Original Article

In Vitro Cytotoxicity and Oxidative Stress Evaluation of Valerian (Valeriana officinalis) Methanolic Extract in Hepg2 and Caco2 Cells


  • Mehtap KARA
  • Ecem Dilara ALPARSLAN
  • Ezgi ÖZTAŞ
  • Özlem Nazan ERDOĞAN

Received Date: 10.11.2020 Accepted Date: 19.02.2021 Turk J Pharm Sci 2021;18(5):604-608 PMID: 34719188


Traditional treatment methods are becoming popular and commonly used in many societies and have become the first treatment option for most people. While some of these methods are helpful, they can interact with medications the patient is taking for another disease and cause a variety of life-threatening risks. Valerian (catweed) plant is used in traditional medicine as a sleep aid due to its sedative effects. Valerian may also exert anticancer effect in vitro.

Materials and Methods:

In this study, the cytotoxicty and oxidative stress effects of valerian root extract were evaluated in human liver hepatocellular carcinoma (Hepg2) and human colorectal adenocarcinoma (Caco2) cell lines. The cytotoxicity was evaluated via the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide test. Total reactive oxygen species analysis was performed via a 2’,7’-dichlorodihydrofluorescein diacetate assay in flow cytometry.


Inhibition concentration 50 values were calculated as 936.6 and 1097.5 µg/mL in the Hepg2 and Caco2 cell lines, respectively. It was observed that valerian root extract did not induce oxidative stress in HepG2 and Caco2 cell lines.


These results indicate that the use of valerian root extract as an alternative method in cancer treatment may not be effective and may cause a risk for public health. On the other hand, it may be safe at recommended tolerated concentrations since it does not cause oxidative stress.

Keywords: Valeriana officinalis, HepG2, Caco2, oxidative stress, MTT


The use of herbal products for the treatment of several diseases has been widespread from the past to the present. In developed countries, herbal remedies are considered over-the counter drugs with strict regulations. However, in developing countries, the use of herbal therapy methods and products lacks control. Most of herbal drugs in the market have not been fully evaluated from the toxicological aspect; thus, these products may cause several adverse effects during therapy of the different body systems.1

Valeriana belongs to Valerianaceae family, which includes approximately 300 species. Valeriana officinalis has been generally used as an alternative traditional medicine for insomnia and depression therapy. This medical plant has sedative, anticonvulsant, hypnotic, and anxiolytic effects. In addition, this plant has treatment potential for gastrointestinal and urinary system problems. The V. officinalis compounds obtained by extraction methods (EMs) include flavonoids, monoterpenes, sesquiterpenes, valepotriates, iridoids, alkaloids, acid-like gamma-aminobutyric acid (GABA), glutamine, and lignans, which affect the central nervous system and have antioxidant and vasorelaxant effects. Given these effects, V. officinalis is a popular herbal remedy choice to cure insomnia, headache, gastrointestinal system, cardiovascular system, and urinary tract problems.2,3,4,5

Valeriana herbal remedy can be effective in cancer patients. Valerian herb ingredients with anticancer effects have been given great concern by the scientific area. However, herbal therapy implementers have been attempting to cure cancer patients with insufficient valerian products.6

In this work, we have observed the cytotoxic and oxidative stress-inducing effects, which play important roles in killing cancer cells, of methanolic extract of V. officinalis on HepG2 and CaCo2 cell lines.


Valeriana officinalis extraction

V. officinalis roots were purchased commercially from a traditional herbal drug store in İstanbul and pulverized in a porcelain mortar. A total of 75 mL methanol was added to 15 g powdered V. officinalis roots and incubated at room temperature in a shaker for 24 h. After the incubation period, the extraction solution was filtered with a Whatman no.1 filter, and methanol was evaporated with fractional distillation.7

Cell culture and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test human liver hepatocellular carcinoma (HepG2- HB-8065™) and human colorectal adenocarcinoma (Caco2- HTB-37™) cell lines were purchased from the American Type Culture Collection (Virginia, USA), and the cells were maintained as per manufacturer’s instructions. Exactly 1000 mg/mL extract was prepared by dissolving V. officinalis roots in 100% dimethyl sulfoxide (DMSO) and stored at +4°C until the experiments. Before the cell treatments, the extract was diluted with DMSO at a final concentration of 1%. Given the selected concentrations for cytotoxicity assay, the root extract was dissolved in a cell culture medium to prepare the desired concentrations. The treatments were performed at a concentration range for 24 h to evaluate the dose-dependent effects. All study experiments were performed in triplicates in three different days.

The HepG2 and Caco2 cells were seeded into 96-well plates (1x104 cells/100 µL cell culture medium/well). After overnight incubation, the cells were treated with V. officinalis extract at the concentrations of 200, 400, 600, 800, and 1000 µM and control for 24 h. In the control group, the final DMSO concentration was 1%. Then, MTT was added into the wells, and the wells were incubated for another 3 h at 37°C in the dark. Optical densities were measured at 570 nm using a microplate reader (Biotek, Epoch, Vermont, USA).8

Total reactive oxygen species (Total-ROS) assay with 2’,7’-dichlorodihydrofluorescein diacetate (H2DC-FDA).

Different pathological conditions were associated with the ROS increase in cells. Thus, changes in the ROS level are detected in basic studies. Given the short half-life of ROS, effective detection methods are important during observations. H2DC-FDA is a non-fluorescent dye, and in the presence of ROS, it returns a green fluorescent with oxidation.9 In the present study, the ROS production was evaluated with H2DC-FDA dye with by a flow cytometer. The 5x105 HepG2 and Caco2 cells in 2 mL medium per well were seeded into six-well plates and incubated overnight. The cells were treated with 100, 200, 100, and 600 µg/mL concentrations. These concentrations exerted a cell viability higher than 70% in the MTT assay for 24 h. The 1% DMSO solution was used as the negative control for the experiment. After 24 h, the cells were washed with phosphate-buffered saline (PBS) twice and incubated with 20 µM H2DC-FDA at 37°C for 30 min. The cells were detached with trypsin-ethylenediaminetetraacetic acid after the incubation period and washed with PBS. Then, the cells were re-suspended with 1% BSA in 150 µL PBS. The fluorescence intensity of 104 cells was measured with an ACEA NovoCyte flow cytometer (San Diego, California, USA), and the results were expressed as the percentage of median fluorescence intensity (MFI %) as previously described.10

Statistical analysis

All the experiments were performed as three replicates; the results were presented as the mean + standard deviation. The statistical comparison results were analyzed using the one-way analysis of variance followed by Tukey’s test for post hoc analysis, and the statistical significance was set at p<0.05 (SPSS, version 21.0, USA).


Cell viability

According to MTT results, the percentage of cell inhibition in Caco2 and HepG2 cell lines with V. officinalis methanolic extract exposure increased and was concentration dependent for 24 h (Figure 1, Table 1). The inhibition concentration 50 (IC50) values were calculated by graph slope formulations. The IC50 values were 939.68 µg/mL for HepG2 cells and 1097.58 µg/mL for Caco2 cells.

Total-ROS induction

The IC30 values of V. officinalis methanolic extract in HepG2 and Caco2 cell lines were calculated for the Total-ROS evaluation. The calculated IC30 values were 600.12 µg/mL for HepG2 cells and 672.95 µg/mL for CaCo2 cells. The 100, 200, 400, and 600 µg/mL concentrations were selected for the Total-ROS analysis to determine the ROS production with a flow cytometer via H2DC-FDA. No statistically significant difference was observed between the concentration groups for MFI for both cell lines. The V. officinalis methanolic extract did not cause an increase in the ROS production after a 24 h exposure (Figure 2).


Valerian belongs to Valerianaceae family, which includes approximately 300 species existing only in Western countries, and contains several different phytochemicals that may have nervous system protection, diuretic, antispasmodic, anthelmintic, antioxidant, antimicrobial, anti-inflammatory, antirheumatic sedative, anticonvulsant, and diaphoretic effects.6 As a medicinal herbal plant, valerian is important in traditional therapy for sleeping and anxiety disorders due to its effects on the GABA A receptor system.11 V. officinalis products are widely used for different type of diseases. In recent years, the usage of valerian products in cancer cure increased due to herbal implementers.2

Several people die worldwide due to different cancer types, and new therapeutic development studies pique the interest of scientists. Oxidative stress induction plays an important role in cancer cell death induction via chemotherapeutics. Oxidative stress has evident effects on cancer cell induction and cancer cell death mechanisms. Oxidative stress may induce cancer cell proliferation via DNA damage or can be a therapeutic strategy to cure cancer via inducing cancer cell death through apoptosis.12,13 In different clinical trials, varied results were reported on valerian species in vitro and in vivo. In human neuroblastoma (SH-SY5Y) cell line, de Oliveria et al.2 reported that aqueous V. officinalis extract exerted a protective role against apoptosis with rotenone exposure. Wang et al.13 showed the antioxidant effects of V. jatamansi on HepG2, human cervix carcinoma, breast cancer (MDA-MB-231), and human umbilical vein endothelial cells. Kakehashi et al.11 reported that V. sitchensis added in drinking water of male F344 rats significantly suppressed the 8-hydroxy-29-deoxyguanosine generation after hepatocarcinogenesis initiation by diethylnitrosamine at 50, 500, and 5000 ppm concentrations. The antioxidant catalase levels and apoptosis increased, and cell proliferation significantly decreased. The apoptosis induction was associated with the suppression of c-myc, Mafb, and cyclin D1 expression and increased expression levels of p21Waf1/Cip1, p53, and Bax. These results indicated that V. sitchensis administration can be associated with carcinogenesis inhibition.11 Zhu et al.14 showed the anticancer effects of V. jatamansi F3 fraction in vitro and in vivo. In MCF-10A cells, the F3 fraction administration increased the intracellular ROS production and induced apoptosis, which play key roles in cancer inhibition. Sudati et al.15 reported that V. officinalis ethanolic extract has an antioxidant effect against different toxic agents in rat brain homogenates. In addition, different studies showed the antioxidant effect of V. officinalis.16,17,18 Tian et al.19 reported that a V. jatamansi ingredient, valtrate, induced apoptosis in MDA-MB-231 and MCF-7 cells and inhibited cell migration. In another study, Han et al.20 reported that valeric acid, one of the important ingredients of valerian root extract, inhibits liver cancer development via histone deacetylase inhibition.

Quan et al.21 isolated different iridoids from V. jatamansi roots, and two of these iridoids showed inhibitory effects on human glioma stem cells. Sen-Utsukarci et al.22 reported that V. alliariifolia ethanolic extracts obtained with different EMs (EM1 and EM2) have a high antioxidant capacity. Additionally, two different ethanolic extracts had different IC50 values (EM1 >200 µg/mL and EM2 <10 µg/mL). Bos et al.23 showed the cytotoxic effects of different ingredients isolated from V. officinalis on human small-cell lung cancer cell and human colorectal cancer cell line (COLO 320) with MTT test. Valerenic acid and its derivatives, such as acetoxyvalerenic acid, hydroxyvalerenic acid, and methyl valerenate, which are obtained from V. officinalis, exhibited extremely low toxicity in both cell lines at the 100-200 µM concentration range.23. In our study, the calculated IC50 values of V. officinalis methanolic extracts were 939.68 µg/mL for HepG2 cells and 1097.58 µg/mL for Caco2 cells. Differences in cytotoxicity results among studies of Valeriana species may depend on the different subtypes of the plant, plant section differences, EM differences, and study cell type.

In our study, the exposure of HepG2 and CaCo2 to the methanolic extract of V. officinalis in all exposure groups did not induce significant ROS production levels. Our results indicate that the methanolic extracts of V. officinalis may not induce cell death in cancer cell lines via oxidative stress induction. These results can be associated with commercial products obtained from different herbalists, which are less effective or ineffective in cancer cells.


In conclusion, the effects of V. officinalis extracts on cancer cells should be analyzed in detail with further studies that include different extraction protocols and with cultivated medical plants to define the anticancer effects of V. officinalis.


The present study was supported by İstanbul University Scientific Research Projects Department (project no: 33246).

Conflicts of interest: No conflict of interest was declared by the authors. The authors alone are responsible for the content and writing of the paper.


  1. Ekor M. The growing use of herbal medicines: issues relating to adverse reactions and challenges in monitoring safety. Front Pharmacol. 2014;4:177.
  2. de Oliveria DM, Barreto G, De Andrade DV, Saraceno E, Aon-Bertolino L, Capani F, Dos Santos El Bachá R, Giraldez LD. Cytoprotective effect of Valeriana officinalis extract on an in vitro experimental model of Parkinson disease. Neurochem Res. 2009;34:215-220.
  3. Hamaidia M, Barez P, Carpentier A, Lebecque S, Miazek K, Paul A, Sriramareddy SN, Staumont B, Danthine S, Deleu M, Frédérich M, Pauw ED, Delaplace P, Delvigne F, Goffin D, Ongena M, Duysinx B, Louis R, Cosse J, Willems L. From Valeriana officinalis to cancer therapy: the success of a bio-sourced compound. Biotechnol Agron Soc Environ. 2016;20(Suppl 1):314-320.
  4. Şen B, Mat A. Chemical and medicinal evaluations of the Valeriana species in Turkey. Istanbul Journal of Pharmacy. 2015;45:267-276.
  5. EMA reports, Assessment report on Valeriana officinalis London, Doc. Ref. EMEA/HMPC/167391/2006. Last Accessed Date: 29.11.2007. Available from:https://www.ema.europa.eu/en/documents/herbal-report/superseded-assessment-report-valeriana-officinalis-l-radix_en.pdf
  6. Nandhini S, Narayanan KB, Ilango K. Valeriana officinalis: a review of its traditional uses, phytochemistry and pharmacology. Asian J Pharm Clin Res. 2018;11;36-41.
  7. Pandey A, Tripathi S. Concept of standardization, extraction and pre phytochemical screening strategies for herbal drug. J Pharmacogn Phytochem. 2014;2:115-119.
  8. Oztas E, Abudayyak M, Celiksoz M, Özhan G. Inflammation and oxidative stress are key mediators in AKB48-induced neurotoxicity in vitro. Toxicol In Vitro. 2019;55:101-107.
  9. Wu D, Yotnda P. Production and detection of reactive oxygen species (ROS) in cancers. J Vis Exp. 2011:3357.
  10. Kara M, Oztaş E, Ozhan G.  Acetamipride induced cyto-and genotoxicity in pancreatic cell line. Turk J Pharm Sci. 2020;17:474-479.
  11. Kakehashi A, Kato A, Ishii N, Wei M, Morimura K, Fukushima S, Wanibuchi H. Valerian inhibits rat hepatocarcinogenesis by activating GABA(A) receptor-mediated signaling. PLoS One. 2014;9:e113610.
  12. Matés JM, Segura JA, Alonso FJ, Márquez J. Oxidative stress in apoptosis and cancer: an update. Arch Toxicol. 2012;86:1649-1665.
  13. Wang F, Zhang Y, Wu S, He Y, Dai Z, Ma S, Liu B. Studies of the structure-antioxidant activity relationships and antioxidant activity mechanism of iridoid valepotriates and their degradation products. PLoS One. 2017;12:e0189198.
  14. Zhu Z, Shen W, Tian S, Yang B, Zhao H. F3, a novel active fraction of Valeriana jatamansi Jones induces cell death via DNA damage in human breast cancer cells. Phytomedicine. 2019;57:245-254.
  15. Sudati JH, Fachinetto R, Pereira RP, Boligon AA, Athayde ML, Soares FA, de Vargas Barbosa NB, Rocha JB. In vitro antioxidant activity of Valeriana officinalis against different neurotoxic agents. Neurochem Res. 2009;34:1372-1379.
  16. Yoo DY, Jung HY, Nam SM, Kim JW, Choi JH, Kwak YG, Yoo M, Lee S, Yoon YS, Hwang IK. Valeriana officinalis extracts ameliorate neuronal damage by suppressing lipid peroxidation in the gerbil hippocampus following transient cerebral ischemia. J Med Food. 2015;18:642-647.
  17. Sudati JH, Vieira FA, Pavin SS, Dias GR, Seeger RL, Golombieski R, Athayde ML, Soares FA, Rocha JB, Barbosa NV. Valeriana officinalis attenuates the rotenone-induced toxicity in Drosophila melanogaster. Neurotoxicology. 2013;37:118-126.
  18. Santos G, Giraldez-Alvarez LD, Ávila-Rodriguez M, Capani F, Galembeck E, Neto AG, Barreto GE, Andrade B. SUR1 receptor interaction with hesperidin and linarin predicts possible mechanisms of action of Valeriana officinalis in Parkinson. Front Aging Neurosci. 2016;8:97.
  19. Tian S, Wang Z, Wu Z, Wei Y, Yang B, Lou S. Valtrate from Valeriana jatamansi Jones induces apoptosis and inhibits migration of human breast cancer cells in vitro. Nat Prod Res. 2020;34:2660-2663.
  20. Han R, Nusbaum O, Chen X, Zhu Y. Valeric acid suppresses liver cancer development by acting as a novel HDAC inhibitor. Mol Ther Oncolytics. 2020;19:8-18.
  21. Quan LQ, Hegazy AM, Zhang ZJ, Zhao XD, Li HM, Li RT. Iridoids and bis-iridoids from Valeriana jatamansi and their cytotoxicity against human glioma stem cells. Phytochemistry. 2020;175:112372.
  22. Sen-Utsukarci B, Taskin T, Goger F, Tabanca N, Estep AS, Kessler SM, Akbal-Dagistan O, Bardakci H, Kurkcuoglu M, Becnel J, Kiemer A, Mat A. Chemical composition and antioxidant, cytotoxic, and insecticidal potential of Valeriana alliariifolia in Turkey. Arh Hig Rada Toksikol. 2019;70:207-218.
  23. Bos R, Hendriks H, Scheffer JJ, Woerdenbag HJ. Cytotoxic potential of valerian constituents and valerian tinctures. Phytomedicine. 1998;5:219-225.