Environmental protection is a necessity to enhance health, safety and environment. Over the decades, in the Niger Delta, Nigeria, the environment has been threatened by different human activities, especially, petroleum exploration and exploitation or oil and gas flare and secondhand smoke or soot. The resultant effect is the emission of toxic petroleum environmental pollutants into the atmosphere. Exposure to petroleum associated environmental pollutants has been suggested to pose serious threats to human life [1-8]. Bio-surveillance of the content of petroleum pollutants in biological samples revealed that cadmium among other heavy metals is one of the toxic environmental pollutants that can cause damage to various organs in the body [2,3,4,9,10]. Cadmium has no known physiological role in humans but its exposure occurs through ingestion as well as inhalation [4, 11, 12,13,14]. It accumulates in the body for a long time and its concentration gradually increase several years after exposure, having a very long biological half-life in order of 20 years and above [11-13].

Available literature suggests that cadmium toxicity induce stimulation of oxidative stress leading to decrease in antioxidant enzymes but causes a rise in reactive oxygen species (ROS), and lipid peroxidation [11-13]. An association between cadmium exposure marker (blood) and atherogenic changes in lipid profile has been suggested [11-13]. Although oxidative stress has been assumed to be the principal molecular basis underlying cytotoxicity caused by cadmium, the mechanism of cadmium induced toxicity remains not clearly elucidated. It has been speculated that cadmium causes cellular damage by modulation of plasma membrane diffusion potential with resultant increase in cellular uptake of pollutants [15-17] which subsequently results to toxicity [3,4,9] primarily through the generation of ROS, which causes single-strand DNA damage and disrupts the synthesis of nucleic acids and proteins [11-13]. However, there is hitherto dearth of information regarding the mitigating potential of Justicia carnea in amelioration of cadmium-induced toxicity. 

Justicia carnea form part of the most important medicinal plants in traditional diets in Nigeria, with a wide spectrum of actions and applications reported [18,19,20,21]. The whole plant as well as specific parts (root, stems, leaves and flowers) of plant extract and its bioactive constituents have been used for the treatment and management of a large number of human ailments. The aim of the present study was to determine Justicia carnea hydro-alcohol leaf extract remediation and detoxification efficacy in cadmium-induced oxidative cellular damage, hyperglycemia and hyperlipidemia in male wistar rats. It is also necessary to find the type of bioactive compounds at molecular level, through phytochemical GC-MS analysis, in the plant leaves of Justicia carnea, for the understanding and establishment of its phytomedicine and phytotherapy potentials in multi-food-drug action.

Plant identification 

Fresh leaves of Justicia carnea were collected from a farm within the milieus of Choba community, Rivers State. Plant was identified and authenticated in the herbarium for Plant Science and Biotechnology, University of Port Harcourt.

Extract preparation 

Prior to use, the leaves of Justicia carnea were washed and dried for about 4 hours using laboratory oven (Heratherm Advanced Protocol Scientific). Hydroethanolic extract (1:4 v/v) was prepared following the procedures previously reported [22-27].

Study design 

In this study, male wistar rats were randomized into 8 groups of 5 rats per group. Group 1 served as control received distilled water.  Cadmium was used to induce toxicity in groups 2, 6, 7 and 8. Cadmium groups received daily oral administration of 10mg/kg of cadmium chloride (CdCl2). Groups 3, 4 and 5 received 100mg/kg, 150mg/kg and 200mg/kg body weight of Justicia carnea extract, below the LD50 [20] respectively. Furthermore, groups 6, 7 and 8 also received 100mg/kg; 150mg/kg and 200mg/kg body weight of Justicia carnea extract respectively daily using oral gavage. The animals were allowed free access to rat chow and water ad libitum. The experiment lasted for 6 weeks (42days).

        Blood glucose was monitored weekly using the on-call plus glucometer [28-33]. After 6 weeks, the animals were sacrificed and blood samples were collected via cardiac puncture, for the determination of the oxidative stress markers; Superoxide Dismutase (SOD), Catalase (CAT), Glutathione Peroxidase (GPx), and Malondialdehyde (MDA)according to standard methods as previously established [34-40].

        Beside lipid profile analysis - high density lipoprotein (HDL), low density lipoprotein (LDL), total cholesterol (TC) was also carried out as previously described [41-43].

        Gas Chromatography - Mass Spectrometry (GC-MS) analysis of the Justicia carnea hydroethanolic leaf extract was carried out following the instructions of the manufacturers as previously described [44-45]

Ethical approval

Institutional ethical approval was obtained and the study was in strict adherence to the proper and cautious use of animals. 

Statistical analysis

Statistical package for social sciences (SPSS) version 22.0 was used for all statistical analysis. Data were presented in tables and graphs. Continuous variables were expressed as mean ± standard error of mean. Differences between groups were assessed by graph pad v.6.01 using one way ANOVA and considering p-value less than 0.05 (P<0.05) as significant. Percentage change relative to control was also recorded.

Environmental exposure to petroleum heavy metal pollutants is a serious problem in Niger Delta (Nigeria), starting from 1956, where many people are exposed to petroleum exploration and exploitation or oil and gas flares and secondhand smoke or soot heavy metal contaminated air, water and soil. It has become necessary to provide basic information of heavy metal poisonings and design an educational and prophylactic plan to substantially reduce the body burden of heavy metal toxicity. 

Table 1: Qualitative phytochemical composition of Justicia carnea

Table 2: GC-MS physio-therapeutic chemical compounds found in Justicia carnea

Table 3: Justicia carnea modulation of cadmium-induced oxidative stress parameters

Groups	Catalase(mmo/l) ± sem	% difference	Glutathione peroxidase (ug/ml)  ± sem	% difference	Malondialdehyde (mmo/l) ± sem	% difference	Superoxide dismutase (mmo/l) ± sem	% difference
Control	5.04 0.16		0.09   0.01		0.43   0.02		0.43   0.04
CdCl2 Only	3.300.06	34.5	0.05   0.01	44.4	0.48   0.02	-11.6	0.27   0.02	37.2
100mg JC	5.48ab  0.09	-8.7	0.06ab 0.01	33.3	0.43b 0.37	0	0.49b 0.04	-13.9
150mg JC	5.51b 0.05	-9.3	0.08ab   .01	11.1	0.27ab 0.01	37.2	0.52ab 0.01	-20.9
200mg JC	6.25ab 0.20	-24	0.08ab 0.01	11.1	0.35ab 0.05	18.6	0.52b 0.05	-20.9
100mg (JC+CdCl2)	4.16b 0.37	-2.3	0.07ab 0.01	22.2	0.33ab 0.02	28.2	0.45b   0.01	-4.6
150mg (JC+CdCl2)	4.47ab 0.02	11.3	0.09b 0.01	0	0.39ab 0.02	9.3	0.41b 0.02	4.7
200mg (JC+CdCl2)	4.54ab 0.25	15.9	0.10ab 0.01	-11.1	0.37ab 0.01	13.9	0.55b 0.01	18.6

Key: N= 5, a = mean significant differences relative to the normal control, b = mean significant difference relative to the negative control at p<0.05. 

Table 4:  Justicia carnea dose-time (weeks) dependent modulation of cadmium-induced high blood glucose

Key: a= mean significant difference relative to the normal control, b = mean significant difference relative to the negative control at p<0.05.

The present study therefore may be informative and helpful to achieve the purpose of managing all aspects of heavy metal (cadmium) poisoning. 

Preliminary phytochemical screening using similar methods of previous studies [22,46,47] revealed that Justicia carnea hydromethanolic leaf extract contains the presence of bioactive constituents, in the following order of abundance – flavonoids and phenols > alkaloids, glycosides, carbohydrate and reducing sugar > saponins, tannins and terpenoids as summarized in (Table 1). The most predominant phyto-constituents was flavonoid and phenol in agreement with previously reported [20,21,48].

The Gas Chromatography - Mass Spectrometry (GC-MS) analysis of hydromethanolic Justicia carnea leaf extract further validated the qualitative phytochemical data. The GC-MS spectrum confirmed the presence ofvarious components with different retention times as illustrated    in (Figures 1 and 2).   Unlike the GC-MS ethanol analysis which confirmed only 6 compounds [18] in Justicia carnea, the present study identified a total of 9 physio-pharmacological compounds, representing 98.9% of total hydromethanolic extract composition of the entire GC-MS chromatogram as shown in (Table 2). This is a reflection of the superiority of hydro-alcohol [22] over other solvents in medicinal plant extraction studies. Importantly, at the molecular level, GC-MS analysis identified hexadecanoic acid (50.90%), methyl tetradecanoate (24.34%) and 9-octadecenoic acid (17.75%) as the abundant physio-therapeutic compounds in Justicia carnea hydromethanolic leaf extract. Beside toxic normal hydrocarbon- nonadecane (0.13%) in hydromethanolic extract of the leaf of Justicia carnea was identified, which have not been reported previously in the Justicia carnea samples.

able 3 depicts that cadmium significantly caused an increase in the activities of catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD) and a decrease in malondialdehyde (MDA) in comparison with the positive control by 34.5%, 44.4%, 37.2% and 11.6% respectively. Administration of Justicia carnea alone or in combination with cadmium in a dose-dependent, significantly decreased malondialdehyde (MDA) (Table 3 columns 6 and 7) compared to control by, 28.2% and14% (or 23% compared to negative control) 

respectively. This result suggests that Justicia carnea is effective in protecting cells against cadmium-induced

Figure 1. GC-MS chromatogram of -Tetradec-11-en-1-yl 2,2,3,3,4,4,4-heptafluorobutanoate in Justicia

Table 5: Justicia carnea modulation of cadmium induced changes in lipid profile

Key: N= 5, a= mean significant difference relative to the normal control, b = mean significant difference relative to the negative control   at p<0.05. 

Figure 2. GC-MS chromatogram of Methyl tetra decanoate in Justicia Carnea

ROS production and resultant oxidative stress. Heavy metal like cadmium promotes reactive oxygen species and lipid peroxidation, suppression of antioxidant defence systems [11-13]. The pro-oxidative character of cadmium was probably reversed by co-administration of the extract as shown for 200mg/kg. Phenolic acids may function as antioxidants. Flavonoids are a class of phytochemicals with free radical scavenging properties [10-12]. Our result is also suggestive that Justicia carnea has the ability to antagonize the toxic effects of cadmium possibly through the antioxidant therapy attributable to high phenolic and flavonoids content [18,21,48] The result showed that combination of cadmium and Justicia carnea or Justicia carnea alone resulted in a decrease in lipid peroxidation, an indication of sensitivity of Justicia carnea, perhaps acting as a chelating agent against cadmium-induced cellular toxicity.

Time – dose dependent blood glucose lowering potential of Justicia carnea alone or in combination with cadmium are as depicted in (Table 4). Cadmium caused a marked increase in blood glucose level by 23% (Table 4 column 7]. Justicia carnea alone or in combination with cadmium improved blood glucose level after 6 weeks (Table 4 column 7). Cadmium can accumulate and result in toxicity to the kidney, liver, lungs, brain, testes, heart and central nervous system, and more so, even cause anaemia, and many more [2-4] [11-13].  Some plant-derived bioactive agents can improve blood glucose homeostasis [20,21,38,49,50]. Fasting blood glucose was significantly, progressively and dose-dependently reduced after independent and combined treatment with cadmium and Justicia carnea hydromethanolic extract. In other words, a combination of Justicia carnea and cadmium reduced cadmium toxicity effects in the body and thereby stimulate blood glucose production. Justicia carnea bioactive constituents may probably have increased the sensitivity of response of the hormone-secreting cells of pancreatic islets to blood glucose.

As depicted in (Table 5), cadmium caused significant increase in both low-density lipoprotein (LDL), and total cholesterol (TC) but significantly decreased high density lipoprotein (HDL) relative to control by 7%, 27.6% and 22.4% respectively. The level of HDL was significantly increased in the extract treatments alone and also in combination with cadmium. This significant positive improvement was dose dependent as summarized in (Table 5). Some plant-based bioactive agents can improve lipid profile [51]. Oleic acid (1.15%) component of the extract revealed by GCMS has the ability to mop up LDL and improve healthy serum level of lipid profile markers [52].

This study reported the officiousness of Justicia carnea hydromethanolic leaf extract in phytoremediation and detoxification of cadmium induced toxicity -oxidative cellular damage, hyperlipidemia and hyperglycemia in experimental rats. From the results, it is evident that the medicinal relevance of Justicia carnea can be traced to the abundant varieties of the biologically therapeutic compounds in the plant that plausibly could be therapeutically beneficial for preventing the deleterious health effects in humans resulting from exposure to heavy metals; including cadmium. These predominant exposure pharmacological compounds include hexadecanoic acid, methyl tetradecanoate, 9-Octadecenoic acid, flavonoids and phenols

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