Comparison and Evaluation of Different Seed Extracts of Trachyspermum ammi for Immunomodulatory Effect on Cell-Mediated Immunity through Delayed-Type Hypersensitivity Assay Skin Thickness Method.

OBJECTIVE: The aim and objective of this study was to find the immunostimulant and immunomodulatory effect of T. ammi seed extracts.
METHODS: Seeds of T. ammi were extracted using three different solvents n-hexane, chloroform, and methanol by using soxhlet apparatus. To assess the immunomodulatory effect, delayed-type hypersensitivity (DTH) assay method was used and by the DTH assay, the effect of T. ammi on the skin thickness of rats was estimated. To find the exact dose for administration, acute toxicity test was performed using crude methanolic extract at a dose of 400, 800, 1600, and 3200mg/kg. After acute toxicity test, 500mg/kg dose was determined as safe for therapeutic effect and immunomodulatory effect was evaluated at this dose. Dose of 500mg/kg was administered to Wistar rats daily for 14 days and skin thickness of rats was measured at 24, 48, and 72h.
RESULTS: Results were obtained from six groups of rats, which were positive control group, negative control group, and the groups receiving the test drugs. Standard drug was the combination of sodium selenite, vitamin E, and sodium chloride and it showed more positive results as compared to that of test drug. Furthermore, among the three extracts, methanol extract showed more effectiveness on skin thickness.
CONCLUSION: There was a meaningful difference was observed between the skin thickness of rats which shows that T. ammi have good immunomodulatory as well as immunostimulant activity.

INTRODUCTION

For decades, different herbal preparations have been used to lessen the diseases by modulating the response of the immune system. There are various plants that have been used to regulate the immune system. Since long, diseases have been cured by altering the immune responses because the mechanisms and etiology of diseases are interlinked to the immunity system.[1] There are many medicinal plants that can alter the immune response by effecting on granulocytes, macrophages, and white blood cells. Various biological complexes and series of actions are carried out by the immune system to protect the body from disease, but whenever there is any irregularity in these complexes or actions, certain immune disorders such as cancer, immunodeficiency, inflammatory disease, and autoimmune disorders may occur.[2]

The process of alteration of the immune system of an organism by intervening in its normal functions is called immunomodulation; if immune response improves then it will be called as immune stimulant or booster, which generally indicates provocation of the immune system. Immunosuppressants are implicit or they minimize the resistance of the body to combat against certain conditions such as stress, infection, and environmental factors. Sometimes immunosuppressants have been administered to patients to suppress their immune system, especially in case of organ transplant to avoid graft rejection.[3] Immune systems of the human body consist of two primitive types, which are cell-mediated and humoral immunity. Together these two systems defend human body from germs. When a pathogen enters the body, humoral system produces antibody; in contrast, cell-mediated immunity resides on the T cells along with B cells, which contributes in the protection of the human body.[4]

There are five different categories of hypersensitivity reactions, which are I, II, III, IV, and V. These reactions based on antibody–antigen reaction and type I, II, III, along with V are known as “immediate” reaction, whereas type IV reactions are termed as “delayed-type hypersensitivity” (DTH) because of the delayed interactions of T lymphocytes with antigen. The DTH reaction includes T helper (TH) cells such as CD4+ as well as T cytotoxic cells such as CD8, which are activated by the cellular system.[5] The action of various agents on cell-mediated and humoral immunity was analyzed through a variety of tests and techniques. Humoral immunity reactions were promoted by an interaction of antigen with B lymphocytes. As a response of antigen humoral and cellular system of the body releases antibody in plasma to fight with these pathogens and which intern increase immunity and this cell mediated immunity measured through delayed type hypersensitivity (DTH) method.[6]

The proliferation of the natural killer cells, T lymphocytes, and macrophages can escalate cell-mediated immunity.[7] DTH needs specific identification of antigen presented through triggered T lymphocytes that finally proliferate and discharge cytokines, which result in vasodilation, boost up vascular permeation, activation, and aggregation of macrophages, enhancing phagocytic action toward foreign substances for extended killing that raises the lytic enzyme concentration.[89] Immunomodulatory agents discriminately activate the humoral or cell-mediated immunity by the provocation through T cells (TH1 or TH2) subsequently. Immunomodulatory drugs with no side effects are required to activate the immune system as a prophylactic measure to avoid various disorders.[10]

Trachyspermum ammi belongs to Apiaceae family having more than 3700 species and 434 genera. It is 16th largest family in flowering plants. T. ammi is indigenous to Egypt and it is grown in dry, open, ruderal regions.[1112]

A study carried out on n-hexane extracts and acetone extracts of T. ammi showed that the extract of n-hexane contains 39.1% thymol, which was the main component, including 30.8% p-cymene, 0.8% terpinene-4-ol, 23.2% γ-terpinene, and 1.7% β-pinene, whereas acetone extract of T. ammi revealed the existence of 18 different compounds, which account for 68.8% of the entire amount. The main compound was 39.1% thymol followed by 1.6% p-cymene, 2.6% γ-terpinene, 9.6% linoleic acid, 0.1% xylene, 1.6% palmitic acid, and 10.4% oleic acid. On one hand, n-hexane extract yielded approximately 31.80% of oil from the seed of T. ammi, on the other hand, ethanolic extract yielded approximately 28% of the oil. The lipid neutral components from oil are monoglycerides, sterols, diglycerides, hydrocarbons, free fatty acids, esters, and triglycerides, whereas lipid polar component contains phosphatidylcholines and phosphatidylethanolamines.[13] Another study revealed that the seeds of T. ammi also contained yellow, crystalline flavones, glucosides, steroid-like substance 6-O-β-glucopyranosyloxythymol, and a yield of 25% oleoresin and 12% of volatile oil components, that is, p-cymene, β-pinene, thymol, α-, and γ-terpinene.[14]

According to a study carried out by researchers, 100 g of ground T. ammi herb contains niacin (2.1 mg), potassium (1.38 mg), riboflavin (0.28 mg), carbohydrate (24.6 g), minerals (7.9 g), thiamine (0.21 mg), sodium (0.443 mg), water (7.4 g), calcium (1.525 g), protein (17.1 g), iron (27.7 mg), and food energy (363 cal). In this study, steam distillation was used to extract T. ammi oil from its seed. This extracted oil was divided into two types, essential oil and nonessential oil. According to this study, T. ammi seeds contained 26% fatty oils, 3–4% essential oil, p-cymene, phenols, and terpenes. There were around 27 components of essential oil of which thymol was abundant (61%) and others included β-pinene (4%–5%), p-cymene (15.6%), dipentene (4%–6%), camphene, γ-terpinene (11.9%), and myrcene. Besides thymol, a liquid hydrocarbon called cymol or cymene was also present in the oil.[15]

The oil obtained from T. ammi exhibits aflatoxin detoxification,[16] gastroprotective, digestive stimulant,[17] abortifacient,[18] nematicidal,[19] antispasmodic, hepatoprotective, bronchodilatory, antihypertensive,[20] antifilarial[21] antiplatelet aggregatory,[22] ameliorative,[23] hypolipidemic,[24] anthelmintic,[25] diuretic, anti-lithiasis and anti-inflammatory effects.[26] Seeds of ajwain have a therapeutic application such as antiseptic, expectorant, and carminative.[2728] T. ammi is used in conventional medicines for curing neurological disorders, rheumatic arthritis, joint pain, and it also reduces inflammation; this study was performed to verify this conventional use.[2629]

MATERIALS AND METHODS

Plant material collection

Seeds of T. ammi were purchased from local market in July 2015 and authenticated by Dr. Iqbal Niazi (Department of Botany, University of the Punjab, Lahore, Pakistan), and voucher specimens of the plant were deposited under voucher no. 175216. The seeds were completely dried under daylight and ground to make a fine powder and stored in an amber glass bottle.

n-Hexane extraction

Approximately 150 g of dried powdered seeds of T. ammi were measured and extracted with 1.5 L of n-hexane. The material was packed into a thimble and placed in Soxhlet apparatus for extraction and fitted with a round-bottom flask of 2 L with n-hexane. Extraction was carried out for 72 h until the solvent in Soxhlet thimble becomes colorless. The extracted material was allowed to cool, and rotary evaporator was used for the removal of the solvent. After the removal of solvent, a sticky black gummy extract was obtained. Which was placed in a pre-weighed sterilized petri dish, covered with aluminum foil and extract was stored in refrigerator.

Chloroform extraction

Dried grounded residue obtained after n-hexane extraction was further treated with 1.5 L of chloroform. The material was packed again in thimble and extraction was carried out by placing in Soxhlet apparatus. The flask was filled with 2 L of chloroform, and extraction was carried out until the solvent appeared colorless. The extracted material was allowed to cool at room temperature and then transferred into the rotary evaporator for removal of the solvent. After the removal of solvent, a sticky black gummy extract was obtained. Which was placed in a pre-weighed sterilized petri dish, covered with aluminum foil and extract was stored in refrigerator.

Methanol extraction

After the chloroform extraction, residues of seeds of T. ammi were treated with methanol. Extraction was performed by following the aforementioned procedure, and the extract was stored in pre-weighed clean petri dishes, covered with aluminum foil and the liquid extract was stored in the refrigerator for further study.

Ethical considerations

Before the commencement of the study, the following ethical approvals was sought from the institutional ethics research committee of the University of Lahore, and registration number specified for this study was IAEC-2016-14-A.

Acute toxicity test

Acute toxicity includes those adverse effects that occur after administration of drug either in single or multiple doses by any route of administration, and for determination of toxicity, acute toxicity tests were carried out to check any adverse effect that occurred on the specific dose for the specific time.

Procedure

Wistar rats were divided into two groups, each containing four rats and they were kept in the animal house at 25°C. Group 1 was the control group that was given normal saline at the dose of 10 mL/kg. Group 2 rats were administered methanolic extract of T. ammi. The dose of extract was prepared in four different concentrations, which were 400, 800, 1600, and 3200 mg/kg body weight. The drug was administered to rat through oral route with the help of syringe equipped with oral gauge. After 4 h, the rats were examined for any possible adverse effect and after 24 h, all the groups were examined again to check the death rate, results obtained were compared with the control group.[30]

Delayed-type hypersensitivity assay activity

Preparation of immunomodulator

Calin et al.[31] described the preparation of immunomodulator, it includes sodium selenite (1 g), vitamin E (15 g), and sodium chloride (9 g) in 1000-mL solution. Thus, immunomodulator was prepared according to Calin et al.[31] by adding the ingredients in distilled water.

Effect of T. ammi on cell-mediated immunity

T. ammi was evaluated for its effect on cell-mediated immunity by DTH assay. In this study, rats were divided into six groups, each containing four rats. Immunomodulator (sodium selenite + vitamin E + sodium chloride) was given to group 1, which was the positive control group, whereas negative control groups 2 and 3 were given sterile water for injection and methanol solvents in which drug was dissolved, respectively. Groups 4, 5, and 6 were given n-hexane, methanol, and chloroform extract at a dose of 500 mg/kg, respectively. All three extracts were dissolved in methanol and immunomodulator was dissolved in sterile water for injection. Dinitrochlorobenzene (DNCB) was applied topically in all the groups except the two negative control groups. These negative groups were only applied acetone. Doses were administered to all groups for 8 days. On the 2nd day of activity, all rats were shaved from the right side to uncover the skin. The thickness of skin from the right side was measured with digital vernier caliper and the shaved skin was marked with the help of a permanent marker. Then 2% of 0.1 mL DNCB was applied on the skin, it acts as a sensitizing agent. In contrast, negative control was applied only 0.1 mL acetone.

On the 14th day of activity, rat’s skin was shaved from the left side to expose the skin and the same procedure was repeated, but the only difference was in the dose of DNCB and acetone, wherein 0.2 mL of 2% DNCB and acetone was applied topically on the left side. Now the thickness of skin was measured for the next 24, 48, and 72 h with a digital vernier caliper.[32]

RESULTS

Acute toxicity test of T. ammi seeds was performed on Wistar rats to check its adverse reaction, physiological behavior, and safety. The crude extract up to 3200 mg/kg was administered to the rats, two deaths occurred after 24 h. Yet at the dose of 1600 mg/kg, side effects such as skin allergy (red spot on skin), difficulty in breathing, and convulsions were observed. At the dose of 800 mg/kg, much less side effects occur, and it was found that the side effects of seed extract were dose dependent, which increased when the dose increased. No side effect was noted at the dose of 400 mg/kg. So, drug dose of 500 mg/kg was determined as safe for therapeutic effect.

Delayed-type hypersensitivity

In this study, the effect of T. ammi on cell-mediated immunity was investigated through DTH reaction by measuring the skin thickness of rats, which received T. ammi extracts, and the results are shown in Table 1. Furthermore, results obtained after activity were evaluated through one-way analysis of variance (ANOVA) to check the significance, and the outcomes are shown in Table 2.

Table 1

Skin thickness measurement of rats and their comparison within groups

Treatments	Before applying DNCB	After 24 h	After 48 h	After 72 h
Sterile water for injection + acetone	2 ± 0.14	2.1 ± 0.0	2.2 ± 0.014	2.3 ± 0.014
Methanol	2 ± 0.01	2 ± 0.02	1.9 ± 0.02	2 ± 0.01
Methanol extract	1.9 ± 0.01	2.9 ± 0.03**++†	2.8 ± 0.01††	2.7 ± 0.07†
n-hexane extract	2 ± 0.16	2.7 ± 0.08**+++†††	2.6 ± 0.01*+††	2.5 ± 0.02**+++
Chloroform extract	1.9 ± 0.11	2.7 ± 0.04†	2.6 ± 0.04†	2.6 ± 0.007†††
Immunomodulator	1.9 ± 0.02	3 ± 0.11**++†	3.7 ± 0.04**++††	3.2 ± 0.12*++†††

All values are mean ± SD

*P < 0.05, **P < 0.01, ***P < 0.001, when other groups were compared with group of rats administered with sterile water for injection + acetone, was compared with control

+P < 0.05, ++P < 0.01, +++P < 0.001, when other groups were compared with group of rats administered with extracts of the test drug

†P < 0.05, ††P < 0.01, †††P < 0.001, when other groups were compared with group of rats administered with sterile water for injection + DNCB

Table 2

One-way analysis of delayed-type hypersensitivity through analysis of variance

Treatment time before and after with DNCB	Variance	Sum of squares	df	Mean square	F	Sig.
Before the second exposure of DNCB	Between groups	0.018	3	0.006	35.14	0.002
	Within groups	0.007	4	0.0001
	Totals	0.019	7
24 h after second exposure of DNCB	Between groups	2.58	5	0.505	54.023	0.000
	Within groups	0.049	6	0.007
	Total	2.57	11
48 h after second exposure of DNCB	Between groups	3.12	8	0.784	50.3	0.000
	Within groups	0.0512	7	0.016
	Total	3.20	12
72 h after second exposure of DNCB	Between groups	1.066	3	0.534	6.888	0.003
	Within groups	1.635	4	0.023
	Total	2.701	7

df = degree of freedom, Sig. = significance

Table 2 showed one-way ANOVA analysis of DTH among control and treated groups. This table provides an overall view of significant difference among control and treated groups. No significant difference was observed before applying DNCB (P > 0.05), but 24, 48, and 72 h after applying DNCB, a significant difference was observed among control and treated groups (P < 0.001).

DISCUSSION

The results obtained from this study state that T. ammi extracts showed an increase in the thickness of skin as compared to the negative control group, and results for all the three extracts noted in the table described that skin thickness of the rat was increased by applying DNCB and in contrast, before applying DNCB, no meaningful difference in skin thickness was observed. After applying DNCB, skin thickness was checked after 24 h, and a meaningful difference in skin thickness was observed for rats among all groups. After 48 h, skin thickness of rat also increased in groups that were administered immunomodulators and methanol extract. After 72 h, skin thickness of rat remained significantly different when compared with the control group. The chloroform and n-hexane extract groups showed similar result on the thickness of skin, whereas the methanolic extract group showed increased thickness as compared to n-hexane and chloroform extract groups, and immunomodulatory group showed more skin thickness as compared to all the other groups. In the immunomodulatory group, DNCB was applied on the skin of rats that was previously shaved, and skin thickness was measured with the help of digital vernier caliper. Skin thickness was measured before and after applying DNCB at 24, 48, and 72 h. DTH is mediated by T cells that are further divided into helper CD4 and cytotoxic CD8 cells. A DTH reaction is managed by T1 helper and CD4 cells, which are differentiated by interleukin (IL)-12 along with IL-18 released by microphages present on the site of DTH. This results in granulomatous inflammation, a principle pathological problem, which shows characteristic of DTH and increases skin thickness. It was thought that α-pinene and carvacrol present in oil of T. ammi acts as an immunostimulant.[33] Some other studies conducted on the seed extracts of medicinal plants such as Argyreia speciosa, Ocimum sanctum, and Trigonella foenum-graecum L. showed immunomodulatory effect.[343536] In another study, aqueous extracts of Carcum copticum, that is, ajwain plant were found to have immunostimulatory activity.[37] The compound found in essential oil also gave rise to perilous upsurge in IgG antibiotics in serum. Cell-mediated immunity had been also dosage dependent and it was boosted with dosage. The increased response to the proliferation was supported by an increase in CD8+ subset, it could be attributable to an increase in proliferation of both the CD4- CD8+ (Cytotoxic T cells) and the CD4+ CD8+ cells. Although the exact mechanism is still unknown.[3338]

CONCLUSION

Results obtained from this study showed that seeds of T. ammi also have immunomodulatory activity and act as a stimulant on cell-mediated immunity. Although the most potent immunomodulatory effect was shown by methanolic extract, to develop an immunostimulant drug of natural origin, further studies are required to identify and separate constituents that are responsible for this effect.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.