Indigo Extract Research Articles

Physicochemical properties and antibacterial activity evaluation of new indigo extract from Baphicacanthus cusia (Nees) Bremek prepared by chemical conversion method

The effect of acidolysis time, hydrochloric acid concentration, ozonization time and pH on the yield of indigo were investigated by single factor experiment using dried leaves of Baphicacanthus cusia (Nees) Bremek (DLB) as raw material. On this basis, the technological condition for preparing new indigo extract (NIE) by directional chemical conversion method was optimized by orthogonal experiment. Then, the physiochemical properties of NIE were analyzed by combustion assay, nitric acid assay, thin layer chromatography identification assay, moisture detection and water-soluble pigment observation assay. Moreover, UPLC-Q-TOF-MS/MS technology was utilized to compare chemical composition difference between the NIE and traditional indigo naturalis (TIN). Finally, NIE emulsion was prepared by ultrasound assisted high pressure homogenization method and its stability, antibacterial activity as well as antibacterial mechanism were evaluated. The experimental results showed that optimum process for preparing NIE were hydrochloric acid concentration of 2 %, ozonization time of 3 min, pH of 7 and acidolysis time of 1 h; Under this condition, the content and yield of indigo in the NIE were significantly improved, which could reach up to 42.6 ± 0.28 % and 2.26 ± 0.04 %, separately. NIE possessed similar physiochemical properties with TIN described in Chinese pharmacopoeia 2020, and had more diverse active ingredients than TIN. The prepared NIE emulsion demonstrated excellent stability, whether at dark or bright place, and no matter at low temperature (4 ℃) or room temperature (25 ℃), the particle size, PDI, pH and indigo content of which displayed little variation. In addition, the NIE emulsion owned good inhibitory activity against S. aureus, and its MIC and MBC values were 40 and 80 μg/mL, respectively. Moreover, NIE emulsion exhibited antibacterial effect by influencing cell membrane integrity, Ca2+-Mg2+-ATPase activity on the cell membrane and intracellular Ca2+ levels of S. aureus. All of above results will lay the foundation for the green and sustainable utilization of Baphicacanthus cusia (Nees) Bremek.

Convenient preparation of indigo from the Ieaves of Baphicacanthus cusia(Nees) Bremek by enzymatic method and its MALDI-TOF-MS and UPLC-Q-TOF/MS analysis

The manufacturing of indigo naturalis requires prolonged leaf soaking and lime stirring; the resulting indigo purity is less than 3.00% and the yield of indigo (measured in stems and leaves weight) is less than 0.50%, making it unsuitable for use in industrial procedures like printing and dyeing. An enzymatic method of creating indigo without the requirement for lime was investigated in order to generate high purity indigo. Single factor tests were performed to optimize the enzymatic preparation conditions. The findings showed that 60 °C, pH 5.5, 200 mL of leaves extract containing 0.45 mg/mL indican, and a 4:1 ratio of the acidic cellulose (activity: 9000 U/mL, liquid) to indican were the ideal parameters for enzymatic preparation. The yield of indigo was 40.32%, and the contents of indigo and indirubin were 37.37% and 2.30%, respectively. MALDI-TOF-MS in positive ion mode and UPLC-Q-TOF-MS in both positive and negative ion modes were used to analyze indigo extracts from Baphicacanthus cusia(Nees) Bremek by enzymatic preparation. It has been discovered that 13 alkaloids, 5 organic acids, 3 terpenoids, 3 steroids, 2 flavones, and 7 other compounds are present in indigo extracts. The presence of the indigo, indirubin, isorhamnetin, tryptanthrin, indigodole B, and indigodole C determined by UPLC-Q-TOF-MS was verified by MALDI-TOF-MS analysis. The enzymatic preparation of indigo extracts kept the same chemical makeup as conventional indigo naturalis. Thermal analysis and SEM morphology were used to confirm that there was no lime in the indigo extract. During the enzymatic process, Baphicacanthus cusia (Nees) Bremek was employed more effectively, increasing the yield and purity of indigo.

"Purplish Blue" or "Greenish Grey"? Indigo Qualities and Extraction Yields from Six Species.

Indigo quality is determined by its indigotin content. Another quality indicator is colour. For an evaluation of species, indigo samples from Indigofera tinctoria, Indigofera suffruticosa, Indigofera arrecta, Persicaria tinctoria, Strobilanthes cusia and Wrightia laevis cultivated in Austria and China were visually classified and analysed spectrophotometrically and using a L*a*b* measuring device. In addition to a standardised hot-extraction method without lime, some samples were extracted simulating traditional methods at ambient temperatures using lime. The highest indigotin contents were achieved with Indigofera arrecta (55%, Austria) and Strobilanthes cusia (56%, China). There were no statistically significant differences between the indigo extraction yields of the species cultivated in Austria, but Indigofera arrecta and Persicaria tinctoria had statistically significantly higher indigotin extraction yields than Indigofera tinctoria and Indigofera suffruticosa. From the species extracted in China, Strobilanthes cusia showed higher values in all parameters than Indigofera tinctoria, Indigofera suffruticosa and Wrightia laevis. Compared with the standardised method, the method simulating local practice yielded more indigo but had a lower indigotin content; the indigotin extraction yields did not differ greatly. L*a*b* values enabled precise estimations of the indigotin content, making it an interesting option for quality control, as inexpensive, easy-to-handle L*a*b* measuring instruments have become available.

Assessing the Impact of Sepiolite-Based Bio-Pigment Infused with Indigo Extract on Appearance and Durability of Water-Based White Primer.

The objective of this study is to evaluate how two varying amounts of sepiolite-based powder, infused with indigo extract, affect the appearance and durability of a water-based, white primer. To examine the influence of this eco-friendly pigment on the coatings' overall appearance, assessments were performed for color, gloss, and surface roughness. Additionally, the coatings were investigated through optical and electron microscopic observations, to evaluate the distribution of the pigment within the polymer matrix. The effect of the pigment on the coating's durability was assessed through accelerated tests, including exposure in a salt spray chamber and a UV-B chamber. These tests aimed to evaluate the emergence of defects and changes in the appearance of the samples over time. Furthermore, the impact of different quantities of sepiolite-based powder on the coating's ability to act as a barrier was assessed using liquid resistance tests and contact angle measurements. These evaluations aimed to understand how the coating responded to various liquids and its surface properties concerning repellency or absorption. In essence, this study underscores the considerable influence of the eco-friendly pigment, demonstrating its capacity to introduce unique color and texture variations in the paint. Moreover, the inclusion of the pigment has enhanced the coating's color stability, its ability to act as a barrier, and its overall durability when exposed to harsh environments.

Indigo production goes green: a review on opportunities and challenges of fermentative production.

Indigo is a widely used dye in various industries, such as textile, cosmetics, and food. However, traditional methods of indigo extraction and processing are associated with environmental and economic challenges. Fermentative production of indigo using microbial strains has emerged as a promising alternative that offers sustainability and cost-effectiveness. This review article provides a critical overview of microbial diversity, metabolic pathways, fermentation strategies, and genetic engineering approaches for fermentative indigo production. The advantages and limitations of different indigo production systems and a critique of the current understanding of indigo biosynthesis are discussed. Finally, the potential application of indigo in other sectors is also discussed. Overall, fermentative production of indigo offers a sustainable and bio-based alternative to synthetic methods and has the potential to contribute to the development of sustainable and circular biomanufacturing.

Topical application of indigo-plant leaves extract enhances healing of skin lesion in an excision wound model in rats.

This study aims to evaluate the pharmacological role of indigo extract in accelerating the wound healing in a rat model. Female Sprague-Dawley rats were anesthetized with ketamine (30 mg/kg, i.p.) and the full thickness of the marked skin was then cut carefully and wounds were left undressed. Indigo extract (5%) in PBS was applied topically twice daily until healing was complete. A control group of rats was treated with povidone-iodide (Betadine®). Rats treated with phosphate buffer saline were used as a negative control group. The rate of wound healing was assessed daily. Histopathological examination of skin sections were qualitatively assessed by independent evaluators. The inflammatory and apoptotic markers were assessed in skin tissue homogenates using ELISA. Histopathology data showed that applying indigo to skin wounds enhanced the healing process, resulting in a significant decrease in dermal inflammation in comparison to untreated rats. Topical application of indigo significantly increased antioxidant enzyme activities with reduced malondialdehyde (MDA) levels in wound tissues. The levels of matrix metalloproteases-2 and -9 were significantly lower with an accompanied increase in the level of TGF-β1 in skin tissues from rats treated with indigo compared to the control group treated with PBS. The antioxidant and anti-inflammatory properties of indigo leaf extract accelerate the healing of skin injuries.

Indigo plant leaf extract inhibits the binding of SARS-CoV-2 spike protein to angiotensin-converting enzyme 2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses its S1 spike protein to bind to angiotensin-converting enzyme 2 (ACE2) on human cells in the first step of cell entry. Tryptanthrin, extracted from leaves of the indigo plant, Polygonum tinctorium, using d-limonene (17.3 µg/ml), is considered to inhibit ACE2-mediated cell entry of another type of coronavirus, HCoV-NL63. The current study examined whether this extract could inhibit the binding of the SARS-CoV-2 spike protein to ACE2. Binding was quantified as cell-bound fluorescence intensity in live cell cultures in which canine kidney MDCK cells overexpressing ACE2 were incubated with fluorescein-labeled S1 spike protein. When indigo extract, together with S1 protein, was added at 8,650x and 17,300x dilutions, fluorescence intensity decreased in a dose- and S1 extract-dependent manner, without affecting cell viability. When 4.0-nM tryptanthrin was added instead of the indigo extract, fluorescence intensity also decreased, but to a lesser degree than with indigo extract. Docking simulation analyses revealed that tryptanthrin readily bound to the receptor-binding domain of the S1 protein, and identified 2- and 7-amino acid sequences as the preferred binding sites. The indigo extract appeared to inhibit S1-ACE2 binding at high dilutions, and evidently contained other inhibitory elements as well as tryptanthrin. This extract may be useful for the prevention or treatment of SARS-CoV-2 infection.

Analysis of bacterial flora of indigo fermentation fluids utilizing composted indigo leaves (sukumo) and indigo extracted from plants (Ryukyu-ai and Indian indigo)

Indigo is a fabric dye that requires reduction by microbial activity or chemical reagents to render it soluble in water. Sources of indigo for fermentation are primarily divided into composted indigo-containing plants and indigo extracted from plants. To elucidate the factors responsible for bacterial diversity, and for sustaining reduced state of indigo in different preparations, this study assessed fermentation-derived fluids using composted plant leaves, sukumo, and extracted indigo (Ryukyu-ai paste, and Indian indigo cake) prepared using different procedures. Regardless of the indigo source, obligate anaerobic bacteria, including the families Proteinivoraceae and Tissierellaceae, predominate (16.9-46.1%), suggesting their high affinity for this fermentation ecosystem (hyperalkaline and low redox potential). Moreover, bacterial communities in sukumo fermentations are more diverse than those from indigo extracts with the diversity tending to increase based on the fermentation period. Our results further suggest that the microbiota composition in sukumo fermentation is associated with the various bacterial nutrients derived from sukumo, including seed microorganisms. In addition, the debris derived from sukumo can reduce the pH stress experienced by the microorganisms. Further, regardless of 5.4 years difference in the fermentation age, the bacterial flora in two Ryukyu-ai batches exhibit similar features with low microbial diversities. The uniformity of the nutrient, along with the simple, yet strong, bacterial network in Ryukyu-ai fluids may be responsible for the stable bacterial flora composition. Taken together, these results indicate that the microbiota in indigo fermentation is highly influenced by the seed culture, the nutrient derived from raw materials, and the fermentation conditions.

Island blues: indigenous knowledge of indigo-yielding plant species used by Hainan Miao and Li dyers on Hainan Island, China

BackgroundHistorically, indigo-yielding plant species were important cash crops from Central Asia to the southern United States and Central America. Indigo-dyed textiles were widely traded along the legendary Silk Road that linked China to Europe. Today, due to the labor-intensive nature of indigo extraction at the household level, lifestyle changes and the widespread availability of commercially produced indigo paste, traditional indigo extraction methods have declined in villages. Yet Li textile weavers on Hainan Island are internationally recognized as producers of indigo-dyed textile using warp ikat techniques. In contrast, Hainan Miao weavers produce indigo-dyed textiles using batik (wax resist) techniques. The aim of this study was to document the indigenous knowledge on indigo-yielding plant species used by both Hainan Miao and Li people on Hainan Island, China.MethodEthnic uses were documented during three field surveys, through a questionnaire survey of 193 respondents, comprising 144 Hainan Miao and 49 Li traditional dyers. Mention index (QI), Availability index (AI), and Preference ranking (PR) of each indigo-yielding plant species were calculated to screen out plant resources with potential development value.ResultsFive indigo-yielding plant species (from four plant families and four genera) were historically used by Hainan Miao and Li dyers. However, just four species are still in use. Strobilanthes cusia was the main indigo source for Hainan Miao dyers. Li dyers also commonly use Indigofera species (I. tinctoria and I. suffruticosa) for indigo extraction. Wrightia laevis is less commonly used as a contemporary indigo source. Indigo extraction by steeping in water to which lime is added to increase the pH is sharing by the five indigo-yielding plant species. Strobilanthes cusia had the highest QI, AI and PR values in Hainan Miao villages. Indigofera tinctoria had the highest QI and AI values, but Indigofera suffruticosa was preferred by Li dyers.ConclusionIn the process of modernization and urbanization, some Hainan Miao and Li dyers retain the traditional indigo extraction methods. We found that Strobilanthes cusia and Indigofera tinctoria have the most potential for sustainable indigo production in the future. Furthermore, this study documents the details of extraction method from Wrightia laevis for the first time and the use of Ricinus communis seeds in that process. As one of the last places globally where Wrightia laevis is still used for indigo production, the may also be a nice market among textile collectors and museums that keeps the tradition of Wrightia laevis production and use for indigo extraction alive.

Identity blues: the ethnobotany of the indigo dyeing by Landian Yao (Iu Mien) in Yunnan, Southwest China

BackgroundIndigo-dyed textiles have been central to the cultural identity of Landian Yao (literally “blue clothes Yao”) people in Southwest China for centuries, driving a significant local market for naturally dyed indigo cloth. In the past two decades, local indigo production for traditional textiles has declined for several reasons: Firstly, the younger generation of Landian Yao has shifted to using western style jeans and T-shirts. Secondly, due to its labor-intensive nature. In contrast, at a global scale, including in China, there has been a revival of interest in natural indigo use. This is due to a growing awareness in the fashion industry about human and environmental health issues related to synthetic dye production. Ironically, this new awareness comes at a time when traditional knowledge of indigo dyeing is being lost in many places in China, with weaving and use of natural dyes now limited to some remote areas. In this study, we recorded indigo dyeing processes used by Landian Yao people and documented the plant species used for indigo dyeing.MethodsField surveys were conducted to the study area from September 2015 to November 2016, supplemented by follow-up visits in July 2018 and November 2018. We interviewed 46 key informants between 36 and 82 years old who still continued traditional indigo dyeing practices. Most were elderly people. Semi-structured interviews were used. During the field study, we kept a detailed account of the methods used by Landian Yao dyers. The data were then analyzed by using utilization frequency to determine the best traditional recipe of indigo dye extraction. All the specimens of documented species were collected and deposited at the herbarium of Kunming Institute of Botany.ResultsOur results showed that indigo dyeing was divided into two main steps: (1) indigo pigment extraction and (2) dyeing cloth. The general procedures of indigo dye extraction included building or buying a dye vat, fermentation, removal of the leaves of indigo producing plant species, addition of lime, oxygenation, followed by collection, and the storage of the indigo paste. The procedures of dyeing cloth included preparing the dye solutions, dyeing cloth, washing, and air drying. It is notable that Landian Yao dyers formerly only performed the dyeing process on the goat days in the lunar calendar from June to October. After comparing the range of local indigo extraction methods, our results showed that the following was best of these traditional recipes: a indigo-yielding plant material to tap water ratio of 30 kg: 200 l, lime 3 kg, a fermentation time of 2–3 d, aeration by agitation for up to 60 min, and a precipitation time of 2–3 h. Our results show that 17 plant species in 11 families were recorded in the indigo dyeing process. With the exception of the indigo sources, only Dioscorea cirrhosa Lour. and Artemisia argyi H.Lév. & Vaniot were previously recorded in dyeing processes. Other species given in this paper are recorded for the first time in terms of their use in the indigo dyeing process. In the study area, Landian Yao men were in charge of indigo dye extraction, and the women were responsible for dyeing cloth.ConclusionsThe Landian Yao has completely mastered the traditional indigo dyeing craft and are one of the well-deserved identity blues. Indigo production from plants using traditional methods is a slow process compared to synthetic dyes and is not suitable for modern and rapid industrial production. Therefore, our study records the detailed information of traditional indigo dyeing to protect and inherit it. Strobilanthes cusia (Nees) Kuntze is the main indigo source in Landian Yao that is widely used in the world and can be commercially exploited as an indigo plant. For commercial and environment benefits, we suggest that producing natural indigo for the commercial market is a good choice.

Optimization of Extraction Methods and Dyeing Standarization of Nila Leaves (Indigoferatinctoria Linn.) as Natural Dyes

Indigosol is commonly used as a synthetic dye to generate blue color. However, synthetic dyes are detrimental to health and environment because they contain heavy metals. Nila plant (Indigoferatinctoria Linn.) contains indican glucoside which produces indoxyl exotic blue gold color and known as indigo. Indigo produce the dull-colored, when applied to cotton material. This study aims to get the optimize extraction method and standard dyeing of nila as cotton material natural dye, in order to generate sharper blue color. Nila leaves were extracted by variation time of maceration at 12, 24, 36, 48 hours and variation boiling temperature at 70, 80, 90 °C in aquadest, with pH 11 and 13. The indigo level was measured using spectrophotometer at λ 611 nm. The optimal result were applied to prima and primissima cotton material and then tested in fade resistance to leaching with staining scale (SNI-08-0285-1998) and color depth with spectrophotometer method with λ 380 - 780 nm. This study showed that in maceration and boiling method, pH significantly affected the indigo level. The treatment of 12-hour maceration at pH 11 and boiling at 80-90°C at pH 11 were the most optimal treatment to produce sharp blue color, both in prima and primissima cotton material. The best scale of fade resistance was 3-4, which was indigo extract with 12-hour at pH 11 in the maceration method and applied in primissima cotton. It can be concluded that maceration method generates sharper dye than the boiling method in primissima cotton material.

明治時代の阿波藍 (「日本資本主義成立期の諸問題」 : 四國地方を中心として)

明治時代の阿波藍 (「日本資本主義成立期の諸問題」 : 四國地方を中心として)

Extraction of Indigo from Some Isatis species and Dyeing Standardization Using Low-technology Methods

Fresh leaves of four Isatis species culture form of I. tinctoria L and wild forms of I. buschiana Schischkin, I. candolleana Boiss. (endemic) and I. tinctoria L. subsp. corymbosa. (Boiss.) were used for indigo production. Dyes were extracted by fermentation and hot water application. The extracted dyes were optimized with different pH and reducing agents. Results showed that the dye from hot water application produced the desired dying quality at pH 11. Reducing agent concentrations had no significant effect on color quality. Dark blue and blue colors were obtained from I. tinctoria and I. candolleana extracts although I. tinctoria subsp. corymbosa and I. buschiana produced mostly yellow-gray colors. Light, dry and wet rubbing fastness values varied between 3 and 3/4 while washing fastness was between 2 and 4/5. The highest indigo amounts were determined spectrophotometrically as 4.19 mg/g and 2.53 mg/g in I. tinctoria and I. candolleana, respectively. Results also showed that harvesting season was important for indigo production and the highest indigo amount was observed in mid-June.

Comparison of extraction methods for the analysis of Indigofera tinctoria and Carthamus tinctorius in textiles by high performance liquid chromatography

The extractions of indigo (Indigofera tinctoria L.) and safflower (Carthamus tinctorius L.) red and yellow from wool fibres are investigated using high performance liquid chromatography with photo-diode-array detection (HPLC–PDA) which is frequently coupled to Mass Spectrometry (HPLC–PDA–MS) for identification purposes. The efficiencies of dimethyl sulfoxide (DMSO) and dimethyl formamide (DMF) to extract indigo and safflower from wool, are compared for a wide range of temperature (from 40 to 120°C) and treatment time (from 1 to 30min). Extraction procedures are investigated by measuring integrated HPLC peak areas of selected dyestuff components, evaluating simultaneously the effects of solvent (DMSO and DMF), treatment temperature (T) and time (t). Thus, cross-influence of the different extraction parameters (solvent, T and t) is taken into account. Indigotin, isatin and indirubin are monitored to evaluate the extraction of indigo. Carthamin, a decomposition product of carthamin, apigenin, safflomin A, 6-hydroxykaempferol-3-O-β-D-glucoside, anhydrosafflor yellow Ct1 and Ct4 are monitored to investigate the extractions of safflower red and yellow. Overall, it is reported that DMSO gives better extraction yields than DMF.The best conditions for the extraction of the marker compounds of the three dyes are summarized as follows. For indigotin, treatments of the wool with DMSO at 80°C for t>5min or at 120°C min for t=1min give the best yield. Longer treatment (t>1min) at 120°C results to decomposition of indigotin. For carthamin, treatments of the wool with DMSO at 80°C for t>5min or at 120°C min for short t (<5min) give the best yield, considering that longer (>5min) treatment at 120°C results to decomposition of the marker compound. Finally, for safflomin A treatments of the wool with DMSO at 100°C for t>10min or at 120°C min for t=5min give the best result.

Antioxidant and binding properties of methanol extracts from indigo plant leaves

AbstractThis study was conducted to clarify the effect of ultra-pressure treatment on the extraction of bioactive compounds from indigo plant leaves (Polygonum tinctorium Lour.) and on their properties. Leaves were harvested the in mid-August, 2013, from Naju City (Korea), and treated using two methods: ultra-pressure (550 MPa, 3 min) and hot-air (70°C, 24 h). Then, the leaves were ultrasonically extracted with methanol. The content of indirubin in leaves treated with ultra pressure and hot air was (535.55 ± 26.14) mg kg−1 and (52.63 ± 6.45) mg kg−1, respectively, and that of tryptanthrin was (165.55 ± 8.74) mg kg−1 and (153.00 ± 7.62) mg kg−1, respectively. Polyphenolic content in the leaves extract was (127.24 ± 13.67) mg kg−1 after the ultrapressure and (88.22 ± 5.33) mg kg−1 after the hot-air treatment. The content of flavonoids was (2298.67 ± 83.27) mg kg−1 after the ultra-pressure and (3224.00 ± 21.45) mg kg−1 after the hotair treatment. Di(phenyl)-(2,4,6-trinitrophenyl)iminoazanium (DPPH) radical scavenging activities of the indigo extract obtained by ultra-pressure and hot-air treatment methods at the concentration of 1 mg mL−1 were (80.25 ± 0.73) % and (66.54 ± 2.35) %, respectively, and 2,2′-azinobis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radical scavenging activities were estimated as (90.14 ± 0.79) % and (64.45 ± 8.97) %, respectively. The methanol leaf extract after ultra-pressure treatment exhibited higher binding properties to human serum albumin in comparison with catechin and conventional treatments. Consequently, it was assumed that the ultra-pressure treatment is an effective method for the extraction of bioactive compounds from indigo leaves.

쪽 선발계통의 생육특성 및 색소함량 차이

나주재래종과 새로이 선발된 나주2호의 지역별 재배시험 결과, 초장은 나주2호가 다소 컸으며, 분지수는 나주재래종이 나주2호 보다 많은 경향이었다. 엽면적은 나주2호가 더 넓었으며, 엽장폭비도 나주2호가 더 크게 나타나 둥근 잎 특성의 안정성을 보였다. 두 계통의 지상부 생중과 건중은 큰 차이가 없었으나, 지상부 전체에 대한 엽의 무게 비율은 나주2호가 더 높아 색소수율면에서 유리하였다. 인디고(indigo) 성분과 니람(泥藍) 함량은 나주2호가 나주재래종 보다 높게 나타났으며 실크로 생엽 이용 염색을 한 결과 청색을 더 많이 나타냈다. 따라서 엽 수량이 높고 색소 품질이 좋은 나주2호가 쪽 재배에 유리한 계통으로 판단되었다. Indigo crop, Persicaria tinctoria H. Gross is an annual plant containing natural colorant, the blue dye indigo, and local cultivars had been cultivated to produce natural indigo for textile dyeing in Korea since ancient times. Naju No. 2 is a new mass-selected line from the mother population, Naju Local cultivar. In this study, two breeding lines of Naju Local and Naju No. 2, have been cultivated in four different locations, the South regions of Korea, to compare plant growth and yield characteristics between two lines. Naju No. 2 was higher in plant height, and Naju Local has more 1st branches. Naju No. 2 has larger leaf area and higher width/length ratio of leaf, showing the round leaf type as morphological stable character without regional differences. Though there was considerable regional variation in fresh and dry top weight of harvested plant, the significant difference of plant weight between two lines were not shown. The ratio of leaf to total shoot of dry weight of Naju No. 2 was higher than one of Naju Local, indicating that Naju No. 2 has better yielding of colorant which is synthesized mostly in leaf. Naju No. 2 contained more Niram (crude indigo extract) and indigo, and showed much blueness at dyeing of silk using fresh leaves than Naju Local. We concluded that a new line, Naju No. 2 could be a superior cultivar due to having higher leaf yield and better quality of natural colorant than Naju local cultivar.

Partial characterization of indigo (Polygonum tinctorium Ait.) plant seeds and leaves

The aim of this study was to assess the contents of indigo's bioactive compounds, its antioxidant and anticancer activities in acetone, hexane and DMSO extracts and to compare the overall bioactivity with another more used medicinal plant named prolipid. It was found that the contents of the bioactive compounds in the studied extracts from different parts of indigo plant varied (P<0.05): the significantly highest content of polyphenols and flavonoids was in DMSO extract of prolipid, flavanols – in acetone extract of brown seeds (P<0.05 in both cases) and tannins – in DMSO extract of green leaves, but not significantly (P>0.05). Also the level of antioxidant activity was different: the highest antioxidant activity of all studied samples was in prolipid: according to ABTS, FRAP and CUPRAC tests in DMSO extract (P<0.05 in all 3 cases), and only in acetone extract according to DPPH was not significant (P>0.05). The correlations between polyphenol compounds and the antioxidant activities were relatively high. DPPH kinetic measurements were used to compare and distinguish the antiradical activity among indigo extracts by multivariate analysis. The FT-IR spectroscopy evaluated the presence of polyphenols. The interaction between DMSO polyphenol extracts of indigo plant and BSA showed that indigo has a strong ability as other medicinal plants such as prolipid to quench the intrinsic fluorescence of BSA by forming complexes and was measured by 3-dimensional fluorescence (3D-FL). The highest anticancer activity was in prolipid in concentrations of 800μg/mL against Calu-6, following by indigo brown leaves. In conclusion, organic extracts of indigo brown leaves were analyzed for their antioxidant and anticancer activities and compared with prolipid, using polyphenols composition, antioxidant activities and fluorescence properties. The indigo ability to quench the intrinsic fluorescence of BSA, relatively high content of phenolic compounds and anticancer properties can be used as medicinal plant.

English

Aqueous and organic basidiocarp extracts of the edible mushroom&nbsp;Lactarius indigo&nbsp;were evaluated for their antibacterial and cytotoxic effects. 10, 20 and 30 mg of organic extracts were tested against&nbsp;diarrheagenic&nbsp;Escherichia coli&nbsp;strains (EIEC, EPEC, ETEC-LT and ETEC-ST),&nbsp;Pseudomonas aeruginosa,&nbsp;Enterobacter cloacae,Staphylococcus aureus&nbsp;and&nbsp;Salmonella&nbsp;enterica. 10 mg of hexane extract showed activity against ETEC-LT (18.8 mm zone of inhibition) and&nbsp;P. aeruginosa&nbsp;(10.5 mm). All levels of the ethyl acetate extract inhibited all the strains, with stronger activity against EIEC (19.0 mm) and&nbsp;P. aeruginosa&nbsp;(21.0 mm) at 30 mg. Methanol extract inhibited all bacterial growth, but&nbsp;E. cloacae. 100 &mu;g/ml of aqueous extract showed antiproliferative activity against MCF7 cells, but not on HeLa, A549 and normal bovine mammary epithelium cells. Methanol and ethyl acetate extracts inhibited proliferation of HeLa cells (50 to 1000 ng/ml) but increased proliferation of A549 (100 ng/ml), as did methanol extract (500 ng/ml). Methanol extract did not inhibit normal rabbit serum fibroblast cells, while hexane and ethyl acetate extracts showed an inhibitory effect with 50 and 100 ng/ml, respectively, less than in proliferation of HeLa cells. These results show that&nbsp;L. indigo&nbsp;basidiocarps contain substances with antibacterial and cytotoxic activities. &nbsp; Key words:&nbsp;Lactarius indigo&nbsp;extracts, antibacterial activity,&nbsp;cytotoxic activity.

Determination of natural colorants in plant extracts by high performance liquid chromatography

The determination of the colouring compounds apigenin (1), lawsone (2), juglone (3) and indigotin (4) in plant extracts using HPLC - UV/VIS methods is reported. The methods were applied to the analysis of 1-4 in ethanolic and propylene glycolic extracts originating, respectively, from chamomile (Chamomilla recutita [L] Rauschert, Asteraceae), henna (Lawsonia inermis L., Lythraceae), walnut (Juglans regia L., Juglandaceae) and natural indigo (Indigofera sp., Fabaceae). In the case of the indigo extracts, an optimized acid hydrolysis was applied. HPLC separations were performed on a Hypersil ODS RP18 column using linear gradient elution programs. The detection limits for 1-4 were 0.11, 0.6, 0.10, 0.089 ?g mL-1, respectively. The procedure did not involve any sample ?clean -up? methods. The amounts of the colouring compounds ranged from 0.006 mg mL-1 (3) to 0.13 mg mL-1 (4) in the ethanolic extracts and from 0.22 mg mL-1 (2) to 1.44 mg mL-1 (4) in propylene glycolic extracts. The proposed HPLC methods are advantageous in terms of sample preparation and the selective separation of the compounds. The plant dye extracts are commonly used in hair colouring formulations. The results indicate that the methods developed may serve for the quantitative control of dying plants and cosmetic products.