Journal of Environmental Treatment Techniques  
2021, Volume 9, Issue 1, Pages: 196-202  
J. Environ. Treat. Tech.  
ISSN: 2309-1185  
Journal web link: http://www.jett.dormaj.com  
https://doi.org/10.47277/JETT/9(1)202  
The Anti-Cancer Property of Mumie as Natural  
Product on Human Cervical Cancer Cell Line  
(
HeLa)  
Azin Tavassoli, Malihezaman Monsefi  
Biology Department, College of Sciences, Shiraz University, Shiraz, Iran  
Received: 15/08/2020  
Accepted: 25/10/2020  
Published: 20/06/2021  
Abstract  
Mumie is a natural component found in some mountains, such as the Himalayas, as well as in some mountainous of Iran. It contains  
of humic and phenolic compounds that have antioxidant and anti-cancer properties. Therefore, in this study, anti-cancer and antioxidant  
properties of mumie were examined on Human Cervical Cancer Cell Line (HeLa). HeLa cells and normal fibroblasts (NIH) were cultured  
in DMEM/F12 with mumie at concentrations of 0, 100, 200, 300, 400, 500 and 1000 µg/ml for 24 and 48 h. The bioviability of these  
cells were evaluated using MTT assay. Chromatin condensation and apoptosis of these cells were examined using acridine orange and  
2 2  
aniline blue staining respectively. Antioxidant property of mumie on NIH cells was evaluated by 10 mM H O and neutral red test. MTT  
assay revealed bioviability of HeLa cells decreased but chromatin condensation increased in concentration of 100 μg/ml mumie treated  
culture. Apoptosis of the HeLa cells were observed in 100 μg/ml mumie treated culture. Mumie did not affect the bioviability, chromatin  
condensation and apoptosis of NIH cells but 500 and 1000 μg/ml concentrations were toxic and induced cell death. The cell cultures in  
different concentrations of mumie after 24and 48 h showed the similar results. NIH cells bioviability increased in 500 and 1000 μg/ml  
concentrations of co-culture of H O and mumie that confirmed the antioxidant property. It concluded that even low concentrations of  
2 2  
mumie could destroy HeLa cells without any side effect on normal cells. Therefore, it can be used for cervical cancer treatment but  
further research is needed.  
Keywords: Apoptosis, Cervical cancer, Mumie  
1
the natural substances that individuals have utilized as a healing  
1
Introduction  
compound for a long time. It is a brownish, rocky exudate  
found in some mountainous areas. The chemical content of the  
mumie is mostly originated from organic humic substances and  
plants (7). Other mumie components include phenolic  
compounds and some minerals such as fatty acids, albumin,  
sterols, amino acids, 3,4 benzocoumarin, aromatic carboxylic  
acids and polyphenols. The efficacy of mumie has been proven  
for treating diseases such as anemia, diabetes, high cholesterol,  
joint and bone disorders, and chronic pain (8). Also, it mainly  
used to treat people with weakness, inflammation, bone  
fracture, bleeding and wound healing (9). Based on (10)  
administration of 200600 mg/dose of shilajit in mice results in  
morphological and phagocytotic changes in peritoneal  
macrophages. (11) showed that shilajit can diminish gastric  
ulcer record by increasing mucus barrier. They also proved that  
shilajit has anti-inflammatory effect in carrageenan-induced  
acute pedal edema, granuloma pouch and adjuvant-induced  
arthritis in rats.  
Cancer burden rise to 18.1 million infection and 9.6 million  
deaths worldwide in 2018 (1). Common cancer treatments such  
as chemotherapy, radiation therapy, and hormone therapy have  
numerous side effects. Some typical side effects of  
chemotherapy are vomiting, constipation or diarrhea and hair  
loss (2). In recent years, increasing the new cases of cervical  
cancer among all types of cancer has become a major medical  
concern. However, the treatment of intermittent cervical cancer  
disease remains ineffectual to a great extent (3). Consequently,  
researchers are attempting to find more effective remedies to  
cure this type of cancer. One of the approaches that has been  
taken into consideration of numerous scientists today, is the  
utilization of natural compounds like herbs. The use of herbs in  
disease treatment has existed for thousands of years in many  
parts of the world. Plants are one of the most important natural  
sources for cancer treatment because many of them are capable  
to inducing apoptosis pathway and blocked in cancer cells (4).  
Approximately, 50ꢀ% of the modern anticancer drugs have  
been originated from the natural products. Therefore, the use of  
natural products in the development of new drugs has been a  
great interest for researchers. For the cancer treatment,  
Traditional Chinese Medicine has been used natural products  
as adjuvant therapy in conjunction with chemotherapy and  
radiotherapy for inhibiting the toxic effects of the treatments,  
as well as improving overall efficacy (5, 6). Mumie is one of  
There are many researches for healing effects of mumie in bone  
fracture. Also, some researcher has been reported anti-cancer  
property of mumie but we did not found its effect on cervical  
cancer. Considering the potency of mumie in curing all above  
mentioned medical complications, the aim of the present study  
was to investigate the anti-cancer properties of mumie on  
human cervical cancer cells (HeLa).  
Correspondence author: Monsefi Malihezaman, Prof. of Anatomy, Biology Department, College of Sciences, Shiraz University  
Shiraz-Iran. Tel: 0098-713-3167357, Fax: 0098-713-2280916, Email address: monsefi.g@gmail.com and  
monsefi@susc.ac.ir.  
196  
Journal of Environmental Treatment Techniques  
2021, Volume 9, Issue 1, Pages: 196-202  
method score 1 to 4 represents light blue nuclei or normal cells,  
semi-dense nuclei, dens nuclei and dark blue nuclei or  
compressed cells respectively.  
2
Materials and methods  
1
.2 Extract preparation  
Black mumie were purchased from a commercial source in  
Kerman, Iran. Mumie were powdered and 0.01 g were solved  
in 12.5 ml culture media in a water bath for 10 min. Mumie  
extract were vortexed for 10 minutes and sterilized with 0.45  
µm syringe filter. The concentrations of 50, 100. 150, 200, 250,  
5.2 Acridine orange/Ethidium bromide (AO/EB) staining  
AO/EB staining is used for evaluation of nuclear  
5
morphology in apoptotic cells. The number of 5×10 cells were  
cultured in each well of 24 well plate and then were treated with  
different doses of mumie for 24 and 48 h. After the treatment  
period, HeLa and NIH cells were harvested and rinsed with  
PBS. The pellets were resuspended in AO/EB solution  
including 5 μl of AO (Merck, Germany) and 5 μl of EB  
3
00, 400, 500, 1000, 2000 and 3000 µg/ml were prepared. After  
pilot study, the concentrations of 100. 200, 300, 400, 500 and  
1
000 µg/ml were selected.  
2
.2 Cell culture  
(
SinaClon, Iran). After 10 min, the cells were observed using a  
The effects of mumie on the human cervical cancer cell line  
fluorescence microscope (Nikon Eclipse-E600) and  
photographs were taken at ×100 magnification using a digital  
camera (Nikon, Japan). Acridine orange is a vital dye and stains  
both live and dead cells. Ethidium bromide stains only cells that  
have lost membrane integrity. Live cells will appear uniformly  
green. Early apoptotic cells stain green and contain bright green  
dots in the nuclei as a consequence of chromatin condensation  
and nuclear fragmentation. Late apoptotic cells also incorporate  
ethidium bromide and therefore stain orange, but, in contrast to  
necrotic cells, the late apoptotic cells show condensed and often  
fragmented nuclei. Necrotic cells stain orange, but have a  
nuclear morphology resembling that of viable cells, with no  
condensed chromatin. The nuclei of 100 cells in each  
concentration of each well were scored based on the percentage  
of cells nuclei color as normal cells, early apoptotic cells and  
apoptotic cells.  
(
HeLa) were evaluated. A NIH cell line from the fibroblast of  
Swiss albino mouse embryo tissue as a normal cell was cultured  
with the same method to compare with the cancer cell line.  
These cells were cultured in DMEM/F12 (Dulbecco's Modified  
Eagle Medium, Gibco, USA) containing 10% fetal bovine  
serum (FBS, Gibco, USA) and 5% Penicillin/Streptomycin  
(
Gibco, USA). The cells were incubated at 37°C and 5% CO2.  
The media were changed every 2 days. After 80-90%  
confluency on the flask, the cells were passaged using Trypsin  
0
.25% (Bio-IDEA Iran).  
Cells number and viability were estimated using a  
haemocytometer under a light microscope after vital staining  
with trypan blue (Sigma-Aldrich, USA). Number of 1×10 cells  
were grown and maintained in 100 µl per each well of 96 well  
plate and 5×10 cells in 200 µl per each well of 24 well plate.  
4
5
2
All cells were incubated at 37°C with 5% CO for 24 and 48 h.  
Each concentration was repeated in 3 wells of well plate and in  
three well plates separately.  
6
.2 H  
To investigate the antioxidant properties of mumie, H  
was added to various concentrations of mumie. H causes  
cell death by oxidation stress and DNA damage. Antioxidant  
substances were reduced H activity in the cells. The number  
2 2  
O assay and Neutral red staining  
2 2  
O
2 2  
O
3
.2 MTT assay  
To evaluate the cytotoxicity effects of mumie on the HeLa  
cells, MTT colorimetric assay was applied. Briefly, 1104  
2 2  
O
5
of 5×10 cells were cultured in each well of 24 well plate and  
then were treated with different doses