Immunostimulant activity of Bergenia Extracts
Lenka Tumova1, Helena endrychová1, Doris Vokurková2
1 Department of Pharmacognosy, Faculty of Pharmacy Hradec Králové, Charles University, Sokolská, Hradec Králové, Czech Republic
2 Departmen of Clinical Immunology and Allergology, Faculty of Medicine and University Hospital, Charles University, Sokolská, Hradec Králové, Czech Republic
|Date of Submission||13-Sep-2017|
|Date of Acceptance||20-Oct-2017|
|Date of Web Publication||14-Aug-2018|
Department of Pharmacognosy, Faculty of Pharmacy Hradec Králové, Charles University, Heyrovského 500 05, Hradec Králové
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Bergenia species contain various therapeutically important compounds such as phenolic compounds-arbutin, bergenin, tannins, gallic acid, flavonoids, minerals, and many others. Bergenia plants show antibacterial, antiviral, anticancer, antidiabetic, diuretic and immunostimulant activities. Materials and Methods: Bergenia leaf extracts from Bergenia crassifolia, Bergenia ciliata, and Bergenia x ornata on lymphocyte activation using flow cytometry method were investigated. Their activation was then monitored with the help of the increasing fluorescence intensity. Results: By activating of cells, there has been an increase in the amount of the established antibodies. Bergenia extracts significantly stimulated the expression of CD69 on lymphocytes in the final concentration of 3.13 and 6.25 mg/ml. The values of stimulation indices of B. crassifolia and B. x ornata extract significantly statistically did not differ. Conclusion: The values of stimulation indices of B. crassifolia and B. x ornata extract significantly statistically did not differ.
Keywords: Bergenia, immunostimulant activity, lymphocytes
|How to cite this article:|
Tumova L, endrychová H, Vokurková D. Immunostimulant activity of Bergenia Extracts. Phcog Mag 2018;14:328-32
- The ex-vivo activation of human immune cells, as determined by cell surface CD69, was reported for the first time in Bergenia extracts.
| Introduction|| |
Bergenia genus belongs to Saxifragaceae family is one of the very important medicinal plants. Over 30 species of this genus is known at present in the world. Therapeutically, interesting compounds are present not only in roots, stems but also in leaves of this plant. Due to the content of various primary and secondary metabolites, many biological activities were found there. Bergenia contains polyphenols such as arbutin, bergenin, catechin, gallic acid, acylated gallic compounds, (+)-afzelechin, and leukocyanidin. Other bioactive ingredients are also flavonoids (kaempferol and quercetin), quinones (aloe-emodin, physcion, and hydroquinone) and others (volatile oils, polysaccharides, carotenoids, amino acids, sterols, and mineral elements).,, Bergenia plants show antibacterial, antiviral, anticancer, antidiabetic, diuretic and immunostimulant activities.,,,,, Leaf extracts of Bergenia plants on the specific immune response was tested in mice in vivo(with a minimum amount of arbutin around 18%). Extracts have led to the normalization of the content of immunocompetent cells in the spleen of the test animals. The extracts decreased the manifestation of the inflammatory process by preventing the accumulation of lymphocytes and a reduction in the ability of cells to produce cytokinins. Polysaccharide bergenan was isolated from the freshly collected green leaves of Bergenia crassifolia. Its administration in the form as the solution to mice (2 mg/ml) for 3 weeks showed an increase in the immune response.,
Transmembrane glycoprotein CD69 is the current activating antigen arranging timely transmembrane signals. Maximal expression achieves already after 18—24 h after stimulation. CD69 has a great importance in the immune response.
In this study, the effect of lyophilized leaf ethanol Bergenia extracts (BCR 2, BCL 2, and BOR 1) on lymphocyte activation by the flow cytometry method was investigated. By activating of cells, there has been an increase in the amount of the established antibodies. Their activation was then monitored with the help of the increasing fluorescence intensity.
| Materials and Methods|| |
Leaves of B. crassifolia L. (Fritsch.), Bergenia ciliata Sternb., and Bergenia x ornata were obtained from the Botanical Garden of Faculty of Horticulture, Mendel University, Brno.
This method modified according to the work of the Cheel monitored the impact of leaf lyophilized ethanol extracts of B. crassifolia (BCR 2), B. ciliata ( BCL 2), and B. x ornata (BOR 1) on the activation of the CD69 cells using flow cytometry. Testing was conducted on a 96-hole microtiter plate. For testing, the peripheral blood with heparin from three healthy donors was used. A volume of 100 μl of this blood was incubated with 100 μl of X-VIVO™ media and with the tested extracts diluted by the growing concentration and control. Extracts were prepared from 0.8 mg lyophilizate with 10 μl of DMSO (concentration in the solution to 0.5%) and 1 ml X-VIVO™ medium. As blank a solution of 100 μl X-VIVO™ medium with 100 μl of blood was used. The positive control was prepared from 100 μl of blood and 100 μl of PHA (phytohemaglutinin in the concentration of 10 μg/ml X-VIVO™ media). As a control, 0.5% solution of DMSO was also prepared. The samples before the actual analysis were filtered through a microfilter (0.2 μm). The final concentration of the tested substances was in the range of 0.2—0.8 mg/ml and 2—50 mg/ml. The microtiter plates were incubated at 37°C for 24 h in the presence of 5% CO2. After the incubation, 40 μl of the suspension was stirred with fluorescent-unstable antibodies (CD69 PE). The analysis was used by flow cytometer Cytomics FC500 (Beckman Coulter), data were analyzed using the CXP analytical software (Coulter Electronic). Activation of lymphocytes (90% T-cells) was tracked with the help of the increasing mean fluorescence intensity (MFI), which was caused by the expression of CD69 on the cell surface. Fluorescent signals were recorded logarithmically as the amplified signals. The values of activation were expressed as activation indexes (AI), which were obtained by comparing the fluorescence intensities of the test samples and the control and stimulant indexes (SI) corresponding to the ratio of the values of the activated sample to the negative control (eliminating thus the variability of the donors). A positive immune cellular response was defined as AI ≥2. The measurement of the immunostimulant activity of selected Bergenia plant extracts conducted at the Institute of Clinical Immunology and Allergology at University Hospital in Hradec Králové.
Significant differences between values were determined by one—way analysis of variance ANOVA. For determining pair-wise differences of means, the Tukeyś test was performed' P ≤ 0.05). Data are presented as the means ± standard deviation of three experiments.
| Results and Discussion|| |
Bergenia extracts significantly stimulated the expression of CD69 on lymphocytes in the final concentration of 3.13 and 6.25 mg/ml. On the contrary, higher concentration of extracts of 12.5; 25 and 50 mg/ml were toxic for the cell and they so did not stimulate the expression of CD69. Low concentration (0.2—0.8 mg/ml) was on the contrary too weak to stimulate the expression of CD69 [Figure 1]. The values of stimulation indices of B. crassifolia [Figure 2]a and B. x ornata [Figure 2]b extracts significantly statistically did not differ.
|Figure 1: The effect of Bergenia extracts at the final concentration of 0.20–6.25 mg/ml Bergenia x ornata (a) and Bergenia ciliata (b) on the activation of lymphocytes corresponding to the expression of CD69. Because of the elimination of the donors variability the resulting values are expressed using the stimulus indices (%). As a positive control phytohemaglutinin was used. Values are the average of three experiments ± standard deviation. Lymphocytes were identified on the basis of the direct scattering (FS) and side scattering (SS). Activated cells (lymphocytes) have been identified in the circulate gate (c)|
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|Figure 2: The effect of the Bergenia extracts at the final concentration of 1.56–6.25 mg/ml Bergenia crassifolia (a), Bergenia x ornata (b) and Bergenia ciliata (c) on the activation of lymphocytes corresponding to the expression of CD69. Because of the elimination of the donors variability, the resulting values are expressed using the stimulus indices (%). As a positive control phytohemagglutinin (PHA) was used. Values are the average of three experiments ± standard deviation. Lymphocytes were identified on the basis of the direct scattering (FS) and side scattering. Activated cells (lymphocytes) have been identified in the circulate gate (d)|
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The highest activation of lymphocytes reached the B. x ornata extract at a concentration of 6.25 mg/ml [Figure 2]b and [Figure 1]a. The histograms in [Figure 3] depict a statistically significant stimulation of the CD69 expression on lymphocytes using leaf Bergenia extracts in concentrations of 1.56—6.25 mg/ml, compared to a blank sample (X-VIVO™ medium) and positive control (PHA).
|Figure 3: The dependence of lymphocyte activation on the concentration of ethanol Bergenia extracts (1.56–6.25 mg/ml). Histograms capture the expression of CD69 (%) on the surface of lymphocytes as compared to positive control (PHA), a blank sample (X-VIVO™ medium) and DMSO|
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As is apparent from the literature, there is so far no study compared the effect of Bergenia extracts on the activation of lymphocytes. As reported by Popov et al., a profound immunostimulant effect and fagocytic activity exhibit a polysaccharide bergenan isolated from the leaves of B. crassifolia. In the work of the Nazir et al., immunomodulatory effect of bergenin and norbergenin on TH1 (inflammation TH cells capable of direct activities) and TH2 (activation of B-lymphocytes) there have been shown.
| Conclusion|| |
The ex vivo activation of human immune cells, as determined by cell surface CD69 expression, was reported for the first time in Bergenia extracts. By activating of cells, there has been an increase in the amount of the established antibodies. Bergenia extracts significantly stimulated the expression of CD69 on lymphocytes in the final concentration of 3.13 and 6.25 mg/ml. The values of stimulation indices of B. crassifolia and B. x ornata extracts significantly statistically did not differ. The values of stimulation indices of B. crassifolia and B. x ornata extract significantly statistically did not differ.
Financial support and sponsorship
This work was supported by SVV 260416.
Conflicts of interest
There are no conflicts of interest.
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