In-vitro cytotoxicity in relation to chemotypic diversity in diploid and tetraploid populations of Gentiana kurroo Royle.

ETHNOPHARMACOLOGICAL RELEVANCE: Gentiana kurroo is a multipurpose critically endangered medicinal herb prescribed as medicine in Ayurveda in India and exhibits various pharmacological properties including anti-cancer activity. The species is rich repository of pharmacologically active secondary metabolites together with secoiridoidal glycosides. AIM OF THE STUDY: The study aimed to investigate the chemical diversity in different populations/cytotypes prevailing in G. kurroo to identify elite genetic stocks in terms of optimum accumulation/biosynthesis of desired metabolites and having higher in-vitro cytotoxicity potential in relation to chemotypic diversity. MATERIAL AND METHODS: The wild plants of the species were collected from different ranges of altitudes from the Kashmir Himalayas. For cytological evaluation, the standard meiotic analysis was performed. The standard LC-MS/MS technique was employed for phytochemical analysis based on different marker compounds viz. sweroside, swertiamarin, and gentiopicroside. Different tissues such as root-stock, aerial parts, and flowers were used for chemo-profiling. Further, the methanolic extracts of diploid and tetraploid cytotypes were assessed for cytotoxic activity by using MTT assay against four different human cancer cell lines. RESULTS: The quantification of major bioactive compounds based on tissue- and location-specific comparison, as well as in-vitro cytotoxic potential among extant cytotypes, was evaluated. The comprehensive cytomorphological studies of the populations from NW Himalayas revealed the occurrence of different chromosomal races viz. n = 13, 26. The tetraploid cytotype was hitherto unreported. The tissue-specific chemo-profiling revealed relative dominance of different phytoconstituents in root-stock. There was a noticeable increase in the quantity of the analyzed compounds in relation to increasing ploidy status along the increasing altitudes. The MTT assay of methanolic extracts of diploid and tetraploid cytotypes displayed significant cytotoxicity potential in tetraploids. The root-stock extracts of tetraploids were highly active extracts with IC50 value ranges from 5.65 to 8.53 µg/mL against HCT-116 colon cancer. CONCLUSION: The chemical evaluation of major bioactive compounds in diverse cytotypes from different plant parts along different altitudes presented an appreciable variability in sweroside, swertiamarin, and gentiopicroside contents. Additionally, the concentrations of these phytoconstituents varied for cytotoxicity potential among different screened cytotypes. This quantitative difference of active bio-constituents was in correspondence with the growth inhibition percentage of different tested cancer cell lines. Thus, the present investigation strongly alludes towards a prognostic approach for the identification of elite cytotypes/chemotypes with significant pharmacological potential.

Rapid temporal changes in root colonization by arbuscular mycorrhizal fungi and fine root endophytes, not dark septate endophytes, track plant activity and environment in an alpine ecosystem.

Fungal root endophytes play an important role in plant nutrition, helping plants acquire nutrients in exchange for photosynthates. We sought to characterize the progression of root colonization by arbuscular mycorrhizal fungi (AMF), dark septate endophytes (DSE), and fine root endophytes (FRE) over an alpine growing season, and to understand the role of the host plant and environment in driving colonization levels. We sampled four forbs on a regular schedule from June 26th-September 11th from a moist meadow (3535 m a.s.l) on Niwot Ridge, Rocky Mountain Front Range, CO, USA. We quantified the degree of root colonization by storage structures, exchange structures, and hyphae of all three groups of fungi. AMF and FRE percent colonization fluctuated significantly over time, while DSE did not. All AMF structures changed over time, and the degree of change in vesicles differed by plant species. FRE hyphae, AMF arbuscules and AMF vesicles peaked late in the season as plants produced seeds. AMF hyphae levels started high, decreased, and then increased within 20 days, highlighting the dynamic nature of plant-fungal interactions. Overall, our results show that AMF and FRE, not DSE, root colonization rapidly changes over the course of a growing season and these changes are driven by plant phenology and seasonal changes in the environment.

[Quality regionalization study on Gentianae Macrophyllae Radix].

The 79 samples of Gentianae Macrophyllae Radix were collected based on the distributed information by document literature. Based on sample information, and using the regression model of Gentianae Macrophyllae Radix index component and environmental factors, and combined with the prediction results of ecological suitability by MaxEnt and principal component analysis results of index component, the space distribution of Gentianae Macrophyllae Radix was estimated with the spatial analysis function of ArcGIS. The results showed that it had a higher comprehensive quality in south of Shaanxi, south of Gansu, middle of Sichuan and southeast of Xizang. The study results were coinciding with the producing regions of Gentianae Macrophyllae Radix. It can provide reference for Gentianae Macrophyllae Radix resource conservation, development and utilization.

[Ecology suitability study of Chinese materia medica Gentianae Macrophyllae Radix].

This paper is aimed to predict ecology suitability distribution of Gentianae Macrophyllae Radix and search the main ecological factors affecting the suitability distribution. The 313 distribution information about G. macrophylla, 186 distribution information about G. straminea, 343 distribution information about G. dauricaand 131 distribution information about G. crasicaulis were collected though investigation and network sharing platform data . The ecology suitable distribution factors for production Gentianae Macrophyllae Radix was analyzed respectively by the software of ArcGIS and MaxEnt with 55 environmental factors. The result of MaxEnt prediction was very well (AUC was above 0.9). The results of predominant factors analysis showed that precipitation and altitude were all the major factors impacting the ecology suitable of Getiana Macrophylla Radix production. G. macrophylla ecology suitable region was mainly concentrated in south of Gansu, Shanxi, central of Shaanxi and east of Qinghai provinces. G. straminea ecology suitable region was mainly concentrated in southwest of Gansu, east of Qinghai, north and northwest of Sichuan, east of Xizang province. G. daurica ecology suitable region was mainly concentrated in south and southwest of Gansu, east of Qinghai, Shanxi and north of Shaanxi province. G. crasicaulis ecology suitable region was mainly concentrated in Sichuan and north of Yunnan, east of Xizang, south of Gansu and east of Qinghai province. The ecological suitability distribution result of Gentianae Macrophyllae Radix was consistent with each species actual distribution. The study could provide reference for the collection and protection of wild resources, meanwhile, provide the basis for the selection of cultivation area of Gentiana Macrophylla Radix.

[Analysis of commercial specifications and grades of wild and cultivated Gentianae Macrophyllae Radix based on multi-indicative constituents].

The study is aimed to analyze the commercial specifications and grades of wild and cultivated Gentianae Macrophllae Radix based on multi-indicative constituents. The seven kinds of main chemical components containing in Gentianae Macrophyllae Radix were determined by UPLC, and then the quality levels of chemical component of Gentianae Macrophyllae Radix were clustered and classified by modern statistical methods (canonical correspondence analysis, Fisher discriminant analysis and so on). The quality indices were selected and their correlations were analyzed. Lastly, comprehensively quantitative grade division for quality under different commodity-specifications and different grades of same commodity-specifications of wild and planting were divided. The results provide a basis for a reasonable division of specification and grade of the commodity of Gentianae Macrophyllae Radix. The range of quality evaluation of main index components (gentiopicrin, loganin acid and swertiamarin) was proposed, and the Herbal Quality Index (HQI) was introduced. The rank discriminant function was established based on the quality by Fisher discriminant analysis. According to the analysis, the quality of wild and cultivated Luobojiao, one of the commercial specification of Gentianae Macrophyllae Radix was the best, Mahuajiao, the other commercial specification, was average , Xiaoqinjiao was inferior. Among grades, the quality of first-class cultivated Luobojiao was the worst, of second class secondary, and the third class the best; The quality of the first-class of wild Luobojiao was secondary, and the second-class the best; The quality of the second-class of Mahuajiao was secondary, and the first-class was the best; the quality of first-class Xiaoqinjiao was secondary, and the second-class was the better one between the two grades, but not obvious significantly. The method provides a new idea and method for evaluation of comprehensively quantitative on the quality of Gentianae Macrophyllae Radix.

[Influences of Base Fertilizer Dosage and Root Size on Quality and Yield of Gentiana crassicaulis Root].

OBJECTIVE: In order to optimize the base fertilizer dosage and root size of Gentiana crassicaulis in Ludian,Yulong, Yunnan,the differences in yield and quality of Gentiana crassicaulis caused by different base fertilizer dosage and root size were investigated. METHODS: The variation in fresh weight, dry weight, drying rate, length, diameter, branches as well as the contents of gentiopicroside, loganin acid, alcohol-soluble extract and total ash were determined. The ideal distance grey relational degree comprehensive evaluation method was applied to make a comprehensive evaluation on the quality and yield of Gentiana crassicaulis. RESULTS: There were big differences in yield and quality of Gentiana crassicaulis among different base fertilizer dosage and root size. 2 000 kg/667 m2 of manure, 20 kg/667 m2 of superphosphate, 100 kg/667 m2 of plant ash and mix size of transplanting roots showed the best comprehensive quality and the highest yield. CONCLUSION: 2 000 kg/667 m2 of manure, 20 kg/667 m2 of superphosphate,100 kg/667 m2 of plant ash, high quality transplanting roots and stagger certain depth to transplant are highly recommended to produce high quality and high yield of Gentiana crassicaulis in Ludian, Yulong, Yunnan.

[Fertilizer Effect on Yield and Quality of Gentiana crassicaulis].

OBJECTIVE: To find the best fertilizer formula of yield-quality-fertilizer effect, and to establish a new comprehensive evaluation-fertilizer effect method. METHODS: Grey related degree analysis and DTOPSIS method were combined to comprehensive evaluate the 11 indexes of 14 fertilizer formulas of nitrogen, phosphorus and potassium, in order to explore the best dosage and ratio. RESULTS: The fertilizer requirements of the factors which contribute to the yield and quality of Gentiana crassicaulis were different and the most appropriate rates of nitrogen, phosphorus and potassium fertilizer were 21.49~42.03 kg/667 m2, 49.53~65.75 kg/667 m2 and 26.34~ 42.97 kg/667 m2, respectively. CONCLUSION: The fertilizer formula in this study can be widely used in the standardized plantation of Gentiana crassicaulis.

[Study on Dormant Culture and Plant Regeneration via Cluster Buds of Gentiana straminea].

OBJECTIVE: In order to provide scientific basis for micropropagation and cryopreservation of Gentiana straminea,plantlets were regenerated from dormant buds by cluster buds. METHODS: Based on MS medium, dormant buds were inoculated in mediums containing different type and concentration of cytokinin and auxin for inducing cluster buds. 1/2MS medium with different concentration of auxin were used for inducing root. RESULTS: The medium of MS with 2.0 mg/L 6-BA, 0.01 mg/L NAA,30 g/L sucrose and 7 g/L agar was suitable for cluster buds' primary culture and subculture. The cluster buds inducing rate was 93. 3%. Multiple shoot clumps multiplication factor was 5.6. The medium of 1/2MS with 2.0 mg/L IAA, 0.5 mg/L IBA, 15 g/L sucrose and 7 g/L agar was suitable for root induction, its inducing rate was 93.5% with plantlets growing well. CONCLUSION: Plantlets regenerated from dormant buds of Gentiana straminea via cluster buds are established in this study.

[Study on Embryonic Development of Four Species of Gentiana (Gentianaceae)].

OBJECTIVE: To study the embryonic development of Gentiana straminea, G. robusta, G. crassicaulis and G. tibetica. METHODS: The seed germination rates, length and width of embryos, starch grains and chloroplasts were observed and analyzed by statistic software. RESULTS: The seed germination rates of the four species were all high. The increase of the length of embryo depended mainly on the increase of the length of hypocotyl. Starch grains were stored in cells and chloroplasts appeared in cotyledons. The process of germination was divided into eight periods. CONCLUSION: The embryo growth characteristics of the four species are recognized, and the results can be used to study the in situ conservation, genetic breeding and cultivation of Sect. Cruciata.

[Varieties textual research and usage investigation of white flos gentianae, a Tibetan medicine].

OBJECTIVE: To clarity the original plants and the main application varieties of White Flos Gentianae. METHOD: Herbal textual research, wild specimen collection, investigation and collection of the samples from Tibetan hospital, Tibetan pharmaceutical factory and medical material market were carried out simultaneously to identify the original plants of White Flos Gentianae. RESULT: The results of varieties textual research and specimen identification showed that Gentiana szechenyii, G. purdomii and G. algida were in accord with the record of Tibetan herbal textual The three species above were the original plants of White Flos Gentianae. The identification of 20 batches samples showed that G. szechenyii was the main application variety. The other varieties were only used in Tibetan hospitals. All the samples above were flowering branches. CONCLUSION: It was necessary to strengthen the research on variety systematization of White Flos Gentianae make a further discussion on the taxonomy position of G. purdomii, G. algida and the white flos population. Its was also nessary to establish and improve the quality standard of different variety based on the principle of "one species, one name". The quality specification of White Flos Gentianae should be established and improved to standard clinical utilization and produce feeding. More study of resources investigation and cultivation of G. szechenyii should be carried on to meet the demand of produce and clinic.

[Optimum harvest study of Gentiana crassicaulis in Ludian].

The paper is aimed to study the difference in yield and quality at different harvest time and determine the optimum harvest of planting Gentiana in Ludian traditional harvest period. The authors analyzed the variation in fresh weight, dry weight, dry discount rate, length, diameter, volume and the content of gentiopicroside, loganin acid, alcohol-soluble extract and total ash and made a comprehensive appraisal of yield, appearance quality and intrinsic quality by gray relational distance ideal Comprehensive Evaluation method. The results showed that there is a big difference in yield and quality both 2-year-old and 3-year-old Gentiana harvested in traditional harvest period and the comprehensive evaluation more better when harvested more later. It can be seen, Gentiana harvested the later had a better yield and quality in Ludian traditional harvest period. The harvest of Gentiana can be appropriate delayed depending on the particular circumstances of production.

[Determination of polysaccharides content of Gentiana farreri from different producing areas based on anthrone-sulfuric acid method].

Study a method for the detemination of the content of polysaccharides in Gentiana farreri, and analysis of the content of polysaccharides from different producing areas. The results showed that using the anthrone-sulfuric acid method, simple operation, accurate result. Sample was measured at 620 nm absorbance after anthrone-sulfuric acid color, at this wavelength, solution absorption and glucose showed a good linear relationship; The linearity was in the range of 0.01-0.07 g x L(-1) (r = 0.996 7). The recovery rate was 99.41%, with RSD of 2.0%. Considering the experimental conditions, to determine the solid-liquid ratio 1:60, extracting time 50 min, concentration of ethanol 80%. The mass fraction of polysaccharides was the highest to reached 0.743% in G. farreri from Gansu Xiahe. This experiment has laid a good foundation for further study on G. farreri.

[Callus cultivation and determination of gentiopicroside from Gentiana macrophylla].

OBJECTIVE: To explore and improve the utilization efficiency of Gentiana macrophylla resources. METHODS: The germination of Gentiona macrophylla seeds were used as explants to induce calluses; Cultured on various culture media and condition for callus induction and subculture by test; The content of gentiopicroside in callus was determined by HPLC. RESULTS: The optimum medium for callus was MS +2,4-D (1.0 mg/L) + 6-BA (0.5 mg/L), and MS +2,4-D (1.0 mg/L) +6-BA (0.5 mg/L) + NAA (0.5 mg/L) was the optimum for subculture,the optimum time of subculture was 30 d; The accumulated gentiopicroside reached the highest level with 0.78 mg/g DW when entered 28-30 d. CONCLUSION: The optimized culture method of callus of Gentiana. macrophylla is obtained; The growth curve of callus of Gentiona macrophylla turns on "S" appearance and the accumulated gentiopicroside increases along with the callus appreciation.

[Species and ecological control of disease on cultivated Gentiana rigescens in Yunnan].

OBJECTIVE: To find out variety of the fungal diseases of cultivated Gentiana rigescens and provide important basis for prevention. METHODS: The diseases were diagnosed based on field investigate, symptoms observation, pathogen isolation, determination the size of morphological and verification following the Koch's Postulate procedures. RESULTS: Anthracnose (Colletotrichum gloeosporioides), grey mould (Botrytis cinerea), brown spot (Alternaria tenuis), rust (Aecidiumpers), circular spot (Pestalotiopsis), leaf blight (Stemphylium, Ascochyta, Pleospora) and nematodes (Heterodera spp., Meloidogyne spp.) were found on Gentiana rigescens. Anthracnose was the first main disease, the diseased plant rate was over 40% and disease severity was 4 - 5 degree and second disease was rust, incidence of rate was less 10% and other diseases rate was not enough 2%. CONCLUSION: All these diseases on Gentiana rigescens are reported for the first time and Gentiana rigescens is the new host plant of the diseases.

An in vitro perspective to cholinesterase inhibitory and antioxidant activity of five Gentiana species and Gentianella caucasea.

The dichloromethane (DCM), ethyl acetate (EtOAc), and methanol extracts from the leaves, roots, and flowers of the five species of Gentiana (Gentiana asclepiadea, Gentiana cruciata, Gentiana olivieri, Gentiana septemfida, and Gentiana verna) and Gentianella caucasea were investigated for their inhibitory activity against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) and antioxidant effect using 2,2-diphenyl-1-picrylhydrazyl radical scavenging, metal-chelation capacity, and ferric-reducing antioxidant power assays. Total phenol and flavonoid contents of the extracts were determined spectrophotometrically. The presence of some characteristic compounds found in Gentiana species (gentiopicroside, swertiamarin, isoorientin, isovitexin and vitexin) was analyzed in the extracts by thin layer chromatography. The flower DCM extract of G. verna exerted the highest inhibition against AChE (53.65 ± 1.03%), whereas the root EtOAc extract of G. cruciata was the most effective in BChE inhibition assay (50.72 ± 0.75%) at 100 µg ml⁻¹. The extracts of G. verna were also found to be more active in the antioxidant tests.

[Macroscopic identification of cultivated and wild Gentiana macrophylla and preliminary screening for high quality germplasm].

OBJECTIVE: To investigate the difference of macroscopic characteristics between cultivated Gentiana macrophylla and wild one, analyze the correlation among its main quality traits and preliminary screen cultivated Gentiana macrophylla from different habitats based on quality traits. METHODS: Measured and obtained characteristic index, including main root length, lateral root number, taproot diameter, residue stem number and dry weight, and analyzed data by SPSS 12.0 statistical software. RESULTS: The main characteristics of cultivated Gentiana macrophylla were all better than those of the wild one. The characteristics was sequenced as follows: lateral root number > dry weight > main root lengthy > taproot diameter > residue stem number in order of difference. There were remarkable positive correlations among main quality traits. The optimal germplasm was located in Yulinpu of Liufengguan Village, Nanxing Town, Feng County in Shaanxi Province. CONCLUSION: Cultivated Gentiana macrophylla is relatively robust, having more branching roots with light colour of surface and section compared with the wild one. The paper provides suggestions for the revising of macroscopic characteristics of Gentiana macrophylla in the next edition of pharmacopoeia; The data offer scientific reference for screening excellent germplasm, introduction expansion and reasonable utilization of resources as well.

[Study on seed morphology of the section Cruciata Gaudin].

OBJECTIVE: To provide a scientific evidence for the identification of the section Cruciata Gaudin. METHODS: Seed morphology of the section Cruciata Gaudin in different regions from Gansu were studied by means of scanning electronic microscopy (SEM). RESULTS: The seeds of section Cruciata Gaudin had relative conformability in terms of shape,size and ornamentation. But the reticulate cell of Gentiana dahurica was obviously wider than those of other species. CONCLUSION: This study provides some useful information for classification and identification of section Cruciata Gaudin.

[Correlation and path analysis on agronomic traits of Gentiana rigescens].

OBJECTIVE: The interrelation of yield and agronomic traits of Gentiana rigescens was studied for the germplasm and breeding variety of this species. METHOD: Twelve agronomic traits, root diameter, root length, root number, root biomass, stem diameter, plant height, the first branch number, leaf length, leaf width, leaf length/leaf width ratio, calyx length, and calyx number of G. rigescens from 26 wild populations in Yunnan were determined for correlation analysis, multiple stepwise regression analysis and path analysis. RESULT: Correlation analysis showed that there were significantly positive correlation between the traits of aboveground part and the length, diameter, number, and biomass of the root. Multiple stepwise regression analysis showed that length, width, and number of root, plant height, the first branch number, and the calyx number were the main factors that affected the root biomass. Path analysis showed that the diameter, length, and number of the root, the stem height, and the first branch number had a direct positive effect on the root biomass. CONCLUSION: The traits, such as high and strong stem, high number of first branch number and shrubby shape could be selected for the breeding and high yielding of G. rigescens.

[Study on the quality and genetic diversity of Gentiana macrophylla pall of different habitats].

OBJECTIVE: To study the quality and genetic diversity in Gentiana macrophylla of different habitats for controlling the quality of Gentiana macrophylla. METHODS: Main characteristics and microscopic identification were adapted to identify Gentiana macrophylla; HPLC method was applied for analysising the contents of Gentiana macrophylla. RAPD method was applied to study genetic structure and genetic diversity of Gentiana macrophylla. RESULTS: The experiment showed that the Gentiana macrophylla of different habitats have different main charactericstics, microscopic identifications and contents, and effective ingredient contents of gentiopicroside. The levels of population genetic diversity of various groups followed by HUANGZHONG County population (K) > HUAN County population (J) > ZiWU Mountain population (H). CONCLUSION: The study of external morphology, internal structure, active ingredient content and genetic level of Gentiana macrophylla from different areas provides a scientific basis for protecting wild species and ensuring the quality in introducing and cultivating Gentiana macrophylla.

[Study on seed biological characteristics of Gentiana crassicaulis].

OBJECTIVE: To study seed biological characteristics of Gentiana crassicaulis. METHOD: The samples were collected from five localities, and the morphological observation, germination test and viability TTC test were carried out. RESULT: The seed morphological characters of all samples were similar to each other. The viability of all samples was similar to each other, but the germination rate of wild seed was the highest in all samples after stored for 8 months. Also, the seed energy of wild seed and sample 2008XR02 was higher than other three samples. There was no obvious correlation between thousand seed weight and germination rate. CONCLUSION: The quality of wild seed and sample 2008XR02 was superior than that of the other three samples. The results can be used for basic data of in situ conservation and germplasm breeding of G. crassicaulis.