New aspects of Saccharomyces cerevisiae as a novel carrier for berberine
© Salari et al.; licensee BioMed Central Ltd. 2013
Received: 22 June 2013
Accepted: 31 August 2013
Published: 20 December 2013
Berberine was encapsulated in yeast cells of Saccharomyces cerevisiae as novel carriers to be used in different food and drug industries. The microcapsules were characterized by differential scanning calorimetry (DSC), fourier transform infra red spectroscopy (FT-IR) and fluorescence microscopy. The encapsulation factors such as plasmolysis of yeast cells which affects the % encapsulation yield were studied.
Fluorescence microscopy showed the yeast cells became fluorescent after encapsulation process. DSC diagram was representing of new peak for microcapsule which was not the same as berberine and the empty yeast cells peaks, separately. FTIR spectrums of microcapsules and yeast cells were almost the same. The plasmolysed and non plasmolysed microcapsules were loaded with berberine up to about 40.2 ± 0.2% w/w.
Analytical methods proved that berberine was encapsulated in the yeast cells. Fluorescence microscopy and FTIR results showed the entrance of berberine inside the yeasts. DSC diagram indicated the appearance of new peak which is due to the synthesis of new product. Although plasmolysis caused changes in yeast cell structure and properties, it did not enhance berberine loading in the cells. The results confirmed that Saccharomyces cerevisiae could be an efficient and safe carrier for active materials.
KeywordsBerberine Encapsulation Saccharomyces cerevisiae yeast cells
Microencapsulation is a process that nowadays developed in many industries. Encapsulation has a lot of advantages. The encapsulated compound can be released in a controlled way in different systems. Besides, it stabilizes the active materials against oxygen or other molecules by wall material as a physical barrier . The stability and release properties of microcapsules are dependent on cell wall properties [2, 3]. However, the new or novel coatings are needed due to the cost and legal limits in the food industries .
Saccharomyces cerevisiae yeast cell can be mentioned as an ideal carrier due to its food-grade and low cost characteristics. Unlike other carriers, it does not depend on active ingredients for its synthesis. We could culture this kind of carriers as much as needed without any excess expenses. Besides, its membrane phospholipids act like liposome structure and have been used for encapsulation of different molecules, hydrophobic and hydrophilic, like resveratrol [5–8].
It possesses the external thick cell wall which composes of a beta glucan network and a small amount of chitin associated with a mannoprotein layer . These cell wall properties make S. cerevisiae such a kind of sustained release drug delivery system. The mechanical characteristics of the yeasts structure allow them to load different molecules.
Barberry (Berberis vulgaris L. family Berberidaceae) grows in Asia and Europe. The plant is famous worldwide for its medicinal properties . Berberine is the most significant alkaloid of this plant which is responsible for its beneficial effects. Berberine is mostly accumulated in the root. Berberine as an isoquinoline alkaloid is a member of protoberberines class . Berberine shows a lot of pharmacological effects .
Berberine shows antiplatelet effects and it is also used in treatment of congestive heart failure (CHF) [13–16]. Extracts are used to cure various inflammatory diseases such as lumbago and rheumatism and to reduce fever . The reports represented immunosuppressive effect of berberine in the tubulointerstitial nephritis model. Peng et al. showed the antianxiety properties of berberine . Berberine can be mentioned as an expectorant due to its ability to increase mucin release .
Berberine has been used to treat infectious diarrhea and gastroenteritis in China in the past . Berberine is defined as an effective drug to cure acute diarrhea which is due to Escherichia coli or Vibrio cholerae[20, 21]. Berberine shows significant antibacterial and antifungal activity against broad spectrum of microorganisms [22–28].
Berberine’s poor water solubility and susceptibility to environmental conditions prevent its application in many industries. The goal of this study was to evaluate the new aspect of yeast cells of S. cerevisiae as an encapsulation carrier for berberine. Moreover, the berberine microcapsules were studied by different analytical methods such as fluorescence microscopy, (DSC) and (FT-IR).
Preparation of plasmolysed yeast cells, non plasmolysed yeast cells and berberine microcapsules
The yeast cells (commercially Bakers S. cerevisiae) were cultured in soybean casein digest broth medium (Himedia, India) for 10 hours  in shaker incubator (20 rpm) (JTSL 20, Iran). The medium was centrifuged and the yeast cells were washed for three times with deionised water. The final cells were classified in two groups. First group as non plasmolysed cells was directly freeze dried. The second one as plasmolysed cells was plasmolysed in different concentration of sodium chloride solutions. The yeast cells suspensions were provided in three different flasks. Each flask contained 10%, 20%, 30% w/w NaCl solutions, respectively. Then the flasks were stirred at 200 rpm for 72 h. The plasmolysed cells were centrifuged and washed three times with deionised water to remove impurities. At last, they have been freeze-dried. The freeze-dried plasmolysed and non plasmolysed yeast cells were studied as carriers .
Two kinds of freeze dried cells (plasmolysed and non plasmolysed) (100 mg) were suspended in two flasks containing 50 ml berberine solutions (500 mM). Berberine hydrochloride was obtained from China (XI AN Rongsheng biotechnology CO., LTD). The flasks were stirred at 200 rpm for 72 h and then centrifuged (6000 rpm, 20 min). The precipitants were washed three times to remove the free berberine. Then the berberine loaded microcapsules were freeze dried.
Analytical methods for confirmation of berberine encapsulation
Three methods were performed for confirmation of encapsulation process including fluorescence microscopy (color CCTV camera, model No.MV-CP470/G), fourier-transform Infrared Spectroscopy (FT-IR) (model. Spectrum two) and differential scanning calorimetry (DSC) (Mettler Toledo).
Fluorescence microscopy images were obtained from empty freeze-dried yeast cells and the yeast cells which encapsulated berberine. Besides Fluorescence microscopy, DSC and FT-IR studies were carried out to confirm the encapsulation of berberine in yeast cells.
In DSC studies, five types of freeze-dried samples were studied: empty yeast cells, berberine powder,physical mixture of berberine and empty cells, cells loaded with berberine (microcapsules) and plasmolysed empty yeast cells. To prepare a DSC sample, a definite amount of each sample was placed in 50 μl closed aluminium pans. The scan rate was 10°C/min between 0 and 400°C. The IR spectra (KBr disc method) of all the above mentioned samples were also obtained using an FT-IR spectrophotometer [8, 10].
Results and discussion
Analytical studies of freeze-dried plasmolysed and non plasmolysed yeast cells
The contributions of hydroxyl vibrations of carbohydrates and NH vibrations of proteins can be observed in a band at 3750–3000 cm-1 in IR spectra .
Region 3050–2700 cm-1 shows us the information about lipid composition of yeast cells. Plasmolysed yeast cells indicated a minor increase of their absorption intensities due to the changes in the length of membrane lipid chains in comparison with non plasmolysed yeast cells absorption peaks in 2959 and 2925 cm-1 (Figure 2) . These two peaks are due to the vibrations of methyl and methylene groups. Plasmolysis effects on cell wall and membrane proteins could be seen in different regions. According to protein degradation by plasmolysis, we faced some changes in peak intensities in the region 1700–1550 cm-1. After plasmolysis, some changes happened in the absorption bands at 1659 and 1553 cm-1, which referred to protein amide I and amide II and carbon-nitrogen vibrations of yeast cells due to the shift of the degradated proteins to an unfolded state. Degradation of one part of yeast proteins in the cell membrane and the cell wall could be detected in the band region from 1530 to 1385 cm-1.
Plasmolysis caused the cell wall polysaccharides degradation which could be detected by changes in the IR region from 1156 to 768 cm-1 (Figure 2).
Hypophosphite vibrations are originated from nucleic acid molecules, so we could find the effect of plasmolysis on nucleic acids degradation as the 1240 cm-1. Nucleic acid molecules are placed inside the cell so the presence of their IR spectra are indicative of cells which are not completely emptied by plasmolysis (Figure 2).
Finally the results show us that plasmolysis causes disorganisation to the cell plasma membrane and thickness of cell wall but higher concentrations of NaCl solutions showed no additional disruptions in the yeast cell.
Plasmolysis enhances the intracellular space so it improves the capacity of carriers for encapsulation process . Nowadays, yeast cells plasmolysis with NaCl  were mainly used for the encapsulation of different active materials.
The results indicated that the %encapsulation yield values were statistically similar (p > 0.05) amongst plasmolysed and non plasmolysed cells which confirms other researches results . The average %encapsulation yield was 40.2±0.2%. No differences were observed in the %encapsulation yield amongst plasmolysed yeast cells when exposed to 10%, 20% or 30% w/w NaCl.
Analytical studies for confirmation of berberine encapsulation
Figure 1 showed the DSC thermograms of non-plasmolysed yeast cells, berberine, physical mixture of berberine and yeast cells as well as berberine microcapsules and plasmolysed yeast cells.
The new peak that appeared at 289°C which differed from the peak of empty yeast cells and physical mixture showed that the new product was produced. This Tm variation was due to decrease of Van der Waals interactions happened to membrane as a result of berberine’s integration. On the other hand, berberine influenced the bilayer organization by interaction of its polar groups with membrane polar groups. So the membrane became more fluid and Tm of microcapsules was lowered in comparison with empty yeast cells.
The characteristic absorption bands of berberine are shown in Figure 5. The IR spectra of empty yeast cells are explained before. The 3298 and 1654 cm-1 bands are due to the phenolic and carbonyl groups, respectively. The absorption peak at 1545 cm-1 shows us the carbonyl and carbon-carbon vibration. The peak at 1451 cm-1 refered to carbon-hydrogen vibration. The absorption peak at 1238 cm-1 is indicative of phenolic vibration. The methoxy groups are responsible for the 1080 cm-1 band. The aromatic carbon-hydrogen group vibrates at 914 cm-1.
The IR spectrum of the physical mixture of berberine and yeast cells was a combination of the IR spectra of empty yeast cells and berberine, separately. The IR spectra of the microcapsules was relatively the same as the spectrum of the empty yeast cells. These spectra could prove the fact that berberine was placed inside the yeast cells. This observation confirmed Shi et al. [5, 7] studies. However we had some peak disappearances at 914 cm-1 and variation in the absorption bands of 1654 and 1545 cm-1 as a result of berberine-protein interactions in microcapsules spectra.
In this study, S. cerevisiae were introduced as a novel carrier for berberine as a model. It has shown that yeast microcapsules were loaded with berberine up to 40.2 ± 0.2% w/w. Three analytic methods indicated that berberine located inside the cells mainly by hydrophobic interactions with the yeast cell membrane and cell wall.
This work was supported by a grant from Mashhad University of Medical Sciences Research Council, Mashhad, Iran. The authors wish to thank the authorities in Mashhad University of Medical Sciences, Biotechnology Research center and School of Pharmacy for their support. This project was part of Ph.D student thesis in School of Pharmacy. The authors have no conflicts of interest that are directly relevant to the content of this manuscript.
- Madene A, Jacquot M, Scher J, Desobry S: Flavour encapsulation and controlled release: a review. Int J Food Sci Tech. 2006, 41: 1-21.View ArticleGoogle Scholar
- Gibbs BF, Kermasha S, Alli I, Mulligan N: Encapsulation in the food industry: a review. Int J Food Sci Nutr. 1999, 50: 213-224. 10.1080/096374899101256.View ArticlePubMedGoogle Scholar
- Khodaverdi E, Khalili N, Zangiabadi F, Homayouni A: Preparation, characterization and stability studies of glassy solid dispersions of indomethacin using PVP and isomalt as carriers. Iran J Basic Med Sci. 2012, 15 (3): 820-32.PubMed CentralPubMedGoogle Scholar
- Gouin S: Microencapsulation: industrial appraisal of existing technologies and trends. Trends Food Sci Tech. 2004, 15: 330-347. 10.1016/j.tifs.2003.10.005.View ArticleGoogle Scholar
- Shi G, Rao L, Yu H, Xiang H, Yang H, Ji R: Stabilization of photosensitive resveratrol within yeast cell. Int J Pharmaceut. 2008, 349: 83-93. 10.1016/j.ijpharm.2007.07.044.View ArticleGoogle Scholar
- Bishop JRP, Nelson G, Lamb J: Microencapsulation in yeast cells. J Microencapsul. 1998, 15 (6): 761-773. 10.3109/02652049809008259.View ArticlePubMedGoogle Scholar
- Shi G, Rao L, Yu H, Xiang H, Pen G, Long S: Yeast-cell based microencapsulation of chlorogenic acid as a water-soluble antioxidant. J Food Eng. 2007, 80: 1060-1067. 10.1016/j.jfoodeng.2006.06.038.View ArticleGoogle Scholar
- Chow C, Palecet P: Enzyme encapsulation in permeabilized Saccharomyces cerevisiae cells. Biotechnol Progr. 2004, 20: 449-456.View ArticleGoogle Scholar
- De Nobel JG, Klis FM, Munnik T, Priem J, Van Den Ende H: An assay of the relative cell wall porosity of Saccharomyces cerevisiae, Kluveromyces lactis and Schizosaccharomyces pombe. Yeast. 1990, 6: 483-490. 10.1002/yea.320060605.View ArticlePubMedGoogle Scholar
- Shamsa F, Ahmadiani A, Khosrokhavar R: Antihistaminic and anticholinergic activity of barberry fruit (Berberis vulgaris) in the guinea-pig ileum. J Ethnopharmacol. 1999, 64: 161-166. 10.1016/S0378-8741(98)00122-6.View ArticlePubMedGoogle Scholar
- Mazzini S, Bellucci MC, Mondelli R: Mode of binding of the cytotoxic alkaloid berberine with the double helix oligonucleotide D(AAGAATTCTT). Bioorg Med Chem. 2003, 11: 505-514. 10.1016/S0968-0896(02)00466-2.View ArticlePubMedGoogle Scholar
- Imanshahid M, Hosseinzadeh H: Pharmacological and therapeutic effects of berberis vulgaris and its active constituent, berberine. Phytother Res. 2008, 22: 999-1012. 10.1002/ptr.2399.View ArticleGoogle Scholar
- Fatehi-Hassanabad Z, Jafarzadeh M, Tarhini A, Fatehi M: The antihypertensive and vasodilator effects of aqueous extract from Berberis vulgaris fruit on hypertensive rats. Phytother Res. 2005, 19: 222-225. 10.1002/ptr.1661.View ArticlePubMedGoogle Scholar
- DerMaderosian A: The Review of Natural Products. 2001, St. Louis, Missouri: Facts and ComparisonsGoogle Scholar
- Chiou WF, Chen J, Chen CF: Relaxation of corpus cavernosum and raised intracavernous pressure by berberine in rabbit. Br J Pharmacol. 1998, 125: 1677-1684. 10.1038/sj.bjp.0702249.PubMed CentralView ArticlePubMedGoogle Scholar
- Zeng XH, Zeng XJ, Li YY: Efficacy and safety of berberine for congestive heart failure secondary to ischemic or idiopathic dilated cardiomyopathy. Am J Cardiol. 2003, 92: 173-176. 10.1016/S0002-9149(03)00533-2.View ArticlePubMedGoogle Scholar
- Yesilada E, Küpeli E, Berberis crataegina DC: Root exhibits potent anti-inflammatory, analgesic and febri-fuge effects in mice and rats. J Ethnopharmacol. 2002, 79: 237-248. 10.1016/S0378-8741(01)00387-7.View ArticlePubMedGoogle Scholar
- Lee CJ, Lee JH, Seok JH: Effects of baicalein, berberine, curcumin and hesperidin on mucin release from airway goblet cells. Planta Med. 2003, 69: 523-526.View ArticlePubMedGoogle Scholar
- Lin SS, Chung JG, Lin JP: Berberine inhibits arylamine N-acetyltransferase activity and gene expression in mouse leukemia L 1210 cells. Phytomedicine. 2005, 12: 351-358. 10.1016/j.phymed.2003.11.008.View ArticlePubMedGoogle Scholar
- Rabbani GH, Butler T, Knight J, Sanyal SC, Alam K: Randomized controlled trial of berberine sulfate therapy for diarrhea due to enterotoxigenic Escherichia coli and Vibrio cholerae. J Infect Dis. 1987, 155: 979-984. 10.1093/infdis/155.5.979.View ArticlePubMedGoogle Scholar
- Rabbani GH: Mechanism and treatment of diarrhoea due to Vibrio cholerae and Escherichia coli: roles of drugs and prostaglandins. Dan Med Bull. 1996, 43: 173-185.PubMedGoogle Scholar
- Birdsall TC, Kelly GS: Berberine: therapeutic potential of an alkaloid found in several medicinal plants. Altern Med Rev. 1997, 2: 94-103.Google Scholar
- Khosla PK, Neeraj VI, Gupta SK, Satpathy G: Berberine, a potential drug for trachoma. Rev Int Trach Pathol Ocul Trop. 1992, 69: 147-165.Google Scholar
- Freile ML, Giannini F, Pucci G: Antimicrobial activity of aqueous extracts and of berberine isolated from Berberis heterophylla. Fitoterapia. 2003, 74: 702-705. 10.1016/S0367-326X(03)00156-4.View ArticlePubMedGoogle Scholar
- Kaneda Y, Torii M, Tanaka T, Aikawa M: In vitro effects of berberine sulphate on the growth and structure of Entamoeba histolytica, Giardia lamblia and Trichomonas vaginalis. Ann Trop Med Parasitol. 1991, 85: 417-425.PubMedGoogle Scholar
- Mahady GB, Pendland SL, Stoia A, Chadwick LR: In vitro susceptibility of Helicobacter pylori to isoquinoline alkaloids from sanguinaria canadensis and Hydrastis Canadensis. Phytother Res. 2003, 17: 217-221. 10.1002/ptr.1108.View ArticlePubMedGoogle Scholar
- Ghosh AK, Bhattacharya FK, Ghosh DK: Leishmania donovani: Amastigote inhibition and mode of action of berberine. Exp Parasitol. 1985, 60: 404-413. 10.1016/0014-4894(85)90047-5.View ArticlePubMedGoogle Scholar
- Seki T, Morohashi M: Effect of some alkaloids, flavonoids and triterpenoids, contents of Japanese-Chinese traditional herbal medicines, on the lipogenesis of sebaceous glands. Skin Pharmacol. 1993, 6: 56-60. 10.1159/000211087.View ArticlePubMedGoogle Scholar
- Lord PG, Wheals AE: Variability in individual cell cycles of Saccharomyces cerevisiae. J. Cell Sci. 1981, 50: 361-376.PubMedGoogle Scholar
- Paramera EI, Konteles SJ, Karathanos VT: Microencapsulation of curcumin in cells of Saccharomyces cerevisiae. Food Chem. 2010, 125: 892-902.View ArticleGoogle Scholar
- Laroche C, Gervais P: Achievement of rapid dehydration at specific temperatures could maintain high Sacharomyces cerevisiae viability. Appl Microbiol Biot. 2003, 60: 743-747.View ArticleGoogle Scholar
- Burattini E, Cavanga M, Dell Anna R, Malvezzi Campeggi F, Monti F, Rossi F: An FT-IR microspectroscopy study of autolysis in cells of the wine yeast Saccharomyces cerevisiae. Vib Spectrosc. 2008, 47: 139-147. 10.1016/j.vibspec.2008.04.007.View ArticleGoogle Scholar
- Korber DR, Choi A, Wolfaardt GM, Caldwell DE: Bacterial plasmolysis as a physical indicator of viability. Appl Environ Microb. 1996, 62 (11): 3939-3947.Google Scholar
- Barik A, Mishra B, Shen L, Mohan H, Kadam RM, Dutta S: Evaluation of a new copper (II)–curcumin complex as superoxide dismutase mimic and its free radical reactions. Free Radical Bio Med. 2005, 39: 811-822. 10.1016/j.freeradbiomed.2005.05.005.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.