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.
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 . 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   . 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 .
In Africa, the majority of HIV patients rely on traditional herbal medicine (THM) for management of side effects and other primary health care needs  . 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 . Many patients take a broad range of natural health products (NHPs) in addition to their conventional therapeutic products . 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  . 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 .
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 surface-active agents, NRTIs, NNRTIs, integrase inhibitors and protease inhibitors . Alkaloids derivatives herbal remedies (e.g. Ancistrocladus korupensis) from tropical liana plant inhibit reverse transcriptase and HIV induced cell 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 . 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 .
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 .
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   . The seeds of N. sativa are the source of the active ingredients of the plant .
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 .
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 .
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 .
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 .
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.
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 -40°C 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  . The weight of the dry extract was expressed as percentage of the total mass of dry plant powder to determine the percentage yield.
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 grinding the tablets into fine powder and dissolved in distilled water.
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.
The experimental animals used in this study were 60 albino Wistar rats of both sexes, each weighing 150–200g and aged three months. All rats were maintained under the controlled conditions of temperature (25 ± 2°C), 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.
A modified method  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.
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, ethylene-di-amine-tetra-acetic (EDTA), to prevent adhesion proteins (coagulation factors) in cell-cell and cell-matrix interactions .
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.
The data (expressed as mean ± SEM) were analyzed by one way ANOVA followed by Tukey–Kramer post hoc test using SPSS software version16.0 program. P values less than 0.05 were considered to be statistically significant.
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%.
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|
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 .
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 .
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  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  and N. sativa  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 .
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  that revealed administration of fixed dose combinations of antiretroviral drugs had no sub-acute hematotoxic effects. According previous study  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.
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.
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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