Cytotoxicity of Bulgarian spring water probiotics (probiotic Laktera Nature®) on colon carcinoma cell line

Abstract

Colorectal carcinoma is a malignant disease, from which more than 1 million people worldwide suffer. Over the past two decades, its social significance attracts more attention. Colorectal cancer is the second leading cause of death from malignancy and the third most frequently diagnosed malignancy. It has been estimated that nutrition is the main reason for the high incidence of cancer. Probiotics are live microorganismswith proven health benefits in a variety of gastrointestinal diseases, and disorders outside the gastrointestinal tract. In this study, we wanted to explore cytotoxicity and anti-proliferative activity of the new probiotic Laktera Nature® on non-cancerous cell lines – 3T3 and BJ and colon adenocarcinoma cell line – HT-29. Probiotic Laktera Nature® has weak direct cytotoxicity against 3T3 and BJ cells, but inhibits their proliferation in a dose-dependent manner. On carcinoma cell line – HT-29, in low concentration range (2-125 µg/ml) probiotic Laktera Nature® stimulates proliferation; at high concentrations (250-1000 µg/ml) we detected concentration-dependent inhibition of proliferation. Probiotic Laktera Nature® has shown promising primary screening results, which should be clarified as mechanisms in further works.

Kaloyan Georgiev – Department of Preclinical and Clinical Sciences, Sector Pharmacology and Toxicology, Faculty of Pharmacy, Medical University Varna, Marin Drinov str. 55, 9002 Varna, Bulgaria. Correspondence to Dr. KaloyanGeorgiev, Department of Preclinical and Clinical Sciences, Sector Pharmacology and Toxicology, Medical University, Marin Drinov 55, 9002 Varna, Bulgaria. Phone: +359898343274, E-mail: kalgeorgiev@hotmail.com

Marieta Georgieva – Institute of Experimental Morphology, Pathology and Anthropology with Museum, Bulgarian Academy of Sciences, Sofia, Bulgaria

Maria Peneva – Medical Centre “Evrozdrave” Sofia, Sofia, Bulgaria

Georgi Alexandrov – Military Medical Institute Academy – Sofia, Sofia, Bulgaria

Application of Lactobacillus Bulgaricus in Clinical Practice

Georgi Alexandrov¹, Maria Peneva²

Abstract

Introduction: Lactic acid fermentation has been known to mankind for thousands of years. Ceramic pots dating back to 6500 years BC, containing residues of fermented dairy products, were found during archeological excavations in the territory around the Black sea. The first written evidence of lactic acid fermentation is by Hrodotus (V Century BC). He wrote that the Thracians (the population inhabited the present Bulgarian land) prepared special fermented dairy foods which were „a gift from Gods”. In 1905 Lactobacillus Bulgaricus was isolated from Bulgarian yoghurt. In 2010 N. Alexandrov and D. Petrova isolated Lactobacillus Bulgaricus from pure spring water from the mountain Stara planina in Bulgaria for the first time in the world. They think that there is a relation between probiotics, the human individual bacterial system, diseases and longevity of life. Beneficial effects of different strains of Lactobacillus Bulgaricus are scientifically proven on over 2500 patients. Probiotics with Lactobacillus Bulgaricus were used in the Space program Intercosmos.

Materials and Methods: During our clinical practice in 625 patients with different diseases we have applied probiotic formulas, containing different strains of Lactobacillus Bulgaricus. All strains of Lactobacillus Bulgaricus were repeatedly multiplied on the territory of Bulgaria.

Results and Discussion: In cases of alcohol intoxication, liver damage is leading, so it is used as a model of chronic liver injury. A double blind randomized study was carried out in 32 patients with alcoholic disease. 17 of them have taken the probiotic formula, containing strains L. Bulgaricus DMR1 and DMR2 at a daily dose of 30 g (over 1 billion CFU/g) and also a combination of that probiotic formula and an extract of silymarin (600 mg/24h) for a period of 6 months as a part of a complex treatment regimen. Adding that nutritional therapy to the main treatment leads to a significantly more efficient normalization of the values of ASAT, ALAT, GGTP and of the ultrasound image of liver and to a restoration of the functions of the liver cell in patients with alcoholic disease. Similar results were obtained when the same daily dose of probiotic foods, containing L. Bulgaricus DWT1, was given to other 78 patients with alcoholic disease and L. Bulgaricus Lb4 – given to 152 patients with heroin addiction and 208 patients with viral hepatitis.

A 28 year old patient with decompensated liver cirrhosis (diagnosed in Germany) and referred for liver transplantation was administered probiotic formula, containing strains L. Bulgaricus DMR1 and DMR2 at a daily dose of 60 g (over 1 billion CFU/g) for a period of 6 months, as a nutritional therapy added to the basic treatment.

We applied a probiotic formula, containing strains L. Bulgaricus DMR1 and DMR2 at a daily dose of 60 g (over 1 billion CFU/g) for a period of 6 months, as a nutritional therapy added to the basic treatment of a patient of 28 years with decompensated liver cirrhosis (diagnosed in Germany) and referred for liver transplantation. The patient avoided the liver transplantation and has recovered almost completely both clinically and in regard of her blood and instrumental tests and restored her working ability.

We carried out a comparative observation of the effects of 3 strains of Lactobacillus Bulgaricus DWT1, DMR1 and DMR2 all in combination with Bulgarian rose oil in 77 patients with proven by ELISA method food allergies. 23 of them have taken a daily dose of a probiotic formula, containing L. Bulgaricus DMR1 and DMR2 (over 1 billion CFU/g) and 40 mg Bulgarian rose oil. The other 54 patients have taken a daily dose of a probiotic formula, containing L. Bulgaricus DWT1 (over 1 billion CFU/g) and 40 mg Bulgarian rose oil. The duration of probiotic intake was 2 months. All patients have stopped the consumption of the proven food allergens and have not taken any antihistamine medications. For a period of 2 to 6 weeks depending on the severity of the manifestations the classic symptoms of allergy and the newly established by us symptoms of food allergy such as hair loss, dandruff, periodontal disease and changes in the prolactin levels in women, were put under control. We have not observed a statistically significant difference regarding the anti-allergic effects of the probiotic formulas, described above.

Local vaginal application of a probiotic formula, containing L. Bulgaricus DMR1 and DMR2 at a daily dose of 500 mg (over 1 billion CFU/g) in 32 women with vaginal discharge as a part of the anti-infective treatment shows a significant increase in the normal lactobacillaceae flora when the probiotic is applied during and 5 days after the anti-infective treatment of vaginal discharge. Similar results were obtained in case of application of the same daily dose of probiotic formulas, containing L. Bulgaricus DWT1 in 12 women and L. Bulgaricus Lb4 in 33 women with vaginal discharge.

Conclusion: Therapy with probiotic formulas, containing strains Lactobacillus Bulgaricus DMR1, DMR2 and DWT1 alone or in combination with plant extracts and oils, as a part of a complex treatment regimen leads to significantly more effective recovery of the liver functions, vaginal flora and putting the food allergy symptoms under control.

The beneficial effects of the probiotic microorganisms are mainly determined by their subspecies as the various strains show certain prominent features.

A proportional relationship is observed between the beneficial effect of the probiotic and the dose and period of its administration.

Keywords: probiotic, Lactobacillus Bulgaricus, allergy, liver, vaginal application

¹ – Military Medical Academy – Sofia, Bulgaria

² – Medical Centre “Evrozdrave” Sofia, Bulgaria

Gabriela Kehayova¹, Marieta Georgieva¹, Kaloyan Georgiev², G. Aleksandrov³, M. Peneva⁴

¹ Department of Pharmacology, Toxicology and Pharmacotherapy, Faculty of Pharmacy, Medical University of Varna

² Department of Pharmaceutical Technologies, Faculty of Pharmacy, Medical University of Varna

³ Department of Abdominal Surgery, Military Medical Academy, Sofia, Bulgaria

⁴ MC Evrozdrave, Sofia, Bulgaria

Probiotics are live microorganisms, which have a beneficial effect on human health and contribute to the intestinal balance. In 1905 the Bulgarian Dr. Stamen Grigorov discovered the microorganism Lactobacillus bulgaricus in yogurt. Later Russian physiologist Ilya Mechnikov concluded that the secret of longevity of Bulgarians lies in eating yogurt. Lactobacillus bulgaricus is the only microorganism, named after a particular geographic territory. It reproduces only in the territory of Bulgaria. Nowadays probiotics are subject of increasing interest because of their proven hepatoprotective, immunostimulating, antioxidant and anti-tumor effect. For the first time in the world, Professor Nikola Aleksandrov managed to isolate natural probiotic bacteria from water source in Stara Planina. The probiotic association contains wild strains of lactic bacteria.

In the present experiment, we first investigated a hepatoprotective effect of a new strain of Lactobacillus bulgaricus DWT1. We used 30 male Wistar albino rats. The rats were treated within 14 days with Laktera Nature probiotic, and on the 12th day an acute liver damage was induced with a high dose of ethanol. After laboratory and histological analysis of rat blood and liver, we found that animals receiving only ethanol had extensive necrosis zones and elevated liver enzymes. In the Laktera Nature pretreated and then subjected to ethanol groups, ALT and alkaline phosphatase levels were significantly lower than those in the ethanol group. Histologically, a preserved liver structure was established. The present experiment demonstrates the hepatoprotective effect of Laktera Nature. It can improve significantly the treatment of the patients suffering from liver disease.

Keywords: probiotics, ethanol, liver damage, hepatoprotective effect

Medical University Prof. Dr. Paraskev Stoyanov, Varna, Bulgaria, Scripta Scientifica Pharmaceutica, ISSN 2367-6000, volume 4, 2017, supplement 2, page 51