Keywords

12.1 Introduction

Plants have been a very important part of human civilization since the ancient times when man was learning to survive. Humans utilized plants and plant products to fulfill all the most important necessities for their nourishment, and their existence is extremely reliant on primary producers, mostly plants. Near about 5000 plant species have been widely utilized by humans as food. Besides fulfilling primary needs, the plant species have been utilized as medicines for various human illnesses (Sullivan and Shealy 1997). Such plants are called traditional medicinal plants and have played the most important roles in our ancient system of medicine. Those plants have been provided hypothesis and become primary source of modern drug discovery by the pharmaceutical industry. The World Health Organization has prepared a list of over 21,000 species of plants that are in use as traditional medicines around the world, with more than 80% of the world’s population believing on plant-based medicines for their primary health care (Gurib-Fakim 2006).

Among a total of 21,000 medicinal plant species, the family Lamiaceae (also mint family) is known as the biggest family explored for the discovery of bioactive secondary metabolites (Wink 2003). This family is represented by 7200 species belonging to 236 genera (Brauchler et al. 2010). Many species belonging to this family are greatly aromatic and mainly produce oil that evaporates in low temperature due to the existence of peripheral glandular structures (Giuliani and Bini 2008). Among them, the genus Marrubium L. has attracted most of the researchers around the world, due to the presence of active chemical and pharmacological properties. This genus normally consists of approximately 40 species. Among these, Marrubium vulgare L. is a perennial herb found along road sides, and in waste areas.

12.2 Methodology

Data related to Marrubium vulgare were collected from published articles using Google Scholar, SciFinder, PubMed, and Science Direct. Books and journals available in library were also consulted while preparing this manuscript. More than 160 articles were studied of which 105 important references are included in this chapter. The present chapter is formulated in such a way so that it becomes easy for researchers to get up to date information on this particular plant, whether related to its phytochemistry or pharmacology.

12.3 Morphological Description

Marrrubium vulgare (Fig. 12.1) height varies from 25 to 45 cm. Leaves of the plants vary from 2.0 to 5.0 cm in length along with very dense surface, mostly sheltered in downy hairs. They also possess blunt and rounded tips. The stems mostly contain branched and woody base. These woody stems are quadrangular and very tightly covered with hairs. Fruits or nutlets are enclosed by calyx. Seeds are brown or black and vary from 1 to 2.5 mm in length (Halvorson 2003).

Fig. 12.1
figure 1

Habit and morphology of Marrubium vulgare (left side: habit of whole plant; middle: root parts used as medicine; right side: leaves)

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12.4 Traditional Uses of Marrubium vulgare

Marrubium vulgare is utilized as traditional medicine to treat different types of ailments. Conventionally, the leaves of the plants have been used to get relief from joint pain, chest infection, inflammation, inflamed eyes, cough and cold, and nighttime blindness. This plant is also used as a purgative agent and widely used as a bitter tonic and appetizer, and is also helpful in expulsion of fetus (Kirtikar and Basu 1996). It is also used as a herbal tea due to its stimulating and antispasmodic properties (Yamaguchi et al. 2006). It is also used in curing head-pain, and the chemicals in it contain diterpene labdane and marrubiin (Piccoli and Bottini 2008; Karioti et al. 2003). It is also used to treat liver problems and flu (Balme 1982; Grieve 1984; Chevallier 1996; Lorenzi and Matos 2002; Sahpaz et al. 2002a). The leaves and stems are used as antiseptic, cholagogue, antispasmodic, and as stimulant (Grieve 1984; Chiej 1984; Launert 1981; Lust 1983; Mills 1985; Bown 1995). It is often made into a syrup or candy, though it can be used as a tea (Grieve 1984). Due to bitter nature, this plant species is also helpful in digestive functioning of the stomach (Bown 1995; Chevallier 1996). Traditionally, this herb has been considered helpful for the treatment of persistent fever and cholera (Anonymous 2005). M. vulgare extracts are also used as flavoring agents in food industries in the USA (Bradley 1992; Vincenzi et al. 1995). The volatile oil produced by M. vulgare has a folk status for its use in calming nervous heart. The tiny amount of marrubiin, a labdane diterpene, has the potential to normalize heartbeats. The hot water-extract is used as sweat-inducer, whereas the cold infusion of bitter taste is used as tonic for digestion. The extract of M. vulgare is also useful in curing malaria and reducing fever (McIntyre et al. 1988).

The dry flowering stem is used in treating menstrual irregularities and pain. It is also useful in treating sore wounds. The presence of highly volatile constituents makes it the best stimulant and antihelmintic, as well as useful in amenorrhea, chronic rheumatism, dyspepsia, and hepatitis (Singh and Panda 2005; Haq et al. 2011). M. vulgare extract also showed appetite stimulant effects through bitter receptors (Janssen et al. 2011). This plant species is also used by local people for candy in the province of United Kingdom. The Egyptians and Romans used plant extracts as the antidote for snake bites. The extract assists to destroy cankerworms when sprayed on fruiting trees. It is also used to ease heartburn, in digestion, and to raze worms in human intestine.

Traditionally, people slowly chewed fresh chopped leaves of M. vulgare along with honey to treat cold and sore throat. A candy formulation that contains fresh leaves (four ounces), crushed seeds of cardamom, one teaspoon anise seed, and 250 mL water is used as a standard care for cough in children aged between 5 and 12 years (Barrett 2009). In the Latin American country Brazil, the plant is employed in treating gastrointestinal disorders, inflammation, and respiratory ailments (Meyre Silva et al. 2005; Culpeper 2006). An infusion of leaves is given as an insecticidal (Benedum et al. 2006).

12.5 Phytochemistry

A large number of chemical studies have been carried out on this plant around the world and have confirmed the occurrence of different classes of molecules in M. vulgare, which belongs to diterpenes, sesquiterpenes, flavonoids, phenylpropanoid esters, and essential oils (EOs).

12.5.1 Diterpenoids

Various terpenoids (Fig. 12.2) are characterized from aerial parts of Marrubium vulgare. The first diterpenoid to be isolated is marrubiin, which was first isolated in 1842; since then, a number of research work has been published regarding isolation, structure elucidation, chemical reaction, stereochemistry, and synthesis of this bioactive compounds (Fulkke et al. 1968; Knoss et al. 1997).

Fig. 12.2
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Diterpenoids of Marrubium vulgare

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In a separate research, the low-pressure-based chromatographic method was developed for the marrubiin isolation (Knoss et al. 1997). In the year 1992, Rey and co-workers established stereochemistry of marrubiin by X-ray crystallography (Rey et al. 1992). It has been reported that marrubiin has two rings. Some other diterpenoids like marrubenol, marrubiol, peregrinin, premarrubiin, marrubinone B, and vulgarol have also been isolated from aerial parts of Marrubium vulgare (El Bardai et al. 2003b; Popa et al. 1968; Sahpaz et al. 2002b; Masoodi et al. 2015; Henderson and McCrindle 1969; Popa and Pasechnik 1975). A novel compound, 11-oxomarrubiin, was identified from methanol extract (ME) of whole plant of M. vulgare (Shaheen et al. 2014). Additionally, two new labdane diterpenoids, 3-deoxo-15-methoxyvelutine C and 12(S)-hydroxymarrubiin, were isolated and identified from the extract of methanol from the collection from Srinagar, Jammu and Kashmir (Masoodi et al. 2015).

12.5.2 Flavonoids

Various flavonoids (Fig. 12.3) including aglycones and their glycosides were reported from different plant parts of M. vulgare. These include chrysoeriol, vitexin, isoquercitrin, quercetin 3-O-rhamnosyl-glucoside, apigenin 7-lactate, luteolin, apigenin and its 7-(6″-p-coumaroyl)- glucoside, apigenin-7-O-glucoside, luteolin-7-O-β-D-glucoside, and luteolin 7-lactate (Nawwar et al. 1989; Kowalewski and Matlawska 1978; Rahman 2005).

Fig. 12.3
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Flavonoids from Marrubium vulgare

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In the year 2014, a flavone derivative 3-hydroxyapigenin-4′-O-(6″-O-p-coumaroyl)-β-D-glucopyranoside was isolated from alcoholic extract of M. vulgare (Shaheen et al. 2014). Alkhatib et al. (2010) have reported Ladanein from dichloromethane extract of M. vulgare. In another study, a flavone 7-O-β-glucuronyl luteolin was reported for the first time in aerial part of M. vulgare along with some well-characterized compounds such as 5,6-dihydroxy-7,4′-dimethoxyflavone (ladanein) and 7-O-β-glucopyranosyl luteolin (Pukalskas et al. 2012).

12.5.3 Phenylpropanoid and Phenylethanoid Glycosides

Several phenylpropanoids (Fig. 12.4) like forsythoside B, (+) (E)-caffeoyl-L-malic acid, ballotetroside, acteoside, and arenarioside were isolated from flowering portion of this species (Sahpaz et al. 2002a).

Fig. 12.4
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Phenylpropanoid and phenylethanoid glycosides isolated from Marrubium vulgare

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Two compounds, verbascoside and forsythoside B, were isolated by Pukalskas et al. (2012) from the above-ground portion of this species using gradient solvent of methanol:water:acetic acid (79:20:1). Vulgarcoside A, a diglycoside derivative of the diterpene peregrinol, was isolated from this plant (Pukalskas et al. 2012). Sahpaz and co-workers have reported few phenylethanoid glycosides such as marruboside and acetyl marruboside (Sahpaz et al. 2002a).

12.5.4 Active Compounds in Essential Oil of Marrubium vulgare

To date, several essential oils present in M. vulgare have been studied by different workers across the globe. They include monoterpenes, sesquiterpenes, and esters.

Monoterpenes: Monoterpenes (Fig. 12.5) isolated from Marrubium vulgare includes: pinene, tricyclene, thymol, carvacrol, α-pinene, piperitone, 1,8-cineole, sabinene, limonene, p-cymene, α-terpinolene, camphene, p-fenchene, geranial, a-thujone, citronellyl acetate, β-citronellol, and p-menthane-5,6-dihydroxy-3-carboxylic acid, commonly known as marrubic acid (Saleh and Glombitza 1989, Ahmad et al. 2010, Salama et al. 2012, Abadi and Hassani 2013, Zawislak 2012; Kadri et al. 2011).

Fig. 12.5
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Active constituents under the class monoterpenes of Marrubium vulgare

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Sesquiterpene: From Marrubium vulgare, following sesquiterpenes (Fig. 12.6) have been isolated: β-bisabolene, δ-cadinene, isocaryophyllene, γ-cadinene, E-β-farnesene, β-caryophyllene, germacrene D-4-ol, Germacrene D, Vulgarin, γ-eudesmol, ledene, and transcaryophyllene (Salama et al. 2012; Zawislak 2012; Kadri et al. 2011; Al Ahl et al. 2015; Amer 1993).

Fig. 12.6
figure 6

Sesquiterpene in essential oil of Marrubium vulgare

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12.5.5 Other Chemical Constituents

Besides pentacyclic triterpene ursolic acid, two steroids including β-sitosterol and stigmasterol, and phenolic acids such as gallic and caffeic acids, have also been isolated from this species (Nawwar et al. 1989; Laonigro et al. 1979). Small amounts of alkaloids, betonicine and turicine have also been isolated in separate studies (Baxter et al. 1998; Daniel 2006). Other compounds such as 2-(omega-1)-dimethylalkanes, 3-methylalkanes, 2-methylalkanes, and 3-(omega-9)-dimethylalkanes have been reported from the above-ground parts of the plant (Meyresilva and Cechinelfilho 2010).

12.6 Pharmacological Activities

12.6.1 Anti-inflammatory Activity

Genus Marrubium vulgare is regularly found in European and Mediterranean countries, hence several researches took place in these regions. In folklore, inflammation and neuro-sedative disorders are treated. The hydro-alcoholic extract (HAE) and methanol extract (ME) were studied separately, which exhibited significant anti-inflammatory activities. The ME at a dosage of 200 mg/kg proved anti-inflammatory function against prostaglandin E2 and carrageenan-induced inflammation in Swiss mice (Kanyonga et al. 2011). The HAE of M. vulgare showed both the in vivo and in vitro anti-inflammatory properties (Abbouyi et al. 2013). The study was assessed by investigating its effect on O2 consumption and O2 generation by rat pleural polymorphonuclear leukocytes (PMNs) stimulated with O2. Five major phenylpropanoid esters, (+) (E)-caffeoyl L-malicacid, acteoside, arenarioside, forsythoside B, and ballotetroside, were isolated, characterized, and their anti-inflammatory activity was studied by Sahpaz et al. (2002a). In another research, ME was assessed on isoproterenol (100 mg/kg/day) induced myocardial infarction (MI) in rat model (Yousefi et al. 2014). It was observed that extract administered orally with dosages of 10, 20, and 40 mg/kg/12 h significantly reduces levels of tumor necrosis factor-α (TNF-α) and peripheral neutrophil count. It was also revealed that the anti-inflammatory activity might be attributed to the presence of marrubiin and other glycosidic phenyl propanoid esters that were isolated from methanolic extract.

12.6.2 Analgesic and Antinociceptive Activities

Marrubium vulgare being utilized in traditional medicine to cure a number of diseases, its hydro-alcoholic extract (aerial parts) showed significant analgesic activity (deSouza et al. 1998). The study clearly proves the analgesic strength with inhibitory dose 50% (ID50) at 22.2 and 272.2 mg/kg for the i.p. and p.o., respectively. It was also discovered that these effects may be due to steroids and terpenes. Meyre-Silva et al. (2005) also recorded that M. vulgare had shown strong analgesic properties, which was accredited to marrubiin, a furan labdane-type diterpene. Marrubiinic acid, which was synthesized from marrubiin, and two of its ester derivatives, marrubiinic acid benzyl and methyl esters, showed antinociceptive activity to acetic acid-induced abdominal writhing in mice at doses of 10 mg/kg i.p. and 50 mg/kg orally. Marrubiin was also described to have antinociceptive effect in nociception mice models by DeJesus et al. (2000). In this study, marrubiin was shown to have exhibited dose-dependent antinociceptive effects with 90–900 μmol/kg by p.o. route or 3–90 μmol/kg by i.p. route. The study was completed to assess the antinociceptive effects due to acetic acid-induced, formalin-induced, and capsaicin-induced pains. The results clearly showed that marrubiin is very effective in inhibiting writhing responses (acetic acid-induced) in mice with an ID50 value of 2.2–90 μmol/kg, i.p. For formalin-induced responses, the ID50 values for i.p. and oral routes were found to be 6.6 μmol/kg and 126 μmol/kg, respectively. For capsaicin-induced inflammation, the ID50 value was found to be 28.8 μmol/kg. In 2011, Kanyonga et al. (2011) proved that ME at a dose of 200 mg/kg of M. vulgare exhibited the analgesic activity analogous to the acetylsalicylic acid.

12.6.3 Antiodematogenic Activity

Marrubiin being the main constituent of Marrubium vulgare, the researchers always try to discover its biological importance. In a study done by Stulzer and co-workers on mice model, marrubiin obtained from this species showed considerable and dose-dependent antiodematogenic effects on micro-vascular leakage in mice ears for different phlogistic agents (Stulzer et al. 2006). The percentage inhibition for histamine at 13.84 mg/kg was found to be 73.7%, for carrageenan at 13.61 mg/kg it was 63.0%, and for bradykinin it was 70% at 18.82 mg/kg. However, dextran yields slight inhibition of 32% as a phlogistic agent.

12.6.4 Antispasmodic Activity

Since Marrubium vulgare is used in folk medicine in numerous countries against several diseases, including gastrointestinal disorders, so to prove its biological feature research is being conducted in most of the countries where this species grows naturally. One such study was done in Brazil, where in vitro studies were conducted on aerial parts and roots of M. vulgare for antispasmodic effects on numerous smooth muscles, and it was observed that HAE (50% ethanol) exhibits antispasmodic properties in considerable range (Schlemper et al. 1996). Antispasmodic activity of plant extract may be due to the inhibition of neurotransmitters with a reasonable selectivity toward cholinergic contraction. A considerable difference was observed for bradykinin on guinea-pig ileum at 1 mg/mL of extract.

12.6.5 Gastroprotective Activity

In Brazil, M. vulgare is used traditionally for the management of respiratory and gastrointestinal infections; so on scientific basis, Paula de Oliveira et al. (2011) evaluated a diterpene marrubiin isolated and characterized from ME of this species for gastroprotective activity. It was observed that ME produced a considerable ulcer-protective effect on alcohol-induced mice model at a dose of 50 and 100 mg/kg, and the result was equivalent to the standard drug omeprazole at 30 mg/kg. In case of indomethacin-induced ulcers, methanolic extract exhibited potential activity at a dose of 50 mg/kg and cimetidine showed similar efficacy at a dose of 100 mg/kg. In both ulcer models, marrubiin (at 25 mg/kg) has shown considerable reduction in gastric parameters.

12.6.6 Antihypertensive Properties

Water extract of M. vulgare is extensively used as antihypertensive drug in folk medicine in Morocco. In order to establish its scientific basis, aqueous extract of M. vulgare was evaluated in normotensive Wistar-Kyoto rats/spontaneously hypertensive rats (SHRs) (El Bardai et al. 2001). Oral administration of aqueous extract considerably lowered the observed systolic blood pressure (SBP) in SHRs and inhibited the noradrenaline and potassium chloride (100 mM) induced contractile responses of rat aorta in an in vitro study. These results clearly indicate that hypotensive activity exhibited vascular relaxant activity. Vasorelaxant activities of two secondary metabolites marrubenol and marrubiin were reported in a study from aqueous extract and its cyclohexane fraction (El Bardai et al. 2003a). Marrubenol and marrubiin have inhibited contraction in dose-dependent manner in rat aorta. Marrubenol (inhibitory concentration 50% [IC50] values 7.7 ± 1.9 μM) was found somewhat more effective than marrubiin (IC50 values 24 ± 2.3 μM). In Mexico, for ethnomedical practices, M. vulgare is used as an antihypertensive drug (Jorge et al. 2013).

12.6.7 Antidiabetic and Antihyperlipidemic Activities

Knowing that Marrubium vulgare is traditionally used for management of diabetes, a scientific study was conducted to investigate the hypoglycemic effect on alloxan-induced diabetic rats, and results clearly demonstrated that the ethanol extract (EE) of aerial parts of M. vulgare showed hypoglycemic effect at a dose of 300 mg/kg with percentage inhibition of 30.3% (Novaes et al. 2001). A similar study was done on alloxan albino Wistar rats for the evaluation of antidiabetic and antihyperlipidemic effects of aqueous extract of M. vulgare (Boudjelal et al. 2012). A 50% reduced blood glucose level was observed at a dose of 100 mg/kg, and at 200 and 300 mg/kg, >60% reduction in blood glucose level was observed. In addition, a considerable lowering in total cholesterol, triglyceride, and lipid levels was also observed due to the same extract. These results were found comparable to the market drug glibenclamide.

In the year 2015, a study was conducted in streptozotocin-induced diabetic model to evaluate antidiabetic and antidyslipidemic effects of M. vulgare (Elberry et al. 2015). It was observed that methanolic extract of M. vulgare considerably lowered the glucose level in blood after treatment for 14 days at a single dose of 500 mg/kg/day. Furthermore, methanolic extract also showed a significant increase in tissue glycone and plasma insulin. A rare fatty acid, 6-octadecynoic acid (6-ODA) was identified in methanol extract of M. vulgare and reported to function as PPARγ α agonist (Ohtera et al. 2013). In another study, effectiveness of plant extract of M. vulgare was investigated in cyclosporine A and streptozotocin-induced diabetes mellitus-type 1 in mice (Maraia 2014). Thus, this species was recommended as useful in curing auto-immune diabetes.

12.6.8 Antihepatotoxic Activity

Horehound is used in herbal medicine of Saudi Arabia for the treatment of gastroenteric, respiratory, and inflammatory disorders. In Saudi Arabia, a study was performed to investigate the antihepatotoxic activity of methanolic extract against carbon tetrachloride (CCl4) induced hepatic damage in rats (Elberry et al. 2010). The observation exposed that methanolic extract (500 mg/kg/day) significantly reduced the levels of lactate dehydrogenase (LDH), aspartate transaminase (AST), and alanine transaminase (ALT), suggesting antihepatotoxic effect of M. vulgare. This effect may be attributed partially to the antioxidant activity of the extract. CCl4-induced hepatotoxicity in albino mice was also studied by Ibrahim et al. (2014). The effects of aqueous, ethanol, and petroleum ether extracts from aerial parts of M. vulgare were also evaluated. The results showed that ethanol extract exhibited strong protection against the damage caused by carbon tetrachloride (CCl4).

In another study, first time 12 active compounds isolated from M. vulgare were evaluated for their drug-likeness and hepatoprotective activity in a computer-based in-silico model (Verma et al. 2012). Among the 12 compounds, vulgarin exhibited significant antihepatotoxic activity against CCl4-induced toxicity in Wistar rat model. Akther et al. (2013) also reported hepatoprotective activity of methanolic extract of whole plant of M. vulgare against paracetamol-induced toxicity in Wistar rats (Akther et al. 2013).

12.6.9 Antioxidant Activity

Majority of the Mexican people make teas from leaflets, while roots of some medicinal plants are used to treat various ailments including infections, arthritis, heart disorders, headaches, fever, asthma, and menstrual pain. So, in a study, over 30 medicinal plants were investigated for evaluation of antioxidants (VanderJagt et al. 2002). Among them, it was reported that aqueous extract of leaves of M. vulgare contained about 560 μmol/g Trolox equivalent/g dry weight. In another study methanol extract of leaves showed strong antioxidant activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH; IC50 = 35 μg/mL) radical scavenging (Chedia et al. 2014). Antioxidant activities of acetone extract (AE), deodorized acetone extract (DAE), and deodorized water extract (DWE) from leaves of M. vulgare were tested at 80 °C in rapeseed (Brassica napus) oil (Weel et al. 1999) and found that acetone extract was better as compared with water extract.

Essential oils (EOs) are considered to be naturally occurring antioxidants, so various researches were performed on the essential oils obtained from M. vulgare. In one such research done in Tunisia, it was reported that the antioxidant effects of aerial parts of M. vulgare were produced through their essential oils probably due to the hydroxylated and oxygen-containing compounds (Kadri et al. 2011). Based on the results obtained from DPPH, β-carotene bleaching test, and reducing power assay, it was concluded that EOs obtained from M. vulgare can be used as a natural food preservative and to improve human health as a natural antioxidant. Comparing the antioxidant activity with synthetic butylated hydroxyanisole (BHT), it was revealed that EOs exhibited an IC50 value of 153.84 μg/mL, which was two times greater than BHT (Abadi and Abdellatif 2013). In a study, the antioxidant activities of five compounds (5–6-dihydroxy,7–4′-dimethoxy flavone, 7-O-β-glucopyranosyl luteolin, 7-O-β-glucuronyl luteolin, verbascoside, and forsythoside B) isolated from aerial parts of M. vulgare were examined using DPPH and ABTS+ free radical scavenging assays, and compared with rosmarinic acid (Pukalskas et al. 2012). The effects of forsythoside B and verbascoside were found comparable to that of natural rosmarinic acid in ABTS+ assay. Among the fractions, the n-butanol fraction from which four compounds, except 5–6-dihydroxy,7–4′-dimethoxy flavone, were isolated exhibited highest antioxidant activity in the β-carotene bleaching assay and linolenic acid model. There was another study in which antioxidant capacity was again found in methanol leaf extract of Marrubium vulgare (Amessis-Ouchemoukh et al. 2014). In this study, antioxidant capacities of methanol and acetone extracts were evaluated using DPPH, H2O2, total antioxidant capacity, and iron-reducing power assays. The percentage of DPPH radical scavenging activity of methanol extract in the range of 51.90–97.15% suggests its importance as a natural preservative and in the prevention of oxidative stress-related disorders.

12.6.10 Antimicrobial Activity

Various studies have been done for the evaluation of antimicrobial activities of Maruubium vulgare, the most widely used herb as medicine in Arab counties. Ethanol extracts (EEs) from leaves and flowers of M. vulgare were investigated for their antimicrobial potential using rapid colorimetry (XTT) and viable count method (Al-Bakri and Afifi 2007). The promising antimicrobial activity shown by EE of M. vulgare was against Bacillus subtilis and Staphylococcus aureus, while against Escherichia coli and Pseudomonas aeruginosa it was very weak. Promising activity was also exhibited against S. aureus, Staphylococcus epidermidis, and B. subtilis when methanolic extract of M. vulgare whole plant was evaluated for its antimicrobial activity (Masoodi et al. 2008). With regard to other two strains, that is, P. vulgaris and E. coli, there was a moderate effect. In this study, six bacterial organisms were used and ciprofloxacin was used as a standard drug. In another study, the M. vulgare roots were extracted with ethanol and inhibition assay of biofilm formation was done in methicillin-resistant S. aureus (MRSA; Quave and Smeltzer 2009). The minimum inhibitory concentration (MIC) was determined after 18 h growth using broth microtiter dilution method. A significant inhibition of biofilm formation (IC50 = 32 μg/mL) and adherence (IC50 = 8 μg/mL) was established in extract of roots at dose-dependent manner. A remarkable antimicrobial activity against Staphylococcus aureus and S. epidermidis was also shown by EE of M. vulgare and that study was done in Turkey in which 22 plant species were investigated (Kunduhoglu et al. 2011). The study was done on ethanol, acetone, and ether extracts obtained from leaves, flowers, and stems of 22 plant species.

Helicobacter pylori bacterium is considered the major cause of infection in gastric disorders; a study was undertaken in which anti-Helicobacter pylori activity was investigated (Robles-Zepeda et al. 2011). In the study, methanol extract at a concentration of 10 mg/mL of M. vulgare was evaluated using broth micro-dilution method and MIC was observed at 800 μg/mL. In another study, essential oil of M. vulgare was evaluated for antimicrobial activity and MIC was determined. (Zarai et al. 2011). In yet another study, essential oil was investigated against series of bacterial and fungal strains by agar disk diffusion method. The results showed a considerable antimicrobial activity against Gram positive bacteria with MIC values in the range of 1.12–2.6 mg/mL by zone inhibition assay. However, Gram negative bacteria exhibited resistance against tested sample. MIC was found in the range of 12.5–25 mg/mL in a study where methanolic extract of M. vulgare was evaluated for antibacterial activity against seven pathogenic bacteria (Chedia et al. 2014). In this study, total phenolic content was also determined and it was concluded that antibacterial activity was due to high content of phenolic compounds in the extract. Tuberculosis, one of the serious health problems, has developed resistance to some of the antibiotics, which has enforced researchers to create or discover new drugs.

EOs are considered naturally occurring antioxidants and were evaluated for antibacterial activity against different Gram positive and Gram negative pathogens. In one study, EOs showed varied antibacterial activity against Listeria monocytogenes, P. aeruginosa, Agrobacterium tumefaciens, and Salmonella enterica (Abadi and Abdellatif 2013). By Mueller-Hinton broth dilution method, the MIC values were obtained, which were in the range of 0.1–15 μg/mL. In another study, the essential oils and ethanol extract prepared from the leaves of M. vulgare were studied for antimicrobial activity against 17 clinical strains of Staphylococcus aureus (Bokaeian et al. 2014). Minimum MIC value for the extract was found to be 2.5 mg/mL and maximum MIC value for essential oils was found to be 2.5 mg/mL. This study confirmed the use of essential oils and ethanol extract of M. vulgare as antibacterial agents.

12.6.11 Anticancer Activity

In recent years, the search for naturally occurring anticancer chemotherapeutic drugs continued at a great speed and research focus on M. vulgare in this field is no way behind. Yamaguchi and co-workers (2006) reported antiproliferative effect on colon cancer cells with leaf extract. In this study, methanol extracts upregulated gene (NAG-1) through a trans-activation of the NAG-1 promoter at a dose of 250 μg/mL. This was for the first time that a major compound ladanein isolated from M. vulgare was studied for its cytotoxic activity against drug dasatinib-resistant murine leukemia cell line (DA1-3b/M2BCR-ABL). In the study, ladanein showed nonsignificant (20–40 μM) activity against series of cancer cell lines, including K562, K562R, and 697 human leukemia cell lines, but was found inactive to MOLM13 and human peripheral blood mononuclear cells (Alkhatib et al. 2010). In vitro cytotoxicity of M. vulgare against cancer cell lines was studied in Morocco, Tunisia, and Dubai. In one of the studies, essential oils obtained from M. vulgare were examined using a modified MTT assay for their cytotoxic activity against HeLa cell lines (Zarai et al. 2011). The results depicted that essential oils destroyed HeLa cells by 27% in a concentration of 0.25 mg/mL and 100% cells died at >0.5 mg/mL concentration. A considerable cytotoxic effect with IC50 at 258 mg/mL against tumor cells was also observed. In another study, alcoholic leaves extract and isolated phenolic compounds, viz., acacetin, apigenin, and its glycoside, were tested against U251 (Ehrlich tumor cell lines) and breast cancer line (MCF7). Alcoholic extract and its secondary metabolites showed significant anticancer activity against U251 with effective dose for 50% (ED50) < 20 μg/mL whereas extract and acacetin showed moderate activity against MCF7 with ED50 > 20 μg/mL (Nawal and Atta 2013). Different extracts including methanol, hexane, dichloromethane, and ethyl acetate obtained from aerial parts of M. vulgare were evaluated for cytotoxic activity against tumor cell lines (Belayachi et al. 2013). The results showed that dichloromethane extract of M. vulgare was effective against colorectal cancer cell lines (SW620 cells), acute T-cell leukemia, and mantle cell lymphoma cell line with IC50 at 30 μg/mL.

12.6.12 Antiprotozoal, Molluscicidal, and Mosquitocidal Activities

Marrubium vulgare is also used for treating intestinal disorders. In two separate studies, M. vulgare was reported for antiprotozoal property. In one study, acetone and methanolic extracts of M. vulgare were found to have activity against Entamoeba histolytica and Giardia lamblia with growth inhibition IC50 = 7 at a dose of 12 μg/mL and IC50 = 90 at 34 μg/mL (Ramos-Guerra et al. 2007). Methanolic extracts of this plant species were reported to be effective against Trypanosoma cruzi with percentage growth inhibition between 88% and 100% at a concentration of 150 μg/mL (Molina-Garza et al. 2014).

Regarding molluscicidal and mosquitocidal activities, the volatile oils of M. vulgare were used to evaluate their activities on eggs of Biomphalaria alexandrina and Culex pipiens (Salama et al. 2012). The result clearly showed 100% ovicidal activity at 200 ppm/24 h. Another study stated that methanol extract of M. vulgare leaves are very effective against fourth instar larvae of the mosquito C. pipiens L. (Amel and Selima 2015).

12.7 Conclusion and Future Perspectives

The traditional uses, phytochemistry, and pharmacology of Marrubium vulgare are presented here in this chapter. Regarding chemical constituents, diterpenes, flavonoids, and phenylpropanoids are the major ones identified. Marrubiin is one of the major labdane diterpenes and exists in higher concentration. The pharmacological activities of M. vulgare proved its potential for the development of new efficacious botanicals in future. It is evident from the literature that mostly methanolic extracts of leaves and aerial parts have been studied in detail; however, no substantial work has been carried out, so there is a need to work over other plant parts (root, stem, flowers, seeds) along with different extracts and their fractions. Though the plant contains different classes of secondary metabolites, but only marrubiin, marrubenol, and ladanein were well studied and hence other compounds need to be investigated. Clinical trials should be initiated for this plant, which is crucial for the diagnosis of herbal toxicity and development of plant-based new drugs.