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Effect of combination therapy of HAART and aqueous extract of Nigella sativa

seed on the hematological parameters in rats
Kissi Mudie (kissimudiey at yahoo dot com), Feyissa Challa, Abiy Abebe, Asfaw Debella, Bekesho Geleta, Atsbeha Gebregzabher, Negero
Gemeda
Ethiopian Public Health Institute (EPHI), Addis Ababa, Ethiopia

phytotherapy
african traditional medicine
highly active antiretroviral therapy
hiv infections
nigella sativa

DOI http://dx.doi.org/10.13070/rs.en.1.1063
Date 2014-10-08
Cite as Research 2014;1:1063
License CC-BY

Abstract

Topics

Background: Highly active antiretroviral therapy (HAART) is very effective in suppressing viral

replication and has led to a significant reduction in the mortality rate of the disease and an increase in the
lifespan of HIV/AIDS patients. Since conventional medical care (CMC) co-exists with traditional medicine systems
in many regions of Africa and elsewhere, people may use medicine from one system exclusively or they may
acquire medicine from each health system and use it simultaneously or sequentially. Aim of the study: to
investigate the hematological alterations that may occur in use of combinations of aqueous extract of Nigella
sativa seed and highly active antiretroviral therapy (Lamivudine, Zidovudine and Efavirenz) in rats. Materials and
Methods: sixty rats weighed between 150-200g were randomly divided into six groups and each group comprised
of ten rats. Rats in group I were administered with distilled water. Rats in group II were administered with highly
active antiretroviral therapy only. Rats in groups III - VI were administered 100, 200, 400 and 800 mg/kg Nigella
sativa plus highly active antiretroviral therapy respectively. The treatments were given orally for 28 consecutive
days. On the 29th day, all rats were sacrificed under light diethyl ether anaesthesia; blood samples were collected
for the assessment of hematological parameters. Results: WBC count was significantly increased in animals
treated with Nigella sativa extract plus highly active antiretroviral therapy when compared to the animals in the
normal control group. However; all other hematological parameters did not show any significant difference among
treatments. Conclusion: Highly active antiretroviral therapy has an immune boosting property; the subacute
co-administration of highly active antiretroviral therapy and Nigella sativa have not brought any hematological
changes, indicating that the combination has no any hematotoxic effect.

Introduction
Human Immunodeficiency Virus (HIV) is a retrovirus that is responsible for causing AIDS, a condition in which the
immune system of the infected individual becomes compromised, hence making the patient susceptible to life
threatening opportunistic infections [1]. Highly active antiretroviral therapy (HAART) is very effective in suppressing
viral replication and has led to a significant reduction in the mortality rate of the disease; increase in the lifespan of
HIV/AIDS patients [2] [3] [4]. As conventional medical care (CMC) co-exists with traditional medicine systems in
many regions of Africa and elsewhere, people may use medicine from one system exclusively or they may acquire
medicine from each health system and use it simultaneously or sequentially [5].
In Africa, the majority of HIV patients rely on traditional herbal medicine (THM) for management of side effects and
other primary health care needs [6] [7]. This is because African traditional healers are not only more available and
accessible than health care professionals (HCPs), but also the majority of the local population also strongly believes
in the usefulness and power of traditional medicine [8]. Many patients take a broad range of natural health products
(NHPs) in addition to their conventional therapeutic products [9]. The Ministries of Health of several African nations
currently promote traditional medicines for the treatment of HIV and associated symptoms. In the case of South
Africa, the Ministry of Health is currently promoting the use of traditional medicines for the treatment of HIV and
associated symptoms, and the use of traditional medicines concurrently with antiretroviral treatments [6] [7]. Studies
in South Africa have shown that herbal remedies are good supplements to antiretroviral therapy because of their
immune boosting properties. A study in western Uganda found that 38% of HIV positive patients used traditional
medicines and antiretroviral drugs at the same time for the management of HIV infection [1].
The effectiveness of herbal remedies in HIV infection is not doubtful. There are many classes of herbal remedies that
have been found to inhibit one or more steps in HIV replication. Several natural products based anti-HIV surfaceactive agents, NRTIs, NNRTIs, integrase inhibitors and protease inhibitors [10]. Alkaloids derivatives herbal
remedies (e.g. Ancistrocladus korupensis) from tropical liana plant inhibit reverse transcriptase and HIV induced cell

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fusion. Pentosan poly-sulphate, a carbohydrate derivate inhibits HIV tat regulatory protein that strongly activates
transcription of proviral DNA. A coumarin herbal remedy in the form of canolides from tropical forest tree (Calophyllum
lanigerum) was rated as nonnucleoside reverse transcriptase inhibitor in potency [11]. Some HIV-infected people use
herbs for potential cure or symptom treatment. Some clinical studies have shown that herbal medicines might have
the potential to alleviate symptoms, reduce viral load, and increase CD4+ cells for HIV-infected individuals and AIDS
patients [12].
Some herbal remedies have been documented to be beneficial when used with conventional medicines. Coumarin
derived herbal remedies decreased drug resistance resulting from HIV mutation associated with non-nucleoside
analogue-nevirapine. Some herbal remedies have also shown to decrease toxicity associated with HAART [11].
Nigella sativa (N. sativa) is a dicotyledon belonging to the botanical family of Ranunculaceae of herbaceous plants
and known as black cumin seed. Nigella sativa is cultivated in many provinces of Ethiopia. It grows to a maximum
height of 60cm, has blue flowers and finely divided foliage. Small caraway-type seeds are produced within the
flowers [13] [14] [15]. The seeds of N. sativa are the source of the active ingredients of the plant [16].
Seed of the N. sativa has been used for medicinal purposes for centuries in Asia, Middle East, and Africa. It has
been traditionally used as a natural remedy for a number of ailments that include headache, stomachache, asthma,
chest congestion, hypertension, diabetes, inflammation, cough, bronchitis, fever, dizziness, and influenza and for
general well-being [14].
Phytochemical screening of the seeds of N. sativa have led to the discovery of many active principles of the N.
sativa like: nigellicine, nigellidine, nigellimine-N-oxide, thymoquinone, dithymoquinone, thymohydroquinone, nigellone,
thymol, arvacrol, oxy-coumarin, 6-methoxycoumarin, 7-hydroxy-coumarin, alpha-hedrin, steryl-glucoside as well as
rich amounts of flavinoids, tannins, essential fatty acids, essential amino acids, ascorbic acid, iron and calcium [17].
Nigella sativa has been extensively studied pharmacologically to justify its broad traditional therapeutic value, from
which it was found to have hepatoprotective and immunopotentiating properties. Previous study investigated that
aqueous extract have protected against carbon tetrachloride-induced acute hepatotoxicity through restoration of the
anti-oxidative defense system and down-regulation of the pro-inflammatory pathway [14].
A remarkable case study described an HIV patient who after undergoing treatment with Nigella sativa seed extract
experienced a complete recovery, with no detectable HIV virus or antibodies against HIV in their blood serum, both
during and long after the therapy ended. This study indicated that there are possible therapeutic agents in Nigella
sativa seed that may effectively control HIV infection [18].
Therefore, the general objective of this study is to investigate the effect of combined administration of aqueous
extract of N. sativa seed and highly active antiretroviral therapy on the hematological parameters in rats.
Materials and methods
Plant collection and preparation of the extracts

Seeds of N. sativa were purchased from Goro district, Bale zone 530 kms southeast of Addis Ababa in September
2013. The taxonomic identity of the plant was verified at the Department of Biology, Addis Ababa University. Voucher
specimen of the plant (k-001/2013) was kept at the national herbarium, Science faculty, Addis Ababa University.
The plant material was then carefully washed with distilled water to remove any extraneous materials, dried under
shade at room temperature, grounded to a coarse powder using an electronic grinder and the aqueous extract of the
seeds of the plant was prepared by decoction as follows: 1500mL of distilled water was added to 200g of the
powdered seeds and the mixture was boiled for 15 minutes with continuous stirring. After cooling, the solution was
decanted and the supernatant solution was filtered with 0.1mm2 mesh gauze. The filtrate was transferred into a
petridish and was frozen in a deep freezer overnight. On the next day the freezed extract was allowed to dry in a
freeze dryer (lyophilizer) under vacuum pressure at -40C and lower pressure (133x10-3mbar) for a week to obtain a
freeze dried product. After the extract was dried, it was collected in air tight plastic containers, weighed, labeled and
put in a desiccator for subsequent experiment [14] [19]. The weight of the dry extract was expressed as percentage
of the total mass of dry plant powder to determine the percentage yield.
Preparation of highly active anti-retroviral therapy

The three antiretroviral drugs used for the study (Lamivudine, Zidovudine and Efavirenz) were obtained from
department of pharmacy, Black Lion Hospital, Addis Ababa. The drugs were combined at the doses of 26.46 mg/kg
Lamivudine (3TC), 52.91 mg/kg Zidovudine (ZDV) and 52.91 mg/kg Efavirenz (EFV). The drugs were prepared by

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grinding the tablets into fine powder and dissolved in distilled water.
Extract preparation for the experiment

The graded concentrations of 100, 200, 400 and 800 mg/kg were prepared from N. sativa aqueous extract. HAART
and N. sativa aqueous extract were mixed together before administration. Only fresh drugs (prepared daily) were
used.
Experimental animals preparation

The experimental animals used in this study were 60 albino Wistar rats of both sexes, each weighing 150200g and
aged three months. All rats were maintained under the controlled conditions of temperature (25 2C), humidity, and
light (12 hours of light and dark) in the Animal House of Ethiopian public health institute (EPHI). The animals had free
access to food and clean tap water. The animals were housed in standard environmental conditions in stainless steel
cages. The rats were acclimatised for 7 days before the start of the experiment. During the acclimatization the
animals were fed with standard pelleted rat chow and water ad libitum.
Animal grouping and Drug dose

A modified method [20] was used for this test. In this study, 60 Albino rats were randomly allotted into one of the six
experimental groups, and each group consisted of ten rats:
Group I received only distilled water and served as a normal control.
Group II received only HAART and served as a positive control.
Group III received combination of HAART and (100 mg/kg) N. sativa seed extract.
Group IV received combination of HAART and (200 mg/kg) N. sativa seed extract.
Group V received combination of HAART and (400 mg/kg) N. sativa seed extract.
Group VI received combination of HAART and (800 mg/kg) N. sativa seed extract.
Animals were deprived of food before drug administration after which they were allowed access to food. A volume of
2mL of each treatment was administered for each rat by oral intubation (blunt intragastric catheter or gavage) once a
day in the morning at 9.00 a.m. for 28 consecutive days. The blunt intragastric catheter was cleaned, placed in an
oven and sterilized after each administration to avoid any contamination.
Blood sample collection

At the end of the experiment, animals were fasted overnight and anesthetized with diethyl ether. Immediately each
animal was placed in supine position on operating board. The extremities of the animals were stretched and fixed on
a dissecting board. The abdominal cavity was opened and blood sample was withdrawn by cardiac puncture using
sterile needle of 5ml syringe. The blood samples were placed in a test tube containing anticoagulant, ethylenedi-amine-tetra-acetic (EDTA), to prevent adhesion proteins (coagulation factors) in cell-cell and cell-matrix
interactions [21].
Hematological assay

The SYSMEX XT-1800i automated hematology analyzer measured and calculated hematological parameters
according to the standard procedures developed by National HIV/AIDS Laboratory of Ethiopian Public Health institute
as follows: Hematological analysis was performed in manual mode when the system is in READY status. The blood
samples were mixed manually, labeled and prepared in a test tube. Then the cap was carefully removed so as not to
spatter blood. Then the opened sample tubes were held under the sample probe, so that the probe immersed into the
sample. Then the start switch was pressed and the sample was aspirated. Finally, when the READY LED turned off
(and two short beeps sounded), the sample tube was removed. When the READY LED turned on again, the next
sample was prepared and the same process was repeated.
Statistical analysis

The data (expressed as mean SEM) were analyzed by one way ANOVA followed by TukeyKramer post hoc test
using SPSS software version16.0 program. P values less than 0.05 were considered to be statistically significant.
Results
Percentage yield from plant material

Percentage yield (%Yield) of the crude extract of N. sativa was calculated by the following formula: %Yield = weight
of the aqueous extract obtained / weight of the powder measured for extraction x 100. 29 grams aqueous extract
were obtained from 200 g of dried powder, with a yield of 14.5%.

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Effects of Nigella sativa seed extract on hematological parameters

Different values were obtained for the various hematological parameters of blood (Table 1). All the treated groups
(Group II - VI) showed significant increase (P < 0.05) in total WBC when compared to normal control group (Group I).
The remaining hematological blood parameters did not show any significant differences between the control and
treatment groups. However, RBC count, HGB concentration and HCT percentage slightly increased for rats in treated
groups when compared with the control group. On the other hand, among different doses of N. sativa, slight decrease
in RBC, HGB, HCT and MCV was observed as the concentration increases from 100 to 800 mg/kg.

Parameter

Normal
control

Positive
control

100mg/Kg +
HAART

200mg/Kg +
HAART

400mg/Kg +
HAART

800mg/Kg +
HAART

WBC
(103/uL)

3.280.47

7.830.43*

5.930.53*

8.470.76*

7.950.45*

6.240.12*

RBC
(106/uL)

6.561.00

8.160.25

8.740.11

8.620.17

8.580.22

8.170.12
15.970.14

HGB (g/dL)

15.151.17

15.900.38

16.970.37

16.780.27

16.600.58

HCT (%)

41.305.85

48.351.04

52.101.59

50.780.89

50.832.08

48.030.12

MCV (fL)

63.301.35

59.280.72

59.632.07

58.950.29

59.231.43

58.800.92

MCH (pg)

24.283.25

19.500.18

19.400.45

19.480.19

19.330.40

19.570.35

MCHC (g/dL)

38.184.61

32.880.10

32.600.35

33.080.28

32.670.23

33.270.38

PLT
(103/uL)

9.082.44

7.403.79

5.311.96

5.377.17

8.341.89

5.331.89

LYPHO (%)

70.955.50

70.602.20

70.305.57

70.204.95

46.8023.4

68.174.10

Table 1. Comparison of the Mean SEM of the hematological parameters. * The mean difference is significant at the 0.05 level
when compared to normal control.

Discussion
Hematological assays give vital information regarding the status of bone marrow activity and potential intravascular
effects. They are also predictive indices for evaluating the level of immune system of the body [22].
Measurement of total and differential WBC counts can be used as an indicator of immune toxicity while platelets help
the blood clot at the site of a wound. High platelet counts can be seen following strenuous activity, in some infections
and inflammatory conditions. Extremely low platelet counts can be associated with spontaneous bleeding.
Measurement of hematocrit, hemoglobin and RBC counts can be used to determine anemia which could be due to a
decrease in a total number of erythrocytes, or reduced RBC size (MCV), or reduced hemoglobin amount per
erythrocyte (MCH), or diminished concentration of hemoglobin per total erythrocytes (MCHC), or drop in hemoglobin
or rise in the number of RBCs destroyed [23].
In this study, hematological results of the WBC count in rats treated with aqueous extract of N. sativa and HAART
showed significant increase (P<0.05) when compared to the rats in the normal control group. This finding agrees with
previous work [3] in which the HAART (Lamivudine, Efavirenz and Abacavir) showed significant increment in WBC
count. This elevation of WBC count is an indication of the ability of antiretroviral drugs [3] and N. sativa [16] to boost
the immune system and reduce the risk of an opportunistic infection. Studies in South Africa have also shown that
herbal remedies are good supplements to antiretroviral therapy because of their immune boosting properties [1].
On the other hand, there were no statistically significant differences in all other hematological parameters (RBC,
HGB, PLT, HCT, LYM, MCV, MCH and MCHC) in rats treated with aqueous extract of N. sativa and HAART. This
result indicates that there is no lysis of blood cells and inhibition in blood cells synthesis by either the extract or
HAART. This finding is in line with an earlier report [24] that revealed administration of fixed dose combinations of
antiretroviral drugs had no sub-acute hematotoxic effects. According previous study [25] the non-significant increase
in RBC, HCT and HGB may be due to the activation of erythrocytes induced by the extract which were decreased as
concentration of N. sativa increased from 100 to 800 mg/kg.
Conclusion
This study demonstrated that HAART has an immune boosting property; the subacute co-administration of HAART
and Nigella sativa has not brought any hematological changes, indicating that the combination has no hematotoxic
effect.

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Declarations
Acknowledgment

The authors are grateful for financial support of this study which was provided by Ethiopian Public Health Institute.
Staffs of the directorate of Traditional and Modern Medicine Research sincerely appreciated for their direct and
indirect contribution during this work.

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