性肝损伤功能产品的潜力。     

Antioxidant activity of novel chitin derivative

Novel water-soluble chitin derivative was prepared by chemical modification to evaluate antioxidant activities by free radical scavenging potential using electron spin resonance spin trapping technique. Aminoethyl-chitin (AEC) exhibited free radical scavenging activities against 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl, superoxide, and peroxyl radicals. AEC quenched DPPH and peroxyl radical over 55% and 59% at 4mg/mL, and also suppressed superoixde radical over 58% at 2mg/mL. Especially, AEC was more active against hydroxyl radical, and scavenging ratio was 92.2% at 0.12mg/mL. These results suggested that free amino group in the -CH(2)CH(2)NH(2) plays an important role in the free radical scavenging activity. In addition, cytotoxic effect of AEC was assessed using human lung fibroblast (MRC-5) cell line, and AEC showed less toxic against MRC-5.     

Antioxidant activity of anti-inflammatory plant extracts

The antioxidant properties of twenty medical herbs used in the traditional Mediterranean and Chinese medicine were studied. Extracts from Forsythia suspensa, Helichrysum italicum, Scrophularia auriculata, Inula viscosa, Coptis chinensis, Poria cocos and Scutellaria baicalensis had previously shown anti-inflammatory activity in different experimental models. Using free radical-generating systems H. italicum. I. viscosa and F. suspensa protected against enzymatic and non-enzymatic lipid peroxidation in model membranes and also showed scavenging property on the superoxide radical. All extracts were assayed at a concentration of 100 microg/ml. Most of the extracts were weak scavengers of the hydroxyl radical and C. chinensis and P. cocos exhibited the highest scavenging activity. Although S. baicalensis inhibited the lipid peroxidation in rat liver microsomes and red blood cells, the extract showed inhibitory actions on aminopyrine N-demethylase and xanthine oxidase activities as well as an pro-oxidant effect observed in the Fe3+-EDTA-H2O2 system. The results of the present work suggest that the anti-inflammatory activities of the same extracts could be explained, at least in part, by their antioxidant properties.     

茶多糖的抗氧化活性及对细胞氧化损伤的保护机制

茶多糖是一种从茶叶中提取的酸性糖蛋白,具有良好的抗氧化活性。以自由基清除率为指标,分析皖西南地区夏秋茶多糖的抗氧化活性,基于H₂O₂和EDTA-Fe²⁺建立的外源性羟基自由基(·OH)损伤细胞模型和PMA诱导内源性羟基自由基损伤模型,进一步探讨茶多糖对自由基损伤的修复作用机制。结果表明,茶多糖具有良好的体外抗氧化活性,对DPPH·和·OH均具有较强的清除效果,EC₅₀值分别为209.5和535.2μg·mL^–1,最大清除效率与Vc相当。细胞增殖实验表明,外源性和内源性自由基氧化损伤模型中细胞存活率均随着茶多糖浓度的增加而升高,在茶多糖浓度为800μg·mL^–1时细胞存活率分别高达87.41%和85.84%,且显著高于模型组(47.67%和48.03%)。在修复机制上,利用激光共聚焦显微镜显影细胞内活性氧(ROS)分布以及荧光强度,分析结果显示,与模型组相比,茶多糖对于细胞模型中外源和内源性ROS均具有明显的清除效果,与体外抗氧化实验结果一致。茶多糖在体外表...     

Antimutagenic and antioxidant/prooxidant activity of quercetin

The present study has been performed to evaluate the antimutagenic activity of quercetin, ascorbic acid and their combination against an oxidative mutagen. An effort was also made to correlate this activity to the in vitro antioxidant activity of these agents. Antimutagenicity testing was done in Ames Salmonella Assay system using Salmonella typhimurium TA102 against t-butylhydroperoxide as an oxidative mutagen. In vitro antioxidant scavenging activity was tested for DPPH free radical, superoxide anion, hydrogen peroxide and hydroxyl radical in their specific test systems. Quercetin (0.5-8 nmole/plate) and ascorbic acid (0.1-100 micromole/plate) showed significant effect. Quercetin (4 and 8 nmole/plate) when combined with ascorbic acid (500 nmole/plate) showed an increase in the antimutagenic activity. In vitro antioxidant activity of quercetin was better than ascorbic acid in all the test systems used. The study indicated that the antimutagenic activity of quercetin was not solely accountable by its antioxidant nature. However, in vitro free radical scavenging activity of quercetin correlated well with the antimutagenic activity.     

Agrobacterium rhizogenes-mediated transformation of Picrorhiza kurroa Royle ex Benth.: establishment and selection of superior hairy root clone

A protocol for induction and establishment of Agrobacterium rhizogenes-mediated hairy root cultures of Picrorhiza kurroa was developed through optimization of the explant type and the most suitable bacterial strain. The infection of leaf explants with the LBA9402 strain resulted in the emergence of hairy roots at 66.7% relative transformation frequency. Nine independent, opine and TL-positive hairy root clones were studied for their growth and specific glycoside (i.e., kutkoside and picroside I) productivities at different growth phases. Biosynthetic potentials for the commercially desirable active constituents have been expressed by all the tested hairy root clones, although distinct inter-clonal variations could be noted in terms of their quantity. The yield potentials of the 14-P clone, both in terms of biomass as well as individual glycoside contents (i.e., kutkoside and picroside I), superseded that of all other hairy root clones along with the non-transformed, in vitro-grown control roots of P. kurroa. The present communication reports the first successful establishment, maintenance, growth and selection of superior hairy root clone of Picrorhiza kurroa with desired phyto-molecule production potential, which can serve as an effective substitute to its roots and thereby prevent the indiscriminate up-rooting and exploitation of this commercially important, endangered medicinal plant species. CIMAP Publication No.: 2007-28J     

Anti-ultraviolet radiation effects of Coptis chinensis and Phellodendron amurense glycans by immunomodulating and inhibiting oxidative injury

HPLC analysis proved that Coptis chinensis glycan contained Ara, Man, and Gal. The monosaccharide constituents of Phellodendron amurense glycan were determined by HPLC analysis. HPLC analysis proved that P. amurense glycan contained Ara, Xyl, Glu, and Gal. FT-IR spectrum of C. chinensis glycan and P. amurense glycan showed the characteristic absorption peaks of carbohydrate polymers. Exposure of the human skin to ultraviolet radiation (UVR) leads to depletion of cutaneous antioxidants, regulation of gene expression and ultimately to the development of skin diseases. In the present study, free radical scavenging activity of C. chinensis and P. amurense glycan were evaluated. The photoprotective effect of C. chinensis and P. amurense glycan against UV-induced oxidative damage was also investigated in skin. At the concentration range employed, the two glycans showed strong free radical scavenging activity. Ultraviolet radiation reduced skin antioxidant enzyme and immunity activities in animals. Administration of C. chinensis and P. amurense glycans dose-dependently significantly increased skin antioxidant enzyme and immunity activities in animals. In conclusion, C. chinensis and P. amurense glycans present photoprotective properties, which can be attributed to molecules, such as flavonoids and carotenoids, which act as UV-absorbing molecules and as antioxidants, as well as stimulate immunity activities in animals.     

Thioureas differentially induce rat hepatic microsomal epoxide hydrolase and rGSTA2 irrespective of their oxygen radical scavenging effect: effects on toxicant-induced liver injury

Thioureas have been employed as potent hydroxyl radical scavengers and also inhibit production of oxygen free radicals. The in vitro oxygen radical scavenging effect by N,N'-substituted thioureas including dimethylthiourea (DMT), diethylthiourea (DET), tetramethylthiourea (TMT) and diphenylthiourea (DPT) was assessed by the conversion of phi x-174 DNA from supercoiled DNA to the open circular form or to fragmented DNA. Addition of the N,N'-substituted thioureas to the incubation mixture significantly prevented a single strand breakage of phi x-174 DNA induced by autooxidation of benzenetriol. These thioureas were also effective in preventing degradation of phi x-174 DNA induced by autooxidation of benzenetriol in the presence of ferrous iron. In view of the in vitro radical scavenging effect by the thioureas and the role of reactive oxygen species in the induction of phase II detoxifying enzymes, expression of microsomal epoxide hydrolase (mEH) and rGSTA2 in response to these agents was investigated in the rat liver. Rats treated with each of the alkylthioureas exhibited marked increases of mEH and rGSTA2 mRNA levels with TMT being the most effective. DPT an arylthiourea, however, was minimally active in increasing the mRNAs. Time-course studies revealed that DMT, DET and TMT increased the mRNA levels to the greatest extent at 24 h after a single dose of treatment. The levels of mEH and rGSTA2 mRNA were elevated in a dose-dependent manner by the alkylthioureas. Immunoblot analysis showed that the alkylthioureas induced mEH and rGSTA2 proteins in the liver (0.6 mmol/kg per day, 3 days), which was consistent with the increases in the mRNA levels. DMT, DET or TMT enhanced CCl4-induced liver toxicity, as monitored by plasma aminotransferase activity, although each of the agents alone caused only slight increase in the alanine aminotransferase activity. In contrast to the effects of the alkylthioureas, DPT protected the liver against the toxicant-induced injury. All of the thioureas prevented decreases in the hepatic glutathione level by CCl4. Expression of cytochrome P450 2E1 and P450 2B1/2, which are implicated with metabolic activation of CCl4, was assessed after treatment with the thioureas. P450 2E1 and P450 2B1/2 were differentially induced by the alkylthioureas with the expression of P450 2E1 being inversely related with that of P450 2B1/2. These results showed that N,N'-substituted alkylthioureas were capable of inducing mEH and rGSTA2 in the liver with elevation of the mRNAs, that induction of mEH and rGSTA2 by these alkylthioureas might be mediated by production of the reactive oxygens derived from metabolic activation of the agents irrespective of their radical scavenging effect and that the agents rather enhanced toxicant-induced liver injury with the induction of P450 2E1 or P450 2B1/2.     

Glucans exhibit weak antioxidant activity, but stimulate macrophage free radical activity

Polymeric carbohydrates have been reported to modulate inflammatory responses in vitro and in vivo. Previous reports suggest that certain carbohydrate polymers, such as (1-->3)-beta-D-glucans, may possess free radical scavenging activity. If glucans are free radical scavengers then it might explain, in part, the ability of these ligands to modulate inflammatory responses. The present study examined the free radical scavenging activity of a variety of carbohydrate polymers and the effect of the polymers on free radical levels in a murine macrophage cell line. All of the carbohydrates exhibited concentration dependent antioxidant effects (EC(50) range = 807 to 43 microg/ml). However, the antioxidant activity for the carbohydrates was modest in comparison with PDTC (EC(50) = 0.13 microg/ml) and the carbohydrate concentration required for antioxidant activity was high (x EC(50) = 283 microg/ml). The antioxidant ability of the polymers was greater (p < .05) than their monosaccharide constituents, i.e., dextrose EC(50) = 807 vs. glucan sulfate EC(50) = 43 microg/ml. Coincubation of glucans with murine J774a.1 cells increased free radical levels when compared to controls. Therefore, the weak free radical scavenging activity of glucan polymers cannot explain their modulatory effect on inflammatory responses in tissue culture and/or disease models of inflammation.