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ORIGINAL ARTICLE
Year : 2018  |  Volume : 14  |  Issue : 56  |  Page : 328-332  

Immunostimulant activity of Bergenia Extracts


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 Submission13-Sep-2017
Date of Acceptance20-Oct-2017
Date of Web Publication14-Aug-2018

Correspondence Address:
Lenka Tumova
Department of Pharmacognosy, Faculty of Pharmacy Hradec Králové, Charles University, Heyrovského 500 05, Hradec Králové
Czech Republic
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/pm.pm_423_17

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   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

How to cite this URL:
Tumova L, endrychová H, Vokurková D. Immunostimulant activity of Bergenia Extracts. Phcog Mag [serial online] 2018 [cited 2019 Nov 12];14:328-32. Available from: http://www.phcog.com/text.asp?2018/14/56/328/238887





SUMMARY

  • The ex-vivo activation of human immune cells, as determined by cell surface CD69, was reported for the first time in Bergenia extracts.



   Introduction Top


Bergenia genus belongs to Saxifragaceae family is one of the very important medicinal plants. Over 30 species of this genus is known at present[1] 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).[2],[3],[4] Bergenia plants show antibacterial, antiviral, anticancer, antidiabetic, diuretic and immunostimulant activities.[5],[6],[7],[8],[9],[10] 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.[10] 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.[11],[12]

Immunostimulant activity

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.[13]

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 Top


Plant material

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.

Method

Immunostimulant activity

This method modified according to the work of the Cheel[14] 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.[14] 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.

Statistical analysis

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 Top


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)

Click here to view


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

Click here to view


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.,[15] 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.,[16] 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 Top


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.



 
   References Top

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Zhang Y, Liao C, Li J, Liu X. A review on resource status, bioactive ingredients, clinical applications and biological progress in Bergenia. J Med Plants Res 2011;5:4396-99.  Back to cited text no. 1
    
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Salminen JP, Shikov AN, Karonen M, Pozharitskaya ON, Kim J, Makarov VG, et al. Rapid profiling of phenolic compounds of green and fermented Bergenia crassifolia L. Leaves by UPLC-DAD-qqQ-MS and HPLC-DAD-ESI-QTOF-MS. Nat Prod Res 2014;28:1530-3.  Back to cited text no. 2
    
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Chernetsova ES, Shikov AN, Crawford EA, Grashorn S, Laakso I, Pozharitskaya ON, et al. Characterization of volatile and semi-volatile compounds in green and fermented leaves of Bergeniacrassifolia L. by gas chromatography-mass spectrometry and id-cube direct analysis in real time-high resolution mass spectrometry. Eur J Mass Spectrom (Chichester) 2014;20:199-205.  Back to cited text no. 3
    
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Shikov AN, Pozharitskaya ON, Makarova MN, Makarov VG, Wagner H. Bergeniacrassifolia (L.) fritsch — Pharmacology and phytochemistry. Phytomedicine 2014;21:1534-42.  Back to cited text no. 4
    
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Shi X, Li X, He J, Han Y, Li S, Zou M, et al. Study on the antibacterial activity of Bergeniapurpurascens extract. Afr J Tradit Complement Altern Med 2014;11:464-8.  Back to cited text no. 5
    
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Rajbhandari M, Mentel R, Jha PK, Chaudhary RP, Bhattarai S, Gewali MB, et al. Antiviral activity of some plants used in Nepalese traditional medicine. Evid Based Complement Alternat Med 2009;6:517-22.  Back to cited text no. 6
    
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Spiridonov NA, Konovalov DA, Arkhipov VV. Cytotoxicity of some Russian ethnomedicinal plants and plant compounds. Phytother Res 2005;19:428-32.  Back to cited text no. 7
    
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Bhandari MR, Nilubon JA, Hung G, Kawabata J. Glucosidase and amylase inhibitory activities of Nepalese medicinal herb Pakhanbhed (Bergenia ciliata Haw). Food Chem 2007;106:247-52.  Back to cited text no. 8
    
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Verma P, Gauttam V, Kalia AN. comparative pharmacognosy of Pashanbhed. J Ayurveda Integr Med 2014;5:104-8.  Back to cited text no. 9
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de Oliveira CM, Nonato FR, de Lima FO, Couto RD, David JP, David JM, et al. Antinociceptive properties of Bergenin. J Nat Prod 2011;74:2062-8.  Back to cited text no. 10
    
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Golovchenko VV, Bushneva OA, Ovodova RG, Shashkov AS, Chizhov AS, Ovodov Iu, et al. Structural study of bergenan, a pectin from Bergeniacrassifolia. Bioorg Khim 2007;33:54-63.  Back to cited text no. 11
    
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Churin AA, Masnaia NV, Sherstoboev EY, Suslov NI. Effect of Bergeniacrassifolia extract on specific immune response parameters under extremal conditions. Eksp Klin Farmakol 2005;68:51-4.  Back to cited text no. 12
    
13.
Cheel HJ. Biological Activities and Chemical Content of Glycyrrhiza species. Dissertation Thesis 2010. Faculty of Pharmacy, Hradec Králové, Charles University; 2010.  Back to cited text no. 13
    
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Cheel J, Antwerpen PV, Tůmová L, Onofre G, Vokurková D, Zouaoui-Boudjeltia K, Vanhaeverbeek M, Nève J. Free radical-scavenging, antioxidant and immunostimulating effects of a licorice infusion (Glycyrrhizaglabra L.). Food Chem 2010;122:508-17.  Back to cited text no. 14
    
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Popov SV, Popova GY, Nikolaeva SY, Golovchenko VV, Ovodova RG. Immunostimulating activity of pectic polysaccharide from Bergeniacrassifolia (L.) fritsch. Phytother Res 2005;19:1052-6.  Back to cited text no. 15
    
16.
Nazir N, Koul S, Qurishi MA, Taneja SC, Ahmad SF, Bani S, et al. Immunomodulatory effect of Bergenin and Norbergenin against adjuvant-induced arthritis — A flow cytometric study. J Ethnopharmacol 2007;112:401-5.  Back to cited text no. 16
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]



 

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