Camel ( Camelus dromedarius ) raw milk’s hypotensive roles within chemical induced hypertension model in rats

Abstract

Dromedary camels (Camelus dromedarius) inhabit not only arid areas but are among common domestic animals that are normally kept for numerous uses. It’s raw milk (CM) is believed to have exceptional nutraceutical value in addition to it’s other uses similar to camel itself. This study aimed to evaluate hypotensive efficacy of raw CM within context of chemical induced hypertension model in albino rats. Rats received the chemical; L-NAME (50 mg/kg body weight/day, p.o.) and amlodipine (10 mg/kg/day, p.o.) as negative and positive controls for 4 weeks. Rats of treatment group received concurrently L-NAME (50 mg/kg body weight /day, p.o.) and raw milk of camel at (100, 300, and 500 mg/kg body weight/day p.o.) respectively for 4 weeks. Result showed significant decrease (p < 0.001) in treatment relative to negative control in all measured parameters viz systolic, diastolic and mean arterial blood pressures in accordance to the used dosages. Also elevated levels of liver/kidney biomarkers in negative control became reduced compared to normal and positive controls courtesy of CM treatment. In conclusion, obtained data revealed CM to be effective in controlling hypertension. The bioactive constituents present in CM appeared likely to be responsible for the observed effect of antioxidant action and ACE inhibition. Evidence is thus provided from research findings that raw CM can afford efficient hypotensive effect.

Keywords

Raw camel milk (CM)hypotensive milk composition ACE inhibition

1 Background

Camel milk and its constituent functional ingredients are special and both are of great nutritive value [1]. The superiority of camel milk over bovine milk and milk of other domestic animals is unmatchable and the milk is parallel to human milk in terms of its nutritional value, ostensibly due to high concentrations of many bioactive compounds that are essential for human health which it contained [2], thus in terms of nutrition it is more beneficial [3]. The health benefits of camel milk have been described for a variety of diseases such as diabetes, kidney disease, hepatitis, etc. including overall improved survival [4].

Moreover, the COVID-19 global pandemic has proliferated the increasing recognition of camel milk value amid the ongoing pursuit of immune-boosting foods by consumers during both pandemic period and beyond [5]. The milk of camel is better tolerated than milk of other ruminant animals, thus potentially expanding its consumer appeal [3].

There are essential vitamins, minerals, and immunoglobulins, contained in raw camel milk which provides it with antioxidant, antibacterial, and antiviral properties. The aforementioned properties may reduce oxidative stress among consumers of camel milk, thereby ameliorating many conditions, notably those of the Central Nervous System; such as autism spectrum disorders (ASDs) as outlined in some performed clinical trials [3].

Some unique bioactivities and therapeutic potential of raw camel milk were demonstrated especially against diabetes and allergy [6]. In the past few decades, there were reported widespread market exposure and commercialization of camel milk because of its advertised health benefits parallel to the increasing prevalence of non-communicable diseases (NCDs) and the continuously increasing health consciousness of its consumers [7]. Camel milk is available in the market and can be found in form of many products including powdered camel milk, coffee, cheese, and ice cream that are sold in many developed countries [8, 9].

Because of it’s proposed healing properties and disease prevention mechanisms; camel milk is being used extensively within variety of populations [10]. A very prominent role is being played by camel milk in the treatment of many serious diseases in many parts of the world, due to it’s richness in numerous bioactive components [11]. The antibodies that are present in camel milk were believed by scientists that they could be effective against cancer, Alzheimer’s disease, hepatitis C and HIV/AIDS [12, 13] and [14].

Different research groups have reported therapeutic value and use of camel milk in managing Helicobacter pylori infections, enterocolitis, lactase deficiency in children [15, 16], pulmonary tuberculosis [17], liver cirrhosis [18], and cancer [19]. Additionally, the unique composition of camel milk that includes multiple protective proteins such as lysozymes, immunoglobulins, and lactoperoxidase, has made it similar to human breast milk and it could serve to confers protection against infection and bolster up immunological responses [20, 21].

There is prevalence of chronic diseases such as obesity, cancer, hypertension, and diabetes which are continuously increasing in parallel to the modern lifestyle of people, especially in developed countries [22]. The contribution of natural products in offering valuable source of potent compounds with wide variety of biological activities and novel chemical structures, many of which might pave the way for novel drug development can not be over emphasized [23]. Compounds and many materials from natural sources appeared to present lesser side effects and are better tolerated during their biotransformation, which importantly drives and geared global attention to searching for new drugs from natural sources [24].

Camel milk is just as natural as its meat, urine, hide and skin. The milk of animals are liquid food-grade which plays prominent role in human nutrition because of its rich nutrient composition and accruable beneficial health effects [25].

Even though, emergence of Middle East Respiratory Syndrome-Corona Virus; (MERS-CoV) infection was associated with camels; there are still unresolved questions regarding the zoonotic aspects of MERS-CoV, which is an emerging corona virus [26, 27]. And camel milk was surprisingly possessive of specific antibodies in place; which may also provide coronavirus cross-protection [28].

Consumption of camel milk in general is made without it’s boiling, there by retaining its various biological and immunological properties which are normally lost in the processes of milk preservation [29]. It is normally and widely consumed either as raw/unprocessed or when processed (condensed, pasteurized, powdered, liquid, heat-treated or UHT-treated) milk [30, 31].

Camel milk is used medicinally for centuries by nomadic people and as at 2016 there were many studies that have reported use of camel milk in the treatment of various human diseases [11]. Thus, camel milk can be used to treat Diabetes, food allergies, cancer, Hepatitis B and C, Autism, psoriasis, gastrointestinal disorders, high cholesterol in the blood, strengthening of immune system, tuberculosis and others [11].

Hypertension, on the other hand was described as medical condition in which there is chronic elevation of arterial blood pressure [32]. Many diseases such as coronary heart diseases, atherosclerosis, kidney diseases and stroke [33], and substantial contribution to cerebrovascular complications [34] were made possible courtesy of hypertension; which is well-defined risk factor for the disease. It has become one of the most common preventable causes of premature mortality worldwide and it was estimated to affects approximately 1 billion individuals [32], and it still account for about 12.8% of all annual deaths worldwide [35].

Many classes of antihypertensive drugs were provided by the modern medicine [32]; but irrespective of their presence in large number; however achievement of target blood pressure in clinical practice is realized only in minority of patients. The efficacy of monotherapy to control hypertension is limited arguably arising from further complications which includes presence of other disease conditions like diabetes mellitus in some patients, occurrence of other side effects and recorded history of poor adherence to drug therapy [36].

Herbal remedy and other natural sources were considered alternative and option by people in developing countries with the sole aim of bridging efficacy and cutting therapeutic costs in hypertension control and its complications distinct from the flaws observed using the conventional drugs for hypertension treatment [32].

There are currently multiple studies that reported camel milk as supplemental therapy and alternative option for treatment of a number of disease conditions and also there are increased number of scientific publications that focused on the medicinal potency of camel milk with its special components.

In view of ulterior motive behind upward surge in oral raw camel milk consumption it became imperative to assess and evaluate each claimed folkloric effect bearing in mind induced disease condition in animals is one of several ways incorporated in research efforts. Chemical induced-hypertension; using Ng Nitro-L-Arginine Methyl Ester (L-NAME) was incorporated in this research that involved studied albino rats. Indeed an induced-hypertension in rat models has been one of several methods of investigating antihypertensive agents. There were reported camel milk studies in other forms of induced hypertension including salt-load, surgical operation and etc. Also after evaluating the claimed effect it becomes necessary to examine some metabolic organs usually involved in biotransformation of all ingested materials. Such that safety or otherwise of the investigated material can be affirmed from the histopathological examination.

2 Objective

To examine and assess the roles of and hypotensive efficacy of raw camel milk; CM within context of chemical induced hypertension in albino rats. Nitro-L-arginine-methyl ester (L-NAME) was the chemical used for induction of hypertension Moreover, to evaluate the safety status of raw camel milk from histopathological assessment of liver and heart of the rats that were used in the study.

3 Methods

3.1 Chemicals and reagents

NG-nitro-L-arginine Methyl Ester (L-NAME) used for hypertension induction was procured from Sigma Aldrich (Germany). Reagents, chemicals and kits used in determination of various biochemical parameters in this study were all of analytical grade and are purest quality available. Among kits used they includes Randox; used for alkaline phosphatase determination and Colorimetric bilirubin assay kit (Catalog Number KA4555) from Abnova company used for conjugated bilirubin assay. Optimal temperature used for the kits storage was 2–8°C. Before being used, the kits were left for 1 h at room temperature. All kits were used in accordance with instructions of their manufacturer’s.

3.2 Preparation of CM

Five (5) number of healthy lactating domesticated female camels (Camelus dromedarius) of 4 to 10 years age were tracked and identified at Kwakwalawa village along main road to permanent site of Usmanu Danfodiyo University, Sokoto to provide the needed milk sample. The milk was collected via hand milking from skilled and experienced camel attendant after camels’ udders were thoroughly cleaned and sterilized in the morning time. Gallon-full (5litres) of milk samples from each camel was obtained. The collected milk samples were labeled A-E and transported via frozen ice pack cold chain medium in big vaccine container to University Post Graduate Central Research Lab at main campus within (<4 hours) of its collection where they were all immediately refrigerated at –20°C until required for further use.

3.3 Preparation of experimental rats and hypertension induction

Male Wistar rats (16 weeks old) weighing between 250–300g were purchased from Animal Laboratory Center of Department of Pharmacology and Therapeutics of Ahmadu Bello University, Zaria, Kaduna State, Nigeria. The rats were kept in well-ventilated cages at room temperature (28–30°C). They were maintained on normal laboratory chow (Vita Feeds^®, Grand Cereals, Jos, Nigeria) and were also allowed free access to their drinking water.

Rats were randomly divided into 6 groups of 5 rats per each group and housed in 6 individual cages under a 12-h light-dark cycle at room temperature of 25–30±1°C and relative humidity of 55±5%. The rats were allowed two weeks to acclimatize with their new environment prior to commencement. The research ran for 4 weeks and terminated on day 29 (24h post last treatment). Treatment was as follows: Group 1 (normal control, NC) received only vehicle (Distilled Water) orally daily during treatment period for 4 weeks, they neither had hypertension induction nor any treatment or intervention given to them; Group 2 (hypertensive negative control, HNC) received orally L-NAME only (50mg/kg body weight/day) daily for 4 successive weeks; in addition during treatment period they received commensurate volume of distilled water. Group 3 (reference group or hypertensive positive control, PC) received orally daily amlodipine (10mg/kg/day) concurrently for 4 successive weeks.

Groups 4, 5, and 6 received orally raw camel milk according to their body weight. Group 4 received 100mg/kg/day dosed as (CM1), Group 5 received 300mg/kg/day of raw CM dosed as (CM3) and Group 6 as (CM5) received 500mg/kg/day of raw CM concurrently with L-NAME dose.

All rats irrespective of which cage or group they were are allowed free access to food and tap water ad libitum. All rats except the NC or Group 1 were orally administered 50mg/kg body weight/day dissolved L-NAME during experimental period which made them inducted with hypertension.

3.3.1 Ethics Statement

All animal studies were performed according to the US National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and the experiments with rats was approved by the University Committee for Animal Use and Care and all ensuing experiments have followed the universal guidelines in accordance with procedure of recommendation of US based National Institutes of Health (NIH) (NIH publication for laboratory animal care and use).

Indirect measurement of rat BP (by non-invasive method) was done with tail-cuff procedure; which involved rats tails being inserted in a cuff which is connected with electrosphygmomanometer (Model 179; Blood Pressure Analyzer IITC, Woodland Hills, USA).

Rats’ SBP and DBP for each of involved 6 groups were measured initially before hypertension induction and then measured at 4, 8, 12 and 24 hours after induction/concurrent treatments. Tail cuff is inflated and deflated. Pulsations disappear when cuff is inflated. When cuff is deflated pulsations start appearing, the pressure in cuff equals to systolic pressure. Cuff is attached to tail-cuff sphygmomanometer and BP measured is recorded on chart.

By end of each week (from weeks 1, 2, 3 and 4 (day 28), 12 hours post induction/treatment, SBP and DBP were measured and recorded. On day 29 (after 10 h fasting) rats of all cages were restrained in containers and warmed for 30min to stabilize them at 28°C in thermostatically controlled heating cabinet (UgoBasille, Italy) which is connected to tail-cuff electrosphygmomanometer. The warming/stabilization were done to allow better detection of tail artery pulse, when those tails passed through the cuff. Five measurements were done for each rat. Maximum and minimum values were discarded, and mean average of the remaining three values was calculated as BP for a rat.

3.4 Biochemical assays (kidney and liver function biomarkers)

3.4.1 (a) Preparation of serum

On day 29 (24h post last treatment and 10 h fasting), rats were humanely killed by lightly anesthetizing them with anaesthetic ether (ether/chloroform mixture dipped with cotton wool) to induce rapid unconsciousness and subsequent death without pain or distress. Rats were subsequently sacrificed. Blood sample from inferior vena cava was collected into tubes containing anticoagulant. Blood samples were immediately centrifuged at 4000rpm/10min to separate serum that will be used for measurement of biochemical indices, and plasma was stored at –40°C until it is required for subsequent further analysis.

3.4.2 (b) Biochemical Analyses

Urea was assayed based on [37] method and creatinine was determined by Jaffe’s reaction of [38] method. Electrolytes (sodium, potassium, chloride and bicarbonate) were assayed based on [39]. Liver marker enzymes; Aspartate (AST) and Alanine (ALT) transaminases were assayed using Randox kit according to standard method described by [40]. Using Randox (colorimetric) kit; Alkaline phosphatase (ALP) was determined by [41] method. Total Protein (TP), was assayed by biuret method reported by [42]. Albumin (ALB) was assayed by bromocresol green method in accordance with [43]. Total bilirubin (TB) was measured according to Malloy and Evelyn method reported by [44] while conjugated bilirubin (CB) was determined using standard kits (Merck Specialities Pvt. Ltd., India).

3.4.3 (c) Evaluation of metabolic organs

On final day of research (day 29), after rats were humanely killed via chloroform/ether mixture anesthesia; and after their blood sample was collected into properly labeled specimen bottles. Whole liver, kidney and heart organs were harvested by their quick removal, washed in ice-cold normal saline (1.15% KCl solution), dried and weighed and kept at –80°C until required for further analyses.

The organs were embedded in paraffin wax and processed for obtaining 4μm section and stained for evaluation using Hematoxylin and Eosin. Parts of liver, kidney and heart tissues were placed in 10% neutral buffered formalin for histopathological examination which was done by pathologist in accordance to methods described by [45] and [46].

3.5 Statistical analysis

Data were analyzed by one-way ANOVA and Fisher’s least significant difference test using Statistical Analysis System [47]. Results are expressed as mean±SEM. Values of p < 0.05 were considered to indicate statistical significance.

4 Results

Obtained results from the study are presented as figures in form of histograms for the effects observed and changes in mean values of calculated mean arterial blood pressure (MABP), systolic blood pressure (SBP), and diastolic blood pressure (DBP) against mean values of milk treatment groups CM1, CM3 and CM5 juxtaposed and compared alongside NC, PC and HNC mean values.

Also changes and effects observed in the mean values of Liver and Kidney function parameters for all the groups in the study are presented in various Tables expressed as Mean±SEM.

Liver and heart photomicrographs of some rats that represents their individual groups within various experimental sets were presented from the performed histopathological evaluation including their assessment report. Comparison of some controls with some doses of raw CM administered were made.

4.1 MABP, SBP and DBP HISTOGRAMS AND TABLES

Presented below in various Tables, which are expressed as Mean±SEM were changes and effects observed in the mean values of SBP, DBP and MABP against controls (normal, positive and negative) from beginning to end of 4 weeks, the weekly SBP, DBP and calculated MABP occurrences as well as Liver and Kidney function parameters for all the groups involved in the study.

4.2 Metabolic organs evaluation

(a) The histopathological peculiarities of all control groups for their Heart, Liver and Kidney are presented in Tables 8–10 hereunder.

(b) Characteristics of liver cells histopathology

The histopathological characteristics of liver cells have changed upon administration of raw camel milk as shown in Table 9.

c) Cardiac (heart) cells peculiarities

The peculiarities of cardiac cells histopathology has not remarkably changed even with the different administered graded doses of raw camel milk to rats. All the 3 used doses, the heart picture appeared normal. Summary of the peculiarity of the heart histopathology is presented in Table 10.

Table 8
Histopathological feature of all control groups for Heart, Liver and Kidney

Group Heart Liver Kidney

1 (NC) Normal Mild portal and focal inflammatory cellular infiltration, congested blood vessels andmicrovacuolation Glomerular shrinkage and tubular swelling

2 (HNC) Normal Focal vascular congestion, necrosis with mild inflammatory cellular infiltration Glomerular shrinkage and tubular swelling

3 (HPC) Normal, few hadmild cellular infiltration Microvacuolation with oedema fluid accumulation Glomerular shrinkage, tubular swelling, tubular congestion and haemorrhage

Group	Heart	Liver	Kidney
1 (NC)	Normal	Mild portal and focal inflammatory cellular infiltration, congested blood vessels andmicrovacuolation	Glomerular shrinkage and tubular swelling
2 (HNC)	Normal	Focal vascular congestion, necrosis with mild inflammatory cellular infiltration	Glomerular shrinkage and tubular swelling
3 (HPC)	Normal, few hadmild cellular infiltration	Microvacuolation with oedema fluid accumulation	Glomerular shrinkage, tubular swelling, tubular congestion and haemorrhage

Group 1:–normal control, are normal rats they neither had hypertensive induction nor any treatment given, group 2:- hypertensive negative control, they are hypertensive but were not given any treatment or any intervention, group 3:-hypertensive positive control, they are hypertensives treated with amlodipine 10mg/kg body weight/day.

Table 9

Histopath characteristics of liver cells of rats administered raw camel milk

Doses (mg/kg body weight)	Feature
(100)	Normal
(300)	Many are normal few had focal congestion
(500)	Many are normal. Varied observations, few being normal yet others had severe congestion, haemorrhage and some with congested cortical veins

At various treatment doses (100–500mg/kg body weight/day) of raw camel milk administered to L-NAME induced hypertensive rats; liver tissue is normal with few exceptions among the 300mg and 500mg/kg body weight/day.

Table 10

Cardiac tissue pathological peculiarities of rats administered raw camel milk

Doses (mg/kg body weight)	Feature
(100)	Majority are normal, few cells had slight focal capillary congestion
(300)	All cardiac cells are normal
(500)	Many cells are normal but fewhad capillary congestion

Lesions were observed only in raw camel milk treatment doses (100mg/kg body weight/day) and at (500mg/kg body 8 weight/day).

4.3 The slides of histopath studies

Plate 1

Liver photomicrographs of rats in raw camel milk toxicity studies. (H and E X400). A:-PC:-Positive control, hypertensive rats given amlodipine (10mg/kg body weight/day). It has microvacuolation with oedemafluid accumulation and congestion. B:-CM1:-Induced-hypertensive rats administered 100mg /kg body weight/day of raw camel milk. The liver is normal. C:-CM3:-hypertensive rats given raw camel milk at 300mg/ kg body weight/day. The liver is normal. D: -CM5:-Induced-hypertensive rats administered 500mg /kg body weight/day. The liver has congestion of vessels and microvacuolation.

Plate 3

Heart photomicrographs of rats in raw camel milk toxicity studies. (H and E X400). A:-NC, normal control rats that received only distilled water during the study. The cardic tissue is normal. B:-HNC; hypertensive negative control rats this heart picture is normal. C:-RM; raw camel milk only without any treatment. It has a normal heart.

5 Discussion

In this present study; L-NAME was employed to induce arterial hypertension. Arterial hypertension occasioned by L-NAME administration is associated with deficiency of nitric oxide (NO) [48]. Generalized NO deficiency and progressive increase in BP if prolonged is characteristic of L-NAME induced-hypertension; which is a well-established experimental model [32]. Evidences from Figures 4.0 and 4.1 which are histograms of calculated mean arterial and systolic blood pressures as well as Tables 1, 2 and 3 have variously depicts effects of L-NAME intoxication which was orally administered to rats of different treatment groups and it have affected all the groups but most pronounced was the negative control (HNC) set; which were given only dissolved L-NAME solution orally devoid of any intervention. In the obtained results; rats of HNC group had the highest calculated MABP values (Figure 4.0 and Tables 1 and 5), highest values of measured SBP (Figure 4.1 and Tables 1 and 3) and DBP (Figure 4.2 and Tables 1, 2 and 4). The figurative shape and appearance as well as values of hypertensive negative control (HNC) set had surpassed it’s control peers (normal and positive controls) within contexts of Figures 4.0–4.2. Data of Table 3 which compiled weekly values of measured SBP is of particular interest in the sense that even the group of normal control devoid of any treatment have had slight increase in BP but when compared with the negative control (HNC) that also similarly lacks any form of intervention a worrisome picture with increased BP for each week is depicted. Positive control which counters the increase in BP with amlodipine usage when compared with the 3 categories of CM interventions; it can be seen that use of the latter have progressively succeeded in decreasing overall the elevated BP tilting it back to normalcy with such fidelity of their result value comparable to the effect observed for the positive control drug. CM use for longer time or with increased dosage could potentially change the narrative story in hypertension control.

Fig. 4.0

Calculated MABP histogram versus CM treatments. Key:-MABP:-Mean Arterial Blood Pressure; CM1:-Raw camel milk administered at 100mg/kg body weight/day; CM3:- Raw camel milk administered at 300mg/kg body weight/day; CM5:- Raw camel milk administered at 500mg/kg body weight/day; HNC:-Hypertensive Negative Control, rats given only dissolved L-NAME (50mg/kg body weight/day); NC:-Normal control, rats given only distilled water during treatment period; PC:-Positive control, rats given amlodipine 10mg/ kg body weight/day.

Fig. 4.1

Systolic Blood Pressure (SBP) histogram against Cm treatments. Key:-SBP:-Systolic Blood Pressure; CM1:-Raw camel milk administered at 100mg/kg body weight/day; CM3:- Raw camel milk administered at 300mg/kg body weight/day; CM5:- Raw camel administered at 500mg/kg body weight/day; HNC:-Hypertensive Negative Control, rats given only dissolved LNAME (50mg/kg body weight/day); NC:-Normal control, rats given only distilled water during treatment period; PC:-Positive control, rats given amlodipine 10mg/ kg body weight/day.

Fig. 4.2

Diastolic Blood Pressure (DBP) histogram versus CM treatments. Key:-DBP:-Diastolic Blood Pressure; CM1:-Raw camel milk administered at 100mg/kg body weight/day; CM3:- Raw camel milk administered at 300mg/kg body weight/day; CM5:- Raw camel milk administered at 500mg/kg body weight/day; HNC:-Hypertensive Negative Control, rats given only dissolved L-NAME (50mg/kg body weight/day); NC:-Normal control, rats given only distilled water during treatment period; PC:-Positive control, rats given amlodipine 10mg/ kg body weight/day

Table 1

Effect of chemical L-NAME in (negative control alone) compared with normal control (NC), positive control (amlodipine 10mg/kg) and raw camel milk, CM on circulating blood parameters recovered by invasive method in rats treated for 4 weeks

	SBP (mmHg)	DBP (mmHg)	MABP (mmHg)
Normal Control (NC)	119.4±1.3	78.6±1.44	95.4±1.4
L-NAME only (50 mg/kg, p.o.)	202.8±1.9^***	107.4±1.8^*	146.7±1.8^***
CM (500 mg/kg, p.o.) + L-NAME)	127.6±2.5^# #	79.2±3.0^#	107.3±2.3^# #
PC (Amlodipine (10 mg/kg, p.o.)+L-NAME)	134.0±2.9^# #	89.2±2.4^#	107.7±2.6^# #

Values are expressed as mean±SEM. One-way ANOVA followed post hoc Bonferroni tests. CM:-raw camel milk; L-NAME:-NG-nitro-L-arginine methyl ester; SBP:-systolic blood pressure, DBP:-diastolic blood pressure, MABP:-mean arterial blood pressure, PC:-positive control, ^***p < 0.001 considered as significant comparison between NC and L-NAME (50 mg/kg), ^*p:-also different but with less significance, ^#p < 0.05 compared to L-NAME (50 mg/kg), ^# #p < 0.001 compared to L-NAME (50 mg/kg).

Table 2

Blood Pressure Values (Systolic and Diastolic) of rats from beginning to the end of 4 weeks\\ of concurrent raw camel milk treatment

	NC	M1	M3	M5
Systolic Blood Pressure (mmHg)
0 week	115.8±2.1	145.8±2.5	140.8±2.0	130.8±2.6
4 weeks	119.4ⁿ±1.3	139.2^m±2.7	137.6ⁿ±1.9	127.6ⁿ±2.5
Diastolic Blood Pressure (mmHg)
0 week	87.0±2.1	90.6±2.1	92.0±1.4	78.8±2.7
4 weeks	78.6^m±1.4	96.2^m±2.3	91.8±1.9	79.2±3.0

Values are presented as Mean±SEM. Superscript letters in a row (m and n) indicated a significant difference (p < 0.05). NC:-Normal Control; M1:-100mg/kg; M3:-300mg/kg; M5:-500mg/kg. (M1, 3 and 5:-Raw Camel Milk Concentrations).

Table 3

Weekly Effect of chemical L-NAME compared with raw camel milk administered at (100mg/kg, 300mg/kg and 500 mg/kg) on non-invasive blood pressure (Tail-cuff method) for SBP during 4 weeks treatment

Systolic Blood Pressure, SBP (mmHg)
Groups	Week 0	Week 1	Week 2	Week 3	Week 4
Group 1	115.8±2.1	117.2±2.0	117.6±1.4	116.0±2.2	119.4±1.3
Group 2	191.8±1.6^***	194.6±2.1^***	198.4±1.9^***	200.4±1.9^***	202.8±1.9^***
Group 3	143.0±2.5	140.0±2.9	138.2±2.8^#	136.0±2.9^#	134.0±2.9^#
Group 4	145.8±2.5	147.6±2.4	147.6±2.6	144.0±2.6	139.2±2.7^#
Group 5	140.8±2.0	143.0±2.1	142.2±2.1	140.0±2.1^#	137.6±1.9^#
Group 6	130.8±2.6^#	133.4±2.4^#	135.2±2.4^#	130.0±2.2^#	127.6±2.5^# #

Values are presented as Mean±SEM. Group 1:-normal control (NC), Group 2:-hypertensive negative control (HNC) L-NAME only (50mg/kg), Group 3:-Hypertensive Positive Control (HPC), L-NAME+amlodipine 10mg/kg, Group 4:-raw camel milk for 100mg/kg, Group 5:-raw camel milk for 300mg/kg Group 6:- raw camel milk for 500mg/kg. ^***p < 0.001 considered as significant comparison between Normal Control (NC) and L-NAME (50 mg/kg), ^#p < 0.05 compared to L-NAME (50 mg/kg), ^# #p < 0.001 compared to L-NAME (50 mg/kg).

Table 4

Weekly Effect of chemical L-NAME compared with raw camel milk administered at (100mg/kg, 300mg/kg and 500 mg/kg) on non-invasive blood pressure (Tail-cuff method) for DBP during 4 weeks treatment

Diastolic Blood Pressure, DBP (mmHg)
Groups	Week 0	Week 1	Week 2	Week 3	Week 4
Group 1	87.0±2.1	75.0±1.0	77.4±1.1	78.0±1.3	78.6±1.4
Group 2	104.6±2.2	106.4±2.0^***	106.4±1.7	106.6±2.1	107.4±1.8^***
Group 3	94.4±3.7	88.8±2.6	88.6±2.3	89.0±2.3	89.2±2.4
Group 4	90.6±2.1	95.2±1.8	95.8±2.0	95.4±2.3	96.2±2.3
Group 5	92.0±1.4	91.2±1.5	92.0±2.1	91.4±1.7	91.8±1.9
Group 6	78.8±2.7^#	80.4±2.8^#	79.0±2.7^#	79.6±2.9^#	79.2±3.0^#

Values are presented as Mean±SEM. Group 1:-normal control (NC), Group 2:-hypertensive negative control (HNC) L-NAME only (50mg/kg), Group 3:-hypertensive positive control (HPC), L-NAME+ amlodipine 10mg/kg, Group 4:-raw camel milk treatment for 100mg/kg, Group 5:-raw camel milk treatment for 300mg/kg, Group 6:- raw camel milk treatment for 500mg/kg. ^***p < 0.001 considered as significant comparison between Normal Control (NC) and L-NAME (50 mg/kg), ^#p < 0.05 compared to L-NAME (50 mg/kg).

Table 5

Weekly effect of chemical L-NAME compared with raw camel milk administered at (100mg/kg, 300mg/kg and 500 mg/kg) on non-invasive blood pressure (Tail-cuff method) calculated for MABP during 4 weeks treatment

Mean Arterial Blood Pressure (MABP)
Groups	Week 0	Week 1	Week 2	Week 3	Week 4
Group 1	98.8±2.1	92.4±1.4	94.0±1.2	93.7±1.7	95.4±1.4
Group 2	140.5±1.8^***	142.7±2.0^***	144.3±1.8^***	145.3±2.0^***	146.7±1.8^***
Group 3	114.4±3.2	109.8±2.7^#	109.0±2.5^#	108.4±2.5^#	107.7±2.6^#
Group 4	113.3±2.2	116.8±2.0	117.1±2.2	115.4±2.4^#	113.9±2.4^#
Group 5	112.1±1.7	112.5±1.8^#	112.7±2.1^#	111.4±1.8^#	110.8±1.9^#
Group 6	100.2±2.7^#	102.2±2.5^#	102.2±2.4^#	101.5±3.5^#	99.1±2.7^#

Values are presented as Mean±SEM. Group 1:-normal control (NC), Group 2:-hypertensive negative control (HNC) L-NAME only (50mg/kg body weight/day), Group 3:-hypertensive positive control (HPC), L-NAME+ amlodipine (10mg/kg body weight/day), Group 4:-raw camel milk treatment for 100mg/kg body weight/day, Group 5:-raw camel milk treatment for 300mg/kg body weight/day, Group 6:- raw camel milk treatment for 500mg/kg body weight/day. ^***p < 0.001 considered as significant comparison between normal control (NC) and L-NAME (50 mg/kg body weight/day), ^#p < 0.05 compared to L-NAME (50 mg/kg body weight/day).

The observed elevation in SBP and MABP was seen commonly among all the experimental groups but was more severe and with marked intensity in negative control group; compared to their normotensive peers and this have validated the chemical induction of hypertension. Thus, on hypertension induction our obtained result was in agreement with [32, 50] and [54] whom have all separately reported chronic hypertension in their various previous studies consequent from L-NAME use.

Deficiency of NO is as a result of competitive inhibition of Nitric oxide synthase (NOS) enzyme. L-NAME, is a structural analog of L-arginine, which became metabolized by non-enzymatic hydrolysis into its active form, N omega-nitro-L-arginine (L-NOARG), which binds to endothelial NOS through competitive means [48]. Several explanations were attributed for L-NAME induced arterial hypertension, among which includes due to deficiency of NO that was reported to control coronary vascular tone [49]. This decrease in endothelium dependent NO arterial dilatation is related to risk of coronary ischemia and infarction [50]. Myocardial infarction in rats was caused by chronic elevation of NO synthase enzyme as was found in another study [51]. L-NAME intoxication can results to blockage of NO synthase which may overall leads to increased serum cholesterol level in rats [50]. L-NAME mediated NO inhibition was also reported to accelerate hypertension and induces perivascular inflammation [53].

Investigating hypotensive effectivity of CM within context of chemical induced hypertension was focused in this study; where chronic hypertension in the studied rats was experimentally induced with continuous L-NAME intoxication as pointed out in the preceding paragraphs. Four weeks of (L-NAME) oral administration have resulted in significant elevation (p < 0.001) of SBP which was recorded by tail-cuff method and similar rise in calculated MABP in rats of all groups; both treatment and controls. This BP rise was evidenced from the generated histograms of Figures 4.0 and 4.1 and drawn Tables 1, 2 and 3 earlier explained.

This significant increase (p < 0.001) in SBP (mmHg) and MABP for HNC rats was more pronounced on 2nd, 3rd and 4th weeks of conducted research if the obtained result values for the period were compared with their normotensive peers. From 3rd and 4th weeks, SBP and MABP in rats of CM treatment had decreased significantly (p < 0.05) relative to their HNC counterparts. This obtained result thus indicates that pretreatment with CM for 28 days could have pronounced effect and can ameliorate/counters elevation in blood pressure occasioned by L-NAME intoxication.

The observed decrease in BP values in CM treatment group is dose-dependent. As can be inferred from Table 3; at 100mg/kg body weight, it was only from 4th week of oral treatment, that SBP started decreasing significantly (p < 0.001) but at 300mg/kg body weight dose, decrease in SBP is significant (p < 0.05) from 3rd week and after 4 weeks of CM concomitant treatment. At 500mg/kg body weight dose; SBP decreased with significance (p < 0.001) also from 3rd week of research up wards. Our indicated results in CM treatment group are similar and comparable in terms of their effectiveness to those obtained for rats of positive control group, where amlodipine (10mg/kg body weight) was administered to L-NAME treated rats which leads to significant reduction in SBP from the 1st week continuously to subsequent 2nd, 3rd and 4th weeks of treatment relative to group of HNC rats.

Likewise, DBP too had decreased across the board as can be evident from Table 4; buts it’s decrease and increase was not uniform. In the NC set; that was devoid of any form of intervention it fell from week 1 before beginning to rise and fall again within the subsequent weeks until the end of research. For PC too; which have amlodipine drug intervention it’s results assumed similar random and haphazard changes; to fall then rise and fall again. Only in HNC was the increase continuous but even at that the increase was not statistically significant by the end of research. Within intervention by CM doses too; only CM1 have had progressive increases but there too, the increases were statistically insignificant while both CM3 and CM5 all showed random and non-uniform change.

Studies have demonstrated that blocking the formation of angiotensin II by ACE inhibitors may result in beneficial organ-protective effects in addition to its actions in controlling blood pressure, and this may be explained by the blockade of angiotensin II–induced pro-inflammatory responses and production of reactive oxygen species; ROS [49].

It is a well known fact that natural products show antioxidant activity [55]. The correlation between antioxidant and Soy Protein or its hydrolysates; which also showed similar effect of attenuating hypertension caused by NO deficiency via its ACE inhibition; was earlier demonstrated. This effect also slows progression of renal disease in chronic renal failure rats [56] and thus conforms to similar reports by [58].

In this same CM study, values of liver function parameters for normotensives were lower than their obtained positive control equivalents for ALT, AST, ALP, ALB and TP as evident from Table 6. From the same Table 6; at 100mg/kg body weight CM dose; there was significant decrease (p < 0.001) for ALT, AST, ALP, TP and TB values compared to HNC rats group. At 300mg/kg body weight CM dose; there was similar observed decline (p < 0.001) within normotensive rats for ALT, AST, ALP, TP and TB that were lower than same peers in HNC rats. At 500mg/kg body weight CM dose, decrease (p < 0.001) was seen in AST, ALP, TP and TB compared also to same parameters in HNC rats.

Table 6

Liver function parameters of chemical induced-hypertensive rats administered raw camel milk

Grp	ALT (U/l)	AST (U/l)	ALP (U/l)	ALB (g/dl)	TP (g/dl)	CB (mmol/l)	TB (mmol/l)
NC	73.4±1.6	126.8±1.7	232.0#±1.0	2.8±0.2	5.6±0.5	8.7±0.2	0.1±0.02
HNC	98.7±2.9	189.2^*±2.3	280.5^‡*±1.9	3.7±0.2	7.9±0.2	9.9±0.7	0.9^*±0.04
CM1	76.4^#±1.2	132.2^#±2.2	240.7^#±2.0	2.9±0.1	5.2^#±0.3	8.5±0.4	0 2^#±0.03
CM3	78.7^#±1.1	136.2^#±2.2	241.1^#±2.3	2.9±0.1	5.3^#±0.3	8.7±0.5	0.2^#±0.02
CM5	81.8±1.8	138.4^#±1.5	250.5^#±1.1	3.5±0.2	6.0^#±0.3	8.7±0.1	0.3^#±0.03
PC	75.8±1.4	131.4±1.0	240.4±1.0	3.0±0.1	5.8±0.4	8.6±0.3	0.1±0.02

Values expressed as mean±SEM, (n is 5). One-way ANOVA followed post hoc Bonferroni tests. NC:-normal control, normotensives; HNC:-hypertensive negative control, L-NAME only (50mg/kg body weight/day), PC:-positive control, L-NAME+ amlodipine (10mg/kg body weight/day), CM1:-raw camel milk treatment for 100mg/kg body weight/day, CM3:-raw camel milk treatment for 300mg/kg body weight/day, CM5:-raw camel milk treatment for 500mg/kg body weight/day. ^*p < 0.001 considered as significant comparison between Normal Control (NC) and HNC; (L-NAME only (50 mg/kg body weight/day), by using analysis of variance (ANOVA), (n is 5), ^# (p < 0.05), significantly different from L-NAME (50 mg/kg body weight/day), ^# # (p < 0.001) Significantly different when compared to PC (10 mg/kg body weight/day). L-NAME:-nitro L-arginine methyl ester, ALT:-serum alanine transaminase, AST:-serum aspartate transaminase, ALP:-serum alkaline phosphatase, ALB:-serum albumin, TP:-serum total protein, CB:-serum conjugated bilirubin, TB:-serum total bilirubin or unconjugated bilirubin.

Kidney function test parameters were showed in Table 7; comparing HNC group with normotensives for creatinine, K+ and Cl^- there was significant increase in their values with (p < 0.001). Urea and creatinine for normotensives, however on the other hand were lower than their equivalents in positive control rats. The normotensive values of the remaining indices of Kidney function that comprised Na⁺, K+, Cl^- and HCO₃^- were found to be higher than their peers in positive control.

Table 7

Kidney function parameters of chemical induced-hypertensive rats administered raw camel milk

Group	Urea (mg/dl)	Creatinine (g/dl)	Na⁺ (mmol/l)	K⁺ (mmol/l)	Cl^- (mg/dl)	HCO₃^- (mg/dl)
NC	34.5±0.9	0.2±0.01	153.8±7.9	5.6±0.4	110.0±0.7	88.0±1.0
HNC	53.9±0.7	1.0^*±0.04	171.0±1.1	8.4^*±0.5	133.4^*±0.7	98.8±1.4
CM1	33.7^#±0.9	0.5^#±0.02	154.7±4.9	6.0^#±0.1	110.2^#±0.9	87.0±4.9
CM3	29.2^#±1.1	0.5^#±0.04	156.5±0.9	5.4^#±0.2	110.2^#±0.9	80.0±2.9
CM5	34.5±0.7	0.6^#±0.01	158.9±1.4	6.2^#±0.2	114.0±1.2	78.6^#±3.2
PC	38.4±0.8	0.4±0.01	153.6±6.1	5.5±0.3	108.4±1.3	86.8±0.9

Values are expressed as mean±SEM, (n is 5). One-way ANOVA followed post hoc Bonferroni tests. NC:-normal control, HNC:-hypertensive negative control, L-NAME only (50mg/kg body weight/day), PC:-positive control, L-NAME+ amlodipine (10mg/kg body weight/day), CM1:-raw camel milk treatment for 100mg/kg body weight/day, CM3:-raw camel milk treatment for 300mg/kg body weight/day, CM5:-raw camel milk treatment for 500mg/kg body weight/day. Na⁺:-serum sodium ion concentration, K+:- serum potassium ion concentration, Cl^-:- serum chloride ion concentration and (HCO₃^-):- serum bicarbonate ion concentration. ^*Significantly (P < 0.05) different from normotensive control by using analysis of variance (ANOVA), (n = 5), ^# (p < 0.05), significantly different from L-NAME (50 mg/kg).

Moreover, CM can generate nitric oxide which can stimulate mucus production, which inhibit adherence of neutrophiles to endothelial cells and increase blood flow to gastric mucous membrane. CM contained high level of vitamins C, A, B2 and E and it was rich in Mg and Zn. These vitamins are useful in reducing oxidative stress caused by toxic agent and Mg is essential for absorption and metabolism of vitamins B, C and E. Additionally, Mg significantly enhance antioxidant defense [51], moreover Mg is also required for biosynthesis of glutathione; which prevent damage to cellular components caused by free radicals, peroxides and heavy metals.

Zinc, (Zn) was reported to provide protective effect against cellular toxicity due to palliative effect on oxidative stress and apoptosis, and activation of antioxidant system to decrease lipid peroxides; this was a finding from some study [52].

It is noteworthy that dairy products (of which mammalian milk constitutes) are among the most common dietary sources of l-arginine more readily available than meat, poultry, fish and nuts [53, 54]. Raw CM is considered among dairy product that may be rich in possession of dietary source of l- arginine. Lower intake of l-arginine rich foods such as fish and nuts has been consistently shown to be associated with future cardiovascular risk [55].

The result of present study showed reduction of L-NAME induced hypertension by concurrent amlodipine treatment (positive control) and also by raw CM intervention. This investigation confirmed beneficial effect of raw CM on regression of hypertension. CM as a natural product from dromedary camel have showed antihypertensive activity just like some other natural products too from plant sources which includes Lagenaria siceraria (Molina) Standley (Cucurbitacae) fruit (Bottle gourd) that similarly demonstrated hypotensive effect [56] but in dexamethasone induced hypertension [59]. Thus not only in L-NAME induced hypertension was observed effect of natural product seen in ameliorating increase in blood pressure values. In this investigation CM and amlodipine showed similarity in exhibiting antihypertensive effect.

The obtained results from histopathological examination of metabolic organs; mainly liver, kidney and hearts of some rats used in the research suggest that raw CM is not having any toxic effect on liver and kidneys, but it even have some layers of hepatoprotective effect. Histopathologic analysis has become always a golden standard for evaluating treatment-related pathological changes in tissues and organs [61]. Histopathological analyses are usually done to further confirm the alteration in cell structure of especially metabolic organs. In this study; no abnormal findings were observed on histopathological results of all the organs that were examined. Since the results obtained from blood biochemical indices and histopathological examination have not depicted abnormal findings that could suggest signs of toxicity in all the tested groups, it can safely be concluded that raw CM is both effective and non-toxic.

5 Conclusion

In conclusion, results of the present research have provided evidences that same comparable objective of overall achievement of desired target blood pressure can be attained with concurrent administration of raw CM with dissolved L-NAME solution on one hand and similarly same dissolved L-NAME solution but which was counteracted by hypertensive control drug amlodipine at (10mg/kg body weight/day) thereby having the two simultaneous studies all showing significant reduction in BP values in rats within context of the chemical-induced hypertension. Presence of crude protein in milk composition of CM may appears to contribute to the observed hypotensive activity of CM. Antioxidants too that may be present in most natural products may appear to reduce adverse BP elevation occasioned by L-NAME intoxication. It is thus concluded that CM possesses some level of antihypertensive activity.

Funding statement

The entire work was funded from emoluments of the corresponding author in form of monthly salaries and allowances paid to him as research candidate and an employee of the Federal Government of Nigeria.

Disclosure

Authors of this research article hereby declare that neither the funding sponsors as one entity nor any other group or interest had any role in the research design; in collection and analyses, or interpretation of the data; in writing of the manuscript; and in the final decision taken to publish the results.

Contributions of Authors

This research work was collaboration between all the authors. RAD conceptualized the research, made the study design, performed most of the conducted experiments, did statistical analysis, and wrote both protocol and initial draft of the manuscript. Authors ML, RSUW and AY supervised conduct of the research, collection and arrangement of the obtained data for the study and have offered help with some literature, and have further proofread. All authors have managed literature searches, arrange tables and graphs. All authors have read and approved the final manuscript.

Statement on Conflict of interest

Authors of this article want hereby affirm that they have no any conflicts of interest of whatsoever to declare.

Footnotes

Acknowledgments

The authors hereby acknowledged and sincerely appreciates cooperation and support rendered by laboratory technologists of the Departments of Biochemistry and Molecular Biology, and Veterinary Pathology, UDUS, and Technical staff members of Laboratory Animal Center of Pharmacology and Therapeutics and their counterparts in Physiology Departments of Ahmadu Bello University, Zaria, Kaduna State, Nigeria for various support rendered while carrying out this research work. Dr Ibrahim Abubakar of Department of Veterinary Parasitology, UDUS is specially appreciated for assistance in the statistical analyses aspect.

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