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Biopharmaceutical potential of submerge cultivated higher basidial mushrooms
New Biotechnology · Volume 25S · September 2009
ABSTRACTS
3.2.10
3.2.12
Bioelicitation of secondary metabolites in in vitro cultures of Gymnema sylvestre R.Br.
Expression and functional analysis of rice-produced human insulin-like growth factor-I (hIGF-I)
C. Subathra Devi ∗ , V. Mohana Srinivasan
S.C.K. Cheung 1,∗ , L. Liu 1 , S.S.M. Sun 1 , Q. Liu 2 , L. Lan 1 , J.C.N. Chan 1 , P.C.Y. Tong 1
VIT University, Vellore, India
1
Aspergillus niger cell extract was used as a elicitor. Growth rate and the gymnemic acid production of the elicited and non-elicited cultures of Gymnema sylvestre were investigated. The growth rate of the elicited and non-elicited cultures was not significantly different, but the gymnemic acid yield was very high in elicited cultures. When compared to non-elicited cultures, 8—9-fold increase of gymnemic acids yield in elicited cultures was noticed. 10 g of cell suspension produced in shake flask culture was transferred to 5 l batch fermentor containing 2-l MS medium. Fresh weight was increased 6.5 times after 20 days of culture and the gymnemic acid production achieved was 900 mg/l. After elicitation, 8 times increase of fresh weight was gained. When compared to shake flask culture, 8—9-fold increase in gymnemic acid production was noticed in fermentors. doi:10.1016/j.nbt.2009.06.643
3.2.11 Spirulina as ‘functional food’ M. Ajeesh ∗ , C.P.N. Bohra, N. Gupta, C. Rajasekaran Plant Biotechnology Division, School of Biotechnology, Chemical and Biomedical Engineering, VIT University, Vellore 632014, India
Functional foods are foods that contain multiple ingredient/s and contribute constructive health benefits over other forms of food. They can be food with or enriched with bioactive substances or traditional food with synthesized food ingredients. Spirulina, a filamentous cyanobacterium, with high nutritive value has been under immense research for its role in medication and as well as in Nutraceuticals. Besides inhibitory effect on viral replication and ageing mechanism, Spirulina has been found to have detoxification of undesirable compounds and help reduce oxidative damage done by free radicals in the body. This poster focuses the use of Spirulina as a functional food in our diet. It also accentuates the role of Spirulina metabolites such as amino acid, pigments, vitamins and minerals in imparting improved metabolism and body functions. doi:10.1016/j.nbt.2009.06.644
2
The Chinese University of Hong Kong, Hong Kong Yangzhou University, Hong Kong
Human insulin-like growth factor-I (hIGF-I) is a growth factor that highly resembles to insulin. It is essential for cell proliferation and can be used to treat various endocrine-associated diseases including growth hormone insensitivity syndrome (GHIS) and diabetes mellitus. To meet the increasing demand of hIGF-I in therapeutic uses, we have developed an efficient plant expression system to produce biologically active recombinant hIGF-I (rhIGF-I) in transgenic rice grains. The plant-codon-optimized hIGF-I, under the control of rice seed-specific Gt1 promoter, was introduced into rice via Agrobacterium-mediated transformation. In order to enhance rhIGF-I stability and yield, two protein-sorting sequences, an endoplasmic reticulum (ER)-retention signal (KDEL) and glutelin signal peptide, were used to deliver the recombinant protein to ER for stable accumulation. In vitro functional analysis showed that the rice-derived rhIGF-I could induce membrane ruffling and glucose uptake on L6myc cells in a dose-dependent manner. It was estimated that 1 mg of transgenic rice grain contained equivalent biological activity of 1.6—3.2 nM of commercial rhIGF-I. In vivo oral meal test with rice-derived rhIGF-I was also found to acutely lower blood glucose level in normal C57/BL6 mice. The blood glucose levels of the mice fed with wild type rice were always higher than those fed with rhIGF-I rice throughout the experiment (p < 0.05). Both of the blood glucose levels reached maximum at 15 minutes, with 3.42 ± 0.76 and 2.49 ± 0.35-fold for wild type and rhIGF-I rice respectively (p < 0.01), indicating that rhIGF-I rice was more effective in lowering blood glucose than wild type rice. To conclude, our findings provided an alternative expression system to produce large quantities of functional rhIGF-I that can be a promising substitution of insulin for diabetic treatment. doi:10.1016/j.nbt.2009.06.645
3.2.13 Biopharmaceutical potential of submerge cultivated higher basidial mushrooms Mark Shamtsyan ∗ , Vera Kolesnikov, Anton Klepikov
Spiridonova, Eugenia
Tozik, Boris
St. Petersburg State Institute of Technology (Technical University), St. Petersburg, Russian Federation
From ancient times humans have used mushrooms for their healing potential. The major part of the traditional knowledge of mushrooms bioactive properties has originated from the Oriental countries. Western countries are just discovering the variability and tremendous potential of medical mushrooms.
www.elsevier.com/locate/nbt S285
New Biotechnology · Volume 25S · September 2009
ABSTRACTS
In our studies mushroom fruit bodies were gathered wiled from various geographical zones, were identified and introduced into culture. Submerge mycelia and native liquids of cultivation of basidiomycetes were studied. The results showed that mycelia aqueous extracts, mainly containing beta-glucans, possess various types of marked immunomodulating activities. Immunomodulating effect was also demonstrated, for oral administration of extracts as well feeding animals with extract fortified yogurts and milk. Addition of this extracts to the daily diet results in anti-tumor effects against melanoma B-16 and Erlich’s ascit carcinoma. In addition extracts of submerge mycelia possessed significant antioxidant and Superoxide dismutase like activity. The addition of submerge mycelia of some mushrooms to a high cholesterol diet effectively reduced cholesterol accumulation in the serum and liver of experimental rats redistributing cholesterol in favor of high-density lipoprotein, reduced production of low-density lipoprotein and very low-density lipoprotein, cholesterol, reduced cholesterol absorption and triglyceride level in blood serum. Uptake of mushroom mycelia was also decreasing glycemic indexes of food in streptozotocine induced diabetic rats, thus possessing antidiabetic effect. From the native liquid of several higher basidiomycetes we isolated enzymes with high fibrinolytic activity can also be produced during submerge cultivation of various mushrooms. Intravenous injections of purified enzyme preparations were resulting in complete lyses of thrombus formed in the venues and arteries of the experimental animals. Obtained results are demonstrating high healing potential of mushrooms. doi:10.1016/j.nbt.2009.06.646
3.2.14 Bio-flocculants, a novel approach to safe water treatment using biologically active proteins prepared from seeds A. Murphy 1,∗ , M. Schneider 2 , I.W. Marison 1 1
School of Biotechnology, Dublin City University, Dublin 9, Ireland Laboratoire de Genie Chimique et Biologique (LGCB), Ecole Polytechnique Federale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland
2
Water is an essential component of life. There is an ever-increasing demand for safe clean drinking water. Aluminium sulphate and ferric sulphate, along with polyacrylamide, are chemicals currently employed in water treatment plants as coagulants and flocculants. Polyacrylamide has been found to be toxic to aquatic organisms and is possibly a carcinogenic aluminium has been linked in recent years to the brain disease Alzheimer’s and iron sulphate has links with liver disease. As a result alternative, safe, sustainable methods are required. The aim is to use proteins derived from oleaginous seeds of subtropical plants as a natural flocculating and coagulating agent and in so doing, eliminate the use of harmful chemicals. In our work we have shown that the active coagulating substances present in Moringa oleifera seed extracts are proteinaceous S286
www.elsevier.com/locate/nbt
components. The nature of the extraction medium appears to have little influence on the composition of the proteins extracted but can have a major effect on the yield. The results have been compared to a standard method of treating drinking water. doi:10.1016/j.nbt.2009.06.647
3.2.15 Separation and purification of crude ellagic acid from white flesh of pomegranate fruits as a potent anticarcinogenic A.W. Hamad ∗ , W. Al-Momene Amman, Jordan
Separation and purification of ellagic acid from Jordanian white flesh pomegranate. Studying the effect of ellagic acid in vitro by using cell lines of pharma-cytoma. It was found that the effect depends on the incubation time and concentration. Studying the effect of ellagic acid in vivo by the treatment of squamous cell carcinoma in mice (balb\c type) after synthesized cancer by chemicals carcinogenic. This study consisted two parts—–the first one is the separation of ellagic acid and second part to use the ellagic acid as antioxidant. Pomegranate fruits are widely consumed fresh and in processed forms as juice, jams and wine. Pomegranate white flesh is a rich source of ellagitannins (ETs). These ETs are extracted from the white flesh in significant quantities into the juice due to their hydrophilic properties. The method is to isolate ellagic acid from Fresh pomegranate. The white flesh was removed, mixed with ethanol and then minced by using homogenizer. The homogenized white flesh solution was washed three times with ethanol. The supernatant was taken and placed in incubator at 37 ◦ C for 48 h until become dry material and then ground to fine powder. The second part—–The anti-neoplastic effects of both compounds on the plasmacytoma cells (in vitro) and squamous cell carcinoma (in vivo) occurs in the absence of DNA synthesis. The effect of ellagic acid and cisplatin compound on the activity of malignant cells was studied by two following ways: It was found that ellagic formula and cisplatin was effective in reducing both size and density of malignant cells. In conclusion, our findings indicate that ellagic acid acts by not only enhancing the host immune function but also through a direct alteration of tumor cell growth and production of cytokines. These findings may offer a mechanism of action that could explain the high clinical success and impressive benefits of ellagic acid treatment. doi:10.1016/j.nbt.2009.06.648
ABSTRACTS
3.2.10
3.2.12
Bioelicitation of secondary metabolites in in vitro cultures of Gymnema sylvestre R.Br.
Expression and functional analysis of rice-produced human insulin-like growth factor-I (hIGF-I)
C. Subathra Devi ∗ , V. Mohana Srinivasan
S.C.K. Cheung 1,∗ , L. Liu 1 , S.S.M. Sun 1 , Q. Liu 2 , L. Lan 1 , J.C.N. Chan 1 , P.C.Y. Tong 1
VIT University, Vellore, India
1
Aspergillus niger cell extract was used as a elicitor. Growth rate and the gymnemic acid production of the elicited and non-elicited cultures of Gymnema sylvestre were investigated. The growth rate of the elicited and non-elicited cultures was not significantly different, but the gymnemic acid yield was very high in elicited cultures. When compared to non-elicited cultures, 8—9-fold increase of gymnemic acids yield in elicited cultures was noticed. 10 g of cell suspension produced in shake flask culture was transferred to 5 l batch fermentor containing 2-l MS medium. Fresh weight was increased 6.5 times after 20 days of culture and the gymnemic acid production achieved was 900 mg/l. After elicitation, 8 times increase of fresh weight was gained. When compared to shake flask culture, 8—9-fold increase in gymnemic acid production was noticed in fermentors. doi:10.1016/j.nbt.2009.06.643
3.2.11 Spirulina as ‘functional food’ M. Ajeesh ∗ , C.P.N. Bohra, N. Gupta, C. Rajasekaran Plant Biotechnology Division, School of Biotechnology, Chemical and Biomedical Engineering, VIT University, Vellore 632014, India
Functional foods are foods that contain multiple ingredient/s and contribute constructive health benefits over other forms of food. They can be food with or enriched with bioactive substances or traditional food with synthesized food ingredients. Spirulina, a filamentous cyanobacterium, with high nutritive value has been under immense research for its role in medication and as well as in Nutraceuticals. Besides inhibitory effect on viral replication and ageing mechanism, Spirulina has been found to have detoxification of undesirable compounds and help reduce oxidative damage done by free radicals in the body. This poster focuses the use of Spirulina as a functional food in our diet. It also accentuates the role of Spirulina metabolites such as amino acid, pigments, vitamins and minerals in imparting improved metabolism and body functions. doi:10.1016/j.nbt.2009.06.644
2
The Chinese University of Hong Kong, Hong Kong Yangzhou University, Hong Kong
Human insulin-like growth factor-I (hIGF-I) is a growth factor that highly resembles to insulin. It is essential for cell proliferation and can be used to treat various endocrine-associated diseases including growth hormone insensitivity syndrome (GHIS) and diabetes mellitus. To meet the increasing demand of hIGF-I in therapeutic uses, we have developed an efficient plant expression system to produce biologically active recombinant hIGF-I (rhIGF-I) in transgenic rice grains. The plant-codon-optimized hIGF-I, under the control of rice seed-specific Gt1 promoter, was introduced into rice via Agrobacterium-mediated transformation. In order to enhance rhIGF-I stability and yield, two protein-sorting sequences, an endoplasmic reticulum (ER)-retention signal (KDEL) and glutelin signal peptide, were used to deliver the recombinant protein to ER for stable accumulation. In vitro functional analysis showed that the rice-derived rhIGF-I could induce membrane ruffling and glucose uptake on L6myc cells in a dose-dependent manner. It was estimated that 1 mg of transgenic rice grain contained equivalent biological activity of 1.6—3.2 nM of commercial rhIGF-I. In vivo oral meal test with rice-derived rhIGF-I was also found to acutely lower blood glucose level in normal C57/BL6 mice. The blood glucose levels of the mice fed with wild type rice were always higher than those fed with rhIGF-I rice throughout the experiment (p < 0.05). Both of the blood glucose levels reached maximum at 15 minutes, with 3.42 ± 0.76 and 2.49 ± 0.35-fold for wild type and rhIGF-I rice respectively (p < 0.01), indicating that rhIGF-I rice was more effective in lowering blood glucose than wild type rice. To conclude, our findings provided an alternative expression system to produce large quantities of functional rhIGF-I that can be a promising substitution of insulin for diabetic treatment. doi:10.1016/j.nbt.2009.06.645
3.2.13 Biopharmaceutical potential of submerge cultivated higher basidial mushrooms Mark Shamtsyan ∗ , Vera Kolesnikov, Anton Klepikov
Spiridonova, Eugenia
Tozik, Boris
St. Petersburg State Institute of Technology (Technical University), St. Petersburg, Russian Federation
From ancient times humans have used mushrooms for their healing potential. The major part of the traditional knowledge of mushrooms bioactive properties has originated from the Oriental countries. Western countries are just discovering the variability and tremendous potential of medical mushrooms.
www.elsevier.com/locate/nbt S285
New Biotechnology · Volume 25S · September 2009
ABSTRACTS
In our studies mushroom fruit bodies were gathered wiled from various geographical zones, were identified and introduced into culture. Submerge mycelia and native liquids of cultivation of basidiomycetes were studied. The results showed that mycelia aqueous extracts, mainly containing beta-glucans, possess various types of marked immunomodulating activities. Immunomodulating effect was also demonstrated, for oral administration of extracts as well feeding animals with extract fortified yogurts and milk. Addition of this extracts to the daily diet results in anti-tumor effects against melanoma B-16 and Erlich’s ascit carcinoma. In addition extracts of submerge mycelia possessed significant antioxidant and Superoxide dismutase like activity. The addition of submerge mycelia of some mushrooms to a high cholesterol diet effectively reduced cholesterol accumulation in the serum and liver of experimental rats redistributing cholesterol in favor of high-density lipoprotein, reduced production of low-density lipoprotein and very low-density lipoprotein, cholesterol, reduced cholesterol absorption and triglyceride level in blood serum. Uptake of mushroom mycelia was also decreasing glycemic indexes of food in streptozotocine induced diabetic rats, thus possessing antidiabetic effect. From the native liquid of several higher basidiomycetes we isolated enzymes with high fibrinolytic activity can also be produced during submerge cultivation of various mushrooms. Intravenous injections of purified enzyme preparations were resulting in complete lyses of thrombus formed in the venues and arteries of the experimental animals. Obtained results are demonstrating high healing potential of mushrooms. doi:10.1016/j.nbt.2009.06.646
3.2.14 Bio-flocculants, a novel approach to safe water treatment using biologically active proteins prepared from seeds A. Murphy 1,∗ , M. Schneider 2 , I.W. Marison 1 1
School of Biotechnology, Dublin City University, Dublin 9, Ireland Laboratoire de Genie Chimique et Biologique (LGCB), Ecole Polytechnique Federale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland
2
Water is an essential component of life. There is an ever-increasing demand for safe clean drinking water. Aluminium sulphate and ferric sulphate, along with polyacrylamide, are chemicals currently employed in water treatment plants as coagulants and flocculants. Polyacrylamide has been found to be toxic to aquatic organisms and is possibly a carcinogenic aluminium has been linked in recent years to the brain disease Alzheimer’s and iron sulphate has links with liver disease. As a result alternative, safe, sustainable methods are required. The aim is to use proteins derived from oleaginous seeds of subtropical plants as a natural flocculating and coagulating agent and in so doing, eliminate the use of harmful chemicals. In our work we have shown that the active coagulating substances present in Moringa oleifera seed extracts are proteinaceous S286
www.elsevier.com/locate/nbt
components. The nature of the extraction medium appears to have little influence on the composition of the proteins extracted but can have a major effect on the yield. The results have been compared to a standard method of treating drinking water. doi:10.1016/j.nbt.2009.06.647
3.2.15 Separation and purification of crude ellagic acid from white flesh of pomegranate fruits as a potent anticarcinogenic A.W. Hamad ∗ , W. Al-Momene Amman, Jordan
Separation and purification of ellagic acid from Jordanian white flesh pomegranate. Studying the effect of ellagic acid in vitro by using cell lines of pharma-cytoma. It was found that the effect depends on the incubation time and concentration. Studying the effect of ellagic acid in vivo by the treatment of squamous cell carcinoma in mice (balb\c type) after synthesized cancer by chemicals carcinogenic. This study consisted two parts—–the first one is the separation of ellagic acid and second part to use the ellagic acid as antioxidant. Pomegranate fruits are widely consumed fresh and in processed forms as juice, jams and wine. Pomegranate white flesh is a rich source of ellagitannins (ETs). These ETs are extracted from the white flesh in significant quantities into the juice due to their hydrophilic properties. The method is to isolate ellagic acid from Fresh pomegranate. The white flesh was removed, mixed with ethanol and then minced by using homogenizer. The homogenized white flesh solution was washed three times with ethanol. The supernatant was taken and placed in incubator at 37 ◦ C for 48 h until become dry material and then ground to fine powder. The second part—–The anti-neoplastic effects of both compounds on the plasmacytoma cells (in vitro) and squamous cell carcinoma (in vivo) occurs in the absence of DNA synthesis. The effect of ellagic acid and cisplatin compound on the activity of malignant cells was studied by two following ways: It was found that ellagic formula and cisplatin was effective in reducing both size and density of malignant cells. In conclusion, our findings indicate that ellagic acid acts by not only enhancing the host immune function but also through a direct alteration of tumor cell growth and production of cytokines. These findings may offer a mechanism of action that could explain the high clinical success and impressive benefits of ellagic acid treatment. doi:10.1016/j.nbt.2009.06.648