Chemical constituents and medical benefits of Plantago major

Biomedicine & Pharmacotherapy 96 (2017) 348–360

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Review

Chemical constituents and medical benefits of Plantago major a

a,⁎

MARK a

Muhammad Bahrain Adom , Muhammad Taher , Muhammad Fathiy Mutalabisin , Mohamad Shahreen Amria, Muhammad Badri Abdul Kudosa, Mohd Wan Azizi Wan Sulaimanb, Pinaki Senguptaa,d, Deny Susantic a Department of Pharmaceutical Technology, Faculty of Pharmacy, International Islamic University Malaysia, Jalan Sultan Ahmad Shah, 25200 Kuantan, Pahang, Malaysia b Department of Basic Medical Sciences, Faculty of Pharmacy, International Islamic University Malaysia, Jalan Sultan Ahmad Shah, 25200 Kuantan, Pahang, Malaysia c Department of Chemistry, Faculty of Science, International Islamic University Malaysia, Jalan Sultan Ahmad Shah, 25200 Kuantan, Pahang, Malaysia d National Institute of Pharmaceutical Education and Research (NIPER)- Ahmedabad, Gujrat, India

A R T I C L E I N F O

A B S T R A C T

Keywords: Medicinal benefits Chemical constituents Plantago major

The medicinal benefits of Plantago major have been acknowledged around the world for hundreds of years. This plant contains a number of effective chemical constituents including flavonoids, alkaloids, terpenoids, phenolic acid derivatives, iridoid glycosides, fatty acids, polysaccharides and vitamins which contribute to its exerting specific therapeutic effects. Correspondingly, studies have found that Plantago major is effective as a wound healer, as well as an antiulcerative, antidiabetic, antidiarrhoeal, anti-inflammatory, antinociceptive, antibacterial, and antiviral agent. It also combats fatigue and cancer, is an antioxidant and a free radical scavenger. This paper provides a review of the medicinal benefits and chemical constituents of Plantago major published in journals from year 1937 to 2015 which are available from PubMed, ScienceDirect and Google Scholar.

1. Introduction Throughout human history, people have sought out natural remedies to improve their well-being and treat disease. The therapeutic benefits of medicinal plants have been recognised around the world and much scientific research has been done to prove their efficacy. It has been suggested that the abundance of research was motivated by the increased commercial potential of plant-derived therapeutic agents [1]. In fact, it is estimated that almost half of the current pharmaceutical medicines are derived from plants [2]. One of the main factors driving this trend is the ability of the chemical constituents in certain plants to exert therapeutic effects. For instance, medicinal plants are rich reservoirs of phenolic compounds which act as a potent antioxidant [3]. This paper reviews one of the medicinal plants used in Malaysia known as Plantago major. This plant, called ‘Ekor Anjing’ in Bahasa Malayu, has been used by Malays and Chinese as a tonic, diuretic and coughs mixture [4]. This plant also has many other medicinal properties as it contains numerous bioactive compounds. 2. Materials and methods The sources for this article are the results of the search for the keyword ‘Plantago major’ from a wide range of internet journal ⁎

databases including Scopus; Sciencedirect; Pubmed; Researchgate; Google scholar and Web of Science with a focus on the phytochemical; ethnopharmacological; pharmacological and biological activities of this plant. The data includes all studies; in all languages; published therein from 1937 until 2017. All data have been evaluated and discussed in this paper. This search method was adapted from Farzaei et al. [5]. 3. Taxonomy Plantago major is a perennial medicinal plant which belongs to the genus Plantago and the family of Plantaginaceae [6]. This is a hearty plant with about 265 species distributed all over the world. The name of Plantago major originates from the Latin ‘planta’, which means ‘sole of the foot’ [7]. This is a reference to the broad size of the leaves [8]. Plantago major is known by a number of common names around the world. Plantago major is known as common plantain (English), ‘Plantain majeur’ (French), ‘Breitwegerich’ (German), ‘Tanchagem-maior’ (Portuguese), ‘llantén’ (Spanish), and ‘llantén comú’ (Spanish) [9]. Besides, it is also known as broadleaf plantain (English) [10]. In addition, Plantago major is known as ‘Daun Sendok’ in Indonesia [11], ‘Lisan Al-hamal’ in Arabic [12] and in Swedish it is known as ‘Groblad’ which means ‘healing leaves’ [13]. Plantago major consists of three subspecies; Plantago major subsp.

Corresponding author. E-mail address: [email protected] (M. Taher).

http://dx.doi.org/10.1016/j.biopha.2017.09.152 Received 9 May 2017; Received in revised form 27 September 2017; Accepted 27 September 2017 0753-3322/ © 2017 Elsevier Masson SAS. All rights reserved.

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6.2. Alkaloids

major, Plantago major subsp. intermedia and Plantago major subsp. winteri. The first two, Plantago major subsp. major and Plantago major subsp. intermedia, have been commonly studied. However, while they have identical appearance, they have different habitat requirements [14]; the former can survive through the winter and can be found on rough surfaces and walkways, grassy places and cultivated regions, whereas the latter is less winter enduring and can be found in coastal areas [15]. On the other hand, Plantago major subsp. winteri has been the subject of just a few studies.

Furthermore, Schneider isolated the alkaloids indicain(12) and plantagonin(13)from Plantago major, however the process used to extract the chemical constituents from the plant was not stated [26] (Fig. 2). 6.3. Terpenoids Terpenoids (Fig. 3) have been isolated from the leaves and leaf wax of Plantago major; Pailer and Haschke-Hofmeister isolated loliolid(17) from the leaves [27] and Hiltibran et al., 1953 found ursolic acid(14), oleanolic acid(15), sitosterol acid(16) and 18β-glycyrrhetinic from the leaf wax extract in 95% ethanol [28]. The findings of the latter were supported by Ringbom et al. which also found the same terpenoids from hexane extract [29]. The leaf wax of Plantago major is readily extractable with chloroform, toluene and dichloromethane [30].

4. Characteristics of Plantago major Plantago major consists of a short stem, broad leaves, roots, flowers, fruits and seeds. It has a broad leaves which are usually about 15–30 cm in diameter of oval shape, 4–9 cm wide and 5–20 cm long, and uncommonly up to 17 cm wide and 30 cm long. There are about five to nine prominent veins on the leaves. The flowers are greenish-brown in colour with purple stamens and are small in size. The plant has small oval-shaped seeds which have a slightly unpleasant bitter taste [16].

6.4. Caffeic acid derivatives

5. Distribution of Plantago major

Noro et al. found caffeic acid derivatives (Fig. 4), namely plantamajoside(18) and acteoside(19), also known as verbacoside [31]. They found that the amount of plantamajoside is higher than acteoside in methanol extract. However, from 80% ethanol extract, the amount of plantamajoside is similar to the amount of acteoside. These findings were also proven by Skari et al. [23].

Plantago major was once found primarily in Europe and Northern and Central Asia, and now is widely dispersed throughout the world where it is known as common weed. A study on air pollen content was conducted and found that this species was present in Denmark, Finland, Iceland, Norway, and Sweden as well as their autonomous regions (the Åland Islands, the Faroe Islands, and Greenland) thousands years ago. It was also identified in England in 1672 and has been known in Canada since 1821 [17]. Interestingly, Plantago major was nicknamed ‘white man’s footprint’ by the Indians because it was found in every place Europeans had been [16]. Plantago major is readily found in areas with compacted soil such as roadsides and besides paths. Besides, it is fertilized by the wind and propagates primarily by seeds, which are held on the spikes located above the leaves [18].

6.5. Iridoid glycosides The main iridoid glycosides present in Plantago major are aucubin (20), which Long et al. isolated from the leaves [32]. A number of other iridoid glycosides (Fig. 5) have been isolated from other parts of the plant. While Bianco et al. isolated asperuloside (21) from the flowers [33], a number of studies have shown the presence of iridoid glycosides in the aerial parts of Plantago major. These include the study by Handjieva et al. which found majoroside(22) in the aerial part of Plantago major when extracted in n-buthanol [34], the study by Taskova et al. which isolated both 10-hydroxymajoroside(23) and 10-acetoxymajoroside(24) [35], and the study by Murai et al. that added catapol(25), gardoside(26), geniposidic acid(27) and melittoside (28) to the substances extracted from the aerial part [36].

6. Plantago major chemical constituents Plantago major is an important therapeutic plant which contains a variety of bioactive compounds including flavonoids, alkaloids, terpenoids, phenolic compounds (caffeic acid derivatives), iridoid glycosides, fatty acids, polysaccharides and vitamins. These compounds can be found in nearly all parts of the plant such as the seeds, leaves, flower and roots. The bioactivities of Plantago major are attributed to these chemical constituents.

6.6. Fatty acids Fatty acids (Fig. 6) have also be isolated from the seeds and leaves of Plantago major. Pailer and Haschke-Hofmeister isolated lignoceric acid (29) from the seeds [27]. Besides, the presence of palmitic acid(30), stearic acid(31), oleic acid(32), linoleic acid(33) and linolenic acid(34) was confirmed in Plantago major by using gas-liquid chromatography as well as permanganate oxidation and spectrophotometric techniques [37]. Likewise, myristic acid(35) was isolated from the seeds by Swiatek et al. [38]. Ahmad et al. found a minor occurring component of fatty acids in Plantago major seed oil extracted in petrol which is 9hydroxy-cis-11-octadecenoic acid [39]. In addition, Guil et al. isolated arachidic acid(36) and behenic acid(37) from the leaves [40].

6.1. Flavonoids The presence of flavonoids (Fig. 1) in Plantago major has been widely reported. The main flavonoids present are flavones, including luteolin(1) and apigenin(2) [19,20]. A number of scholars have isolated flavonoids from this plant, including Yuting et al. who isolated baicalein(3), hispidulin(4) and plantaginin(5) [21] and Sanz et al. who isolated scutallarein(6) [22]. Kawashty et al. isolated a broad number of flavonoids from Plantago major in Egypt: luteolin 7-glucoside(7), hispidulin 7-glucuronide(8), luteolin 7-diglucoside(9), apigenin 7-glucoside(10), nepetin 7-glucoside and luteolin 6-hydroxy 4′-methoxy 7-galactoside [19]. Most recently, Skari et al. found homoplantaginin(11) [23]. The presence and content of selected flavonoids from Plantago major methanol extracts has been confirmed by using LC–MS/MS technique [24]. The whole plant (100 g dried plant) has been extracted in hot water (1000 ml) for one hour. The flavonoids isolated from the aqueous extract were aucubin, baicalein, leuteolin, and baicalin, the glucuronide of baicalein [25].

6.7. Polysaccharides Ahmed et al. extracted polysaccharides from the seeds of Plantago major. They also isolated xylose(38), arabinose(39), galacturonic acid (40) in cold water extract and found galactose(41) from hot water extract [41]. This is supported by Gorin and Samuelsen et al. [42,43]. Additionally, Samuelsen et al. isolated glucuronic acid(42), rhamnose (43), galactose and glucose(44) from 50 °C water extract [43] (Fig. 7). Earlier studies by Samuelsen et al. isolated a highly esterified pectin polysaccharide, PMII [44,45]. These findings were followed by 349

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Fig. 1. Flavonoids in Plantago major.

continued examination leading to Samuelsen et al. wherein an anticomplementary acidic arabinogalactan, PMIa composed of arabinose, galactose, rhamnose, and galacturonic acid was isolated from 50 °C water extract [46].

6.8. Vitamins Additionally, Plantago major is a good source of vitamin C and carotenoids. This is confirmed in the study by Zenni and Ogzwewalla where ascorbic acid(45) and β-carotene (provitamin A) were isolated (46) [47]. (Fig. 8).

7. Medicinal benefits of Plantago major The important biological activities of Plantago major are summarized in Table 1.

Fig. 2. Alkaloids in Plantago major.

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Fig. 3. Terpenoids in Plantago major.

Fig. 4. Caffeic acid derivatives in Plantago major.

responsible for would healing is polysaccharides [49]. In addition, the antioxidant compounds present in Plantago major contribute to wound healing. The possible mechanism responsible for this is that it protects cells against destruction by inflammatory mediators which contribute to wound healing. Plantaginin, baicalein and hispidulin are flavonoids which are responsible as antioxidants and free radical scavengers [50].

7.1. Wound healing effects Three different concentrations (0.01, 0.1 and 1.0 mg/mL) of waterbased extract and ethanol-based extract from dried leaves of Plantago major were studied. The results demonstrated that the wound healing activity in the ex-vivo porcine wound-healing model was stimulated by both extracts with a clear pattern of dose and response. Moreover, it was claimed that the activity of wound healing by ethanol-based extracts with a concentration of 1.0 mg/mL had the highest activity compared to the other samples. The same concentration of Plantago major with water-based extract also resulted in increased activity of wound healing but not quite significant compared with the control [48]. Herein, the compound that was responsible for wound healing is the polyphenols. The results of phytochemical analysis showed that the ethanol-based extract contained higher levels of plantamajoside and other polyphenols compared with other extracts. Another compound

7.2. Antiulcerative A study was conducted by Rahimi et al. to screen the activity of Plantago major as anti-ulcerative. The leaves and seeds of the plant were extracted in methanol and the effect of the extract was determined by through studying the effect of ethanol and aspirin-induced gastric ulcers in rat models. The results showed that the Plantago major leaf extract significantly reduced the ulcer index with a curative ratio of 87.50% in ethanol-induced gastric ulcers in rat models. However, the 351

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Fig. 5. Iridoid glycosides in Plantago major.

and aqueous extracts. Studied separately, methanol extract inhibited ulcer formation by 29% and the aqueous extract by 37% [52]. The antiulcerative mechanism and the responsible chemical constituents have not been studied extensively. However, Cogo et al. claimed that Plantago major is capable of inhibiting H. pylori in vitro which is the main cause of digestive system ulcers [53].

seed extract showed no significant effect in the same model. The oral dose of 400 mg/kg Plantago major leaf extract significantly reduced the amount of gastric ulcers. In contrast, the seed extract at the same dose showed no effect on reducing the amount of gastric ulcers. All in all, Plantago major leaf and seed extract displayed the ability to reduce total acidity [51]. Atta et al. also conducted a similar study which tested the anti-ulcer activity of Plantago major leaves on alcohol and aspirin-induced gastric ulcers. The results showed that ulcer formation was significantly inhibited; 40% relative to the control group by combination of methanol

7.3. Antidiabetic The most recent study conducted by Abdulghani et al. found that 352

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Fig. 6. Fatty acids in Plantago major.

remnant pancreatic-cells in diabetic rats. However, the exact chemical constituents responsible for the antidiabetic effect have not been stated. Only some studies claimed that the hypoglycemic effect of Plantago major is due to the presence of flavonoids, sterols and sugars in the dichloromethane extract and tannins in the hexane extract.

Plantago major has a potent antidiabetic activity in streptozocin-induced diabetic rats. Plantago major methanol extract also has a hypoglycemic activity in oral glucose tolerance test (OGTT). OGTT of Plantago major extracted in methanol was studied on overnight fasted normal rats. The concentration for the extract was 500 and 1000 mg/kg per oral dose. The results showed that maximum glucose tolerance in Plantago major extracts after 30 min glucose administration was observed in the higher dose (1000 mg/kg) and minimum glucose tolerance was observed in the lower dose (500 mg/kg). For the effect of Plantago major on blood glucose level of STZ-induced diabetic rats, continuous post-treatment for 14 days with the 1000 mg/kg of Plantago major showed potential hypoglycaemic activity [54]. The mechanism of reducing blood glucose levels in diabetic rats may be caused by enhancing the control of glycemic mechanisms from

7.4. Antidiarrhoeal Plantago major is one of the traditional medicinal plants used in management of diarrhoea [55]. However, there have been very few of scientific studies to confirm the efficacy and activity of Plantago major as treatment of diarrhoea. A study conducted by Atta & Mouneir studied the effect of Plantago major leaves methanol extracts on diarrhoea induced by castor oil, as 353

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Fig. 7. Polysaccharides in Plantago major.

slightly more effective than the lower one. Plantago major ethanol extracts in a concentration of 1.6 mg/mL and produced a transient stimulation followed by an inhibition of the duodenal motility. A higher dose produced rapid relaxation. The initial stimulant effect may be attributed to the presence of irritant substances and may explain the contradiction in the folkloric use of this plant [52]. The antidiarrhoeal effect of Plantago major could be attributed to their content of tannins, flavonoids and alkaloids. Tripathi [56] and Mukherjee et al. [57] suggested that tannins showed antidiarrhoeal action by forming protein tannate which reduces intestinal secretion. Oi

well as the gastrointestinal movement in rats (charcoal meal) and on the motility of duodenum isolated from freshly slaughtered rabbits. A significant antidiarrhoeal effect of Plantago major methanol extracts against castor oil-induced diarrhoea in rats was achieved by 200 mg/kg orally for at least 4 h. The Plantago major methanol extracts given at a dose 400 mg/ kg administered orally showed greater potency than the 200 mg/kg oral dosage. Besides, at doses of 200 and 400 mg/kg, Plantago major methanol extracts also significantly decreased the gastrointestinal motility by significantly decreasing the distance travelled by the charcoal meal in the gastrointestinal tracts. The higher dose was

Fig. 8. Vitamins in Plantago major.

354

Water, methanol, chloroform, hexane

80% ethanol

Acetone, ethanol.

Aerial parts

Leaves

Leaves

355

Hexane

Soxhlet extraction: Dichloromethane Supercritical fluid extraction: ethanol 50% ethanol solution.

80% methanol solution.

Seeds

Leaves

Roots, stem and leaves.

Aerial parts

Aerial parts

Antifatigue

Anti-inflammatory

Ethanol

95% methanol solution

Leaves

Antidiarrhoeal

Methanol

Leaves

Water Methanol, water

Antidiabetic

Leaves Seeds, leaves, roots

Methanol

96% ethanol solution

Aerial parts

Leaves

n.a2

n.a2

Antibacterial

Anticancer

Extract

Plant part

Pharmacological/ biological activity

Table 1 Summary of biological activities of Plantago major1.

In vitro- prostaglandin-endoperoxide synthase 2 In vitro- prostaglandin endoperoxide H-synthase-2 (COX-2) and prostaglandin endoperoxide Hsynthase (COX-1) In vitro-rat liver mitochondria and human liver HepG2 cells. In vitro-human platelet cells

In vivo-mice

In vivo-adult mice

In vivo-STZ induced diabetic rat

In vivo-male Mus musculus Balb/C mice In vitro- MCF-7, MDA-MB-231, HeLaS3, A549, and KB

In vitro-human renal adenocarcinoma (TK-10), human breast adenocarcinoma (MCF-7) and human melanoma (UACC-62) cell lines.

In vitro-9 bacterial species

In vivo- mice infected with Streptococcus pneumoniae

In vitro-Escherichia coli, Bacillus subtilis, Candida albicans

In vitro- Helicobacter pylori

In vitro-neutrophils

Method

Ursolic acid Α-linoleic acid, ursolic acid and oleanolic acid.

IC50 = 130 μM 100 μg/mL COX-1 and COX-1 inhibition

Inhibition:

Crude extract

[82]

[81]

[80]

[73]

[52]

[54]

[71] [79]

[72]

[24] (continued on next page)

Crude extract

30–120 mg/kg

Crude extract

Crude extract

200 mg/kg

Hypoglycaemic in oral glucose tolerance test Decrease in number of soft faecal pellets due to relaxation of gastrointestinal tract. Improved exercise performance by forced swimming test (FST) method and biochemical assays namely serum urea nitrogen and lactate. COX-2 inhibition

0.1 mg/mL

–

Ursolic acid = 6.27–18.33 μg/mL Oleanolic acid = 17.63–100 μg/mL 1000 mg/kg

Inhibit production of reactive oxygen species (ROS) COX-1 and 12-LOX inhibition

Crude extract Ursolic acid, oleanolic acid, aucubin

Luteolin-7-O-β-glucoside

Ethanol extract: 85 mg/mL Total growth inhibition:

[77]

[74]

Pectin polysaccharide, PMII. Crude extract

[75]

[53]

[78]

References

Crude extract

Crude extract

Crude extract containing baicalein and aucubin.

Constituent

Acetone extract: 57 mg/mL

Water extract: Bacillus subtilis (0.4–0.025 g/ mL) Methanol extract: Bacillus subtilis (0.2–0.4 g/mL) Chloroform extract: Escherichia coli (0.2–0.4 g/mL) Hexane extract: Escherichia coli (0.025–0.4 g/mL) 1.2 mg

Aucubin: 50 μg/mL Baicalein: 2 μg/mL 5 mg/mL

Crude extract: 3%

Used Dose/concentration

TK–10 = none MCF–7 = 97 μg/mL UACC–62 = 112 μg/mL 25 μg/mL Methanol extract (seeds) = 153.38–247.41 μg/mL

Inhibition the growth of cancer cells Cytotoxicity effects.

Inhibition the growth of cancer cells

Reduction of number of bacteria in the serum and improvement of mice survival rate. Acetone extract showed effectiveness in MIC for all bacterial species, but ethanol extract only showed effectiveness for 2 bacterial species.

Growth inhibition of H. pylori by disk diffusion method. Growth inhibition of the bacteria by disk diffusion method

Production of reactive oxygen species (ROS)

Mechanism

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70% methanol solution

50% ethanol solution

Ethanol

Whole plant Leaves

Leaves

Leaves

356

2

1

Table format was adapted from Farzaei et al. [5]. n.a = data not available.

Wound healing effects

70% ethanol solution

Aqueous

Renoprotective effect

Antiviral

In vitro- oral epithelial cell (OEC) line H400 Ex vivo- porcine wound healing model

In vivo-Mice of Swiss line

In vivo-Albino Wistar rats.

In vitro- Herperviruses (HSV-1, HSV-2) and adenoviruses (ADV-3, ADV-8, ADV-11)

In vitro- calcium oxalate formation

Methanol

Stems, leaves, roots Whole plants

Antiurolithiatic

In vitro-DPPH assay

Leaves, Seeds

Decrease serum creatinine, potassium and urea concentration Regeneration of epidermis at the wound area Stimulate cell regeneration and proliferation in scratch assay Stimulation of wound healing activity

Improves the cyto-protection against virus infection towards human skin basal cell carcinoma (BCC-1/KMC) cells line

Reduce the area of calcium oxalate crystal

Percentage inhibition of paw oedema. Improve response to pain by tail-flick method. Improve free radical scavenging activity

Inhibition of proinflammatory cytokines such as IL-1α, IL-1β and TNF-α

In-vivo-Sprague Dawley rats.

In vivo-Sprague Dawley rats In vivo-male Swiss albino mice

Mechanism

Method

Ethanol

60% ethanol, 60% methanol, deionized water. Methanol Methanol

Leaves

Seeds Seeds

Extract

Plant part

Antioxidant

Antinociceptive

Pharmacological/ biological activity

Table 1 (continued)

1 mg/mL

10 mg/mL

10% (w/w)

Caeffic acid: HSV-1 (15.3 μg/mL), HSV-2 (87.3 μg/mL), ADV-3 (14.2 μg/mL) Cholorogenic acid: ADV-11 (13.3 μg/mL), HSV-1 (47.6 μg/mL), HSV-2 (86.5 μg/mL), ADV-3 (76 μg/mL), ADV-8 (108 μg/mL) 1200 mg/kg

Crude extract: HSV-2 (1000 mg/mL)

Seeds = 60 ppm 100–250 μg/mL

Leaves = 20 ppm

Crude extract

Crude extract

Crude extract

Crude extract

Caffeic acid, cholorogenic acid

Terpenoid

Linoleic acid, amino acids, phenolic and flavonoids compound

Crude extract Crude extract

Crude extract

COX–1 = 0.65 mg/mL 12-LOX = 1.73 mg/mL 1000 mg/kg

25 mg/kg 400 mg/kg

Constituent

Used Dose/concentration

[48]

[49]

[84]

[83]

[25]

[76]

[69]

[67] [68]

[62]

References

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contrast, the seed extract in ethanol at the same concentration showed very low antioxidant activity (25%). The researchers came to the conclusion that Plantago major ethanol extracts have more active antioxidant activity when compare to cold and hot water extracts, and the leaves in particular showed greater positive effects compared to seeds extracts [69]. Another study conducted by Amic et al. tested the antioxidant activity of the whole plant of Plantago major extracted in methanol. The results showed that the extract at a concentration of 0.8 mg/g had 89.3 ± 1.5% scavenging activity. The responsible compounds that have the scavenging property against DPPH are probably due to hydroxyl groups present in the phenolic compounds of Plantago major. The other suggested chemical constituent responsible for the scavenging activity is the flavonoids present in Plantago major. They possess a 3′, 4′dihydroxy occupied β ring and/or 3-OH group which can attract and decrease the oxygen radicals into neutralized substances like water [70].

et al. claimed that galloyl-tannin isolated from rhubarb was effective in cholera toxin activities including fluid accumulation in ileal loops isolated from rabbits and mice [58]. It was reported that intraperitoneal injection of some alkaloids decreased intraluminal accumulation of fluid [59]. The active principle possessing the antidiarrhoeal effect appeared to be extracted from methanol. In this respect, the methanol extracts were more effective than aqueous plant extracts against castoroil induced diarrhoea [60]. Additionally, the flavonoid content of the plant extracts may also contribute to the antidiarrhoeal effect [61]. 7.5. Anti-inflammatory A recent study was conducted by Hussan et al. [62] to test the antiinflammatory activity of Plantago major leaf extract on acetaminopheninduced liver injury of rats. 1000 mg/kg of Plantago major was extracted in three different solvents such deionized water, 60% ethanol and 60% methanol. The lipoxygenase assay was used to examine the anti-inflammatory activity. (Lipoxygenase is an enzyme that catalyzes arachidonic acid to synthesize leukotrienes which exhibits an important function in inflammation.) The Plantago major extracts displayed an inhibitory effect on leukotrienes with the methanol extracts demonstrating the highest anti-inflammatory activity followed by the ethanol extract [62–65]. The IL-1 and TNF-α which are released by activated macrophages stimulate leukocyte adhesion to endothelial surfaces prior to migration into tissues. IL-1 often expresses synergistic action with TNF-α to initiate cell death. Samuelsen claimed that the anti-inflammatory activity of Plantago major is contributed by iridoid glycosides such as aucubin and flavonoids such as baicalein and hispidulin [16]. Another studied pointed out that hispidulin is effective as a 5-Lipoxygenase inhibitor [66]. A study conducted by Türel et al. reported the anti-inflammatory activity of methanol extract of seeds of Plantago major on carrageenaninduced rat paw oedema. Plantago major showed anti-inflammatory effect however it was less effective than the reference drug in the study (indomethacin) [67]. It is possible that inhibition of COX-2-catalyzed prostaglandin biosynthesis may be responsible for the anti-inflammatory action [29].

7.8. Anticancer A study conducted by Ozaslan et al. tested the anticancer activity of Plantago major against Ehrlich ascites carcinoma in mice. Plantago major leaves were extracted with hot water. Then, the extract was administered orally for ten days in three different concentrations (25 μg/mL, 50 μg/mL and 75 μg/mL). The effect of the extract as an anticancer agent was determined by using pathological investigations. The intestines and the colons of the animals were stained with haematoxileneosin for histological examination. The results from pathological findings found that Plantago major leaves aqueous showed inhibitory effect against Ehrlich ascites carcinoma. The inhibition decreased due to increasing the extract concentration. The most effective concentration was found to be 25 μg/mL dose when compared to the other experimental doses. However, some ingredients of Plantago major have shown toxicity or adverse side effects on lower gastrointestinal tissues, so that the acceptable concentration is from 25 to 50 μg/mL. These promising results propose that Plantago major could be an effective agent for cancer prevention [71]. Another study conducted by Gálvez et al. studied Plantago major extract with methanol to evaluate the cytotoxic activity against three human cancer cell lines, human melanoma (UACC-62), human breast adenocarcinoma (MCF-7) and human renal adenocarcinoma (TK-10) cell lines, using the sulphorhodamine B (SRB) assay in vitro. The results found that Plantago major methanol extract showed a cytotoxic effect on the melanoma (UACC-62) and breast adenocarcinoma (MCF-7) tumor cell lines. The cytotoxic activity of the extract is dependent on the concentration administered. Nevertheless, the extract showed no cytotoxic activity against renal adenocarcinoma (TK-10) cells. Flavonoids, flavone and luteolin in Plantago major are thought to be the bioactive compounds responsible for the cytotoxic activity present in the extract. Although, the exact mechanism responsible for the cytotoxic activity of luteolin-7-O- β-glucoside is not thoroughly understood, it is suggested that topoisomerase-mediated DNA damage is the involved mechanism. Meanwhile, Luteolin-7-O-β-glucoside acts as a potent DNA topoisomerase I poison [72]. Kobeasy et al. also conducted a study of Plantago major as anticancer agent. The plant leaves and seeds were extracted with ethanol, cold and hot water separately. The results found that the inhibition of tumor growth is dose dependent. The greatest effect was showed by the ethanol extracts of Plantago major leaves followed by hot water extract of the leaves [69].

7.6. Antinociceptive A study was conducted by Atta & Abo El-Sooud to examine the activity of Plantago major as antinociceptive. Herein, Plantago major leaves and seeds were extracted separately in methanol. Then, the extracts were studied on mice in an acetic acid-induced writhing and tail flick test. The results found that the seed extract with the oral dose of 400 mg/kg showed significant antinociceptive activity against acetic acid-induced writhes with a protection of 62.3%. The protection rate of the leaf extract at the same doses was decreased by only 48.8%. The plant extract of 200 mg/kg (smaller dose) showed no antinociceptive activity on the pain stimulated by acetic acid. The leaf extract of 400 mg/kg showed significant prolonged effect in the latency to the response of the tail towards heat stimulation, while the smaller dose showed no effect [68]. More needs to be known about the chemical constituents responsible for activity of Plantago major as antinociceptive and the exact mechanism involved in this activity. 7.7. Antioxidant and free radical scavenger A study was conducted by Kobeasy et al. [69] to test the free radicalscavenging activity of Plantago major extracts. Plantago major leaves and seeds were extracted in cold water, hot water and ethanol. Then, the activity of each extracts was determined by using stable 1, 1-diphenyl2-picryl hydrazyl radical (DPPH) in vitro. The results claimed that the Plantago major leaf ethanol extract showed the highest antioxidant capacity even at a low concentration of 20 ppm (78% activity). In

7.9. Anti-fatigue Fatigue is a symptom that is marked by the feeling of tiredness due to vigorous physical activity and usually can lead to muscular pain. A study was conducted by Mao-ye & Li-guo that examined the anti-fatigue 357

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Fig. 9. Summary of medicinal benefits and chemical constituents of Plantago major.

Under electron microscope, it showed the rupture of Gram positive bacteria cell walls and the formation of blebs on Gram negative bacteria [76]. Another study conducted by Metiner et al. examined the antibacterial activity of Plantago major leaves extracted in acetone and ethanol by using the macrodilution liquid (tube) method. Both extracts were studied on nine species of bacteria; Escherichia coli, Bacillus cereus, Bacillus subtilis, Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumonia, Salmonella enteritidis and Proteus mirabilis. The results showed the ethanol extract was only effective against Escherichia coli and Bacillus cereus. In contrast, Plantago major acetone extract showed effect on all of the nine bacteria species with different concentrations [77].

activity of Plantago major (73). Herein, the seeds were extracted in ethanol, and the effect of the extract was studied on forty eight male mice. The anti-fatigue activity of Plantago major seed ethanol extract was determined by using forced swimming test and biochemical assays of blood of the mice. The results showed that the extract prolonged swimming time by increasing glycogen in the tissue (as an energy source) and reducing lactic acid in the blood as well as serum urea nitrogen. (Glycogen acts as source of energy while lactic acid is one of the causes of fatigue.) Therefore, it is proposed that Plantago major seed ethanol extract possesses anti-fatigue activity so that it can be used to enhance endurance exercise capacity. However, more evidence is required to determine the exact mechanisms involved in Plantago major as anti-fatigue agent and the bioactive compounds which are responsible for the effect [73].

7.11. Antiviral 7.10. Antibacterial Plantago major has been used by the Chinese as a traditional medicine to treat the common cold, conjunctivitis and viral hepatitis. A study conducted by Chiang et al. examined the activity of antiviral of Plantago major on herpes virus (HSV-1, HSV-2) and adenoviruses (ADV3, ADV-8, and ADV-11). Certain pure compounds of Plantago major extracts were found to be the important constituents which exhibit antiherpes and anti-adeno virus activities. The phenolic compounds were claimed to have the strongest activities against human herpes viruses and adenoviruses infections. Chlorogenic acid was active against HSV1, HSV-2, ADV-3, ADV-8 and ADV-11, whereas caffeic acid was active against HSV-1, HSV-2 and ADV-3. Caffeic acid and chlorogenic acid showed a broader spectrum of antiviral effect than ferulic acid and pcoumaric acid. This difference is due to the number of hydroxyl groups attach to cinnamic acid moiety. Caffeic acid and chlorogenic acid have two hydroxyl groups while ferulic acid and p-coumaric only have one. In contrast, Plantago major aqueous extract showed only slight anti-

A study was conducted by Hetland et al. on mice to test the antibacterial activity of Plantago major. They found that the soluble pectin polysaccharide (PMII) isolated from Plantago major leaves had defensive effects against systemic Streptococcus pneumoniae serotype 6B [74]. In another study, Velasco et al. found that antibacterial activities of Plantago major leaves and seeds in aqueous, methanol, chloroform and hexane extracts were positive on Escherichia coli, Bacillus subtilis and Candida albicans cultures in different ranges [75]. Besides, a study conducted by Sharifa et al. tested the whole plant aqueous, methanol and ethanol extracts of Plantago major on Bacillus subtilis, Staphylococcus aureus, Candida albicans, Candida tropicalis and Escherichia coli. The results showed that the methanol and ethanol extracts posed bactericidal activity against both Gram positive and Gram negative bacteria at concentrations of 100–200 mg/mL, but there was no activity on yeast. This was proved by using microscopy observation. 358

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