|Ahead of print publication
Anticancer, antioxidant, and hepatoprotective activity of Saussurea lappa, C.B. clarke (qust) on human hepatoma cell line
Shabnam Ansari1, Kazim Hasan2, Sajad Bhat2
1 Department of Biotechnology, Faculty of Natural Sciences, Jamia Millia Islamia, New Delhi, India
2 Department of Interdisciplinary Sciences, Faculty of Natural Sciences, Jamia Millia Islamia, New Delhi, India
|Date of Submission||06-Aug-2019|
|Date of Decision||09-Oct-2019|
|Date of Acceptance||19-Dec-2019|
|Date of Web Publication||09-Oct-2020|
Department of Biotechnology, Faculty of Natural Sciences, Jamia Millia Islam, New Delhi
Source of Support: None, Conflict of Interest: None
Background: Liver cancer is considered as one of the most widespread malignancies across the globe. According to a recent estimate, about 782,000 people were diagnosed with liver cancer, out of which 746,000 people died. Conventional anticancer therapy cannot fulfill all the clinical needs due to accessibility, clinical efficacy, and safety issues. Hence, the need of novel inexpensive drugs from traditional medicine as potential chemotherapeutic agent becomes utmost urgent. Root of Saussurea lappa, C.B. Clarke (Unani name, qust) has been used in the Unani medicine for the treatment of chronic liver diseases (warm-e-jigar sulb) including hepatocellular carcinoma since centuries.
Objective: The objective is to study the anti-cancerous, antioxidant, and hepatoprotective activity of different extracts of SLE in vitro.
Materials and Methods: MTT assay was used to determine the anticancer activity and EC50 of SLEs. Cell viability and cell inhibition were calculated. Apoptosis was studied by DAPI 4',6-diamidino-2-phenylindole (DAPI) staining. Thein vitro hepatotoxicity of CCl4 was produced and hepatoprotective properties of different concentrations of ethanolic (ESL), aqueous (ASL), and hydroethanolic extract of Saussurea lappa (HSL) have been evaluated by measuring cell viability in HepG2 cells.
Results: MTT assay revealed that the molecule reduced the cell viability of HepG2 cancer cells. Test drugs induced apoptosis in a concentration-dependent manner, as indicated by DAPI staining. In addition, ESL, ASL, and HSL also reduced the colony-forming potential of the HepG2 cell. ESL, ASL, and HSL were observed to protect the HepG2 cells from CCl4 induced injury in a dose-dependent manner.
Conclusion: The observed effect substantiated the anti-cancerous, antioxidant, and hepatoprotective activity of SLEs in HepG2 Cells.
Keywords: Liver cancer, qust, Saussurea lappa, unani medicine
|How to cite this URL:|
Ansari S, Hasan K, Bhat S. Anticancer, antioxidant, and hepatoprotective activity of Saussurea lappa, C.B. clarke (qust) on human hepatoma cell line. J Can Res Ther [Epub ahead of print] [cited 2020 Oct 28]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=297625
| > Introduction|| |
Hepatocellular carcinoma (HCC) is a global health concern with estimated 782,000 new HCC cases in 2012 that occurred in less developed regions of the world. According to the World Health Organization, HCC is the fifth most common tumor worldwide and the second most common cause of cancer-related death. In addition, besides, a recent study using the Surveillance, Epidemiology, and End Results registry projects that the incidence of HCC will continue to rise until 2030. Approximately 85% of global HCC cases occur in developing countries including India. Most of the cases occurred in persons 50–80 years of age and most involved whites. The primary risk factor for HCC is cirrhosis as 80% of cases of HCC occur in individuals with cirrhosis and the risk of developing HCC increases as fibrosis increases., Chronic viral infection (with hepatitis C virus or hepatitis B virus or both), alcoholic liver disease, and to a lesser extent, nonalcoholic fatty liver disease are the most common risk factors for the development of HCC.,
Symptoms may include abdominal pain, anorexia, early satiety, weight loss, obstructive jaundice, fever, watery diarrhea, and bone pain (from metastases)., Conventional medicine provides treatment such as surgical (liver transplantation and liver resection), locoregional (ablation, transarterial chemoembolization [TACE]), and systemic (e.g., sorafenib) for HCC depending on its stages. Palliative treatments for HCC are indicated if there is no curative treatment option, four palliative treatments are TACE, systemic chemotherapy, interferon, and hormonotherapy. However, palliative therapy of patients with HCC remains challenging as HCC is highly resistant to systemic therapies. The prognosis of HCC carcinoma remains poor despite early detection screening strategies. The 5-year survival rate is overall 12%, but for the patients who were diagnosed after the beginning of the symptoms, the survival rate is only 0%–10%.,,
On the other hand, Unani medicine provides a rehabilitative approach for the treatment of liver cancer. Unani medicine (Greeco-Arab medicine) is one of the oldest traditional systems of medicine and primarily based on the theories of homors and temperament of Hippocrates (Buqrat 460-377 BC), the “father of medicine,” also “father of Unani medicine.” Later on, Galen (Jalinoos), Rhazes (al Razi), Avicenna (Ibn-e-Sina), Al-Zahrawi, and Ibn Nafis are few of the names who have developed this medicine into an elaborate medical treatment. Unani medicine provides various range of treatment approaches for health maintenance and disease recovery starting from prevention, rehabilitation, drug treatment, and even surgery. Mostly (99%), drugs of herbal origin are utilized for treatment. Various herbal drugs are recorded to be beneficial as liver tonics in Unani medicine. Some of these herbal drugs have not only been substantiated for their potent antiviral, anti-inflammatory, and hepatoprotective activities but also evidenced to relieve disease symptoms., Thus, with this purview, the current study was carried out which aims to evaluate the anticancer, antioxidant, and hepatoprotective activity of root of Saussurea lappa, C.B. Clarke (qust) on the human hepatoma cell line. Qust has been used as a liver tonic in Unani medicine and substantiated their antiviral, hepatoprotective, and immunomodulatory activities in various studies.,,
| > Materials and Methods|| |
Plant material collection and identification
Qust roots were procured from the local market of Khari Baoli Old Delhi, India. The drug was identified by Dr. Sunita Garg, of NISCAIR. The voucher specimen (NISCAIR/RHMD/Consult/2018/3271-72) was authenticated samples as S. lappa, C.B. Clarke.
Preparation of different extract with solvents
Three types of extracts were prepared namely ethanolic extract of S. lappa (ESL), aqueous extract of S. lappa (ASL), and hydroethanolic extract of S. lappa (HSL) (50% w/v). Initially, roots of qust were cleaned for any adulterant and pulverized into powder with waring blender. Twenty-five grams of the powder was soaked in 150 mL of absolute ethanol, milli-q water, and 50% ethanol in 500 mL capacity flasks. The powdered drug was remained socked in the solvent system for 3 days in between gentle shaking four times daily. After 3rd day, the drug was filtered with Whitman filter paper grade four-under gravity. The filtered solution was centrifuged and the supernatant was lyophilized under high vacuum and stored in sterile vials in −20°C refrigerator. Stocks were prepared by dissolving 200 mg in 1 mL of dimethyl sulfoxide (DMSO) during experimentation.
HepG2, human liver hepatoma cells were obtained from hepatitis and liver cancer research laboratory of the Department of interdisciplinary sciences in basic research, Jamia Millia Islamia New Delhi. Cells were grown in standard conditions in cell culture flasks: Supplemented Dulbecco's Modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, 1% nonessential amino acids in cell culture flasks, and incubated in a CO2 incubator in a 5% CO2 and 95% humidity atmosphere. When the cells reached 70%–80% confluency, they were trypsinized, the cell count was done, and the cell viability was tested by trypan blue using a hemocytometer and plated at 3 × 104 cells per well in a 96-well microplate. Assays were done after the cells were attached to the flask.
Anticancer (inhibition) assay in HepG2 cells
HepG2 cells were treated with six different concentrations of ESL, ASL, and HSL starting from 5 mg/mL in serial dilution, and cytotoxicity was measured with the help of MTT assay. After 48 h, the media of treated cells (100 μL) were removed and each well was incubated with 50 μL of MTT at 37°C for 4 h. After incubation, the formazan produced thus produced were solubilized by the addition of 150 μL DMSO. The suspension was placed on a gel-rocker for 20 min, and then the absorbance was recorded at 490 nm by an enzyme-linked immunosorbent assay reader. The results were analyzed in triplicate and the percentage was calculated.
Dosage of working herbal extract
ESL (250, 125, 75.5 μg/mL), ASL (1000, 500, 250 μg/mL), and HSL (1000, 500, 250 μg/mL) were utilized forin vitro DAPI staining, antioxidant, and hepatoprotective evaluation.
DAPI staining for apoptosis
The morphological changes in apoptotic cells were assessed by fluorescent microscopy following DAPI staining. Briefly, HepG2 cells were seeded at a density of 5 × 105 cells/1 ml in 24-well plates, and then grown with three different concentrations of ESL, ASL, and HSL for 24 h in a 37°C incubator. After washing once with Phosphate buffered saline (1x PBS), cells were incubated with 5 mM H2O2 for another 12 h, then fixed in 4% formaldehyde for 1 h and permeated with 0.1% Triton X-100 (Sigma-Aldrich) for 5 min. Next, the DNA-specific fluorochrome DAPI (100 μM, Invitrogen) was applied to each well, after which samples were incubated for 10 min in the dark at room temperature. Finally, the cells were washed three times with × 1 PBS and examined using a confocal fluorescent microscope (Olympus FV3000) at × 400 magnification.
The evaluation of in vitro hepatoprotective effect
For the evaluation of hepatoprotective activity, cells were incubated with above mentioned three different concentrations of ESL, ASL, and HSL for 12 h. Vehicle control cells were incubated with DMEM advanced with DMSO (0.05% v/v). After 12 h, the media was changed with DMEM with 40 mM CCl4 and incubated for 1 h 30 min. To measure the cytotoxicity level of CCl4 in the test and control wells, MTT assay was performed. Cell culture supernatant after cytotoxicity was retrieved and measure for the levels of ALT and AST. The values of enzymes were expressed in IU/L.
Diphenyl-one-picrylhydrazyl radical-scavenging activity
The free radical scavenging effect of SLE's was determined using the stable scavenger two, two-diphenyl-one-picrylhydrazyl (DPPH) with slight modifications of the method described by Brand-Williams et al. Briefly, the hepatoprotective agents were dissolved in ethanol to obtain stock solutions (1000 μg/mL), from which serial dilutions were made. Diluted solutions (0.5 mL of each) were mixed with 0.5 mL of 125 μM DPPH and allowed to react for 30 min. Ultraviolet absorbance was recorded at 517 nm (Multiskan EX; Thermo/LabSystems, Vantaa, Finland). The experiment was performed in triplicate and the average absorption was recorded for each concentration. The same procedure was followed for the quercetin (positive control). The degree of DPPH purple decolorization to DPPH yellow indicated the scavenging efficiency of the extract. Lower absorbance of the reaction mixture indicated higher free radical-scavenging activity. The results were analyzed in triplicate. The half-maximal inhibitory concentration (IC50) value is the concentration of the sample required to inhibit 50% of the DPPH-free radical.
The results were expressed as mean ± standard deviation. Descriptive statistics was used to analyze the mean, standard deviation, variation, and level of statistical significance between groups. When P < 0.05 and P < 0.01, it was considered statistically significant for analysis.
| > Results|| |
EC50 of ESL, ASL, and HSL was 1.10 mg/mL, 2.85 mg/mL and 3.5 mg/mL respectively. All the extracts produced an inhibitory effect on the HepG2 human cancer cell line [Figure 1], [Figure 2], [Figure 3].
|Figure 1: Inhibition of HepG2 cells by ethanolic extract of Saussurea lappa|
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|Figure 2: Inhibition of HepG2 cells by aqueous extract of Saussurea lappa|
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|Figure 3: Inhibition of HepG2 cells by hydroethanolic extract of Saussurea lappa|
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DAPI staining for apoptosis
DAPI was used to stain the chromatin condensation in the nucleus of apoptotic cells. Apoptotic cells showed smaller and brighter nuclei than normal cells. Hydroethanolic extract of Saussurea lappa (HSL) induced cell death by increasing the percentage of apoptotic cells correspondingly to tamoxifen [Figure 4].
|Figure 4: DAPI staining (a) ethanolic extract of Saussurea lappa at concentration of 250 μg/mL, (b) aqueous extract of Saussurea lappa at concentration of 500 μg/mL, (c) aqueous extract of Saussurea lappa at concentration of 1000 μg/mL, (d) hydroethanolic extract of Saussurea lappa at concentration of 500 μg/mL, (e) hydroethanolic extract of Saussurea lappa at concentration of 1000 μg/mL, (f) Control well treated with tamoxifen|
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Measurement of diphenyl-one-picrylhydrazyl (anti-oxidant) activity
The DPPH radical-scavenging activity of ESL, ASL, and HSL was evaluated. The IC50 values were 40.71 ± 0.86 μg/mL, 18.1 ± 0.75 μg/mL, and 10 ± 0.55 μg/mL, respectively. Quercetin (used as a positive control) scavenged DPPH radicals completely, and its IC50 value was 2 μg/mL.
The evaluation of in vitro hepatoprotective effect
HepG2 cells were pretreated with a hepatoprotective agent (ESL, ASL, and HSL) and subsequently exposed to CCl4 to induce damage. Only ASL, HSL at a concentration of 1000 μg/mL or 500 μg/mL significantly (P < 0.001) decreased the levels of AST and AST while at concentration of 250 μg/mL decreases above enzymes at P < 0.01 compared to control wells. Pretreatment with ASL prevents the increase of ALT and AST in a dose-dependent manner (P < 0.01) while ESL only at a concentration of 250 μg/mL decreased ALT and AST levels significantly (P < 0.05).
On MTT assay, pretreatment with HSL and ASL prevented the CCl4 induced injury in treated wells in dose-dependent manner than control wells (P < 0.01). While ESL only at a concentration of 250 μg/mL prevented the cell injury significantly (P < 0.05) when compared with untreated control wells.
| > Discussion|| |
The anticancer activity of S. lappa can be attributed to its constituent “Lappadilactone” which has substantiated cytotoxicity against HepG2, OVCAR-3, and HeLa cell lines. The activity of 500 and 1000 μg/mL of hydroethanolic extract of S. lappa (HSL) in inducing apoptosis of the nucleus of the liver cancer cell line was almost equivalent to tamoxifen, as evidenced on DAPI staining. Jeong et al. observed that costunolide, a sesquiterpene lactone from S. lappa inhibits the VEGER KDR/FLK-1 signaling pathway similar to the finding of Sun et al. (2003), who have observed cytotoxic potential of sesquiterpene lactone from S. lappa roots.,,
The hepatoprotective effect of S. lappa against CCl4 induced cell injury in the HepG2 cell line can be attributed to its hepatoprotective nature evidenced in various animal studies. Yaeesh et al. have also observed that the aqueous extract of shank length has significantly restricted the progression of D-galactosamine (D-GalN) and lipopolysaccharide (LPS)- induced hepatitis in mice (P < 0.05).
| > Conclusion|| |
Aqueous and hydroalcoholic extract of S. lappa showed potential anti-cancerous, anti-oxidant, and hepatoprotective activity on the human hepatoma cell lines. Further studies should be conducted to elucidate the underlying mechanism in apoptosis and cytotoxicity, and substantiating efficacy in humans.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]