Cytogenetic and Cytotoxic study of
Micromeria myrtifolia Extract on Animal and
Human Cancer Cell Line
Khulood W. Al-Samarraei Ebtehal H. Al-Naimy
Raghad K. Al-lihaibi Rafal S. Al-Ani
Biotechnology Research Center/Al-Nahrain University
Abstract
The study was designed to evaluate the cytogenetic effect of
Micromeria myrtifolia methanolic extract and cyclophosphamide
in albino male mice (in vivo). The cytogenetic evaluation included
the metaphase index of bone marrow. Two doses 200 and
400mg/kg of extract and one doses of cyclophosphamide 15mg/kg
were investigated as a positive control. Additionally the cytotoxic
effect of Micromeria myrtifolia on two cancer cell line was carried
out. The chemical detection of the flavonoids, polysaccharides and
alkaloids was also carried out. The chemical detection for active
compounds revealed that the methanol extract was positive for
flavonoids and polysaccharides and it was negative for alkaloids.
Also the result showed that M. myrtifolia caused a significant
increase in metaphase index of mice bone marrow cells in
comparison with the negative control (distilled water) and positive
controls (Cyclophosphamide). The methanolic extract showed
some inhibitory effect on L20B and RD cell line growth rate after
72 hr in comparison with control. From this study we conclude
that the M .myrtifolia extracts were effective in enhancing the
values of immunological parameters by increasing the metaphase
index of mice bone marrow cells and the study prove that the M
.myrtifolia extract has significant cytotoxic activity on two types of
tumor cell lines.
131
.2011 (6)ff
دراسة وراثية خلوية سمية لمستخلص نبات الزوفا
micromeria Myrtifolia في الخلايا السرطانية للانسان والحيوان
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خلود وهيب السامرائي ابتهال حسين النعيمي
رغد كاظم اللهيبي رفل شكيب العاني
مركز بحوث التقنيات الإحيائية/ جامعة النهرين
الخلاصة
أجريت الدراسة لمعرفة التأثير الوراثي السمي للمستخلص الميثـانولي لنبـات
الزوفا عقار السايكلوفوسفومايد في ذكور الفئران البيض، كما تضمنت هـذه الدراسـة
معامل الاقسام الخلوي الاستوائي لخلايا نقي العظم، وقد تم استخدام جرعتان بواقع 200
400 ملغم من المستخلص وجرعة واحدة من عقار الـسايكلوفوسفومايد بتركيـز 15
ملغم/ كغم، فضلا عن التأثير السمي لنبات الزوفا عبر خطين من الخلايا السرطانية، كما
تم اجراء الكشف الكيميائي عن مركبات الفلافينودات والسكريات المتعددة والقلويـدات،
وقد ظهر الكشف الكيميائي احتواء المستخلص الميثانولي على الفلافينودات والسكريات
المتعددة وخلوه من القلويدات، كما أظهرت هذه الدراسة وجود زيادة معنوية في معامل
الانقسام الخلوي الاستوائي لنقي العظم في الفئران مقارنة مع السيطرة السالبة (المـاء
المقطر) والسيطرة الموجب (السايلكوفوسفومايد) وأظهر المستخلص الميثانولي تـأثير
تثبيطي في نمو خطي الخلايا السرطانية (RD) (L20B (بعد مدة تعريض بلغـت 72
ساعة مقارنة مع السيطرة ومن هذه الدراسة نستنتج بان مستخلص نبـات الزوفـا ذو
قابلية على تعديل قيم المعاملات المناعية من خلال زيادة طور الانقسام الاسـتوائي فـي
خلايا نخاع العظم للفئران المختبرية واثبتت هذه الدراسة ان نبات الزوفـة ذو فعاليـة
سمية واضحة في خطي الخلايا السرطانية.
132
.2011 (6)ff
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Introduction
Herbal supplements are dietary supplements that contain herbs,
either singly or in mixture. A herb also called a botanical is a plant or
plant part used for its scent, flavor, and/or therapeutic properties.
Products made from botanicals that are used to maintain or improve
health have been called herbal supplements, botanicals, or
phytomedicines (20).
Herbal remedies and alternative medicines are used throughout
the world, and in the past, herb often represented the original sources
of most drugs. The plant kingdom has provided an endless source of
medicinal plants first used in their crude forms as herbal teas, syrups,
infusions, ointments, liniments and powder (24).
Interest in a large number of traditional natural products has
increased (30). It has been suggested that aqueous and ethanolic
extract from plants used in allopathic medicine are potential sources
of antiviral and anti tumor agents (8). Furthermore, the selection of
crude plant extracts from screening programs has the potential of
being more successful in its initial steps than the screening of pure
compounds isolated from natural products (15).
With respect to the former field ,and over the last two decades,
an expanding body of evidence from epidemiological and laboratory
studies has demonstrated that some edible plants as a whole, or their
identified ingredients, have substantial protective effects on human
mutagenesis and/or carcinogenesis (18). In this regard, a progress was
made to under stand the biochemical mechanisms of dietary and
medicinal anti-mutagens and anti-carcinogens, and the investigators
have broaden the horizons to cover various aspects of
chemoprevention by edible photochemical or their mixtures (29).
The immune system is further related target of the medicinal
plant research. In this context, it is interesting to note that it has been
recognized for several decades that nutrition and health are closely
interrelated, and much research has focused on the nutrition effect on
the immune system and its proper functioning (16). More recently, the
effect of nutrition on chronic degenerative disease has become an area
of intense study, bringing about a shift in the concept of optimal
nutrition away from merely preventing diseases stemming from
nutrient deficiencies to reducing the risk of chronic diseases (13). One
group of nutrients thought to play a vital role in such disease
prevention is antioxidants. Evidence has been accumulating over the
past few years that many plant constituents not previously thought of
133
.2011 (6)ff
as separate nutrients, for example the phenolic compounds, can act as
powerful antioxidants and immune modulators (26).
Micromeria spp. (Labiatae) are perennial herbs or
chamaephytes. The extract of these plants has been reported to have
some medicinal value, for example, the leaves have been reported to
possess anti-inflammatory and antimicrobial effects and are also used
against some other human ailments (inflamed eyes, wounds, skin
infections, stomachache, chest pain, colds, fevers, and others (21).
These plants are Known to be a rich source of essential oil
contents (mono and sesquiterpenes especially thymol, carvacrol), and
flavanoids, to which, the medicinal effects of Micromeria have been
ascribed (28).
This study aimed to extract the active compounds from
Micromeria myrtifolia and tested the cytogenetic and cytotoxicity
effect of the plant.
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Materials and methods
Laboratory Animals:
Albino male mice (Musmusculus) were used to carry out the
investigations of the present study. They were obtained from
Biotechnology Research Center (Al-Nahrain University). Their age
range was 8-9 weeks, and their weight was 23-27 grams at the
beginning of experiments. They were caged in the animal house of the
supplier, in which the temperature was 23-26°C, and a light: dark
periods of 10:14 hours/day.
The animals had free excess to diet (standard pellets) and
drinking water during all experiments.
Plant Extraction:
The plant powder was extracted with methanol solvents,50
grams of the powder were extracted in the solvent at 45°C for three
hours using the soxhlet apparatus. The resulted extract was
concentrated by rotary evaporated at 45°C, and the dry deposit was
obtained, the methanol deposit extract was dissolved in sterile distilled
water to prepare the doses (25).
Experimental Design:
Two doses of Micromeria myrtifolia were used to assess the
cytogenetic effects of extract, and their modulating effects of the drug
cyclophosphomide in albino male mice. Two stages were used in the
134
.2011 (6)ff
First Stage:
In this stage, the cytogenetic effects on mitotic index of
Micromeria myrtifolia and cyclophosphamide were investigated. The
animals were divided into three groups:
1.GroupI: treated with distilled water (negative controls = 4 animals).
2.GroupII: treated with cyclophosphamide at a dose of 15 mg/kg
(positive controls = 4 animals).
3.Group III: treated with two doses of the Micromeria myrtifolia
plant extract (200, 400 mg/kg) (8 animals).
The tested materials were given orally as a single dose 0.1 ml
per a day for 7 days. Then the mice were sacrificed in day 8 for
laboratory assessments. The total numbers of mice in this stage were
Second Stage:
Cell line study of the plant Micromeria myrtifolia was carried
out in Biotechnology Research Center/Al-Nahrain University . In this
study, the preliminary screening on cytotoxic activity of Micromeria
myrtifolia was carried out .
The screening involved the investigation of cytotoxicity of
methanolic extract of Micromeria myrtifolia, then the extract were
evaporated until complete dryness. The screening of cytotoxicity was
carried out on tumor cell lien L20B and RD.
The percentage of growth inhibition was calculated according
to (22), and according to following equation:
Growth inhibition % = ×100 −
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Control Treatment cell
Control
Laboratory Methods:
Mitotic index:
The metaphase index was assessed on somatic cells obtained
from the bone marrow of experimental animal mice, according to a
pre-established method (4), which was based on the following steps:
1. The animal was injected intraperitoneally with 0.25 ml of colchicin
solution with concentration of 1mg/ml, and after two hours,
the animal was sacrificed by cervical-dislocation.
2. The animal was dissected, and femur bone was removed and
transferred to two Petri dishes containing 5 ml of PBS.
135
.2011 (6)ff
3. The femur bone was cleaned from muscles and other tissues, and
both ends were cut. Then, the bone marrow was obtained with
PBS 5 ml using disposable insulin syringe, and collected in a
test tube.
4. The cell suspension of tube was gently pipette, and centrifuged
2000 rpm for 5 minutes.
5. After discarding the supernatant, the cell deposit was suspended in
10 ml of a warm 37°C, hypotonic KCl (0.075M), and
incubated for 30 minutes in a water bath 37°C, with shaking
every 5 minutes.
6. The tube was centrifuged 2000 rpm for 5 minutes, and the
supernatant was discarded.
7. The cell deposit was slowly suspended in 5 ml of cooled fixative
4oC, and incubated for 30 minutes at 4°C.
8. Step 7 was repeated, and the cell deposit was gently suspended in 1-
2 ml of cooled fixative, to prepare a single cell suspension.
9. Few drops 4-5 of the fixed cell suspension were dropped vertically
from a height of about 3 feeton cleaned slides to give chance
for nuclei and chromosomes to spread well.
10. The slides were air-dried, stained with Giemsa stain for 15
minutes, rinsed with distilled water, and left to dry at room
temperature.
11. The slides were examined under oil immersion lens 100X, and at
least 1000 cells (divided and non-divided cells) were scored.
Then, the percentage of metaphase cells (metaphase index)
was calculated according to the following equation:
Metaphase index (%) = x 100
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Number of Metaphase Cells ⎟
⎛
⎞ ⎜
⎝
Total Count
Cell line procedure:
This was applied according to the method adopted by (1).
136
.2011 (6)ff
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Results and Discussion
Metaphase index:
In the present study, only cells at metaphase were scored in
samples of bone marrow and there for the metaphase index was based
on the percentage of these cells. A treatment with Cyclophosphamide
caused a significant reduction in the metaphase index 2.44% as
compared to the negative control 2.76 %. In contrast the two doses of
extract were associated with a significant increased index 6.2% and
9.6% respectively as compared with both negative and positive control
Plant extract preparation is effectively and extensively used for
their medicinal properties, and they have become increasingly popular
worldwide (5).
Interest in a large number of traditional natural products has
increased (32; 30). It has been suggested that aqueous and ethanolic
extract from plants used in allopathic medicine are potential sources of
antiviral and anti-tumor agents (8). Furthermore, the selection of crude
plant extracts from screening programs has the potential of being more
successful in its initial steps than the screening of pure compounds
isolated from natural products (15).
One of the current strategies for drug discovery involved the
study of plant materials based on the ethnobotanical usage. The search
for anticancer drugs, use of a plant or plant materials for the treatment
of certain cancer-related disease can provide a guide for further
studies, this includes, cancer treatment, immune disorders, infectious
diseases, parasitic diseases and viral diseases (9).
The results indicate that Cyclophosphamide with its dose
resulted in the reduction of MI in mouse bone marrow cells. This may
be related to the proteins required for mitosis which were not
produced at the same quantities, or the code was not reached the cell
to induce it to proliferate, or the drug may cause the death of bone
marrow cells (31) or due to defect occurred in the mitotic spindle
composition during cell division (27).
The plant extract were significantly effective in increasing the
metaphase index of bone marrow cells, but the extent effect was dose
dependent. The lymphocytes are originated in the bone marrow
through the lymphoid lineage progenerater, which is the outcome
hemopoitic stem cell proliferation.
The latter outcome was investigated in term of bone marrow
metaphase index, which was significantly increased in mice treated
137
.2011 (6)ff
with the dose of plant extract, and therefore, their absolute peripheral
count was excepted to be increased. M. myrtifolia extract contain
polysaccharides, and in this regard Biringanine and co-workers 2004
(6) suggested that several plant extracts of the family extract have
some therapeutically activities that could be dependent on the extracts
content of polysaccharides, and also it has been recently demonstrated
that polysaccharides isolated from fungal spp. (Ganoderma lucidum)
accelerated the recovery of bone marrow cells and total leucocyte
count in immunosuppressed mice (34).
Many herbs have a long history of use and of claimed health
benefits. However, herbal supplements and botanicals have potent
pharmacologic activity and, consequently, contribute to potential
adverse effects and drug interactions (20).
Table (1): Metaphase index of bone marrow cells (mean ± standard
error) of albino male mice treated with Micromeria myrtifolia
concentrated filtrate, distilled water (negative controls) and
cyclophosphamide drug (positive control).
Groups Dose
Positive Control
(Cyclophosphamide
Drug)
Negative Control
(Distilled Water) 2.76±0.21 0.00 ---09- A
200 First dose 6.2±0.3 147.03 B
Second dose 400 9.6±0.30 34.69 C
Different letters in the same column: significant difference (P ≤ 0.05)
between means.
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Mean ±
Standard
Error %
Treatment
Efficiency
(%)
(mg/kg)
2.44±0.43 -18.44 15 A
138
.2011 (6)ff
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Figure (1): Cell in metaphase stage taken from mice treated with
extract showing normal chromosome.
Cytotoxicity of Plants Extracts on Tumor Cells:
The results of plant extract on their effect on both cell lines
shows there are also a significant differences (P<0.05) between means
of cell viability of each L20B and RD cultures treated with
Micromeria myrtifolia extract(Table 2).Micromeria myrtifolia cause a
significant decrease (P<0.05) in cell viability of L20B and RD cell
line for all concentrations to reach maximum significant decrease at
concentration 1000μg/ml in comparison with the negative
control(Figure 2 ; Figure 3).
However, natural products provide an inexhaustible source of
anticancer drugs in terms of both variety and mechanism of action
The use of herbal supplements by cancer patients in the
preoperative period is prevalent and consistent with the substantial
increase in the use of alternative medical therapies by cancer patients
(14). Anywhere from 25% to 85% of cancer patients are seeking
alternative and complementary nutritiona4l therapies for prevention or
during cancer treatment. The use of these therapies is highest among
patients with breast cancer 80% to 85% (19), prostate cancer 27% to
43% (17), and head and neck cancer 25% (10). In a study of 820
cancer patients receiving chemotherapy or radiation therapy, 29.1%
reported using complementary integrative nutritional therapies that
were not prescribed by their physician (14).
139
.2011 (6)ff
Such findings can be considered important, especially if we
consider that methanol extract of members of Lamiaceae family
showed anti- carcinogenic effects, in vivo and in vitro and the dose
was effective in this regard (3; 11). Equally important, carcinogenesis
is normally preceded by mutation induced by different agents,
especially those that have oxidant effects(33; 2).
The terpens are a class of essential oil terpens that have many
biological and pharmaceutical activities, which can be useful to treat
human disease; for example, volatile terpens as monoterpens
sesquiterpens are known to have several pharmacological activities
including antibacterial, antifungal, antispasmodic (7), also they can be
used as potentiators of anti tumor agents which can increase
bioavailability of an orally administered hydrophopic pharmaceutical
compound by inhibition of cytochrome p450 and /or decreasing of p-
glycoprotein drug transport (12).
Table(2): Cytotoxic effect of Micromeria myrtifolia extract on the
growth of cancer cell line(L20B,RD).
Cell viability (Absorbance)
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M.myrtifolia M.myrtifolia
% (m)
L20B RD
A
0.864
B
0.225
C
0.156
C
0.149
D
0.134
D
0.133
E
0.121
Concentration
Negative control A
0.312
75 B
0.281
125 B
0.277
250 B
0.277
500 C
0.210
750 D
0.136
1000 D
0.134
Differences A,B,C,D,E are significant (p<0.05) to compression
140
.2011 (6)ff
(3)fx א{%w} א b}אhאא
Figure(2): Growth inhibition percentage of Micromeria myrtifoliaon
L20B after 72hr.
Figure(3): Growth inhibition percentage of Micromeria myrtifoliaon
RD after 72hr.
141
.2011 (6)ff
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