Background
Mankind, from antiquity, has continuously searched curing agents to various maladies and practiced the use of different medicinal plants as a remedy thereof. Conforming to some estimates, medicinal plants have served, directly or indirectly, as the source of about 80% of the present day medicines [
1] and have been explored due to having strong pharmacological activity, less adverse pharmacological effects and due to lower costs [
2]. Among the pharmacological activities, antiallergic activity is crucial as the prevalence of allergic diseases is significantly increasing around the planet [
3]. Allergic rhinitis, affecting 5–22% of the global population, is one of the most common allergic diseases in the world [
4]. Helminthic manifestation, due to high prevalence particularly in third-world countries, is a major health problem associated with several severe complications like anemia, malnutrition, dysentery etc. resulting from this infection [
5]. Although the second-generation antihistamine drugs, used for the treatment of allergic rhinitis, itching, urticaria etc., are devoid of the major adverse effects of first-generation antihistamines, nevertheless those drugs have several adverse effects including somnolence, headache, appetite simulation, body-weight gain, cardiac arrhythmia etc. [
6]. Again, most of the currently available anthelmintic drugs, though less frequently, exhibit various adverse effects including fatigue, drowsiness, insomnia, headache, dizziness, rashes, hallucinations, Stevens-Johnson syndrome, tinnitus etc. [
7]. This situation has prompted researchers to investigate new antiallergic and anthelmintic drugs [
8].
Acanthus ilicifolius Linn. (Family: Acanthaceae) is an around 2 m tall shrub, having somewhat elongated to oval shaped leaves [
9], with about 4 cm long, light blue, sessile flowers and up to 3 cm long, brown-colored capsule fruits [
10]. It is a true mangrove species abundantly found in estuaries, from Bangladesh, India to Polynesia of Asian tropics and northern Australia [
11].
Various parts of
Acanthus ilicifolius L. have been used in the treatment of a wide range of ailments. In ethnomedical practices, the leaves of the plant are used, in different parts of the world, for the treatment of asthma, rheumatism, paralysis and snakebite [
12‐
14]. Its roots have been used traditionally as antidote, expectorant, nervine tonic, and in the treatment of general debility, neuralgia, leucorrhoea, asthma, paralysis, dropsy, strangury, rheumatism, and psoriasis [
15‐
18]. Folklore claims for this plant in Indian and Chinese system of traditional medicine are usage as diuretic, blood purifier, aphrodisiac, in treating asthma, backache, diabetes, dyspepsia, excitation, gastralgia, hepatitis, leprosy, leucorrhoea, leukemia, malignant-tumors, neuralgia, paralysis, ringworm, rheumatism, allergic skin diseases, snakebite, stomach pain, and swelled spleen [
15‐
17,
19,
20]. Moreover, the bark-extracted water is used as antiseptic and to treat cold allergy and dermatitis in Thailand [
15,
20].
The plant has been reported to have antioxidant [
21‐
23], anti-inflammatory [
12,
24,
25], antinociceptive [
26], anticancer [
27,
28], antiasthmatic [
29], antimicrobial [
30,
31] antidiabetic [
32], antiprotozoal [
33,
34], cytotoxic (activity of flower extract) [
22], gastroprotective [
35], hepatoprotective [
13,
36], hypercholesterolemic [
37,
38], antihyperglycemic [
39], osteoblastic activity [
40] and activity against neurological disorders [
41].
Scientific investigations claimed the plant to contain a wide range of compounds including, Alkaloids, e.g. Acanthicifoline [
42], Trigonellin [
43], Benzoxazin-3-one [
44], Benzoxazinoid glucosides [
45], 4-O-b-D-glucopyranosyl-benzoxazolin-2(3H)-one [
46]; Flavonoids, e.g., Quercetin, quercetin 3-O-
β-D-glucopyranoside [
44], Acacetin 7-O-
α–L rhamnopyranosyl-(1″‘6″)-O-
β -D-glucopyranoside [
47]; Aliphatic glycosides, e.g., Ilicifolioside B [
48], Ilicifolioside C [
49]; Lignan glycosides, e.g., (+)-Lyoniresinol 3a-[2-(3,5-dimethoxy-4-hydroxy)-benzoyl]-O-
β-glucopyranoside, (8R,7’S,8’R)-5,5′-dimethoxylariciresinol4-
O-
β-D-glucopyranoside, Acanfolioside, Alangilignoside C [
50], (+)-lyoniresinol 2a-O-
α-D-galactopyranosyl-3a-O-
β -D-glucopyranoside, (+)-lyoniresinol 3a-O-
α-D-galactopyranosyl-(1–6)-
β–D glucopyranoside [
51]; Megastigmane and phenolic glycosides, e.g., (Z)-4-coumaric acid 4-O-
β –D glucopyranoside [
48]; (6R,7E,9R)-9-hydroxy-megastigman-4,7-dien-3-one-9-O-
β –D glucopyranoside [
47], 2,6-dimethoxy-p-hydroquinone 1-O-
β -D-glucopyranoside, syringic acid
O-
β-D-glucopyranosyl ester [
50], 5,11-epoxymegastigmane glucoside [
52]; Phenylethanol glycosides, e.g., ilicifolioside A, ilicifolioside D [
48,
53]; Triterpenoids, e.g., lupeol, oleanolic acid and ursolic acid [
44,
54,
55]; Steroids e.g., stigmasterol, stigmast-7-en-3
β -ol, stigmasteryl
β –D glucopyanoside [
44]; Miscellaneous, 7-chloro-(2R)-2-O
-β-D-glucopyranosyl-2H-1,4-benzoxazin-3(4H)-one [
50] etc.
While
Acanthus ilicifolius L. enjoys its traditional uses in curing skin allergies in Thailand in their traditional medicinal system, [
15,
19,
20,
55] or in helminthiasis [
12,
56], or in treatment of cancer and in many other diseases, no scientific evidence is so far available on the antiallergic, anthelmintic or cytotoxic activities of aerial parts of the plant. Moreover, phenolic antioxidant constituents being associated with antiallergic property [
57], due to the presence of lots of phenolic antioxidant constituents in the plant, the present study was designed and conducted to assess the antiallergic, anthelmintic and cytotoxic activity of aerial parts of
Acanthus ilicifolius L. In addition, an acute toxicity study was also carried out to assess the toxicity profile of
A. ilicifolius L. aerial parts.
Discussion
Evaluating the anti-allergic potential of ethanolic extract of aerial parts of
A. ilicifolius L. on TDI-induced allergic rhinitis in Swiss albino mice was the objective of this study. The investigation was carried out on an established animal model in which mice were sensitized and provoked with TDI for inducing the onset of allergy-like symptoms, e.g., sneezing, rhinorrhea, nasal blockage, redness, swelling, scratching (rubbing), differential cell infiltration into tissues of lungs and airways, eosinophilia and release of biochemical substances like interleukins (IL)-4, IL-6 etc. [
62,
63]. In this study, we showed that ethanolic extract of aerial parts of
A. ilicifolius L. exhibits symptomatic relief of allergic rhinitis in mice.
A. ilicifolius L. extract significantly suppressed sneezing and nasal score as shown in Fig.
2a, b, c. It also decreased the nasal rubbing (scratching), but that was not significant. Suppression of these allergy-like symptoms (Fig.
2a, b, c) prompted us to study whether the extract has any effect on cells associated with inflammation and on the total and differential blood cell count in TDI-induced allergy model mice [
8,
63]. In addition, in our study we have found increase in levels of total leukocytes, eosinophils, lymphocytes neutrophils, monocytes and basophils in blood of TDI-control mice (Fig.
3), and treatment with the ethanolic extract at an oral dose of 300 mg/kg and 500 mg/kg and cetirizine (20 mg/kg) reduced the count of these inflammatory cells as compared to TDI-control.
Mention-worthy, increased eosinophils in blood represent an overall state of allergic condition. Moreover, allergy/asthma patients show increased eosinophilia and leukocytosis and this increased eosinophilia and leukocytosis may work as a good cellular biomarker for allergic symptoms [
70,
71]. Therefore, our experimental results (Fig.
3) are consistent with hypothesis and findings presented previously by several researchers [
72‐
74]. In addition, the significant increases in the number of total and differential leukocytes, particularly, increase in the number of eosinophils, neutrophils, lymphocytes, monocytes and basophils, in the blood of the mice in TDI Control group, found in our study, is consistent with previous data reported by researchers who induced asthma using TDI in guinea pig model [
61] and in rat model [
62].
Preliminary phytochemical screening exhibited that
A. ilicifolius aerial part extracts contains alkaloids, flavonoids, glycosides, steroids, tannins, triterpenoids and saponins (Table
1). It has been reported previously that the antiallergic activity of
Gymnema sylvestre R Br is assumed to be due to the presence of tannins, total phenols, and flavonoids [
74]. Histamine signaling, which is the major cause of allergic symptoms, is suppressed by several phytochemicals compounds e.g. tannins, including epigallocatechin-3-O-gallate isolated from green tea [
75]. It has been reported that polyphenolic compounds e.g., gallic acid (3, 4, 5-trihydroxy benzoic acid) suppress various hypersensitivity reactions in mice and inhibit release of histamine and helper T cell cytokines, IL-4, IL-5 and IL-2 form mast cells in various experimental animal model of allergic diseases [
76‐
78]. As
A. ilicifolius extract has higher phenolic content, those could be the compounds responsible for the antiallergic effects of ethanolic extract of aerial parts of
A. ilicifolius in a possibly similar mechanism. Moreover, as gallic acid is a tannin compound in chemical nature, and as
A. ilicifolius extract exhibited presence of tannin in phytochemical screening, antiallergic activity of the extract could be due to the presence tannin contents in it.
Antioxidants reduce intracellular reactive oxygen species production and thus modulate both the function of mast cell as well as gene expression of IL-4 [
79]. It has been reported that the antiallergic activity of the ethanolic extract of
Sanseveiria trifasciata leaves is due to the presence of steroidal saponins, triterpenoids, flavonoids etc. which are known as potent antioxidants [
72]. As the extract of
A. ilicifolius L., in preliminary phytochemical analysis, exhibited presence of all of these compound including flavonoids, and as the extract exhibited free radical scavenging activity and possesses potent antioxidant property [
12,
13,
22,
23], it is possible that these phytoconstituents either alone or combinedly, may have elicited the antiallergic activity observed in our investigation.
According to a previous scientific report, saponins inhibit degranulation of mast cells and thus inhibit histamine signaling and alleviate allergic symptoms [
80]. Thus, presence of saponins in the extract exhibited in preliminary phytochemical analysis, could be responsible for the anti-allergic effect of the extract. In addition, Smita and other researchers have showed that a steroid, stigmasterol (stigmast-5, 22-dien-3
β-ol), in combination with dexamethasone (an antiallergic drug), imparts antiallergic activity which is more potent than the activity of dexamethasone alone. Thus, as
A. ilicifolius L. contains stigmasterol [
44] as well, the ethanolic extract of
A. ilicifolius may impart its antiallergic activity through the similar mechanism [
66].
Accumulating all these data, in conclusion, our experimental findings provide adequate scientific basis for traditional use of A. ilicifolius L. in the treatment of allergic rhinitis. Thus, according to our experimental data, we may hypothesize that prolonged oral administration of the extract might play an important role in the management of TDI-induced allergic rhinitis.
In in vivo studies of anthelmintic activity of plant materials, loss of movement or paralysis and complete destruction or death of live parasites are usually utilized as the parameters [
81‐
83]. In vivo anthelmintic activity of
A. ilicifolius has previously been reported on adult earth worm model and worm parasite of chicken [
56]. In the present work, we established significant anthelmintic activity of aerial parts of the extract of
A. ilicifolius in a concentration dependent manner on freshly collected live parasites (Trematoda and Nematode) from cattle which is consistent with the earlier report by Husori and colleages. Anthelmintic activity of secondary metabolites obtained from plants have been attributed to the presence of alkaloids, flavonoids, steroids, tannins and saponins [
56]. This is consistent with our data (Table
1) for the phytochemical analysis of the plant, as the preliminary phytochemical screening exhibited that
A. ilicifolius extract contains alkaloids, flavonoids, steroids, tannins and saponins.
Though, too little is known regarding the precise mechanism involved with the obtained anthelmintic activity of the extract, a role may have been played in it by the polyphenolics or other biochemical compounds present in the extract. It has been reported, for instance, that by uncoupling oxidative phosphorylation or binding to the glycoprotein on the cuticle of parasites, tannins are capable of interfering with energy generation in helminthes parasites and thus to cause death of the parasites [
84,
85]. Capability to bind with the free protein in the host animal’s digestive tract has been reported as another mechanism of tannins anthelmintic activity. According to some reports, tannins contained in the plant are capable of enhancing the absorption of protein which is obtained through the formation of protein complexes in the rumen, which at the low pH in the small intestine break down later [
86].
Moreover
, flavonoids and saponins have recently been reported to have the capability of exerting anthelmintic activity. Saponins have been described to increase the membrane permeability and pores formation, both of which actions are similar to the mechanism of established anthelmintic praziquantel and toltrazuril [
87,
88]. Saponins and tannins may cause damage to the mucopolysaccharide membrane of worm which results in paralysis and death of those [
89].
Furthermore, the alkaloids and steroids in the extracts may play key role in providing the suppressive effect of sucrose transfer to the small intestine which eventually can reduce glucose support for the worm parasites. These effects provided by alkaloid and steroids together with the antioxidant effects exerted by flavonoids can reduce the production of nitrate to be used in synthesis of protein [
90]. Paralysis and death of parasites can possibly result from the action of alkaloid on CNS of parasites [
91]. In addition, all these phytochemicals and their interaction may possibly have synergistically enhanced therapeutic efficacy of anthelmintic activity of
A. ilicifolius aerial parts extract.
The brine shrimp lethality bioassay is an established, easy, safe, economic and practical method for predicting important pharmacological activities such as enzyme inhibition, ion channel interference, cytotoxic activity and several other bioactivities of synthetic compounds and of plant products [
69,
92‐
94]. The significant correlation between the brine shrimp assay and human solid tumor cell lines in vitro growth inhibition demonstrated by the National Cancer Institute (NCI, USA) has shown the value of this bioassay as a pre-screening tool for antitumor drug research [
95,
96]. According to Meyer et al., natural products’ extracts exhibiting LC
50 ≤ 1000 μg/mL in brine shrimp bioassay have been claimed to possess bioactive constituents [
69]. Several criteria for toxicity in brine shrimp lethality bioassay for synthetic compounds or natural products have been established where the LC
50 values above 1000 μg/mL are non-toxic, between 500 and 1000 μg/mL are weak toxic and below 500 μg/mL are toxic [
97]. In the present study, both the extract and vincristine sulphate showed a gradual increase in mortality rate with the increase of concentration. The LC
50 value for the ethanolic extract of
A. ilicifolius was found to be very low (44.57 μg/mL), signifying that the extract may contain potent pharmacologically active compound(s). The toxicity of plants is mainly attributed to the presence of alkaloids, phenolic compounds, glycosides, steroids, tannins, phlobatannins, terpenoids, flavonoids and saponins [
98‐
101]. This is consistent with our observation as well, as the phytochemical group analysis of the extract exhibited the presence of alkaloid, tannins, flavonoid, steroids and saponins (Table
1). Thus, the principal advantage of the extract with regard to its cytotoxic effect could be exploitation of the cytotoxic potentials of
A. ilicifolius for the development of natural product derived better medication for cancer.