scholarly journals Huperzine A and Huperzine B Production by Prothallus Cultures of Huperzia selago (L.) Bernh. ex Schrank et Mart

Molecules ◽  
2020 ◽  
Vol 25 (14) ◽  
pp. 3262
Author(s):  
Wojciech J. Szypuła ◽  
Beata Wileńska ◽  
Aleksandra Misicka ◽  
Agnieszka Pietrosiuk
Keyword(s):  
Pharmaceutical Industry ◽  
Current Source ◽  
Growth Index ◽  
Dry Weight ◽  
Huperzine A ◽  
Biomass Growth ◽  
Tissue Cultures ◽  
In The Wild ◽  
Very High

This is the first report of an efficient and effective procedure to optimize the biosynthesis of huperzine A (HupA) and huperzine B (HupB) in vitro from Huperzia selago gametophytes. Axenic tissue cultures were established using spores collected from the sporophytes growing in the wild. The prothalia were obtained after 7–18 months. Approximately 90 up to 100% of the gametophytes were viable and grew rapidly after each transfer on to a fresh medium every 3 months. The best biomass growth index for prothallus calculated on a fresh (FW) and dry weight (DW) basis, at 24 weeks of culture, was 2500% (FW) and 2200% (DW), respectively. The huperzine A content in the gametophytes was very high and ranged from 0.74 mg/g to 4.73 mg/g DW. The highest yield HupA biosynthesis at >4 mg/g DW was observed on W/S medium without growth regulators at 8 to 24 weeks of culture. The highest HupB content ranged from 0.10 mg/g to 0.52 mg/g DW and was obtained on the same medium. The results demonstrate the superiority of H. selago gametophyte cultures, with the level of HupA biosynthesis approximately 42% higher compared to sporophyte cultures and 35-fold higher than when the alkaloid was isolated from H. serrata, its current source for the pharmaceutical industry. Moreover, the biosynthesis of HupB was several-fold more efficient than in H. selago sporophytes growing in the wild. HPLC-HR-MS analyses of the extracts identified eight new alkaloids previously unreported in H. selago: deacetylfawcettine, fawcettimine, 16-hydroxyhuperzine B, deacetyllycoclavine, annopodine, lycopecurine, des-N-methylfastigiatine and flabelline.

scholarly journals A new and fast method to obtain in vitro cultures of Huperzia selago (Huperziaceae) sporophytes, a club moss which is a source of huperzine A

2013 ◽  
Vol 82 (4) ◽  
pp. 313-320 ◽  
Author(s):  
Wojciech J. Szypuła ◽  
Paulina Mistrzak ◽  
Olga Olszowska
Keyword(s):  
Developmental Stages ◽  
Therapeutic Potential ◽  
Callus Formation ◽  
Axenic Culture ◽  
Growth Index ◽  
In Vitro Cultures ◽  
Huperzine A ◽  
Fast Method ◽  
Biomass Growth

This study presents a protocol for a fast and effective in vitro axenic culture of <em>Huperzia selago</em> (Huperziaceae Rothm.) sporophytes, a club moss which is a source of huperzine A, an alkaloid of a considerable therapeutic potential extensively investigated for its uses as treatment for some neurodegenerative diseases. The proposed procedure allowed approximately tenfold shortening of the species developmental stages with the omission of the gametophyte stage while the sporophyte mass could be increased tenfold within a 6-month period. The cultures were established using vegetative propagules (bulbils) procured from sporophytes growing in the wild without degrading the habitats of this endangered plant species. Explants underwent surface and internal disinfection to eliminate the epiphytic and endophytic bacteria and fungi. In in vitro cultures, the optimum results were achieved using Moore (Mr) medium without growth regulators or supplemented with 0.015 mg/l IBA and 0.3 mg/l kinetin. These media ensured both viability of the propagules and their further development. The biomass growth index for <em>H. selago</em> sporophytes grown from propagules, determined at 3 months of culture (1 passage) on Mr medium with IBA and kinetin was 650%. At 6 months, the biomass growth index increased to 1114%. Vigorous growth of adventitious roots, especially on Mr medium with the addition of 0.25 mg/l NAA, and callus formation on shoot apices were observed. At 6 months of culture, some sporophytes obtained from the bulbils were used as the initiating material for shoot subcultures, which developed best on Mr medium with IBA and kinetin.

Oestrogen synthesis by bovine foetal placenta at normal parturition

1981 ◽  
Vol 98 (1) ◽  
pp. 112-118 ◽  
Author(s):  
K. Larsson ◽  
W. C. Wagner ◽  
M. Sachs
Keyword(s):  
Tissue Culture ◽  
Conversion Rate ◽  
Tissue Cultures ◽  
Peripheral Plasma ◽  
Conversion Rates ◽  
Phenolic Extraction ◽  
Percentage Conversion ◽  
Very High ◽  
Culture Conversion

Abstract. In vitro conversion of C19-[3H]androstenedione and C21-[3H]pregnenolone steroids to oestrogens was studied in tissue cultures prepared from bovine cotyledons collected 10–240 min after natural parturition. A total of 17 cows was used, 15 had normal parturitions and 2 cows required assistance due to weak and prolonged labour. At collection of tissues, blood samples were drawn from the cows and peripheral plasma oestrogen levels were determined by radioimmunoassay. Percentage conversion to unconjugated and conjugated oestrogens was estimated for each cow from 6 tissue culture replicates for each precursor. The steroids were extracted with toluene and separated by phenolic extraction and column chromatography. The repeatability of conversion rate was very high. Incubation with androstenedione yielded consistently higher conversion rates than incubation with pregnenolone. Independent of precursor the conversion rates, within cows, ranked unconjugated Oe1 > conjugated Oe1 > unconjugated Oe2 > conjugated Oe2. The conversion rates decreased significantly with increasing intervals from parturition to collection of material. Lower conversion rates were found for cultures of tissues from the 2 cows with weak labour when compared to cultures from normally calving cows. The sire of the foetus had an influence on conversion rate in the tissue cultures. The plasma oestrogen concentrations of the cows decreased significantly with an increased interval from parturition to blood-sampling. With the exception of one cow, the tissue culture conversion rates and the plasma oestrogen levels of the cows were significantly correlated.

scholarly journals Differences in huperzine A yield when different amino acids were added for in vitro thallus culture of Huperzia serrata (Thunb)Trev and gene expression analysis

2020 ◽  
Author(s):  
Ye YuXuan ◽  
Tu YiSheng ◽  
Yu Xiao ◽  
Huang Qian ◽  
Yuan Huihui
Keyword(s):  
Gene Expression ◽  
Amino Acids ◽  
Aspartic Acid ◽  
Quantitative Pcr ◽  
Dry Weight ◽  
Huperzine A ◽  
Differential Analysis ◽  
Fluorescence Quantitative Pcr ◽  
Alignment Analysis

Abstract Background：the secondary metabolite of H. serrata, huperzine A (HupA) can be used in the treatment of Alzheimer’s disease and can improve the cognitive function of patients. The use of in vitro culture and secondary metabolism engineering to obtain secondary metabolites is the most effective method to solve a lack of HupA sources and protect H. serrata as a natural resource. This study was based on the in vitro thallus culture conditions for H. serrate, and different concentrations of alkaloid precursor amino acids (lysine, aspartic acid, and trytophan) were added. We found that addition of different amino acids to thallus cultures had different effects on HupA accumulation. Transcriptome sequencing was carried out on thalli with significant differences in the HupA content due to treatment with different amino acids for differential analysis, and real-time fluorescence quantitative PCR was used for validation to examine the functional genes involved in exogenous amino acid regulation of HupA accumulation in thalli.Results：We found that addition of 1 mmol·L−1 aspartic acid (D) solution promoted HupA accumulation, at a level of 84.05 μg·g−1 dry weight (DW), which was 1.29-fold that of the control (CK: 65.15 μg·g−1 DW). Addition of 4 mmol·L−1 lysine (K) solution significantly inhibited HupA accumulation, at a level of 48.42 μg·g−1 DW, which was 0.75-fold that of the control.Transcriptome sequencing-bioinformatics alignment analysis of the aforementioned materials showed that in GO alignment analysis, functions were annotated for 16,258 unigenes. From the statistical analysis of the DEGs of the three groups, we found that there were 1046, 782, and 1586 DEGs for CK vs D, CK vs K, and D vs K, respectively, with D vs K having the most DEGs. DEGs that were enriched in KEGG metabolic pathways and validated by fluorescence quantitative PCR included PANK1, GDH2, APX, HA1, ND4L, and COX1. Conclusions：The above results showed that HupA content differences in D and K treatments were directly proportional to DEGs. Gene expression differences are the molecular basis that affects HupA accumulation in in vitro thallus cultures. PANK1 and GDH2 encode enzymes that synthesize an alkaloid intermediate.

Transgenic prolactin−/− mice: effect of trauma-hemorrhage on splenocyte functions

2005 ◽  
Vol 288 (5) ◽  
pp. C1109-C1116 ◽  
Author(s):  
Takeshi Matsutani ◽  
T. S. Anantha Samy ◽  
Loring W. Rue ◽  
Kirby I. Bland ◽  
Irshad H. Chaudry
Keyword(s):  
Transgenic Mice ◽  
Immune Functions ◽  
Wild Type ◽  
Female Mice ◽  
Nb2 Cells ◽  
In The Wild ◽  
Ifn Γ ◽  
Local Release ◽  
Very High

Prolactin (PRL) is involved in the regulation of immune functions under normal and pathological conditions. Trauma-hemorrhage (T-H) produces profound immunosuppression in male mice but not in proestrus female mice. Administration of PRL in males after T-H, however, restores immune functions. In this study, PRL+/+ and transgenic (PRL−/−) male and female mice were used to assess immune suppression after T-H and to determine the reasons for the hormone's beneficial effect. In vitro lymphoproliferation assay with Nb2 cells showed complete absence of PRL in the circulation of the transgenic PRL−/− mice of both sexes, whereas very high levels of the hormone were detected in the wild-type PRL+/+ mice of both sexes. Moreover, T-H resulted in the appearance of significant levels of the hormone in circulation, but only in PRL+/+ mice. Splenocyte proliferation in male PRL−/− mice was significantly lower than in PRL+/+ mice after T-H. Marginal differences between PRL+/+ and PRL−/− mice were observed in the release of IL-2 and IFN-γ by splenocytes, while the release of IL-10 was significantly higher in PRL−/− than in PRL+/+ mice. A significant observation of our study is the release of a ∼25-kDa protein in the concanavalin A-stimulated splenocytes of male PRL+/+ and PRL−/− mice that was active in the in vitro lymphoproliferation assay with Nb2 cells. It is unlikely that this protein is PRL because it is also present in the splenocyte extracts of PRL−/− transgenic mice. Nonetheless, because control of lymphoid cell proliferation is considered one of the characteristics of the immune system, the local release of this protein may be significant in the differences observed in splenocyte cytokine release after T-H in wild-type as well as transgenic mice.

scholarly journals Enhancement of L-DOPA Production in Micropropagated Plants of two Different Varieties of Mucuna pruriens L., Available in India

2011 ◽  
Vol 21 (2) ◽  
pp. 115-125 ◽  
Author(s):  
Kotisree Lahiri ◽  
Madhumita J. Mukhopadhyay ◽  
Sandip Mukhopadhyay
Keyword(s):  
Nuclear Dna ◽  
Wild Strain ◽  
Dry Weight ◽  
Nodal Segments ◽  
Mucuna Pruriens ◽  
Shoot Bud ◽  
In The Wild ◽  
Micropropagated Plants ◽  
Dopa Concentration

Reports on increment in L-DOPA content in micropropagated plants, regenerated through a simple technique following shoot bud multiplication in four strains of two different varieties of Mucuna pruriens are made. Nodal segments from in vitro grown seedlings were cultured on modified MS with various concentrations and combinations of BAP, 2iP, Kn either alone or with NAA. Highest shoot regeneration from the callus was achieved in modified MS fortified with BAP at 1.33 µM level. The regenerated shoots were rooted in vitro in half-strength of liquid MS supplemented with various levels of NAA (0.54 - 10.7 mM) or IBA (0.49 - 9.85 mM). However, roots of superior quality were obtained at 5.4 mM NAA level after two weeks in culture. The regenerants were acclimatized for 2 - 3 weeks and about 85% survival rate was observed after transferring to the field. Both chromosome number stability as well as stability in nuclear DNA contents of the regenerants of all these strains was recorded with complete absence of aneuploidy. The different strains have revealed two to three-folds increase in L-DOPA contents in the cultured plants. The L-DOPA concentration in leaves of the regenerated plants varied from11.85 - 15.42 mg/g dry weight in all these accessions. However, the highest amount was observed in the wild strain of M. pruriens. This is the first report on enhancement of L-DOPA content in differentiated tissue of cultured plants of both the varieties of M. pruriens.   Key words: L-DOPA, Micropropagation, Mucuna pruriens, Nuclear DNA   D. O. I. 10.3329/ptcb.v21i2.10224   Plant Tissue Cult. & Biotech. 21(2): 115-125, 2011 (December)

scholarly journals Influence of Culture Conditions on In Vitro Asymbiotic Germination of Anacamptis longicornu and Ophrys panormitana (Orchidaceae)

Plants ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 2543
Author(s):  
Myriam Arcidiacono ◽  
Caterina Catalano ◽  
Antonio Motisi ◽  
Maurizio Sajeva ◽  
Francesco Carimi ◽  
...  
Keyword(s):  
Seed Germination ◽  
Nitrogen Source ◽  
Culture Conditions ◽  
Ms Medium ◽  
Symbiotic Germination ◽  
Terrestrial Orchids ◽  
In The Wild ◽  
Asymbiotic Germination ◽  
Very High

This study is the first approach to in vitro asymbiotic germination of two species of Sicilian threatened terrestrial orchids, Anacamptis longicornu and Ophrys panormitana. Seeds were collected in the wild and cultured in two different media—Orchimax medium (OM) and Murashige and Skoog (MS)—and exposed to different photoperiods and temperatures to evaluate the best conditions for the specific stages of development. The germination of A. longicornu was very high on OM (95.5%) and lower on MS medium (21.4%), whereas O. panormitana germinated only on OM medium, with significantly lower percentages (12.0%), compared with A. longicornu. This difference is caused by variation in quality and quantity of nutrients used, primarily by nitrogen source. The results show that temperature and photoperiod widely affect seed germination and development. Although further investigations on asymbiotic and symbiotic germination are needed for the improvement of conservation of Mediterranean terrestrial orchids, our results contribute to the conservation of this group of plants.

Daunorubicin and Platelet Function

1981 ◽  
Vol 45 (01) ◽  
pp. 038-042 ◽  
Author(s):  
M E Pogliani ◽  
R Fantasia ◽  
G Lambertenghi-Deliliers ◽  
E Cofrancesco
Keyword(s):  
Electron Microscope ◽  
Cellular Membrane ◽  
Hemorrhagic Diathesis ◽  
Clot Retraction ◽  
Freezing And Thawing ◽  
Platelet Factor ◽  
High Doses ◽  
Very High ◽  
Platelet Functions

SummaryThe influence of Daunorubicin on some platelet functions in vitro was investigated, using different concentrations of the drug (0.01-0.02-0.04 μg/ml). Daunorubicin was shown to inhibit Collagen and Thrombin induced platelet aggregation and the intensity of inhibition depended on both drug concentration and the time of preincubation.Daunorubicin was also shown to inhibit the release reaction, the platelet prostaglandin pathway and the availability platelet factor 3; the drug at concentrations for clinical use does not damage the platelet membrane, as is the case with the freezing and thawing test, in platelet uptake of 14C-serotonin and as confirmed by the electron microscope. When very high doses (0.16 mg) of Daunorubicin are used, lysis of the platelets can be observed and this is confirmed under the electron microscope by the presence of empty platelets with fractures at the level of the cytoplasmic membrane.Finally, Daunorubicin causes irreversible inhibition of reptilase clot-retraction, even if this is less severe than with Vincristine. Working with gel-filtered platelets, it would appear that the inhibition exercised by the drug on platelet reactions is not caused through modifications in Ca++ metabolism.The authors suggest that Daunorubicin, at the dosages used clinically, induces in vitro thrombocytopathy without damaging the cellular membrane as confirmed by the electron microscope.This impairment of platelet functions could play a part in hemorrhagic diathesis observed during Daunorubicin therapy.

Effects of Heparin Oligosaccharides with High Affinity for Antithrombin III in Experimental Venous Thrombosis

1982 ◽  
Vol 47 (03) ◽  
pp. 244-248 ◽  
Author(s):  
D P Thomas ◽  
Rosemary E Merton ◽  
T W Barrowcliffe ◽  
L Thunberg ◽  
U Lindahl
Keyword(s):  
Antithrombin Iii ◽  
Specific Activity ◽  
High Specific Activity ◽  
Factor Xa ◽  
Blood Levels ◽  
Thrombin Time ◽  
High Affinity ◽  
Very High

SummaryThe in vitro and in vivo characteristics of two oligosaccharide heparin fragments have been compared to those of unfractionated mucosal heparin. A decasaccharide fragment had essentially no activity by APTT or calcium thrombin time assays in vitro, but possessed very high specific activity by anti-Factor Xa assays. When injected into rabbits at doses of up to 80 ¼g/kg, this fragment was relatively ineffective in impairing stasis thrombosis despite producing high blood levels by anti-Xa assays. A 16-18 monosaccharide fragment had even higher specific activity (almost 2000 iu/mg) by chromogenic substrate anti-Xa assay, with minimal activity by APTT. When injected in vivo, this fragment gave low blood levels by APTT, very high anti-Xa levels, and was more effective in preventing thrombosis than the decasaccharide fragment. However, in comparison with unfractionated heparin, the 16-18 monosaccharide fragment was only partially effective in preventing thrombosis, despite producing much higher blood levels by anti-Xa assays.It is concluded that the high-affinity binding of a heparin fragment to antithrombin III does not by itself impair venous thrombogenesis, and that the anti-Factor Xa activity of heparin is only a partial expression of its therapeutic potential.

Controlled Release of Metformin Hydrochloride I. In vitro Release from Physical Mixture Containing Xanthan Gum as Hydrophilic Rate Retarding Polymer

1970 ◽  
Vol 4 (1) ◽  
Author(s):  
Mashkura Ashrafi ◽  
Jakir Ahmed Chowdhury ◽  
Md Selim Reza
Keyword(s):  
Controlled Release ◽  
Xanthan Gum ◽  
In Vitro Release ◽  
Correlation Coefficients ◽  
High Ratio ◽  
Water Soluble ◽  
Metformin Hydrochloride ◽  
Model Drug ◽  
Very High

Capsules of different formulations were prepared by using a hydrophilic polymer, xanthan gum and a filler Ludipress. Metformin hydrochloride, which is an anti-diabetic agent, was used as a model drug here with the aim to formulate sustained release capsules. In the first 6 formulations, metformin hydrochloride and xanthan gum were used in different ratio. Later, Ludipress was added to the formulations in a percentage of 8% to 41%. The total procedure was carried out by physical mixing of the ingredients and filling in capsule shells of size ‘1’. As metformin hydrochloride is a highly water soluble drug, the dissolution test was done in 250 ml distilled water in a thermal shaker (Memmert) with a shaking speed of 50 rpm at 370C &plusmn 0.50C for 6 hours. After the dissolution, the data were treated with different kinetic models. The results found from the graphs and data show that the formulations follow the Higuchian release pattern as they showed correlation coefficients greater than 0.99 and the sustaining effect of the formulations was very high when the xanthan gum was used in a very high ratio with the drug. It was also investigated that the Ludipress extended the sustaining effect of the formulation to some extent. But after a certain period, Ludipress did not show any significant effect as the pores made by the xanthan gum network were already blocked. It is found here that when the metformin hydrochloride and the xanthan gum ratio was 1:1, showed a high percentage of drug release, i.e. 91.80% of drug was released after 6 hours. But With a xanthan gum and metformin hydrochloride ratio of 6:1, a very slow release of the drug was obtained. Only 66.68% of the drug was released after 6 hours. The percent loading in this case was 14%. Again, when Ludipress was used in high ratio, it was found to retard the release rate more prominently. Key words: Metformin Hydrochloride, Xanthan Gum, Controlled release capsule Dhaka Univ. J. Pharm. Sci. Vol.4(1) 2005 The full text is of this article is available at the Dhaka Univ. J. Pharm. Sci. website

Improvement of effectiveness in maize breeding

2006 ◽  
Vol 54 (3) ◽  
pp. 351-358 ◽  
Author(s):  
P. Pepó
Keyword(s):  
Recurrent Selection ◽  
Genetic Manipulation ◽  
Field Testing ◽  
Tissue Cultures ◽  
Maize Breeding ◽  
Breeding Programmes ◽  
Speed Up ◽  
Selection For

Plant regeneration via tissue culture is becoming increasingly more common in monocots such as maize (Zea mays L.). Pollen (gametophytic) selection for resistance to aflatoxin in maize can greatly facilitate recurrent selection and the screening of germplasm for resistance at much less cost and in a shorter time than field testing. In vivo and in vitro techniques have been integrated in maize breeding programmes to obtain desirable agronomic attributes, enhance the genes responsible for them and speed up the breeding process. The efficiency of anther and tissue cultures in maize and wheat has reached the stage where they can be used in breeding programmes to some extent and many new cultivars produced by genetic manipulation have now reached the market.

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