Immunomodulatory Activity, GC-MS Analysis and Pharmakokinetic Potential of Camellia Sinensis

The immunomodulatory activities of medicinal plants are well-known. Medicinal plants found natural components enhance the immune system. However, both the Camellia sinensis is found a large number of bioactive compounds that make it strong to ght against ailments. It plays a signicant role in cell-mediated and humoral immunity. The immunomodulatory activity of C. sinensis was carried out by neutrophil adhesion test, antibody titer and delayed-type hypersensitivity. The present study was aimed to investigate the immunomodulatory activity of methanolic extract of Camellia sinensis in Wistar albino rats. GC–MS analysis was carried out on the potent plant (C. sinensis) to recognize bioactive volatile compounds, for their therapeutic properties, respectively. C. sinensis treated animals showed a signicant outcome at dose 200 mg. GC–MS analysis was carried out of the C. sinensis to recognize bioactive volatile compounds. Out of 20, ve major compounds were found are 2-Pentanone, 4-hydroxy-4-methyl, Caffeine, 1-H Benzimidazole, 2-phenyl, Hexadecenoic acid, 15-methyl, methyl ester, Trans-13-Octadecenoic acid, methyl ester. In this study, C. sinensis has more potential to modulate the immune system in experimental animals. This study provides a substantial way to replace deleterious medicines and provides natural compounds that a part of the lifestyle to get rid of diseases. The highest % area was found is 1-H Benzimidazole 2-phenyl. The pharmacokinetic property of bioactive compound was carried out by SwissADME tool.


Introduction
The immune system plays a signi cant role to ght against foreign bodies (Rahayu et al., 2018). Medicinal plants are an abundant source of bioactive substances that are obliging to enhance the immune system of our body. In Ayurveda, traditional medicinal plants create attention to build up a body defense mechanism against various threatening diseases (Shukla and Mehta, 2015). The notion of immunomodulation relates to the non-speci c activation of the immune system. It is a non-antigen-dependent reaction and triggers the function and activation of macrophages, granulocytes, natural killer cells, lymphocytes, and also the production of various effectors molecules by activated cells (Sumalatha et al., 2012). Immunomodulation is the process by which the immune system enhances towards the prevention and cure of many disorders in the human body such as respiratory disorder, in ammation in skin, cancer and some autoimmune diseases including rheumatoid Animal Treatment After assent from the institutional animal ethical committee and in (Ref. No. GKV/AHF/10/2018), laboratory breed of male Wistar albino rats weighing between 180 g and 200 g were procured from Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, Haryana. Husk (Paddy) was used as bedding and changed at regular interval of time. All animals were housed in polypropylene cages under standard environment maintained at 21±2 ºC and supplied with a standard pellet diet and water ad libitum in the Animal House (Reg. No. 1324/a/10/CPCSEA), Department of Pharmaceutical Sciences, Gurukula Kangri (Deemed to be University), Haridwar.

Neutrophil adhesion test
A neutrophil adhesion test was carried out according to the method of Patel and Ashaq (2010). Rats were pre-treated orally with saline (Group I) as a control and the other 3 groups administered were methanolic extracts of C. sinensis On the 14 th day of treatment, blood samples were collected by puncturing the retro-orbital plexus into heparinized vials and examined for total leucocyte counts (TLC) and Differential leucocyte count (DLC). After initial counts, the same blood samples were incubated with 80 mg/ml of nylon bers for 15 min at 37 o C and again analyzed for TLC. The product of TLC and neutrophil (%) gives the neutrophil index (NI) of the blood sample. Neutrophil adhesion (%) was enumerated as follows: Neutrophil adhesion (%) = NIu -NIt × 100 NIu Where, NIu= Neutrophil index (untreated blood sample) NIt= Neutrophil index (treated blood sample with nylon ber) Haemagglutination antibody (HA) titer Rats of groups I, II, III, and IV were immunized with 0.5 × 10 9 SRBCs/rat by intraperitoneal route. Rats of groups II, III, and IV were pre-treated with methanolic C. sinensis extract for 14days. The day of immunization with SRBCs was referred to as day 0. The animals were treated with plant extracts for 14 more days and blood samples were collected from each rat on day 15 for HA titer. The titer was determined by titrating serum dilutions with SRBC (0.025 × 10 9 cells). The microtitre plates were incubated at room temperature for 2 hours and examined visually for agglutination reaction. The minimum concentration of serum showing haemagglutination was expressed as HA titer (Fulzele et al., 2003).

Delayed type hypersensitivity (DTH) response
The delayed-type hypersensitivity response was determined by following Bin-Hafeez et al., 2003). Six animals per group were immunized with 1× 10 9 SRBC/rat, subcutaneously. On the fth day, all the animals were again immunized with 1× 10 9 SRBC in the left hind foot. The right footpad injected with the same volume of saline served as control. Plant extract (methanolic extract of C. sinensis) was administered orally. DTH response was measured at 24 h after SRBC challenge on day 5 and expressed as a mean percent increase in paw volume.

GC-MS analysis (bioactive compounds)
The GC-MS analysis of methanolic extracts of C. sinensis was carried out by Pradhan and Dubey, 2021.
Interpretation of results of GC-MS was conducted using the database of the National Institute Standard and Technology (NIST 2.0).

Statistical analysis
The signi cancy of data were analyzed statistically by using one-way ANOVA followed by a post hoc t-test with Bonferroni's comparison. All the value of results were expressed as mean±SD and p< 0.05 was considered signi cant.
Evaluation of Pharmacokinetic Properties of bioactive compound (1-H Benzimidazole 2-phenyl) Pharmakokinetic properties and drug-likeness evaluation of GC-MS compound (highest peak) were carried out using SwissADME a free online web tool used in evaluating ADME properties and drug-likeness of small molecules. Lipinski's rule of ve is helpful at the pre-clinical evaluation discovery of drugs (Tripathi et al., 2019).

Neutrophil adhesion test
A blood sample incubated with nylon bers diminution in the neutrophil count ascribed to the adhesion of neutrophil to the nylon bers. Dosages of 100 mg and 200 mg showed signi cant results (p < 0.05), but dose 200 mg produced more signi cant result (p < 0.001). Therefore, a 200 mg dose was found more effective than the other doses of both the Camellia sinensis (Tables 1).

Haemagglutination titre
The administration of an increased dose of extracts signi cant result was observed at dose 100 mg (p < 0.01) and 200 mg (p < 0.001) of C.sinensis compared with the control group. At dose, 50 mg did not show non-signi cant results when compared with the control (Figure 1).

Delayed type hypersensitivity reaction
The delayed hypersensitivity reaction was observed signi cantly at dose 100 mg (p < 0.01) and 200 mg (p < 0.001) of C. sinensis as compared to control. However, dose 50mg was found non-signi cant compared to control ( Figure 2).

Evaluation of Pharmacokinetic Properties of bioactive compound (1-H Benzimidazole 2-phenyl) by Swiss ADME
The bioactive compounds were identi ed through GC-MS analysis and the pharmacological properties were determined by the SwissADME tool. Molecule ful lls the standard criteria expressed by Lipinski's rule of ve, meaning the activity of the compounds high potency. In a null shell, the property of the molecules are considered according to absorption, low toxicity level, and orally bioavailable. The Bioavailability Radar gives an outline of the drug-likeness of a molecule ( Figure 4). The region denote with pink colour indicates the different properties of the compound (Table: 3; Figure: 4 and 5).

Discussion
The present work was done to assess the immunostimulatory acitivity of C. sinensis in an animal model by studying different parameters such as neutrophil adhesion test, haemagglutination titer, and delayed-type hypersensitivity test. In this study methanolic extract of C. sinensis possesses immunomodulatory properties which enhance both cellular and humoral immunity. The extract was found to be more e cacious at high concentrations (200 mg), but at 100 mg concentration, the extract was quitely effective in modulating the immune system. The plant products aids to modulate the immune system either by stimulation or suppression (Wagner, 1984) and help to use an alternative drug for immune-compromised people. The drugs that stimulate the immune system are called immunostimulant drugs that help to enhance the non-speci c and speci c system such as macrophages, granulocytes, certain T-lymphocytes ( The haemagglutination test correlates with humoral immunity. The C. sinensis extract showed effects on the humoral immune system. The results of C. sinensis were found to be most effective at 200 mg with a value of 469.33 (p < 0.001). Likewise, at 100 mg also C. sinensis was found to be signi cant with a value of 213.33 (p < 0.01). The humoral immune response is composed of B cells with antigens and eventually proliferating into plasma cells that secrete antibodies. Antibodies producing cells bind to antigens, and neutralizes it, and accelerates the elimination of antigens by readily ingesting phagocytic cells (Fulzele et al., 2003). This was indicated by the mean value of the haemagglutination titer that depicted a dose-dependent elevation for the production of antibodies (Nfambi et al., 2015). According to the Coombs and Gell classi cation (1975), the delayed-type hypersensitivity is also known as type IV cell-mediated immune response. This test directly correlates with the cell-mediated response and was found to be signi cant with C. sinensis at doses 100 mg (p < 0.01) and 200 mg (p < 0.001). In DTH, when challenged by the antigen, the T-lymphocytes were converted into lymphoblasts and producing lymphokines that attract more macrophages at the site of reaction. Due to the presence of avonoids, the humoral immune response ampli es by proliferating macrophages and B-lymphocytes for the production of antibodies (Makare et al., 2001). It is used to analyze the skin response after inoculation of the antigen intradermal which depends on antigen-speci c memory T-cells and the results were due to the augmentation of neutrophils and mononuclear cells. T-cell activation recruits the accumulation of macrophages which induces vasodilation, in ammation, and increases vascular permeability (Janeway et al., Goronzy and Weyand, 2007). It also uplifts the phagocytic activity and increases the production of lytic enzymes for the more e cient killing of microorganisms (Janeway et al., 2001). An increase in the DTH response indicates that plant extracts have a stimulatory effect on lymphocytes and accessory cell types required for the expression of the reaction (Mitra et al., 1999). By ndings of immunomodulatory activity, C. sinensis has great potential to enhance the immune system. Whereas with the avail of GC-MS for nding the bioactive compounds that are amenable for boosting the immune system. The possible compounds present in the methanolic extract of C. sinensis by GC-MS analysis. About 20 peaks are observed in the GC-MS study of C. sinensis. The major and minor peaks were discussed ( Table 2). It is explicit that these peaks are for the phytoconstituents (bioactive compounds) present in the plant which are responsible to possess many biological and therapeutic potential. The current study con rmed that the methanolic extract of C. sinensis. possesses potent antimicrobial, antioxidant, anti-in ammatory, anti-cancerous properties.
The pharmacokinetic property and drug-likeness were carried out by Swiss ADME (Table: 3; Figure:  Based on the study, the results prove that the leaves of Camellia sinensis has the potential to enhance the cell-mediated immunity and it may be effective in several immunocompromised clinical conditions.

Conclusions
The immunostimulatory effect produced by Camellia species helps in the stimulation of both cellular and humoral immune system and also increases the total leucocyte counts. This study proves the use of tea beverages shall be helpful to immunosuppressed people by enhancing the immune system. The GC-MS (Gas chromatography-Mass spectrometry analysis depicted the biologically active compounds that are accountable for enhancing the immune system. The prediction of pharmacokinetics potential, bioavailability, drug-likeness and medicinal chemistry friendliness revealed that the bioactive compound present in C. sinensis can be a lead compound for new drug discovery for antimicrobial, immune enhancement and anti-in ammatory phytomedicine. However, it is suggested further in vivo and computational assay for toxicology, pharmacology and experimental bioavailability study for reliving phytocompound to validate the present augury. All values are expressed as mean±Standard deviation of six observations. UTB, untreated blood; NFTB, nylon ber treated blood.Values are statistically signi cant at P < 0.05*, P < 0.001*** when compared with control group.   Figure 1 Haemagglutination titre of Camellia sinensis.