Optimization of Adventitious Root Culture for Production of Biomass and Secondary Metabolites in Prunella vulgaris L.

Adventitious root cultures of Prunella vulgaris L. were established in shaking flask system for the production of biomass and secondary metabolites. Adventitious root cultures were induced from callus cultures obtained from leaf explants on solid Murashige and Skoog (MS) medium containing combination of 6-benzyladenine (BA; 1.0 mg l^?1) and naphthalene acetic acid (NAA; 1.5 mg l^?1). Thereafter, 0.49 g inoculum was transferred to liquid MS medium supplemented with different concentrations of NAA (0.5–2.0 mg l^?1). Growth kinetics of adventitious roots was recorded with an interval of 7 days for 49 days period. Highest biomass accumulation (2.13 g/l) was observed in liquid medium containing 1.0 mg l^?1 NAA after 21 days of inoculation. However, other concentrations of NAA also showed similar accumulation pattern but the biomass gradually decreases after 49 days of inoculation. Adventitious roots were collected and dried for investigation of total phenolics (TP), total flavonoids (TF), and antioxidant activities. Higher TPC (0.995 GAE mg/g-DRB) and TFC (6.615 RE mg/g-DRB) were observed in 0.5 mg l^?1 NAA treated cultures. In contrast, higher antioxidant activity (83.53 %) was observed 1.5 mg l^?1 NAA treated cultures. These results are helpful in up scaling of root cultures into bioreactor for secondary metabolites production.     

Flavones from Callus Tissue of <Emphasis Type="Italic">Iris ensata</Emphasis>

Flavonoids uncharacteristic of intact plants were isolated from callus tissue of Iris ensata and were identified as 5-hydroxy-4′-methoxyflavone, 5-hydroxy-3′-methoxyflavone, and 5-hydroxy-2′-methoxyflavone using PMR and mass spectrometry. It was proposed that the lack of growth of callus tissue after changing cultivation conditions was related to the inhibiting effect of these flavones on cell proliferation. __________ Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 440–442, September–October, 2005.     

Production of camptothecin in the elicited callus cultures of <Emphasis Type="Italic">Nothapodytes nimmoniana</Emphasis> (J. Graham) Mabberly

The biotechnological approach of in vitro cultures elicitation offers an alternative strategy for the production of camptothecin (CPT) in Nothapodytes nimmoniana to mitigate indiscriminate harvest of the endangered natural population for the alkaloid. Yeast extract (YE) and vanadyl sulfate (VS) elicitors were used to enhance the biosynthesis of CPT in hypocotyl-derived callus cultures of N. nimmoniana by cultivation using solid and liquid Murashige and Skoog (MS) medium amended with NAA + BAP (2.0 + 1.0 mg L^?1). Effects of the two elicitors on biomass and CPT production at 6.25, 12.5, 25, 50 and 75 mg L^?1 concentrations using callus cultures from three cell lines were evaluated after 15, 30 and 45 days culture. Yeast extract elicitor treatments showed a linear enhancement effect on biomass and CPT production up to 50 mg L^?1 YE and beyond the concentrations, no significant effect was observed. Enhanced biomass and CPT production were achieved with VS elicitor up to 25 mg L^?1 concentrations but, 50 and 75 mg L^?1 VS had minimal effects on biomass and CPT production in callus sources and incubation duration-dependent manner. The intracellular yield of CPT in liquid media-cultivated cultures at concentrations of the two elicitors was lower when compared to solid media treatments relative control due to the extracellular accumulation but, higher overall production. Accumulation of the biomass showed association with produced CPT in the elicitor treatments and control cultures.     

Modern Trends in the In Vitro Production and Use of Callus,Suspension Cells and Root Cultures of Medicinal Plants

This paper studies modern methods of producing and using callus, suspension cells and root cultures of medicinal plants in vitro. A new solution for natural product production is the use of an alternative source of renewable, environmentally friendly raw materials: callus, suspension and root cultures of higher plants in vitro. The possibility of using hairy root cultures as producers of various biologically active substances is studied. It is proven that the application of the genetic engineering achievements that combine in vitro tissue culture and molecular biology methods was groundbreaking in terms of the intensification of the extraction process of compounds significant for the medical industry. It is established that of all the callus processing methods, suspension and root cultures in vitro, the Agrobacterium method is the most widely used in practice. The use of agrobacteria has advantages over the biolistic method since it increases the proportion of stable transformation events, can deliver large DNA segments and does not require special ballistic devices. As a result of the research, the most effective strains of agrobacteria are identified.     

Scarlet Flax Linum grandiflorum (L.) In Vitro Cultures as a New Source of Antioxidant and Anti-Inflammatory Lignans

In vitro cultures of scarlet flax (Linum grandiflorum L.), an important ornamental flax, have been established as a new possible valuable resource of lignans and neolignans for antioxidant and anti-inflammatory applications. The callogenic potential at different concentrations of α-naphthalene acetic acid (NAA) and thidiazuron (TDZ), alone or in combinations, was evaluated using both L. grandiflorum hypocotyl and cotyledon explants. A higher callus induction frequency was observed on NAA than TDZ, especially for hypocotyl explants, with a maximum frequency (i.e., 95.2%) on 1.0 mg/L of NAA. The presence of NAA (1.0 mg/L) in conjunction with TDZ tended to increase the frequency of callogenesis relative to TDZ alone, but never reached the values observed with NAA alone, thereby indicating the lack of synergy between these two plant growth regulators (PGRs). Similarly, in terms of biomass, NAA was more effective than TDZ, with a maximum accumulation of biomass registered for medium supplemented with 1.0 mg/L of NAA using hypocotyls as initial explants (DW: 13.1 g). However, for biomass, a synergy between the two PGRs was observed, particularly for cotyledon-derived explants and for the lowest concentrations of TDZ. The influence of these two PGRs on callogenesis and biomass is discussed. The HPLC analysis confirmed the presence of lignans (secoisolariciresinol (SECO) and lariciresinol (LARI) and neolignan (dehydrodiconiferyl alcohol [DCA]) naturally accumulated in their glycoside forms. Furthermore, the antioxidant activities performed for both hypocotyl- and cotyledon-derived cultures were also found maximal (DPPH: 89.5%, FRAP 866: µM TEAC, ABTS: 456 µM TEAC) in hypocotyl-derived callus cultures as compared with callus obtained from cotyledon explants. Moreover, the anti-inflammatory activities revealed high inhibition (COX-1: 47.4% and COX-2: 51.1%) for extract of hypocotyl-derived callus cultures at 2.5 mg/L TDZ. The anti-inflammatory action against COX-1 and COX-2 was supported by the IC₅₀ values. This report provides a viable approach for enhanced biomass accumulation and efficient production of (neo)lignans in L. grandiflorum callus cultures.     

Chemical constituents from Piper marginatum Jacq. (Piperaceae)—antifungal activities and kinetic resolution of (RS)-marginatumol by Candida antarctica lipase (Novozym 435)

The leaves of Piper marginatum contain the antifungal compounds 3,4-methylenedioxypropiophenone 1, 2-methoxy-4,5-methylenedioxypropiophenone 2, 1-(3,4-methylenedioxyphenyl)propan-1-ol 3 (marginatumol), 5,4′-dihydroxy-7-methoxyflavanone 4 and 5,7-dihydroxy-4′-methoxyflavanone 5. The absolute configuration of natural marginatumol was determined as (+)-(R)-3 (ee 48%) by comparison of its optical properties with the chiral forms obtained by kinetic resolution of racemic 3 using Candida antarctica lipase (Novozym 435).     

Improved Protocol for Somatic Embryogenesis and Calcium Alginate Encapsulation in Anethum graveolens L.: A Medicinal Herb

An improved procedure has been developed for efficient somatic embryogenesis in Anethum graveolens. Green friable embryogenic callus was obtained from hypocotyl segments on medium augmented with 2,4-dichlorophenoxyacetic acid (2,4-D). The highest embryogenic callus induction frequency of 87 % was obtained on Murashige and Skoog (MS) medium containing 1.13 μM 2,4-D. At lower concentration of 2,4-D (0.34 μM) callus turned dark in color and slow growing. Embryogenic cultures (76 %) responded with a mean number of 43 globular and 18 heart stage embryos. Somatic embryo maturation and subsequent conversion into plantlets took place on MS lacking growth regulators. Maximum number of somatic embryos developed on MS medium was 128.3 (per flask) and a plantlet conversion of 82 % was observed. Calcium alginate beads were produced by encapsulating somatic embryos. Highest percent germination (83 %) was observed on 0.8 % agar solidified MS medium with the plantlets acquiring an average length of 2.1 cm. Encapsulated somatic embryos could be stored at 4 °C up to 60 days with a conversion frequency of 49.3 %. Highest protein and proline content has been observed in embryogenic callus with small globular embryos. During morphological differentiation of the somatic embryos, changes in the antioxidant enzymatic system were observed. Superoxide dismutase (SOD) activity increased during initial stages and decreased catalase (CAT), peroxidase (POD), and ascorbate peroxidase (APX) activities were detected.     

Establishment and Quality Assessment of Tissue Cultures in Glycyrrhiza uralensis Fisch.

In this study, seedling, callus, cell, and adventitious root of Glycyrrhiza uralensis Fisch. have been established. In order to find the best one for producing G. uralensis active constituents, triterpenoid saponins and flavonoids in native root and tissue cultures were determined, and the contents in different G. uralensis materials were analyzed using cluster analysis. The contents of triterpenoid saponins and glycyrrhizic acid in tissue cultures were much lower than that in native G. uralensis. The total flavonoids content we determined in adventitious root was 6.31 mg?g^?1, which was close to that of native root (9.82 mg?g^?1). Based on the cluster analysis, we found that G. uralensis cultures were not suitable for production of glycyrrhizic acid, while adventitious root had a greater capability of flavonoids production comparing to seedling, callus, and cell.     

Effect of Salts (NaCl and Na2CO3) on Callus and Suspension Culture of Stevia rebaudiana for Steviol glycoside Production

Steviol glycosides are natural non-caloric sweeteners which are extracted from the leaves of Stevia rebaudiana plant. Present study deals the effect of salts (NaCl and Na₂CO₃) on callus and suspension culture of Stevia plant for steviol glycoside (SGs) production. Yellow-green and compact calli obtained from in vitro raised Stevia leaves sub-cultured on MS medium supplemented with 2.0 mg l^?1 NAA and different concentrations of NaCl (0.05–0.20 %) and Na₂CO₃ (0.0125–0.10 %) for 2 weeks, and incubated at 24?±?1 °C and 22.4 μmol m^?2 s^?1 light intensity provided by white fluorescent tubes for 16 h. Callus and suspension biomass cultured on salts showed less growth as well as browning of medium when compared with control. Quantification of SGs content in callus culture (collected on 15th day) and suspension cultures (collected at 10th and 15th days) treated with and without salts were analyzed by HPLC. It was found that abiotic stress induced by the salts increased the concentration of SGs significantly. In callus, the quantity of SGs got increased from 0.27 (control) to 1.43 and 1.57 % with 0.10 % NaCl, and 0.025 % Na₂CO₃, respectively. However, in case of suspension culture, the same concentrations of NaCl and Na₂CO₃ enhanced the SGs content from 1.36 (control) to 2.61 and 5.14 %, respectively, on the 10th day.     

Growth kinetics and diosgenin estimation from callus cultures of Costus speciosus (Koen. ex. Retz.)

The present investigation reports the growth kinetics and diosgenin accumulation in callus cultures of Costus speciosus. Effect of explants, media and plant growth regulators was evaluated with respect to callus induction and growth. Out of the two explants viz pseudostem and seed, pseudostem showed maximum callus induction frequency of 90% on MS medium. The fresh weight of callus was maximum (9-folds) on 28th day on 1.0 mg/L picloram containing medium. The callus obtained was white compact hard (WCH). For growth kinetics study pseudostem derived callus was transferred on different media supplemented with 1.0 mg/L picloram. All phases of growth were seen in callus inoculated on all the three media except the absence of stationary phase on MS and SH media. MS medium proved to be the best for maximum biomass accumulation (9-fold) on 28th day of culture and callus in post-exponential phase showed maximum diosgenin accumulation (33 ppm).     

Analysis of Secondary Metabolite Production in Somatic Embryo of Pasak Bumi (Eurycoma Longifolia Jack.)

Pasak bumi (Eurycoma longifolia Jack.) has been known as a plants that can produce secondary metabolites for medicinal purposes such as: aphrosidiac, antimalaria, dysentri, antitumor, etc. Poor seed germination of pasak bumi will affect the avaibility of plant material for drug extraction. Over exploitation of this plant will also reduce plant population in its natural habitat. In vitro culture, i.e. through somatic embryogenesis, therefore, can be used as one of an alternative method for plant regeneration as well as for in vitro metabolite production. Based on this reason, the research has been done with an objective to analyze the presence of secondary metabolite in somatic embryo of pasak bumi. Seed-derived callus was used as an explant. This callus was maintained to proliferate in MS (Murashige&Skoog, 1962) medium supplemented with 2.25 mg/L 2,4-D and 2.0 mg/L kinetin. A half gram of callus from proliferation medium was transferred into the MS liquid medium containing 1.0 or 2.25 mg/L 2,4-D, and 2.0 mg/L BAP or 2.0 mg/L kinetin. Histochemical examination using Jeffrey's reagen and neutral red showed that alkaloid and terpenoid substances were presence in somatic embryo of pasakbumi. In accordance with histochemical test, GC-MS analyses showed that secondary metabolites was also synthesized by non embryogenic callus and the mixture ofembryogenic callus and somatic embryo, although the concentration in the mixture of embryogenic callus and somatic embryo was lower than those in non-embryogenic callus. Secondary metabolites, including 3-[(cyclohexyl-methyl-amino)-methyl]-3H-benzooxazole-2-one (0.06%) and 2-furancarboxaldehyde, 5-(hydroxymethyl) (43.024%) were found in embryogenic callus and somatic embryo. In addition, the mixture of embryogenic callus and somatic embryo also synthesized fatty acid and lipids (52.751%) which was higher than non-embryogenic callus (24.789%). Based on the result, the mixture of embryogenic callus and somatic embryo could produce secondary metabolites, such as alkaloid, terpenoid subtances, and phenol. The concentration of metabolites in the mixture of embryogenic callus and somatic embryo, however, was lower compare to non-embryogenic callus.     

A New Biotechnology Method of Bioelements’ Accumulation Monitoring in In Vitro Culture of Agaricus bisporus

Agaricus bisporus (J.E. Lange) Imbach is one the most popular species of edible mushrooms in the world because of its taste and nutritional properties. In the research, repeatability of accumulation of bioelements and biomass yield in experimentally chosen in vitro culture medium, was confirmed. The in vitro cultures were conducted on the modified Oddoux medium enriched with bioelements (Mg, Zn, Cu, Fe). The aim of the study was to create an effective method of sampling, which enabled non-invasive monitoring of metals concentrations changes in the medium, during increase of biomass in in vitro cultures. The first, indirect method of sampling was applied. The non-invasive probe (a dipper) for in vitro culture was used; hence, the highest biomass increase and metals accumulation were gained. The method also guaranteed culture sterility. The second method, a direct one, interfered the in vitro culture conditions and growth of mycelium, and as a consequence the lower biomass increase and metals’ accumulation were observed. Few cases of contaminations of mycelium in in vitro cultures were observed. The proposed method of non-invasive sampling of the medium can be used to monitor changes in the concentrations of metals in the medium and their accumulation in the mycelium in natural environment. Changes in concentrations of the selected metals over time, determined by the method of atomic absorption spectrometry, made it possible to correlate the obtained results with the specific stages of A. bisporus mycelium development and to attempt to explain the mechanism of sampling metals from the liquid substrate.     

Regeneration and Agrobacterium-mediated transformation of the apomictic species Eulaliopsis binata

Protocols for regeneration and Agrobacterium-mediated transformation of the apomictic species Eulaliopsis binata were developed. Initially, seeds of four genotypes of E. binata were incubated on a callus induction Murashige and Skoog (MS) basal medium supplemented with three concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D). It was found that 36.2 % of explants developed highly friable callus on medium containing 3.0 mg l^?1 2,4-D. Based on frequency of callus induction, the genotype Neixiang was selected for regeneration and transformation. Callus incubated on MS basal medium supplemented with 0.2 mg l^?1 α-naphthalene acetic acid and 6.0 mg l^?1 6-furfuryl-aminopurine developed shoots. Subsequently, Agrobacterium tumefaciens strain EHA105—harboring a plasmid pCAMBIA1381 carrying a hygromycin phosphotransferase (hpt) resistance gene and a synthetic green fluorescent protein (GFP) gene, both driven by the cauliflower mosaic virus 35S promoter—was used for transformation system. Putative transgenic callus was obtained following two cycles of hygromycin selection. Expression of the transgene(s) in putative transgenic callus was analyzed using the GFP detection. Molecular identification of putative transformed shoots was performed by polymerase chain reaction and Southern blot analysis to confirm presence and integration of the hpt gene.     

Shoot Multiplication and Callus Induction of Labisia pumila var. alata as Influenced by Different Plant Growth Regulators Treatments and Its Polyphenolic Activities Compared with the Wild Plant

This study aims to investigate whether the in vitro-cultured L. pumila var. alata has higher antioxidant activity than its wild plant. An 8-week-old L. pumila var. alata nodal segment and leaf explants were cultured onto Murashige and Skoog (MS) medium supplemented with various cytokinins (zeatin, kinetin, and 6-benzylaminopurine (BAP)) for shoot multiplication and auxins (2,4-dichlorophenoxyacetic acid (2,4-D) and picloram) for callus induction, respectively. The results showed that 2 mg/L zeatin produced the optimal results for shoot and leaf development, and 0.5 mg/L 2,4-D produced the highest callus induction results (60%). After this, 0.5 mg/L 2,4-D was combined with 0.25 mg/L cytokinins and supplemented to the MS medium. The optimal results for callus induction (100%) with yellowish to greenish and compact texture were obtained using 0.5 mg/L 2,4-D combined with 0.25 mg/L zeatin. Leaves obtained from in vitro plantlets and wild plants as well as callus were extracted and analyzed for their antioxidant activities (DPPH and FRAP methods) and polyphenolic properties (total flavonoid and total phenolic content). When compared with leaf extracts of in vitro plantlets and wild plants of L. pumila var. alata, the callus extract displayed significantly higher antioxidant activities and total phenolic and flavonoid content. Hence, callus culture potentially can be adapted for antioxidant and polyphenolic production to satisfy pharmaceutical and nutraceutical needs while conserving wild L. pumila var. alata.     

Plant Regeneration Through Callus Organogenesis and True-to-Type Conformity of Plants by RAPD Analysis in Desmodium gangeticum (Linn.) DC.

An efficient plant regeneration protocol was established for an endangered ethnomedicinal plant Desmodium gangeticum (Linn.) DC. Morphogenic calli were produced from 96 % of the cultures comprising the immature leaf explants on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (4.0 mg?l^?1) in combination with 6-benzylaminopurine (BA; 0.8 mg?l^?1). For callus regeneration, various concentrations of BA (1.0–5.0 mg?l^?1) or thidiazuron (TDZ; 1.0–5.0 mg?l^?1) alone or in combination with indole-3-acetic acid (IAA; 0.2–1.0 mg?l^?1) were used. Highest response of shoot regeneration was observed on MS medium fortified with TDZ (4.0 mg?l^?1) and IAA (0.5 mg?l^?1) combination. Here, 100 % cultures responded with an average number of 22.3 shoots per gram calli. Inclusion of indole-3-butyric acid in half MS medium favored rooting of recovered shoots. Out of 45 rooted plants transferred to soil, 40 survived. Total DNA was extracted from the leaves of the acclimatized plants of D. gangeticum. Analysis of random amplified polymorphic DNA using 13 arbitrary decanucleotide primers showed the genetic homogeneity in all the ten plants regenerated from callus with parental plant, suggesting that shoot regeneration from callus could be used for the true-to-type multiplication of this plant.     

Effects of Exogenous Methyl Jasmonate on the Biosynthesis of Shikonin Derivatives in Callus Tissues of Arnebia euchroma

The shikonin derivatives, accumulated in the roots of Arnebia euchroma (Boraginaceae), showed antibacterial, anti-inflammatory, and anti-tumor activities. To explore their possible biosynthesis regulation mechanism, this paper investigated the effects of exogenous methyl jasmonate (MJ) on the biosynthesis of shikonin derivatives in callus cultures of A. euchroma. The main results include: Under MJ treatment, the growth of A. euchroma callus cultures was not inhibited, but the expression level of both the genes involved in the biosynthesis of shikonin derivatives and their precursors and the genes responsible for intracellular localization of shikonin derivatives increased significantly in the Red Strain (shikonin derivatives high-producing strain). The quantitative analysis showed that six out of the seven naphthoquinone compounds under investigation increased their contents in the MJ-treated Red Strain, and in particular, the bioactive component acetylshikonin nearly doubled its content in the MJ-treated Red Strain. In addition, it was also observed that the metabolic profiling of naphthoquinone compounds changed significantly after MJ treatment, and the MJ-treated and MJ-untreated strains clearly formed distinct clusters in the score plot of PLS-DA. Our results provide some new insights into the regulation mechanism of the biosynthesis of shikonin derivatives and a possible way to increase the production of naphthoquinone compounds in A. euchroma callus cultures in the future.     

Optical isomer separation of flavanones and flavanone glycosides by nano-liquid chromatography using a phenyl-carbamate-propyl-β-cyclodextrin chiral stationary phase

In this paper a phenyl-carbamate-propyl-β-cyclodextrin stationary phase was employed for the enantioseparation of several flavonoids, including flavanones and methoxyflavanones by using nano-liquid chromatography (nano-LC). The same stationary phase was also used for the diastereoisomeric separation of two flavanone glycosides. The compounds: flavanone, 2′-hydroxyflavanone, 4′-hydroxyflavanone, 6-hydroxyflavanone, 7-hydroxyflavanone, 4′-methoxyflavanone, 6-methoxyflavanone, 7-methoxyflavanone, hesperetin, hesperidin, naringenin and naringin were studied using reversed, polar organic and normal elution modes. The effect of the nature and composition of the mobile phase (organic modifier type, buffer and water content in the reversed phase mode) on the enantioresolution (R_s), retention factor (k) and enantioselectivity (α) were investigated. Baseline resolution of all studied flavonoids, with the exception of 2′-hydroxyflavanone and naringin, was achieved in reversed phase mode using a mixture of MeOH/H₂O at different ratios as mobile phase. Good results, in terms of peak efficiency and short analysis time, were obtained adding 1% triethylammonium acetate pH 4.5 buffer to MeOH/H₂O mixture. The separation of the studied compounds was also performed in polar organic mode. By using 100% of MeOH as mobile phase, the resolution was achieved for the studied analytes, except for 7-hydroxyflavanone, 2′-hydroxyflavanone, naringenin, hesperidin and naringin. Normal mode was tested employing a mixture of EtOH/hexane/TFA as mobile phase achieving the enantiomeric and diastereomeric separation of only hesperetin and hesperidin, respectively. The use of nano-LC technique for the resolution of flavanones optical isomers allowed to achieve good resolutions in shorter analysis time compared to the results reported in literature with conventional HPLC.     

Datura stramonium hairy roots tropane alkaloid content as a response to changes in Gamborg’s B5 medium

Hairy roots are less susceptible to manipulation by changes in medium composition than callus and cell suspension cultures. However, when the individual components of the Gamborg’s B₅ medium were modified one by one, several of them modified the yield of root cultures. Nitrogen (N) source changes, the absence of phosphorus (P), calcium (Ca), and cobalt (Co), and the increase in the sucrose content of the medium increased the level of alkaloids inDatura stramonium hairy root cultures. The use of ammoniun as N source provoked the alkaloid excretion into the medium. The addition of different auxins inhibited the alkaloid accumulation, but the addition of abscisic acid (ABA) did not change the alkaloid content of the cultures. The use of MeJa increased the content of hyoscyamine to 100%.     

An interlaboratory study as useful tool for proficiency testing of chemical oxygen demand measurements using solid substrates and liquid samples with high suspended solid content

In 2008, the first Proficiency Testing Scheme of Chemical Oxygen Demand (1^stCOD-PT^ADG) was conducted to assess the results obtained for different research groups whose field work is mainly anaerobic digestion. This study was performed using four samples, two solid samples as raw materials and two solid samples to prepare high concentration suspended solid solutions. Invitations were sent to a large number of laboratories, mainly to anaerobic digestion research groups. Finally, thirty labs from sixteen countries agreed to participate, but for different reasons four participants could not send any data. In total, twenty-six results were reported to the COD-PT coordinator.This study showed the importance of continuous participation in proficiency testing (PT) schemes in order to compare the results obtained. Taking into account the lack of a general standard method and high quality certified reference materials (CRMs), the traceability of COD determination is not currently easy to check. In addition, the spread of participants’ results obtained was high and pointed to the advisability of using consensus values due to their unreliability. Therefore, the theoretical oxygen demand (ThOD) values were considered as assigned values for all the samples analysed. On the other hand, in this PT the established standard deviation (ESD) has been determined by the Horwitz modified function.Participants of this 1^stCOD-PT^ADG were asked to give a short report on the analytical method used. Although all the participants used potassium dichromate as their oxidant reagent, their experimental procedures were very different. With the purpose of comparing the results obtained, the different experimental conditions used were classified into five methods, corresponding to two main categories, open and closed reflux. The performance of laboratories was expressed by the z-score, whose value is considered satisfactory when z-score ≤±2. The overall analytical data evaluation showed that 64% of z-scores obtained were outside the accepted limits.     

A Comparative Study of Anti-Candida Activity and Phenolic Contents of the Calluses from Lythrum salicaria L. in Different Treatments

In the study, anti-Candida activity and phenol contents of Lythrum salicaria L. calli and wild species have been evaluated. The seeds of L. salicaria (Lythraceae), collected from Lahidjan City in the north of Iran, were cultured in Murashige and Skoog medium (MSM) with a supplement, gibberellin, to germinate. Callus inductions were performed from segments of seedling on MSM containing different concentrations of plant growth regulators, 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BAP). The activity of calluses extracts, wild plant, gallic acid, and 3,3′,4′-tri-O-methylellagic acid-4-O-β-d-glucopyranoside (TMEG) as the main phenolic compounds against Candida albicans was assessed using cup plate diffusion method. The total phenols contents of calli and wild plant extracts were analyzed using Folin–Ciocalteu reagent. The callus formation in MSM supplemented with various concentrations of 2,4-D and BAP were 0–100 %. Anti-Candida activity of callus extract which obtained from MSM supplemented with 2,4-D and BAP (1 mg?dm^?3) was similar to the wild plant extract. Minimum inhibitory concentration values of gallic acid and TMEG were obtained as 0.312 and 2.5 mg?cm^?3, respectively. Gallic acid equivalent values in all treatments were from 0 to 288 μg GAE mg^?1. Phenolic contents of plant aerial parts (331?±?3.7 μg GAE mg^?1) and the callus, which developed in MSM including 1 mg?dm^?3 of both 2,4-D and BAP, showed the same phenolic value and exhibited anti-Candida extract activity.