Microbe supported enhanced production of Rosmarinic acid of medicinal plants in vitro


Scientific Study, 2017
12 Pages

Excerpt

Contents

Introduction

Material and Methods
Plant material
Suspension media

Preparation of beads of E coli K12

Down stream process for Rosmarinic acid extraction

High Performance Thin Layer Chromatography (HPTLC) Analysis

Infra Red Spectral Studies

Statistical analysis

Results and Discussion

Conclusion

Acknowledgement

References

Abstract

In the described study , the bio-enhancement of Rosmarinic acid( RA) was done successfully in suspension culture of Salvia officinalis and Ocimum sanctum suspended with E coli K12 entrapped alginate beads.It is reported that Tyrosin Amino transferase has a key role in the production of Rosmarinic acid , which is present in E coli K 12. Quantification by HPTLC indicated that the maximum RA content was noted in 4 weeks old (2050 mg/l) in Salvia officinalis suspension culture fed with Tyrosin 10 mg/100ml followed by 4 weeks old Ocimum sanctum suspension culture fed with Tyrosin 10 mg/100ml(715 mg/l), that are about 18 fold and 5 folds enhancement as compared to control. The method for significantly enhancement of RA was reported for the first time.

Key words: Rosmarinic acid ; Salvia officinalis; Ocimum sanctum suspension culture ; E coli K 12 , Tyrosin Amino Transferase, HPTLC

Introduction

Rosmarinic acid (RA) is a water-soluble phenolic acid , which is mainly found in the plant species of Lamiaceae and Boraginaceae (1). RA derived from caffeic acid and (R)-(+)-3-(3, 4-dihydroxyphenyl) lactic acid represents one of the most common caffeic esters in plant material, was originally identified in Rosmarinus officinalis L. and accumulated constitutively (2). .In addition to many essential nutritional components, plants contain phenolic substances, agroup of biologically active non-nutrients RA is one of them .RA possesses various biological activities such as antimicrobial, anti-inflammatory, antimutagenic, improvement of cognitive performance, prevention of the development of Alzheimer’s disease, cardioprotective effects, reduction of the severity of kidney diseases, antioxidant and cancer chemoprevention (3, 4). Salvia officinalis and Ocimum sanctum ( Lamiaceae) have been used extensively in various fields, such as medicine, food industries, as well asperfume and cosmetics productions (Adinee, et al., 2008). Salvia officinalis and Ocimum sanctum are reported to contain Rosmarinic acid (5, 12).It is known that most of the medicinal effects of these plants are related to its active ingredient, (Park et al., 2008). The production of secondary metabolites through a cell culture technology of renowned medicinal plants has been a challenging subject for many researchers. It has been established that cell cultures, obtained from plants of Boraginaceae family such as Lithospermum erythrorhizon and Anchusa officinalis, produce considerable amounts of rosmarinic acid (5, 6). Nasiri et al., 2014 have reported the role of Tyrosine amino transferase(TAT) in the production of Rosmarinic acid in Melissa officinalis.. TAT catalyses the reversible transfer of an amino group between L-tyrosine and L-glutamate using a-ketoglutarate and 4-hydroxyphenylpyruvate as co-substrates. Aminotransferases in general play an important role in nitrogen metabolism and are said to be responsible for the synthesis, in fungi, of the so-called secondary amino acids from the primary amino acids (Echetebu 1982). Tyrosine aminotransferase (TyrB) known as aromatic-amino acid aminotransferase, is a broad-specificity enzyme that catalyzes the final step in tyrosine, leucine, and phenylalanine biosynthesis. Since this important enzyme is present in Escherichia.coli K 12 strain (Keseler et al.,2011). Hence in the present study, Escherichia coli K 12 was used in immobilized form to enhance the Rosmarinic acid production in Salvia officinalis and Ocimum sanctum suspension culture as TAT is known to has extracellular nature.. The used technology is not well documented.

Material and Methods

Plant material

The leaves of Salvia officinalis and Ocimum sanctum collected from green house of Loyola Center for Research and Development, St. Xavier’s college campus. The leaves were washed with teepol , rinsed and subjected for Bovistein ( 1%) treatment for 15 minutes. After washing in running water, these were used for further experiment in aseptic condition.

Suspension media

Zenk production media( Zenk 1975) with 5% sucrose incorporated with Tyrosin (7.5 and 10 mg/100 ml), Jasmonic acid (20, 40 mg/ 100 ml), Caffiec acid (20 and 40 mg/ 100 ml) separately were prepared and autoclaved at 121 o c temperature . Leaves were surface sterilized with 0.1% of mercuric chloride for 3 min. followed by sterilized distilled water separately in aseptic condition. Sterilized leaves( 1 g ) of both of plant spp were macerated in laminar flow and inoculated in to sterilized Zenk media ( 100 ml) supplemented with Tyrosin ( 7.5 and 10 mg/100 ml), Jasmonic acid (20, 40 mg/ 100 ml), Caffiec acid (20 and 40 mg/ 100 ml) separately. pH was 5.6-5.8 before autoclaving.

Preparation of beads of E coli K12

E coli K12 strain was bought from Microbial Type Culture Collection and Gene Bank Institute of Microbial Technology (MTCC), Chandigarh. The lyophilized culture were revived in L B liquid media. 24 hrss old culture of E coli K 12 was taken (20 µl /20 ml) and centrifuged at 8000rpm. Palate was suspended in 1 ml of N broth in aseptic condition and was added to 3.6% Sodium alginate.

Beads were prepared by adding this in 4% cold CaCl2.These prepared beads were washed with sterile distilled water and inoculated in suspension production media of Salvia officinalis and Ocimum sanctum in aseptic condition. These inoculated media were incubated on rotary shaker at 110 rpm at 25 oc and 70 % humidity with 16 hrs of photoperiod and 8 hrs of dark period. Samples were harvested at the time interval of 2, 3 and 4 weeks and extraction for Rosmarinic acid were carried our separately.

Down stream process for Rosmarinic acid extraction

The harvested liquid media for both of the plant spp were filtered by using what man paper no 1 and filtrate were dried on hot plate at 40 [0]c temperature till thick slurry. All the resultant samples were kept in desiccator for further dryness for 24 hrs and put them in -20 deep fridge with 5 ml of 70% ethanol for 36 hrs in each sample. The supernatant of all the samples were pipette out and subjected for dryness at room temperature separately. The dried , weighed various samples of 2, 3 and 4 weeks age of both the plant spp (S. officinalis and O. sanctum)were then subjected for High Performance Thin Layer Chromatographic ( HPTLC) analysis and FTIR spectral studies with standard compound of Rosmarinic acid ( purchased from Sigma Alderich)

High Performance Thin Layer Chromatography (HPTLC) Analysis

HPTLC analysis (Camag software controlled HPTLC System) of various samples were done with standard compound of Rosmarinic acid. Toluene: Ethyl acetate (9:1) was used as mobile phase. The spot corresponding with standard compound of Rosmarinic acid was noted and scanning was done at 285 nm. Rf value was 0.1 was observed. Detection was also done by spraying developed plates with 5%ethnolic ferric chloride. Dark blue color was observed corresponding with standard Rosmarinic acid. The quantitative estimation of Rosmarinic acid in all samples were calculated by using a calibration curve.

Infra Red Spectral Studies

Infra Red Spectral studies of all used samples were carried out with standard compound of Rosmarinic acid . (By using equipment Buck scientific 500).

Statistical analysis

Statistical analysis of obtained Data was done by using t test of Graph ped prism software .

Results and Discussion

Plant Tissue culture has been served as a mean for secondary metabolites production. The strong and growing demand in today's marketplace has refocused attention on in vitro plant materials as potential factories for secondary phytochemical products. Chaturvedi et al., 2009, 2010, 2011, 2013, 2014, 2015 , 2016 have extensively studied the enhancement of secondary metabolites by using different compound in tissue culture of medicinal plants. The treatment of plant cells with biotic and/or abiotic elicitors has been a useful strategy to enhance secondary metabolite production in cell cultures( Karuppusamy et al., 2009). Tyr B gene of E. coli strain K-12 govern the production of Tyrosin Amino Transferase (TAT) . TAT, a broad-specificity enzyme, which catalyzes the transamination of 2-ketoisocaproate, p-hydroxy phenyl pyruvate, and phenyl pyruvate to yield leucine, tyrosine and phenylalanine, respectively . (Huang, 2009).

Hence keeping all the facts in view,in the present study various elicitors were used to bio-enhance the Rosmarinic acid(RA)by using tissue culture technique. The production of RA, a caffiec acid ester compound of medicinal value has been successfully induced by using alginate beads of E coli K12 bacterium in suspension culture of Salvia officinalis and Ocimum sanctum separately supplemented with various elicitors. Enhancement as observed in all the samples but the maximum content was noted in 4 weeks old (2050 mg/l) in Salvia officinalis suspension culture fed with Tyrosin 10 mg/100ml followed by 4 weeks old Ocimum sanctum suspension culture fed with Tyrosin 10 mg/100ml(715 mg/l), that are about 18 fold and 5 folds enhancement as compared to control (Table 1,2)..It is already reported. That this extra cellular enzyme catalyzes the first step of the tyrosine pathway leading to the formation of rosmarinic acid (alpha-O-caffeoyl-3,4-dihydroxyphenyllactic acid) in suspension cultures of Anchusa officinalis L(De-Eknamkul et al., 1887) .This study supports the enhanced production of Rosmarinic acid in our present findings. The another reason for induction of RA content in 4 weeks old culture of both of the plant spp is may be due to given stress to E coli K 12 bacterium by immobilization, that lead to enhance the production of Tyrosin . It is well reviewed that Tyrosin has a key role in production of Rosmarinic acid as precursor compound in Biosynthetic pathway.( Fig 3.).The main advantage of the developed technology is the use of natural resource for the overproduction of RA. E coli (k-12) can be multiplied into number of copies in short time period in laboratory condition, which made the developed technology as economically feasible and affordable source for RA production.

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Details

Title
Microbe supported enhanced production of Rosmarinic acid of medicinal plants in vitro
Author
Year
2017
Pages
12
Catalog Number
V355224
ISBN (eBook)
9783668426948
ISBN (Book)
9783668426955
File size
727 KB
Language
English
Notes
Dr Pratibha chaturvedi is Women scientist GOI and National Professor, visiting scientist of Haffkine Institute, Mumbai. She won several awards such as INSA, IDMA and Bharat Gaurav award. She has published 33 research papers and 4 books from National and International repute publication. Prof Dr Pushpa Khana and Prof Dr Abhay Chowdhary are the names of her mentors. She is involved in the research activities of secondary metabolits, tissue culture, proteomics, plant physiology, Medicinal plants, Plant molecular biology, Plant Biochemistry, pharmacognosy for the last 13 years with many aspects.
Tags
enhanced, rosmarinic, salvia, ocimum, using, hptlc, rosmarinic acid, medical plants, in vitro, salvia officinalis, ocimum sanctum, e coli K 12, alginate beads
Quote paper
Dr. Pratibha Chaturvedi (Author), 2017, Microbe supported enhanced production of Rosmarinic acid of medicinal plants in vitro, Munich, GRIN Verlag, https://www.grin.com/document/355224

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