Molecular and Cellular Biochemistry 275: 1–6, 2005. c�Springer 2005

Cytoprotective and antioxidant activityof Rhodiola imbricata against tert-butylhydroperoxide induced oxidative injuryin U-937 human macrophages

Kanupriya, D. Prasad, M. Sai Ram, R. Kumar, R.C. Sawhney,S.K. Sharma, G. Ilavazhagan, D. Kumar and P.K. BanerjeeDefence Institute of Physiology and Allied Sciences, Delhi, India

Received 1 June 2004; accepted 25 November 2004

Abstract

The present study reports cytoprotective and antioxidant activity of aqueous and alcoholic extracts of Rhodiola imbricata rhizomeon tert-butyl hydroperoxide (tert-BHP) induced cytotoxicity in U-937 human macrophages. There was an increase in cytotoxicityand apoptosis significantly in the presence of tert-BHP over control cells. The tert-BHP induced cytotoxicity can be attributedto enhanced reactive oxygen species (ROS) production which in turn is responsible for fall in reduced glutathione (GSH)levels; further there was a significant decrease in mitochondrial potential and increase in apoptosis and DNA fragmentation.Both aqueous and alcoholic extracts of Rhodiola rhizome at a concentration of 250 µg/ml were found to inhibit tert-BHPinduced free radical production, apoptosis and to restore the anti-oxidant levels to that of the control cells. The alcoholic extractof Rhodiola showed higher cytoprotective activities than aqueous extract. These observations suggest that the alcoholic andaqueous extracts of Rhodiola have marked cytoprotective and antioxidant activities. (Mol Cell Biochem 275: 1–6, 2005)

Key words: antioxidant activity, apoptosis, cytoprotection, oxidative stress, Rhodiola imbricata

Introduction

Recently, there has been a growing interest in establishingthe therapeutic potentials of medicinal plants as antioxidantswhich help in reducing free radical induced tissue injury[1–3]. Free radicals have been implicated in a number ofpathophysiological processes such as alzheimer’s disease,atherosclerosis, apoptosis and cancer [4]. The aging is alsobelieved to be due to excessive production of reactive oxygenspecies (ROS) [5]. Many plants such as Emblica officinalis,Curcuma longa, Santalum album, Swertia chirata Buch-Hamhave been reported to posses antioxidant activity [6]. Themedicinal importance of some plant species has been proved

Address for offprints: M. Sairam, Department of Experimental Biology, Defence Institute of Physiology and Allied Sciences, Lucknow Road, Timarpur,Delhi-110054, India (E-mail: sairam64@rediffmail.com)

by the appearance of many drugs as patented products indeveloped countries. The use of plant extracts for medici-nal purposes seems to be more natural, less expensive andwithout side effects. The biological activities of these plantsare due to vitamins, flavonoids and polyphenolic compoundswhich posses antioxidant activities [7].

Rhodiola sp, a perennial herb may have originated inthe Sino Himalayas, Nepal, Qingha, Xizang. It is a mem-ber of family Crassulaceae that grows in drained soil at analtitude of 2700–5000 m and known for long to possessimmense medicinal potential. The extracts of the R. sacrahave active oxygen scavenging activities [8] and reported

2

to improve learning and memory [9]. It has been reportedthat R. rosea has potent anti-fatigue and anti stress ac-tivity [10]. It has also been shown to have anticancer,antioxidant and immune enhancing and stimulating sexualactivity [11, 12]. Some of the bioactive compounds such ashydroquinone, caffeic acid, protocatechuic acid, gallic acid,epigallocatechin 3-O-gallate, 3-O-galloylepigallocatechin-(4β→8)-epigallocatechin 3-O-gallate, heterodendrine,4-hydroxybenzoic acid, 3,4-hydroxycinnamic acid ofR. sacra have been characterized [8]. About more than70 species of Rhodiola have been reported to exist [13].Rhodiola has been used as tonic, hemostatic and for contu-sions in traditional Chinese medicine for thousands of years.In Tibet, Rhodiola is commonly used for promoting bloodcirculation and relieving cough [14].

However, most of the literature published in Russia andChina is mainly carried out on R. rosea and no authenticatedinformation is available on the bioactivity of R. imbricata.Keeping the above facts in view, the present study was de-signed to evaluate the cytoprotective and antioxidant activityof Rhodiola imbricata using human macrophages U-937 asmodel system.

Materials and methods

Plant material/drug preparation

Rhodiola imbricata rhizome/root was collected from hilly re-gions of western Himalayas, in the month of September. Thefresh roots were cleaned and dried under shade. The extrac-tion was carried out using powdered root (10 g) by refluxingwith 500 ml-distilled water for 10 h. The supernatant wasfiltered and the crude extract was dried in vacuum and frac-tionated using 90% ethanol. Then the ethanolic extract wasdried in vacuum and lyophilized for further use.

In vitro experiments

U-937 human macrophages were obtained from NationalInstitute of Immunology (NII), New Delhi, India. Unlessotherwise mentioned, the oxidative stress was induced byexposing the cells to 250 µM tert-butyl hydroperoxide(tert-BHP) for 1 h.

Determination of cytotoxicity

The cells were incubated with tert-BHP and 250 µg/ml ofaqueous or alcoholic extract of Rhodiola overnight in CO2

incubator at 37◦C. Cytotoxicity was studied by using neutralred as described earlier by Sairam et al. [15]. Briefly, 10 µl ofneutral red dye (0.1%) was added to the cells and incubated

at 37◦C in CO2 incubator for 45 min. Later the cells werecentrifuged, washed thrice with saline followed by additionof 200 µl of ethanol–acetic acid (50:1) solution. The opti-cal density was then measured at 570 nm using (MolecularDynamics) ELISA reader.

Measurement of reactive oxygen species (ROS)

It was determined by using 2,7-dichlorofluorescindiacetate(DCFH-DA) as described by Cathcart et al. [16]. The cells(1 × 106 cells/ml) were incubated with DCFH-DA (20 µM)for 30 min. The cells were then washed, incubated with tert-BHP (500 µM) with and without Rhodiola extracts for 90min. After incubation the fluorescence was measured by spec-trofluorimeter (Varian, U.S.A.) with an excitation at 485 nmand emission at 530 nm. ROS production was also monitoredflow cytometrically and the data was analyzed by using FACSCalibur Flow Cytometer equipped with Cell Quest Software(Beckton Dickinson, U.S.A.).

Determination of anti oxidant status

After incubation, the cells were lysed by adding 200 µl oflysis buffer (10mM Tris, 20 Mm EDTA, 0.25% Triton X-100 pH 8). The glutathione peroxidase (GPx) activity wasdetermined in cell lysate using RANDOX Kits (U.K.) as permanufacturer’s instructions.

The reduced glutathione (GSH) levels in the cells weredetermined fluorimetrically by the method of Hissin and Hilf[17].

Determination of mitochondrial transmembrane potential

After incubation of cells with tert-BHP and Rhodiola extract,10 µl of Rhodamine 123 (10 µg/ml) was added to the cellsand incubated for 30 min. The cells were washed three timeswith saline and fluorescence was measured using spectroflu-orimeter (Varian, U.S.A.) with an excitation at 485 nm andemission at 530 nm [18].

Determination of apoptosis

The cells were incubated with tert-BHP (500 µM) for 1 hand apoptosis was determined by using Apoptosis DetectionKit (BD Pharmingen), as per manufactures instructions. Thecells were washed twice with saline and then resuspended in1X binding buffer at a concentration of 1×106 cells/ml 100 µlof the solution containing 1 × 105 cells/ml was transferred toa 5 ml culture tube and added FITC labeled Annexin V (7 µl)

3

and Propidium iodide (10 µl). After incubating the cells for15 min in dark, 400 µl of binding buffer was added and thecells were analyzed by flow cytometer (Becton Dickinson,U.S.A.).

Alkaline single cell gel electrophoresis or Comet assay

The Comet assay was used to assess single strand breaks ofDNA as described by Tice et al. [19]. Microscope slides werepre-coated with agarose (0.1%). The cells were exposed totert-BHP (500 µM) for 1 h. The cells were then washed inPBS and 100 µl of cell suspension ( 3.5×104 cells) was mixedin warm low gelling agarose (0.75%) (Sigma IX-grade) andpipetted onto the slides, and put immediately on ice to allowthe agarose to solidify. The slides were placed in cold ly-sis solution (SDS 2.5%, Sodium sarcosinate 1% and EDTA25 mM, pH 9.5) for 15 min at room temperature. After wash-ing the slides in distilled water, the slides were placed inhorizontal electrophoresis tank (BIORAD) containing elec-trophoresis buffer (Tris 90 mM, Boric acid 90 mM, EDTA 25mM, pH 8.3) and electrophoresed for 5 min at 2 V/cm. Theslides were washed in distilled water, stained with propidiumiodide (25 µg/ml) and cells were then visualized using HundsFluorescence Microscope. One hundred nuclei on each slidewere scored and given a score from 0 (undamaged nuclei)to 4 (severely damaged nucleus) and scored randomly as perthe method described by Collins et al. [20].

Results

Cytotoxicity

The cytoprotective effect of aqueous and alcoholic extracts ofRhodiola on tert-BHP induced cytotoxicity is shown in Ta-ble 1. Addition of tert-BHP (250 µM) resulted in enhancedcytotoxicity compared to control cells as determined by de-creased neutral red dye uptake. Aqueous and alcoholic ex-tracts of Rhodiola significantly inhibited the cytotoxicity in-duced by tert-BHP and their activity is better than Vit. C(Table 1).

ROS production and mitochondrial integrity

Addition of tert-BHP to the macrophages resulted in signifi-cant increase in free radical production measured in terms offluorescence of DCFH-DA (Fig. 1) Free radical productionwas also analyzed flow cytometrically, (Fig. 2). Further therewas a marked reduction in mitochondrial transmembrane po-tential in presence of tert-BHP. Aqueous and alcoholic ex-tracts of Rhodiola inhibited tert-BHP induced free radical

Table 1. Effect of Rhodiola on tert-BHP induced cytotoxicity in macrophages

Groups Live cells (%)

Control 100 ± 7.2

tert-BHP 68 ± 9.2a

Rh (aq) 98 ± 8.4

Rh (aq) + tert-BHP 85 ± 8.1b

Rh (alc) 97 ± 6.7

Rh (alc) + tert-BHP 89 ± 7.5b

Vit C + tert-BHP 80 ± 6.9b

Note. Values are expressed as mean ± S.D.; p < 0.05 .aVs control.bVs tert-BHP.

Fig. 1. Effect of Rhodiola on free radical production and mitochondrialtransmembrane potential in macrophages in the presence of tert-BHP.

production significantly and their activity is comparable tothat of Vit.C. Further, the extracts also inhibited tert-BHPinduced fall in mitochondrial transmembrane potential whileVit. C had little effect.

Antioxidant status

There was a significant decrease in GSH and GPx levels inmacrophages exposed to tert-BHP compared to control cells.Addition of aqueous and alcoholic extracts of Rhodiola hadno effect on GSH levels. However, GPx levels were main-tained at higher levels similar to that of control. (Figs. 3 and 4)

Apoptosis

Addition of tert-BHP (500 µM) to the macrophages resultedin a significant increase in cells undergoing apoptosis. Therewas six times increase in early apoptotic cells (Annexin-Vpositive, B) and three times increase in late apoptotic cells(Annexin-V plus PI positive, C) when cells treated with

4

Fig. 2. Flow cytometric analysis-effect of Rhodiola on tert-BHP inducedfree radical production.

Fig. 3. Effect of Rhodiola on GSH levels in the presence of tert-BHP.

Fig. 4. Effect of Rhodiola on GPx levels in the presence of tert-BHP.

Fig. 5. Effect of Rhodiola on apoptosis induced by tert-BHP.

tert-BHP compared to control cells. Interestingly aqueousand alcoholic extracts of Rhodiola significantly lowered thepercentage of early apoptotic cells and late apoptotic cells,the later having much higher activity (Fig. 5).

Comet assay

Exposure of cells to 500 µM tert-BHP for 1 h resulted in anappreciable increase in DNA single strand breaks. Pretreat-ment of cells with aqueous or alcoholic extracts of Rhodiolasignificantly inhibited DNA single strand breaks induced bytert-BHP (Figs. 6 and 7).

Fig. 6. Effect of Rhodiola on DNA single strand breaks induced by tert-BHP.

5

Fig. 7. Photomicrographs of comet assay depicting various stages of DNA damage.

Discussion

Tert-BHP, an organic hydroperoxide, is a useful model com-pound for the study of mechanism of oxidative cell injury[21]. Organic hydroperoxides are formed as a result of oxy-gen abstraction from peroxyl radicals. Tert-BHP may then de-compose to other alkoxyl and peroxyl radical that acceleratelipid peroxidation chain reactions [22]. This decomposition isaided by metal ions and their complexes. Several studies haveshown that exposure to tert-BHP results in oxidative stress,producing free radicals which in turn results in reduced in-tracellular GSH levels [23, 24]. In the present study, additionof tert-BHP to macrophages resulted in a significant increasein cytotoxicity and decrease in mitochondrial integrity whilepre-treatment of cells with R. imbricata significantly inhib-ited cytotoxicity and maintained mitochondrial integrity. Inthis regards, our studies fall in confirmation with earlier stud-ies by Hongjingtian et al. [25], who reported that Rhodiolahas anti-hypoxic effect and improved efficiency of mitochon-drial respiration in skeletal muscle which is attributed to theincrease of the oxygen supply and decrease in oxygen con-sumption in the respiratory cells.

Fluorimetric and flow cytometric studies using DCFH-DAhave revealed a marked increase in ROS generation in thepresence of tert-BHP. When the cells were incubated withextracts of Rhodiola, the free radical production was signifi-cantly inhibited and its efficacy was comparable to that of Vit.C which in turn was responsible for observed cytoprotection.Mizue ohsugi et al. [8] also reported the scavenging activi-ties of the constituents of R. sacra against superoxide anionradical (·O2

−) and hydroxyl radical (·OH) in cell free sys-tems. A recent study of 19 compounds isolated from R. sacrashowed that these have strong scavenging activities againstsuperoxide (·O2

−) and hydroxyl radicals (·OH)). This wasattributed to the presence of gallic acid, 4-hydroxycinnamicacid, hydroxybenzoic acid etc. [8]. In the present study, thecells pre-treated with aqueous and alcoholic extracts of R.imbricata maintained higher GPx levels but could not re-store reduced glutathione levels in the presence of tert-BHP.To answer the inability of Rhodiola to maintain higher GSH

levels, the Glutathione reductase (GR) levels in cell lysateswere determined. We found that the extracts of Rhodiola wereunable to maintain GR levels (data not shown) in the pres-ence of tert-BHP which explains the low levels of GSH cells.Earlier studies had shown that flavanoids like flavonol, 6 hy-droxyflavonol, 7 hydroxyflavonol protect the neuronal cellsfrom glutamate induced oxidative stress by preventing cal-cium influx [26]. These flavanoids had no effect on GSHmetabolism nor they have any anti-oxidant activity. Theyhad shown that cells treated with glutamate have ROS andGSH levels indistinguishable from those in cells dying fromglutamate toxicity. Despite accumulating high ROS and lowGSH levels, these cells were able to survive in the presenceof flavonol and other related compounds. Further, flavanoidslike Galanin and Kaempherol protect the cells from oxidativestress by scavenging ROS without effecting the GSH levels.In this regard, our results fall in agreement with these find-ings. In contrast, animal experiments showed that treatinganimals with R. imbricata prevented ROS mediated damageand lipid peroxidation by increasing the levels of GSH andsuper oxide dismutase (SOD) levels [13]. However, more ex-perimentation is required to answer the observed variationbetween in vitro and in vivo experimentation.

To know whether the alcoholic or aqueous extracts ofRhodiola imbricata have anti-apoptotic activity, experimentswere conducted by pre-treating cells with 250 µg/ml ofRhodiola extract and exposing them to 500 µM tert-BHP for1 h. To differentiate the early apoptotic cells from late apop-totic and necrotic cells, we have used FITC labeled annexin Vand propidium iodide. Annexin-V binds to Phosphatidyl ser-ine moieties (which get externalized during apoptosis) and iscommonly used to detect early apoptotic cells while PI stainsnucleus of cells whose membrane has been extensively dam-aged or dead cells. In the present study, we found that thereis a significant increase in early and late apoptotic cells whenthe cells were treated with 500 µM tert-BHP for 1 h. Fur-ther, there was a significant increase in single strand DNAbreaks as revealed by comet assay. However, pretreatment ofcells with aqueous and alcoholic extracts of Rhodiola sig-nificantly inhibited both apoptosis and single stranded DNA

6

breaks induced by tert-BHP. Earlier Mook-Jung et al. [27]reported that neuro-protective effects of the constituents ofRhodiola sacra against beta-amyloid (Aβ) toxicity and H2O2

induced oxidative injury which was attributed to their anti-oxidant and anti-apoptotic activity. However in this study,the apoptosis was induced by staurosporine but they stud-ied the apoptosis by MTT reduction assay which is not ameasure of apoptosis but is commonly used for studying cy-totoxicity. However, we found that the aqueous and nobreakalcoholic extracts of R. imbricata rhizome could inhibit apop-tosis and single stranded DNA breaks induced by free radi-cals very effectively in macrophages using well authenticatedtechniques.

In conclusion, the extracts of Rhodiola have a marked cy-toprotective activity against tert-BHP induced cytotoxicity.The ability of Rhodiola to inhibit the free radical produc-tion and apoptosis induced by tert-BHP could be responsiblefor the observed cytoprotective activity. Experiments are inprogress to characterize bioactive principles of Rhodiola im-bricata and their immunomodulatory activity.

References

1. Eastwood MA: Interaction of dietary antioxidants in vivo: How fruitsand vegetables present disease? QJM 92: 527–530, 1999

2. Siddique MS, Eddeb F, Mantle D, Mendelow AD: Extracts of Ginkgobiloba and Panax ginseng protect brain proteins from free radical in-duced oxidative damage in vitro. Acta Neurochirurgica Suppl 76: 87–90,2000

3. Koleva II, Van Bleek TA, Linssen JP, De-Groot A, Evstatieva LN:Screening of plant extracts for antioxidant activity: A comparative studyon three testing methods. Phytochem Anal 13: 8–17, 2002

4. Halliwell B: Antioxidants and human disease: A general introduction.Nutr Rev 55: s44–s52, 1997

5. Meydani SN, Wu D, Santos MS, Hayek MG: Antioxidants and immuneresponse in aged persons: Overview of present evidence. Am J Clin Nutr62: 1462S–1476S, 1995

6. Scartezzini P, Speroni E: Review on some plants of Indian traditionalmedicine with antioxidant activity. J Ethnopharmacol 71: 23–43, 2000

7. Tapiero H, Tew KD, Ba GN: Polyphenols: Do they play a role in theprevention of human pathologies? Biomed Pharmacother 56: 200–207,2002

8. Mizue Ohsugi, Wenzhe Fan, Koji Hase, Quanbo Xiong, YasuhiroTezuka, Katsuko Komatsu, Tsuneo Namba, Tomhiro Saitoh, KenjiTazawa, Shigetoshi Kadota: Active-oxygen scavenging activity of tra-ditional nourishing-tonic herbal medicines and active constituents ofRhodiola sacra. J Ethnopharmacol 67: 111–119, 1999

9. Ming HQ, Xia GC, Zhang RD, Zhongcaoyao: Advanced research onRhodiola. Chinese Herbal Medicines 19: 229–234, 1988

10. Darbinyan V, Kteyan A, Panossian A, Gabrielian E, Wikman G, WagnerH: Rhodiola rosea in stress induced fatigue: A double blind cross over

study of a standardized extract SHR-5 with a repeated low dose regimenon the mental performance of healthy physicians during night duty.Phytomedicine 7(5): 365–371, 2000

11. Furmanova M, Skopinska-Rozewska E, Rogala E, Malgorzata H: Rho-diola rosea in vitro culture: Phytochemical analysis and antioxidantaction. Acta Societis Botanicorum Poloniae 76(1): 69–73, 1998

12. Bolshakova IV, Lozovskaia EL, Sapezhinskii II: Antioxidant propertiesof a series of extracts from medicinal plants Biofzika 42(2): 480–483,1997

13. Siu Po Ip, Chun Tao Che, Po Sing Leung: Association of free radicals andthe tissue renin-Angiotensin system: Prospective effects of Rhodiola, agenus of Chinese herb, on hypoxia induced pancreatic injury. J Pancreas2(1): 16–25, 2001

14. Peng JN, GE YC, Li XH: Studies on chemical constituents of Rhodiolafastigita. Acta Pharmacuet Sin 31: 798–800, 1996

15. Sairam M, Anju B, Pauline T, Prasad D, Kain AK, Mongia SS, SharmaSK, Singh B, Singh R, Ilavazhagan G, Kumar D, Selvamurthy W: Effectof Kombucha tea on chromate (VI) induced oxidative stress in albinorats. J Ethnopharmacol 71: 235–240, 2000

16. Cathcart R, Schwiers E, Ames BN: Detection of picomole levels of hy-droperoxides using fluorescent dichlorofluorescein assay. Ann Biochem134: 111–116, 1983

17. Hissin PJ, Hilf R: A fluorometric method for determination of oxidizedand reduced glutathione in tissues. Ann Biochem 74: 214–226, 1976

18. Jiang T, Acosta D: An in vitro model of cyclosporin induced nephro-toxicity. Toxicol Appl Phamacol 20: 486–495, 1993

19. Tice RR, Andrews PW, Hirai O, Singh NP: The single cell gel (SCG)assay: An electrophoretic technique for the detection of DNA damagein individual cells. In: C.M. Witmer (ed.) Biological and Reactive In-termediates IV. Plenum Press, New York, 1990. pp. 157–164

20. Collins AR, Ai-guo M, Duthie SJ: The kinetics of oxidative DNA dam-age (strand breaks and oxidized pyrimidines) in human cells. Mutat Res336: 69–77, 1995

21. Altman SA, Zastawny TH, Randers L, Lin Z, Lumpkin JA, Remacle J,Dizdaroglu M, Rao G: Tert-butyl hydroperoxide mediated DNA basedamage in cultured mammalian cells. Mutat Res 306: 35–44, 1994

22. Baker M, He S: Elaboration of cellular DNA breaks by hydroperoxides.Free Radical Biol Med 11: 563–572, 1991

23. Dubuisson ML, De Wergifosse B, Trouet A, Baguet F, Marchand-Brynaert J, Rees JF: Antioxidative properties of natural coelenterazineand synthetic methyl coelenterazine and synthetic methyl coelenter-azine in rat hepatocytes subjected to tert-butyl hydroperoxide inducedoxidative stress. Biochem Pharmacol 60(4): 471–447, 2000

24. Suzuki Y, Apostolova MD, Cherian MG: Astrocyte cultures from trans-genic mice to study the role of metallothionein in cytotoxicity of tert-butyl hydroperoxide. Toxicol 145: 51–62, 2000

25. Hongjintian In Zheng HZ, Dong ZH, She J: Modern study of traditionalChinese medicine. Vol 6, 1st edn, Beijing Xue Yuan press, Beijing, 1997,pp. 5658–5678

26. Ishige K, Schubert D, Sagara Y: Flavanoids protect neuronal cells fromoxidative stress by three distinct mechanisms. Free Radical Biol Med30(4): 433–446, 2001

27. Inhee Mook-Jung, Hee Kim, Wenzhe Fan, Yasuhiro Tezuka, ShigetoshiKadota, Hisao Nishijo, Min Whan Jung: Neuroprotective effect of con-stituents of the oriental crude drugs, Rhodiola sacra, R. sachalinensisand Tokaku-joki-to, against Beta-amyloid toxicity, oxidative stress andapoptosis. Biol Pharm Bull 25(8): 1101–1104, 2002