Evaluation of the Effect of Hydroethanolic Root Extract and Solvent Fractions of Cyphostemma adenocaule (Steud. ex A. Rich) Descoings ex Wild & Drummond on Cell-Mediated Immune Response and Blood Cell Count in Mice

Introduction Cyphostemma adenocaule (Steud. ex A. Rich) Descoings ex wild & Drummond (Vitaceae) has been used in traditional medicine for the management of various immunological and hematological disorders in different areas of Ethiopia and the rest of the world. In Ethiopia, the plant is used for the management of enlarged spleen, rabies virus, helminthic infection, snake bite, and various types of tumors. Objective To evaluate the effect of hydroethanolic root extract and solvent fractions of Cyphostemma adenocaule on cell-mediated immunity (delayed-type hypersensitivity), organ index (spleen and liver), and blood cell count in Swiss albino mice. Materials and Methods Acute oral toxicity test was conducted using nulliparous and nonpregnant Swiss albino mice following OECD 425 limit test method. Delayed-type hypersensitivity model was used to evaluate the effect on cell-mediated immunity. The experimental animals were divided into twelve groups which were sensitized and challenged with sheep red blood cells on day 0 and day 7, respectively. Levamisole 50 mg/kg was used as stimulant control, whereas cyclophosphamide 30 mg/kg was used as suppressant control. Hydroethanolic root extract (100 mg/kg, 200 mg/kg, and 400 mg/kg), aqueous fraction (100 mg/kg, 200 mg/kg, and 400 mg/kg), and n-butanol fraction (100 mg/kg, 200 mg/kg, and 400 mg/kg) were administered for seven days. The paw volume was measured using a digital plethysmometer before challenge and 24 hours after challenge. Blood was collected, and organs (spleen and liver) were isolated from each challenged mouse to determine blood cell count and organ index, respectively. Results No mortality and noticeable behavioral changes were observed among all mice receiving hydroethanolic root extract and solvent fractions at a dose of 2000 mg/kg. Hydroethanolic root extract and solvent fractions of Cyphostemma adenocaule showed enhancement of delayed-type hypersensitivity, organ index, and blood cell count. Hydroethanolic root extract at a dose of 400 mg/kg showed the highest and statistically significant stimulation of delayed-type hypersensitivity (0.123 ± 0.010) and blood cell count compared to the vehicle. Conclusion Hydroethanolic root extract and solvent fractions of Cyphostemma adenocaule showed a stimulatory effect on cell-mediated immunity and hematopoiesis.


Introduction
Alteration of the structure and functions of the immune system leads to suppression or stimulation of the immune response.
e immune system which can be innate or adaptive is responsible for the protection of the body from infection and other immunological disorders [1]. e whole plant and phytoconstituents of many plants of various taxonomic classes exhibited immunomodulatory properties to an antigenic material that gets access to the body of living cells. Cyphostemma adenocaule (Vitaceae family) is an herbaceous climber widely distributed in Ethiopia [2]. In traditional medicine, C. adenocaule has been used for the treatment of various illnesses such as malaria, helminthic infection, cancer, tumors, snake bite, rabies, and anthrax [3][4][5][6]. Earlier scientific studies showed that C. adenocaule has in vitro antiplasmodial [7], antioxidant [8], and deworming [9] effects. e anti-infective, antitoxin, and antitumor activity of this plant might be due to its effect on the cellular immune system and accessory cell types. erefore, this study aimed to demonstrate the effect of hydroethanolic root extract and solvent fractions of C. adenocaule on cell-mediated immunity, blood cell count, and organ index in Swiss albino mice. , and cyclophosphamide (Cadila health care Ltd., India) were used. All the chemicals, reagents, and standard drugs were of laboratory/analytical grade. Sheep blood and Alsever's solution were also used during the study.

Experimental Animal.
Female nulliparous and nonpregnant Swiss albino mice aged 8-12 weeks were used for the acute toxicity study, whereas for the main study, healthy, Swiss albino mice of either sex (weight 25±5 g, and 6-8 weeks of age) obtained from the animal house of the School of Pharmacy, College of Health Sciences, Mekelle University, were used. e mice were housed in a laboratory of 12/12 hr light/dark cycle with the provision of a commercial pellet diet and water ad libitum. Before beginning the experiment, the animals were acclimatized to the laboratory condition for a week.

Collection and Authentication of the Plant.
e plant sample was collected from Angacha riverside and authenticated by Mr. Getnet Chekol (Botanist, University of Gondar, Ethiopia), and a sample specimen (voucher DA01/2018) was deposited in the herbarium of the university. e roots of the plant were collected, washed with tap water, and shade-dried.

Extraction and Fractionation.
Shade-dried roots were powdered with an electric grinder of which one kilogram of the powder was macerated with 70% (v/v) ethanol (1 : 5 ratios) for 72 hours which was then filtered with a muslin cloth and Whatman filter paper No. 1 to remove any insoluble matter. e marc was remacerated twice with the same solvent for 72 hours [8]. All the filtrates were dried in an oven at 37°C. 60 gram of the dried extract was dispersed in water (1 : 5 ratios) and fractionated by successively treating the dispersed medium with n-hexane and n-butanol in triplicates. e three fractions (aqueous, n-butanol, and n-hexane) were dried in an oven at 37°C.

Sheep Red Blood Cell (SRBC) Preparation and
Standardization. Fresh blood was collected from the external jugular vein of sheep and mixed with freshly prepared Alsever's solution (2% dextrose, 0.8% sodium citrate, 0.055% citric acid, and 0.42% sodium chloride) in 1 : 1 proportion.
e collected blood was centrifuged at 2500 rpm for 10 min to separate the plasma and washed thrice in pyrogenfree normal saline (0.9% w/v) with subsequent centrifugation.
e SRBCs were suspended in normal saline, and their count was adjusted to a concentration of 1 × 10 8 cells in 1 mL of sheep blood which was used as T-lymphocyte-dependent antigens for immunization and challenge of the mice [10,11].

Acute Oral Toxicity
Testing. Female nulliparous and nonpregnant Swiss albino mice were used for the study for each extract and solvent fraction. e test was performed following the Organization for Economic Cooperation and Development (OECD) guideline 425 using limit test method [12].

Animal
Grouping and Dosing. SRBC-sensitized and challenged mice were randomly grouped into twelve groups of six mice each that received the respective dose of vehicle/ standard drug/extract/solvent fractions as shown in Table 1.

Delayed-Type Hypersensitivity (DTH).
All groups of mice were immunized with 0.1 mL of SRBC (i.p) on day 0 and received an oral dose of respective treatment for 7 days. On the 7th day, one hour after administration of the respective dose, the paw volume of the left leg of each mouse was measured with a digital plethysmometer and challenged with a subcutaneous injection of 0.05 mL of SRBC into the hind footpad of the same leg. e volume of the challenged paw was measured 24 hours after the challenge. e difference in paw volume between the pre-and postchallenge was taken as a measure of DTH response, and the percentage change in paw volume was determined using the following equation [13,14]: PV: paw volume of mice receiving each treatment dose and vehicle.

Effect on Blood Cell Count and Organ Index.
At the end of the treatment period of the DTH model, blood was withdrawn from the retro-orbital plexus of all groups of mice to determine blood cells. Following blood withdrawal, the spleen and liver were isolated and weighed immediately to determine their relative index using the following equation [11,15]:

Statistical Analysis.
e data from each group were analyzed using SPSS version 20 software. Results were presented as mean ± SEM (standard error of mean). e difference of treatment group means from each other and the control mean values was determined by using one-way ANOVA followed by Tukey post hoc multiple comparison test, and p values <0.05 were considered significant.

Acute Oral Toxicity Test.
e acute toxicity study showed that no mortality and noticeable behavioral change were observed in all mice that received 2000 mg/kg of the hydroethanolic root extract and solvent fractions (aqueous and n-butanol fractions).

Delayed-Type Hypersensitivity Test.
In DTH test, groups treated with levamisole, hydroethanolic root extract, and the solvent fraction of C. adenocaule showed a dose-dependent increase in DTH response compared to the vehicle ( Table 2).
is enhancement in DTH response indicated the stimulation of cell-mediated response to SRBC which could be due to the activation of the production of lymphocytes and accessory cell types which play the central role in DTH response. It might also be due to the production of memory T lymphocytes during sensitization with SRBC that are

Organ
Index. e hydroethanolic root extract and solvent fractions of C. adenocaule induced an increase in organ index compared to the vehicle ( Table 2) which might be attributable to the stimulatory effect of treatment on the growth and development of organs. Hydroethanolic root extract at a dose of 400 mg/kg induced the highest spleen index (5.359 ± 0.174). e increase in organ index might also be due to the stimulatory effect of treatment on proliferation and differentiation of bone marrow stem cells that migrate to and settle within the organ for interaction with the incoming antigen, thereby increasing the mass of the organ [20].

Blood Cell Count.
In the present study, Hydroethanolic root extract and solvent fractions of C. adenocaule increased the blood cell count compared to the vehicle (Table 3) which might be due to stimulation of the proliferation and differentiation of hematopoietic stem cells (HSCs) in the bone marrow giving rise to functioning blood cells [21]. It might also be due to the reported antioxidant property (IC50 of 38.42 μg/mL) of the study plant that prevents cells from oxidative damage [8]. Hydroethanolic root extract revealed the highest blood cell count compared to solvent fractions of comparable dose which might be accredited to the presence of a larger quantity of antioxidant phytoconstituents at higher concentrations compared to their existence in solvent fractions of comparable dose, thereby enhancing hematopoiesis through synergistic effect [22]. is finding agreed with the finding of Ojogbane and his coworkers who demonstrated the enhancement of WBC count with the administration of aqueous extract of C. glaucophilla leaves [23].

Conclusion
From this study, it can be concluded that hydroethanolic root extract and solvent fractions of C. adenocaule had stimulatory properties on the cell-mediated immune response.
Data Availability e data generated and/analyzed during the study will be available from the corresponding author upon reasonable request.

Conflicts of Interest
e authors declare that there are no conflicts of interest.