Hematological, biochemical, and histopathological study of the plant extract of Terminalia chebula Reitz., Aloe vera Linn., and Tamarindus indica Linn. on animal model: A comparative study.
Scientifically Aloe vera named as A. vera Linn., A. vera belongs to Liliaceae family, and it is in nature xerophytic, succulent, perennial, green pea color plant. Worldwide, well-known plant A. vera is a popular for its usefulness. A. vera applied externally in the cure of several skin disorder such as in burns, eczema, and cuts.  It is purported that juice from A. vera reduces inflammation and pain.  It has antibiotic and antiseptic stuffs which make it extremely valued in treating scratches and wounds. It has been frequently used in the treatment of 1st and 2nd degree burns, as well as suntans poison of oak, ivy, and sumac, infections, and eczema. Aloe has been promptly used as a therapeutic agent for wounds, ulcers, cancer, and many more disorders. [9,10] A. vera contains more than seventy active constituents such as minerals, sugars, enzymes, amino acids, and salicylic acids. [11-14]
Tamarindus is a member of family Leguminosae. Tamarindus indica Linn. mainly known as Tamarind tree. It is one of the another significant versatiles tropical fruit tree in the Indian subcontinent. T. indica is used as antimicrobial, antiseptic, antiviral, diabetes, diarrhea, eye inflammation, sore throat, sores, sprains, swelling (joints), urinary stones, etc. [15-17] T. indica is active components, such as cardiac glycosides, compounds phenolic,  L-(-) mallic acid,  mucilage, tartaric acid, arabinose, xylose, pectin, galactose, uronic acid, and glucose. [19,20] The ethanolic extract of T. indica contains fatty acids also and various trace elements such as cadmium, copper, iron, sodium, manganese, magnesium, potassium, arsenic, calcium, phosphorus, zinc, and lead which is important for our body in very low amount. 
Phytochemical analysis of T. chebula Reitz. reveals the presence of tannins, flavonoids, saponins, and cardiac glycosides. Despite the numerous use of all three plants is mainly used as anti-inflammatory, analgesic, and antipyretic. Steroid narcotic and nonsteroidal anti-inflammatory drugs are used at the present time. However, the available drugs have decreased popularity against inflammatory conditions due to numerous adverse effects. As an example, steroidal drugs are used as anti-inflammatories due to their precise mechanisms of action and also the same mechanism of actions is responsible for their adverse effects as well. Leukotriene inhibition and other basal physiological function inhibiting phenomena are used for anti-inflammatories by steroidal drugs. Hypertension as adverse effects noted due to the resemblance of these drugs to the steroid hormones. The nonsteroidal drugs have fewer adverse effects than the steroidal, but it includes gastrointestinal bleeding and improper clotting of blood. Therefore, researchers are looking for alternative treatment options that have least adverse effects and full of efficacy and must be comparable to a standard drug. To fulfill this demand currently, researchers are looking for alternate source of drug apart from synthetics one. The best alternative is an herbal crude drug that is commonly used in rural part of India as a folk medicine. Research work plan in the present study for safety and potency after repeated dosing schedule has not been fully scrutinized in a comprehensive manner. As per World Health Organization 2008 there is huge demand of herbal medicinal products hence assessment and standardization is required of all kind of herbal preparations including T. chebula Reitz., A. vera L., and T. indica Linn. Investigation was projected to determine oral acute and subacute toxicity profile of the ethanol leaf extract of T. chebula Reitz., A. vera L., and T. indica L. in experimental rats. It was also investigated for their hematological, biochemical, and histopathological changes in liver and kidney tissues after the treatment.
MATERIALS AND METHODS
On the basis literature appraisal, three plants are selected for the studies:
* T. chebula Reitz.
* A. vera Linn.
* T. indica Linn.
Afterward collection of all three plants and it was acknowledged and authenticated by a pharmacognosist and a letter receipt specimen (No. IU/PHAR/HRB/7/10) was submitted at the Department of Pharmacognosy, Integral University, Lucknow, Uttar Pradesh, India. Aqueous extractions of all these three plants were done according to standard procedure.
Preparation of Plant Extract
T. chebula Reitz.
T. chebula Reitz. fruit was utilized for the experimentation. T. chebula Reitz. shade-dried fruits were taken for the preparation of extract. A fine powder in an electric grinder was grinded. Approximately 890 g of crushed processed material was hauled out with 80% ethanol at a temperature of 60[degrees]C for near about 48 h. The mixture was segregated. Condensed solvents, transferred in watch glass in an abundant surface area to make it more compressed and permit the remaining solvent to vaporize. Whereas the compressed filtrate transferred into a viscid concentrate, it was observable that we create the crude ethanolic extract. The ethanolic extract was more vaporized to dehydration to obtain the dried out ethanolic extract. Harvested extract percentage obtained 12.1% weight/weight in compare to the actual air-dried powder.  The extract was lastly deposited in airtight holder at 2-8[degrees]C for more uses throughout the study.  The dose was accustomed as per dosing schedule following standard technique.
A. vera Linn.
For extraction with water of A. vera leaves first wash away in cold water then spikes round the leaves were removed using a blade after that the leaves were cut up by knife. 200 g of the cut up material were assorted in distilled water and blended in an electric blender for 3 min to obtain 400% (weight/volume) extract. The melded material was pressed in a muslin cloth. The filtrate was freeze-dried at -50[degrees]C beneath vacuum using a lyophilizer and kept at -20[degrees]C till utilize. Numerous dose ranks of A. vera were prepared by reconstituting the extract at concentration of 1% (w/v). 
T. indica Linn.
The extraction was performed using softening technique.  The coarse powder of T. indica seeds (100 g) was imperiled to maceration for 72 h at room temperature using 500 ml methanol. After sieving of the extract, the solvent was removed by evaporation technique to acquire a powdered residue. After that during experiment dose was adjusted as per dosing schedule following standard procedure.
Qualitative Analysis of Phytochemical Constituents
Primary chemical tests were conducted for all plant extracts to distinguish diverse phytochemical compositions present in the plants extract as per the protocol described. 
Acute and Subacute toxicological study were conducted on male and female Wistar albino rats. Animals were familiarized adapted to laboratory surroundings for 14 days before the investigation. Throughout familiarization, accommodation rats were kept in cages with pellet diet and clean water. All methods in the present work were done as per the Institutional Animal Ethics Committee guidelines. Working protocols were approved by the Institutional Animal Ethics Committee with reference no. SUG/Pharm/2015/II/22.
Acute Toxicity Study
The Organisation for Economic Co-operation and Development (OECD), 2002 method was considered. Either sex wistar rats 180-200 g weight were taken for the same study divided into 2 groups. Three sets of three rats in every one cage were administered 100, 600, and 1000 mg/kg all three plant extracts, respectively, through oral route. Surveillance for indications of toxicity and mortality was done for 24 h with exceptional care given to the first 4 h. 2000, 3000, and 5000 mg/kg dose administered for the three plant extracts and monitored to all three groups of three rats as earlier stated and daily for 7 days for indication of late toxicity.
Subacute Oral Toxicity Study
OECD, 1995 guidelines were considered for this study. A total of 24 rats (12 each as male and female) weighed and grouped in four of three each as male and female rats in each group (the [male] rats were separated from the [female] rats) for mentioned experiment. The animals were assembled depend on three different treatment doses of the leaf extract and one control (distilled water) group. The rats were orally given daily with extract of T. chebula at doses of 100, 200, and 400 mg/kg and distilled water for 28 days. Same dosing schedule and number of groups of animal taken for rest two plant extracts of T. chebula Reitz. and A. vera L. on experiment rats. The treatment doses in lieu of the therapeutically active dose which was the most effective dose detected in the evaluation of the analgesic, anti-inflammatory, and antipyretic.  Finally, after 28 days treated period, all the rats were destitute of pellet but given free access to water for 24 h before being sacrificed under inhaled standard (isoflurane, dose - 4-5% for induction and 1-2% for maintenance) anesthesia. Cardiac pinhole into ethylenediaminetetraacetic acid and non-heparin containing test tubes used for blood samples for hematological and biochemical investigation correspondingly. The organs were cut down, measured and macroscopic study was done.
Heparinized test tubes were used for the collection of blood sample. Estimation of white blood cells (WBC), red blood cells and platelets performed by visual methods (Dacie, 1991).
The blood sample has been collected in the non-heparinized tubes. Blood has been centrifuged at 3000 rotation per min for 10 min. The separated serum and ultimately liver and kidney homogenates (20%) were investigated for enzymes. Reitman and Frankel (1957) method is used for alanine aminotransferase and aspartate aminotransferase. Alkaline phosphatase was analyzed by Bessey et al., 1946. Protein and creatinine by Gornall et al., 1949, and Barterls et al. (1972) techniques used, respectively.
Conventional hematoxylin-eosin technique is used for histopathological examines of the liver and kidneys.
Statistical analysis was articulated as mean. The mean values also expressed along with [+ or -] standard error mean. One-way analysis of variance (ANOVA) was executed to compare the variances between two or more means. A mean dissimilarity was considered statistically significant when P < 0.05.
Acute Oral Toxicity
The extract of all three plants did not produce any untoward effect in animal at the different dose levels. No changes in their behavior and stool texture of the animals. No mortality detected after 7 days of treatment. The [LD.sub.50] values for all the plants were found to be >5000 mg/kg for oral administration.
Effect of the Extract on Hematological Parameters
Figures 1-3 show the effects of all the three plant extracts on the blood parameters during subacute study. Non-significant variation noticed in all other hematological parameters and persists within normal physiological norms after the 28 days treatment period.
Effect of the Extract on Biochemical Parameters
The effect on biochemical parameters is presented in Figures 4-6 after oral administration of subacute dose of all the three plant extracts. All the biochemical parameters were found to be normal levels in all the cases including control treatment.
The histological investigation of the sections of liver tissue and kidney tissue of control and treated groups indicated a normal histological structure in Figure 7. No gross pathological lesions were observed even for higher dose treatment. It also showed no deformities in the color or texture when compared these organs in comparison to control.
Since long time, human being has used different medicinal plants. All over the world accepted for the management of different types of ailment.  For the evaluation of the therapeutically active plants for their activity and finding of any toxic effect of the substances is taken into consideration. Besides number of therapeutic usefulness of all the three plant extracts, a comparative knowledge related to the toxicological effect not been screened. So that, the acute and subacute toxicity of all three plant extracts in rats is conducted to rule out toxicological effects.
After oral treatment >5000 mg/kg, no any mortality was observed for all the three plant extracts. The food, water taken, and behavior were did not change throughout the study period in comparison to control group. It was also observed that the meal-taking behavior such as food and water was freely taken by the investigating animals. When exposed to all the three plants extract which recommend that the extract was did not caused any changes in food metabolism in these experimental animals. Hematological analysis was performed for all the three plant extracts treated and their outcome indicates no any significant effects when compared with control group. No any alterations in renal and hepatic functions were observed for any possibility of pathological changes after biochemical analyses of serum. Furthermore, it was observed that slight increase in blood volume and hemoglobin content. These changes may be due to the nutritional substances in the extract as previously reported by other researchers also. 
The blood parameters evaluation usually carried out to find out the adverse effect of any foreign molecule, along with therapeutic constituents in plantsextract. [29,30,31] Meanwhile, it is also mandatory to examine as alteration in the blood composition. These alterations are mainly indicative to toxicological effect to human being. Here, results are derived from animal studies.  The liver and kidney function exploration is an important factor for the toxicological assessment of drug and plant extracts as it is well-known that both are vital organs for the survival of animal.  WBC is the indicator of defense which fights against infection, inflammation, or organ damages. Moreover, no any such countable alteration observed in the neutrophils, lymphocytes, and monocytes when studied for all the three plant extracts treated animals. All the three plant extracts were not displayed any countable alterations for all the biochemical values at different dose level. It means no toxicological effect on rats liver and kidney function.  The results of the present study indicated all the vital organs such as kidney, liver, lungs, spleen, and heart were not altered throughout the 28-day treatment period. Therefore, it could be concluded that all three plant extracts are not toxic to vital organs of the said groups in comparison to control group in rats.
Major outcomes from this present study suggest that all the three plant extracts do not having any such toxic effects which will lead to compromise their therapeutic application. Long-term use of these herbal drugs showed the effects which recommended all three plant extracts do not exhibit any toxic effects in terms of hematological and biochemical parameters of rats. The outcomes also indicated that A. vera extract may have immune system modulating effect. It too recommended that all the three plant extracts having defensive potential to hepatic and nephrotic cell. All our above findings indicated that all the three plant extracts not showed adverse effects and are non-toxic for therapeutic purposes.
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Mohammad Daud Ali (1), Mohammad Arif Naseer (1), Mohammad Aamir Mirza (2), Md. Sarfaraz Alam (3)
(1) Department of Pharmacology, School of Pharmacy Adarsh Vijendra Institute of Pharmaceutical Sciences, Shobhit University, Gangoh, Saharanpur, Uttar Pradesh, India, (2) Department of Pharmaceutics, School of Pharmaceutical Education & Research, Jamia Hamdard New Delhi, India, (3) Department of Pharmaceutics, School Of Pharmacy, OPJS University, Churu, Rajasthan, India
Correspondence to: Mohammad Daud Ali, E-mail: email@example.com
Received: January 02, 2019; Accepted: January 23, 2019
Source of Support: Nil, Conflict of Interest: None declared.
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|Title Annotation:||RESEARCH ARTICLE|
|Author:||Ali, Mohammad Daud; Naseer, Mohammad Arif; Mirza, Mohammad Aamir; Alam, Md. Sarfaraz|
|Publication:||National Journal of Physiology, Pharmacy and Pharmacology|
|Date:||Mar 1, 2019|
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