molecules Plectranthus amboinicus (Lour.) Spreng: Botanical, Phytochemical, Pharmacological and Nutritional Significance

Mallappa  Kumara Swamy

Plectranthus amboinicus (Lour.) Spreng. is a perennial herb belonging to the family Lamiaceae which occurs naturally throughout the tropics and warm regions of Africa, Asia and Australia. This herb has therapeutic and nutritional properties attributed to its natural phytochemical compounds which are highly valued in the pharmaceutical industry. Besides, it has horticultural properties due to its aromatic nature and essential oil producing capability. It is widely used in folk medicine to treat conditions like cold, asthma, constipation, headache, cough, fever and skin diseases. The leaves of the plant are often eaten raw or used as flavoring agents, or incorporated as ingredients in the preparation of traditional food. The literature survey revealed the occurrence 76 volatiles and 30 non-volatile compounds belonging to different classes of phytochemicals such as monoterpenoids, diterpenoids, triterpenoids, sesquiterpenoids, phenolics, flavonoids, esters, alcohols and aldehydes. Studies have cited numerous pharmacological properties including antimicrobial, antiinflammatory, antitumor, wound healing, anti-epileptic, larvicidal, antioxidant and analgesic activities. Also, it has been found to be effective against respiratory, cardiovascular, oral, skin, digestive and urinary diseases. Yet, scientific validation of many other traditional uses would be appreciated, mainly to discover and authenticate novel bioactive compounds from this herb. This review article provides comprehensive information on the botany, phytochemistry, pharmacology and nutritional importance of P. amboinicus essential oil and its various solvent extracts. This article allows researchers to further explore the further potential of this multi-utility herb for various biomedical applications.

molecules Review Plectranthus amboinicus (Lour.) Spreng: Botanical, Phytochemical, Pharmacological and Nutritional Significance Greetha Arumugam, Mallappa Kumara Swamy * and Uma Rani Sinniah * Department of Crop Science, Faculty of Agriculture, Universiti Putra Malaysia, Serdang, Selangor, Darul Ehsan 43400, Malaysia; greetha82@yahoo.co.uk * Correspondence: swamy.bio@gmail.com (M.K.S.); umarani@upm.edu.my (U.R.S.); Tel.: +60-1-7248-4528 (M.K.S); +60-3-8947-4839 (U.R.S.) Academic Editor: Christopher W. K. Lam Received: 30 January 2016 ; Accepted: 8 March 2016 ; Published: 30 March 2016 Abstract: Plectranthus amboinicus (Lour.) Spreng. is a perennial herb belonging to the family Lamiaceae which occurs naturally throughout the tropics and warm regions of Africa, Asia and Australia. This herb has therapeutic and nutritional properties attributed to its natural phytochemical compounds which are highly valued in the pharmaceutical industry. Besides, it has horticultural properties due to its aromatic nature and essential oil producing capability. It is widely used in folk medicine to treat conditions like cold, asthma, constipation, headache, cough, fever and skin diseases. The leaves of the plant are often eaten raw or used as flavoring agents, or incorporated as ingredients in the preparation of traditional food. The literature survey revealed the occurrence 76 volatiles and 30 non-volatile compounds belonging to different classes of phytochemicals such as monoterpenoids, diterpenoids, triterpenoids, sesquiterpenoids, phenolics, flavonoids, esters, alcohols and aldehydes. Studies have cited numerous pharmacological properties including antimicrobial, antiinflammatory, antitumor, wound healing, anti-epileptic, larvicidal, antioxidant and analgesic activities. Also, it has been found to be effective against respiratory, cardiovascular, oral, skin, digestive and urinary diseases. Yet, scientific validation of many other traditional uses would be appreciated, mainly to discover and authenticate novel bioactive compounds from this herb. This review article provides comprehensive information on the botany, phytochemistry, pharmacology and nutritional importance of P. amboinicus essential oil and its various solvent extracts. This article allows researchers to further explore the further potential of this multi-utility herb for various biomedical applications. Keywords: Plectranthus amboinicus; Indian borage; pharmacology; biological activities; nutrition; phytochemistry; botany; medicinal plant 1. Introduction At present, plant-based medicines are widely employed in various public health practices throughout the globe as they are safe and cost-effective, and efficiently combat various deadly diseases and help in maintaining good health [1]. Herbal medicines are very commonly used in Unani, Ayurveda, Sidda, folk and other traditional practices of healthcare management [2]. According to the estimation by the World Health Organization, about 80% of people on the globe are still dependent on traditional herb-based medications due to their low cost, easy accessibility and likely negligible side effects in comparison to allopathic medicines [2–4]. Certainly, many of the leading active drug molecules of plants and their derivatives used presently in allopathic medicine are mainly due to the understanding of traditional medical practices for curing diseases [2]. Modern drug discovery research is governed by natural plant-based compounds and their products, followed by synthetic chemical Molecules 2016, 21, 369; doi:10.3390/molecules21040369 www.mdpi.com/journal/molecules Molecules 2016, 21, 369 2 of 26 drugs. Currently, natural products are considered as a major source of medicaments and, hence, they are extensively used by pharmaceutical industries. This has led towards increased global demand for medicinal plants in the modern era of natural medicine, leading to exploration and exploitation of new plant sources for their medicinal properties [3,5,6]. The Lamiaceae members of plant species belonging to commercially important genera, such as Plectranthus, Salvia, Ocimum and Mentha, are attributed with a rich diversity of ethnobotanical benefits. More than 300 species of Plectranthus are reported all over the tropical and warm regions of the old world, including Asia, Africa and Australia [7]. In over 85% of the literature, documentation of Plectranthus is on the therapeutic values of this genus followed by its nutritional and horticultural properties attributed to its aromatic nature and essential oil producing capability [8,9]. Plectranthus amboinicus (Loureiro) Sprengel is one of the most documented species in the family Lamiaceae. P. amboinicus, also commonly known as Indian borage, and is a fleshy, succulent herb famous for its distinct oregano-like flavor and odor. It is one of the most cited species in the Lamiaceae family, especially for its medicinal properties, accounting for 68% of all customary applications of this genus [10]. This herb is widely used by indigenous people of tropical rain forests, either in folk medicine or for culinary purposes. This is mainly due to its natural production of an essential oil with high amounts of bioactive compounds such as Carvacrol [11], Thymol [12] β-Caryophyllene, α-Humulene, γ-Terpinene, p-Cymene, α-Terpineol and β-Selinene, identified in the oil component of its leaves [13,14]. These biochemical components exhibit various biological properties [15,16] and are widely used in folk medicine to treat conditions like cold, asthma, constipation, headache, cough, fever and skin diseases. The leaves of the plant are often eaten raw or used as flavoring agents, or incorporated as ingredients in the preparation of traditional food. The chopped leaves are also used as a substitute for sage (Salvia officinalis) in meat stuffing [17]. With this background, the present review was undertaken to present complete facts on the multifarious medical benefits of P. amboicus. This review is a compiled survey of information on various aspects of P. amboinicus including botany, distribution, wild relatives, phytochemistry, medicinal and nutritional properties and other benefits. The available information was retrieved from many search engines including ScienceDirect, Google Scholar, PubMed, Scopus and SciFinder etc. 2. Botanical Description 2.1. Taxonomy P. amboinicus (Loureiro) Sprengel is a member of the family, Lamiaceae. or mint family. The paleotropical oil-rich genus, Plectranthus belongs to the subfamily Nepetoideae. It comprises about 300 species of annual or perennial herbs or subshrubs which are often succulents [18]. Many species of Plectranthus have economical and medicinal values. Among them, P. amboinicus is one the most important aromatic medicinal succulent plants that possess distinctive smelling leaves with short soft erect hairs. 2.2. Morphological Feautures P. amboinicus is a succulent shrub with a tendency for climbing or creeping. It can reach over 1 m in height and even more in width in the wild [18–20]. This sprawling large succulent herb is fleshy and highly aromatic. The fleshy stems grow about 30–90 cm, either with long rigid hairs (hispidly villous), or tomentose (densely covered with soft, short and erect hairs, pubescent) [18,19]. Leaves are undivided (simple), broadly ovate to suborbicular with a tapering tip (ovate) and very thick; they are pubescent (thickly studded with hairs), with the lower surface possessing the most numerous glandular hairs, giving a frosted appearance [20]. The taste of this leaf is pleasantly aromatic with an agreeable and refreshing odor. Flowers are on a short stem (shortly pedicelled), pale purplish in dense whorls at distant intervals in a long slender raceme. Flowers have a bell shaped calyx and the throat is smooth inside with two lips, the upper lip being ovate and thin, the lower lip having four narrow teeth. The corolla is pale purplish and five times longer than the calyx, with a short tube, inflated Molecules 2016, 21, 369 3 of 26 throat and short lips [19,20]. Fruit nutlets are smooth, pale brown in color, 0.7 mm long and 0.5 mm wide. P. amboinicus rarely flowers and seeds are difficult to collect [19]. 2.3. Origin, Wild Relatives and Geographical Distribution The name Plectranthus derives from the Greek words “plectron”, meaning spur, and “Anthos”, meaning flower, in reference to the spur-shaped flowers of some members of the genus [21]. Due to lack of precise morphological features to distinguish species within the genus Plectranthus and its closely associated genera, numerous taxonomic problems with the naming of species have resulted in misplacement of species in some closely linked genera such as Coleus, Solenostemon and Englerastrum [10]. The species P. amboinicus was originally classified under the genus Coleus but was moved to the Plectranthus genus, although both names are sometimes seen in the literature today. This species also has the greatest number of synonyms (Table 1). The synonyms of P. amboinicus include P. aromaticus Roxb., Coleus aromaticus Benth. and C. amboinicus Lour. [10]. P. amboinicus is of unknown origin, but is possibly from Africa and India [18], and it has since been distributed and cultivated pantropically. The type specimen of the species was collected in Amboina, Moluccas, resulting in its species name amboinicus [22]. Taxonomic revisions of Plectranthus have taken place on a regional rather than international basis and thus have contributed to a misunderstanding of the same species [10]. The traditional uses around the world are represented in Table 1. Table 1. Vernacular names and traditional uses of Plectranthus amboinicus commonly used by locals in their respective countries. Country Vernacular Names Traditional Uses Barbados Poor man’s pork, Broad leaf thyme Folk medicine, Culinary Cambodia Sak dam ray Folk medicine, Culinary China Da shou xiang Folk medicine, Home garden Cuba orégano; orégano de Cartagena Folk medicine, Culinary Fiji Rhaivoki, Sage Folk medicine, Culinary Germany Jamaika thymian Folk medicine, Culinary Guyana Thick leaf thyme, broad leaf thyme Folk medicine, Culinary India Indian Borage, Pashan Bhedi, Karpooravalli, Patharchur Folk medicine, Culinary, Home garden Indonesia Torbangun, Daun Kutjing Folk medicine, Culinary, Home garden Malaysia Daun bangun-bangun, Pokok bangun-bangun Folk medicine, Culinary, Home garden Philippines Latai, Suganda, Oregano Folk medicine, Culinary, Home garden Puerto Rico Puerto Rican oregano brujo, Cuban oregano Folk medicine, Culinary South Africa Sup mint, French thyme, Indian mint Folk medicine, Culinary, Home garden Thailand Hom duan huu suea, Niam huu suea Folk medicine, Culinary USA Indian Borage, Country borage, Spanish thyme, Mexican mint, French thyme, Indian mint Culinary, Home garden Vietnam Can day la Folk medicine, Culinary West Indies French thyme, Spanish thyme, Broad-leaf thyme Folk medicine, Culinary 2.4. Cultivation P. amboinicus is a fast-growing plant usually propagated by stem cuttings. This preferred propagation through vegetative means is because it rarely seeds or sets seed [23]. The herb grows easily in a well-drained, semi-shaded location. It is found to grow well under tropical and subtropical locations. It was also found to adapt well in cooler climates if grown in a pot and brought indoors, or moved to a warm, sheltered position during winter [19]. The plant should be watered only sparingly. P. amboinicus grows best in rich, compost soil with neutral pH and high humidity, but if there is excess Molecules 2016, 21, 369 4 of 26 water in the ground its roots might start to rot. On the other hand, it copes well with severe droughts, as it has lots of water stored in its succulent flesh. It also survives well with severe heat and scorching sun, as well as strong shade, but grows best under partial shade. For all those reasons, it is very easy to grow indoors and that is why it is becoming more and more popular as a house plant in northern Europe. P. amboinicus cannot withstand temperatures lower than 0 ˝ C and is stressed even when it is colder than 10 ˝ C. In general, very meager information is available on the methods/practices used for commercial cultivation and harvesting of this herb. 3. Phytochemistry A large and growing body of literature has investigated the chemical composition and pharmacological properties of P. amboinicus. The literature survey has emphasized the occurrence of different classes of phytocompounds including 76 volatiles and 30 non-volatile compounds. More recently, interest among phytochemists and biologists has focused on the isolation of specific bioactive compounds of P. amboinicus and comprehending their pharmacological importance. However, the chemical profile and the accumulation pattern of bioactive constituents in different parts of the plant and their essential oil content varies depending on various parameters, such as geographical features, climate and different stages of plant material collection [2]. Moreover, the method of extraction and identification can also lead to variations in phytochemical composition. Presently, only a few reports have stated the isolation and authentication of individual compounds of P. amboinicus. Therefore, correct identification, isolation and quantification of phytocompounds is very much necessary in order to understand their pharmacological and biological significance. P. amboinicus is reported to contain several classes of phytochemicals including monoterepenoids, diterpenoids, triterpenoids, sesquiterpenoids, phenolics, flavonoids and esters. The details of these bioactive constituents are discussed in this section. 3.1. Volatile Composition of P. amboinicus The essential oil obtained from the leaves and stem explants was shown to contain a total of 76 volatile constituents. The essential oil contained a copious quantity of the two major phenolic compounds, namely, carvacrol and thymol, which are pharmaceutically appreciated for various culinary properties. The quality as well as quantity of chemical compounds occurring in the essential oil is directly related to its biological functions. P. amboinicus oil is rich in oxygenated monoterpenes, monoterpene hydrocarbons, sesquiterpene hydrocarbons and oxygenated sesquiterpenes [10]. The leaf essential oil of P. amboinicus is particularly rich in phenolic monoterpenes such as Thymol and Carvacrol, which are speculated to exert various pharmacological properties [10,17,20,24]. Table 2 represents the details of these volatile compounds, while Figure 1 depicts the structural details of some of these major compounds. A hydro distillation method using a Clevenger type apparatus for 3–4 h is commonly employed for extracting P. amboinicus essential oil. However, volatile constituents of P. amboinicus leaf obtained by using hexane extraction, steam distillation and supercritical CO2 extraction methods were shown to have a chemical difference [25]. The hexane extraction method produced the highest oil yield (6.52%) compared to steam distillation (0.55%) and supercritical CO2 extraction methods (1.40%). It was also noticed that there was a difference in aroma of the extracts which was attributed to qualitative and quantitative dissimilarity in chemical composition. In another study, a solid phase micro extraction (SPME) method showed the presence of eucalyptol in P. amboinicus leaves [17,26]. The volatile constituents of P. amboinicus leaves collected from Uganda were extracted with head space solid phase microextraction (HS-SPME) and their analysis using the gas chromatography–mass spectroscopy (GC-MS) electron impact ionization method revealed the presence of Linanol (50.3%) as the major component [27,28]. The other volatiles observed were Carvacrol (10.3%), Geranyl acetate (11.75), Nerol acetate (11.6%), γ-Terpinene (3.2%), p-Cymene (2.9%), Nerol (2.3%), α-4-Carene (1.3%), Caryophyllene (1.2%) and β-Myrcene (0.8%). Molecules 2016, 21, 369 5 of 26 Table 2. The known volatile constituents of P. amboinicus. Compound Name Formula Plant Origin/Part Method References GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS [29–32] [12–14,29,32–38] [29,30] [13,29,34,36,38] [29,31] [29–31,38] [14,35] [29,36] [29] [29,31,36] [14,29,30,34,35,39] [13,14,29,30,32,34–37] [31,37] [14,29,31,35,38] Monoterpene hydrocarbons δ-3-Carene p-Cymene Limonene β-Myrcene Ocimene α-Phellandrene β-Phellandrene α-Pinene β-Pinene Sabinene α-Terpinene γ-Terpinene α-Terpinolene α-Thujene C10 H16 C10 H14 C10 H16 C10 H16 C10 H16 C10 H16 C10 H16 C10 H16 C10 H16 C10 H16 C10 H16 C10 H16 C10 H16 C10 H16 India, Malaysia, Morocco, Mauritius/Leaf Brazil, India, Cambodia, Malaysia, Venezuela/Aerial parts, Leaf India, Mauritius/Leaf Cambodia, India, Venezuela /Leaf Morocco/Leaf India, Comoros, Mauritius, Venezuela/Leaf India/Leaf India, CambodiRa /Leaf India/Leaf Cambodia,India, Morocco /Leaf India, Mauritius/Leaf Brazil, Cambodia, Malaysia, Mauritius India, /Leaf Morocco, Brazil/Leaf India, Comoros, Venezuela/Leaf Camphor C10 H16 O Carvacrol C10 H14 O Carvone 1,8-Cineole Eugenol Geraniol Linalool Methyl carvacrol Methyl eugenol α-Terpineol Terpinen-4-ol Thymol Thymol methyl ether C10 H14 O C10 H18 O C10 H12 O2 C10 H18 O C10 H18 O C11 H16 O C11 H14 O2 C10 H18 O C10 H18 O C10 H14 O C11 H16 O Comoros, Malaysia, Mauritius/Leaf Cambodia, India, Malaysia, Mauritius, Venezuela/Aerial parts, Leaf, Flower India/Leaf India/Leaf Cambodia, India/Leaf Mauritius/Leaf Comoros, Mauritius /Leaf India/Leaf Cambodia/Leaf India, Comoros, Venezuela /Leaf Brazil, India, Mauritius /Leaf Brazil, Cambodia, India, Venezuela/Aerial parts, Leaf Brazil/Leaf α-Amorphene Aromadendrene trans-α-Bergamotene trans-β-Bergamotene γ-Cadinene δ-Cadinene α-Calacorene cis-Calamenene β-Caryophyllene γ-Caryophyllene α-Copaene α-Cubebene (E,Z)-α-Farnesene Germacrene D γ-Gurjunene Humulene α-Muurolene Patchoulene β-Selinene β-Sesquiphellandrene C15 H24 C15 H24 C15 H24 C15 H24 C15 H24 C15 H24 C15 H20 C15 H22 C15 H24 C15 H24 C15 H24 C15 H24 C15 H24 C15 H24 C15 H24 C15 H24 C15 H24 C15 H24 C15 H24 C15 H24 Cambodia/Leaf Brazil, India/Leaf Brazil, Comoros, India, Venezuela /Leaf, Aerial parts, Flower Cambodia/Leaf Cambodia/Leaf India, Cambodia/Leaf India/Aerial parts Cambodia/Leaf Brazil, India, Venezuela /Leaf, Flower India/Leaf Comoros, India /Leaf India/Leaf, Aerial parts France /Leaf Cambodia/Leaf India/Aerial parts Brazil, Cambodia, India, Morocco, Venezuela/Leaf, Aerial parts Cambodia, France, Mauritius/Leaf India, Mauritius/Leaf India, Comoros/Leaf Cambodia/Leaf Oxygenated monoterpenes GC-MS [30–32] GC-MS [12–14,28–30,32–35,38–43] GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS [29] [12,26,27,29,41] [29,36,39–41,44] [30] [30,31] [29] [29,36] [29,31,38] [12,13,29–31,37,39] [12–14,29,33–35,37–41,44,45] [37] GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS [36] [34,37] [13,31,33,37,38] [36] [36] [13,29] [33] [36] [13,29,31,33,34,37,38,41] [42] [29,31] [13,33] [29,46] [36] [33] [14,31,33,35–38,45] [30,36,46] [30,42] [14,31,35,44] [46] Sesquiterpene hydrocarbons Molecules 2016, 21, 369 6 of 26 Table 2. Cont. Compound Name Formula Plant Origin/Part Method References GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS [13,14,29,33,35,36,38,39,44] [14,35] [14,35] [14,35] [29] [14,35] [29] [12,39] Oxygenated sesquiterpenes Caryophyllene oxide β-Cedrene epoxide β-Copaen-4-α-ol 1-Epi-cubenol β-Eudesmol β-Himachalene oxide Humulene oxide Spathulenol C15 H24 O C15 H24 O C15 H24 O C15 H26 O C15 H26 O C15 H24 O C15 H24 O C15 H24 O India, Cambodia, Venezuela /Leaf, Aerial parts India/Aerial parts India/Aerial parts India/Aerial parts India/Leaf India/Aerial parts India/Leaf India/Leaf Others (Terpenes, phenylpropanoids, esters, fatty acids, alcohols, aldehyde) 1,2-Benzenediol 4-(1,1 dimethylethyl) Chavicol Methyl chavicol α-Corocalene Dihydro carveol Durohydroquinone 1,4 Eicosadiene Ethyl Salicylate (Z)-1,3-Hexadiene (Z)-3-Hexen-1-ol Methyl octanoate 1-Octen-3-ol Oleic acid 2-Phenyl ethyl tiglate Phytol Squalene Tetradecanal 3,7,11,15–Tetramethyl-2-hexadecen-1-ol Thymol acetate Trans-sabinene hydrate Undecanal C10 H14 O2 India/Leaf GC-MS [44] C9 H10 O C10 H12 O C15 H20 C10 H18 O C10 H14 O2 C20 H38 C9 H10 O3 C6 H10 C6 H12 O C9 H18 O2 C8 H16 O C18 H34 O2 C13 H16 O2 C20 H40 O C30 H50 C14 H28 O C20 H40 O C12 H16 O2 C12 H20 O2 C11 H22 O India/Leaf India/Aerial parts India/Aerial parts India/Aerial parts India/Leaf India/Leaf India/Leaf France /Leaf France/Leaf India/Aerial parts India, Mauritius, Venezuela/Leaf India/Leaf India/Aerial parts India/Leaf India/Leaf India/Aerial parts India/Leaf India/Leaf India/Aerial parts India/Aerial parts GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS GC-MS [27,40] [14,33,35] [33] [14,35] [44] [44] [27,40] [46] [29,46] [14,35] [29,30,38,39] [44] [14,35] [44] [44] [14,35] [44] [29,39] [14,35] [14,35] Molecules 2016, 21, 369 7 of 26 The chemical constituents of P. amboinicus essential oil differed with the collected samples from diverse geographical places. In India, P. amboinicus essential oil was reported to possess volatiles such as Carvacrol (43.1%), Thymol (7.2%), Eugenol (6.4%), Chavicol (5.3%) and Et-salicylate (3.2%) [40], which varied from the constituents observed by Baslas and Kumar [41] with Thymol (41.30%), Carvacrol (13.25%), 1,8-Cineole (5.45%), Eugenol (4.40%) and β-Caryophyllene (4.20%). Likewise, analysis of essential oil obtained from wild growing P. amboinicusΆ plants in Bangalore, India showed the presence of 36 compounds [29]. The major compounds identified were Carvacrol (53%–67%), p-Cymene (6.5%–12.6%), β-Caryophyllene (7.4%), Caryophyllene oxide (2.2%) and α-Terpinene (5.9%–15.5%). Ά ΅ For the first time, there was an occurrence of the compounds eugenol and methyl eugenol in Indian (Andra Pradesh) P. amboinicus oil [12]. Figure 1. Cont. Molecules 2016, 21, 369 8 of 26 Figure 1. The structures of some of the volatile chemical constituents. Figure 1. The structures of some of the volatile chemical constituents. GC and GC-MS techniques have indicated the occurrence of Thymol (94.3%), followed by Carvacrol (1.2%), 1,8-Cineole (0.8%), p-Cymene (0.3%), Spathulenol (0.2%) and Terpinen-4-ol (0.2%) as the major constituents of Indian P. amboinicus leaf essential oil. Investigation of volatiles of P. amboinicus collected from Mysore, Karnataka, India showed the existence of Carvacrol (70%), β-Caryophyllene (6.2%), p-Cymene (5.6%) and α-Terpinolene (5.3%) as the main components [13]. The volatile composition of aerial parts and flowers of P. ambonicus growing in Belgaum, Western Ghats region of North West Karnataka, India have been studied by Joshi et al. [33] using gas chromatography outfitted with a flame ionization detector (GC-FID) and GC-MS. The results revealed the occurrence of Molecules 2016, 21, 369 9 of 26 12 components from aerial parts oil and 4 constituents from flower oil and represented 94.29% and 90.25% of the total oil, respectively. In both the essential oils, the major compound observed was Carvacrol (50.98% in flowers and 77.16% in aerial parts oils). They also noticed the chemotype nature of the plants with minor quantities of α-Calacorene, Methyl chavicol and α-Corocalene from aerial parts oil. However, P. amboinicus collected from Western Ghats (Siruvani hills), Tamilnadu, India contained only 14% of Carvocrol. Other chief constituents observed were Thymol (18%), cis-Caryophyllene (18%) and p-Cymene (10.83%) [34]. Yet, the presence of chemical variation in the leaf essential oils had been evident from the plants collected from the same state [42,44]. A total of 10 volatile compounds were identified with dominant constituents as Carvacrol (50.7%), β-caryophyllene (13.1%) and patchoulane (8.7%) [42], while the occurrence of Carvacrol was not evident from the study of Uma et al. [44]. They reported the occurrence of 3-Methyl-4-isopropyl phenol (31.70%) as the major constituent followed by Squalene (10.07%), Caryophyllene (2.36%) and Phytol (8.44%). Similarly, the essential oil of P. amboinicus leaves obtained from the Cuddalore district of Tamilnadu, India was shown to contain a total of 26 compounds, with Carvacrol (28.65%) and Thymol (21.66%) observed as the major compounds [14]. Some of the other major components included α-Humulene (9.67%), γ-Terpinene (7.76%), Undecanal (8.29%), p-Cymene (6.46%), α-Terpineol (3.28%), β-Selinene (2.01%) and Caryophyllene oxide (5.85%). Likewise, plants growing in the same locality were shown to contain 26 compounds and among them Carvacrol (29.25%), Thymol (21.66%), Undecanal (8.29%) and α-Humulene (9.67%) were the major compounds [35]. About 28 volatile compounds were revealed from the aerial parts of P. amboinicus cultivated in Uttarakhand, India by using GC and GC-MS analysis. Among the 16 identified compounds, Thymol (83.39%) was the major component. The other important components observed were 1-Octen-3-ol, Caryophyllene oxide, Terpine-4-ol, trans-Caryophyllene, Eugenol and α-Cadinol [39]. Likewise, chemical investigation of essential oil of P. amboinicus collected from Himalayan foothill and mid-hill regions of India were reported by Verma et al. [47]. They identified 44 constituents representing 86%–99% of the oil composition using GC and GC-MS analysis. Some of the major components observed were Thymol (57.7%–66.4%), γ-Terpinene (5.8%–11.7%), p-Cymene (4.1%–14.2%), (E)-Caryophyllene (2.6%–3.6%), Caryophyllene oxide (1.3%–1.5%) and 1-Octen-3-ol (1.0%–1.8%). Essential oil of P. amboinicus from Mauritius was characterized by Gurib-Fakim et al. [30] using capillary GC and GC-MS analysis and the results revealed Carvacrol (41.3%) and Camphor (39%) as the dominant volatiles. The leaf essential oil obtained from Martinique, France was found to contain Carvacrol (72%) as a chief phenolic component along with newly identified compounds such as (Z)-1,3-Hexadiene (0.1%), (E,Z)-α-Farnesene (0.2%), (Z)-3-Hexenol (0.6%), α-Muurolene (0.2%) and (E,E)-α-Farnesene (0.2%) [46]. P. amboinicus oil from Cambodia was shown to contain largely Thymol (57.4%). Other major constituents such as Carvacrol (13.5%), p-Cymene (5.2%) and γ-Terpinene (5.6%) were also noticed [36]. Likewise, 64.3% of Thymol was identified from Brazilian P. amboinicus leaf essential oil [37]. Other major compounds identified were p-Cymene (10.3%), β-Caryophyllene (2.8%) and γ-Terpinene (9.9%). Liquid-solid chromatography (LSC), Gas-liquid chromatography (GLC) and GC-MS analysis of P. amboinicus plants from Cuba revealed Carvacrol (64%) as the major component among the identified 13 terpene hydrocarbons and seven oxygenated compounds [43]. Similarly, Velasco et al. [38] identified 15 volatile constituents in the essential oil of P. amboinicus collected from Venezuela and the major component observed was Carvacrol (65.2%). Capillary GC and GC-MS analysis of P. amboinicus essential oil from the Archipelago of Comoros was carried out by Hassani et al. [31] and the results showed the existence of Carvacrol (23.0%), Camphor (22.2%), δ-3-Carene (15.0%), γ-Terpinene (8.4%), p-Cymene (7.7%) and α-Terpinene (4.8%) as the major constituents of the oil. The essential oil from Uganda consisted of Linalool (50.3%), Carvacrol (14.3%), Nerol acetate (11.6%) and Geranyl acetate (11.7%) as the major components as revealed by GC-MS analysis [40]. Essential oil of P. amboinicus leaves from Serdang, Malaysia was shown to contain Carvacrol (19.29%), 3-Carene (20.78%) and Camphor (17.96%) as the major volatile constituents [32]. Molecules 2016, 21, 369 10 of 26 The essential oil yield and its main chemical constituents are also influenced by the environmental factors and different seasons. Mallavarupu et al. [29] have revealed that the quality of essential oil will be superior when collected during September. The oil content was found to be higher in the plants harvested during September in comparison to the plants harvested during May. Moreover, the compositions also differed among the two harvest times. About 68% of oxygenated monoterpenes, 11% of sesquiterpenes and 3.3% oxygenated sesquiterpenes were found in the oil distilled from the September harvest time, whereas, the oil obtained from the May harvest comprised higher amounts of monoterpenes (35.7%). The major compounds such as Carvacrol (67.0%), β-Caryophyllene (7.4%), α-Humulene (2.1%) and Caryophyllene oxide (2.2%) were found to be higher in oil obtained during September, while the oil distilled during May showed the presence of p-Cymene (12.6%), γ-Terpinene (15.5%), Carvacrol (53.0%) and β-Caryophyllene (4.3%). A GC-MS study was carried out by Roja et al. [45] to identify the chemical constituents of P. amboinicus oil from the leaves of the tissue culture plants, in vitro root cultures as well as parent plants. The results revealed the presence of similar volatile constituents, though the parent plants and root cultures contained 21 compounds in comparison to only 15 compounds noticed in the tissue culture plants. The concentration of Thymol compound was found to be 0.012, 0.29 and 0.009% (fresh weight) in the tissue culture plants, in vitro roots and the parent plants, respectively. They also suggested that thymol and cis-Caryophyllene can be produced from the tissue culture plants as oil yield and Thymol concentration was found to be similar to the parent plants. Interestingly, the root cultures had a three times higher Thymol content after a period of four weeks of day growth. This suggested the possible use of root cultures for producing Thymol and cis-Caryophyllene on a large scale. 3.2. Non-Volatile Chemical Constituents of P. amboinicus A total of 30 non-volatile constituents have been identified from P. amboinicus according to our literature survey [17,48]. These non-volatile chemical components included phenolic acids, flavonoids, monoterpene hydrocarbons, sesquiterpene hydrocarbons, oxygenated monoterpenes and esters. The details of these phytocompounds are exemplified in Table 3 and some of the important and major compound structures are illustrated in Figure 2. The chloroform extract of P. amboinicus air dried leaves was subjected to fractionation using the silica gel column chromatography technique to separate non-volatile compounds [49]. Later, the isolated compounds were identified by using ultra violet (UV), 1-D nuclear magnetic resonance (NMR) and 2-D NMR spectroscopy as three flavones, namely, Cirsimaritin, Salvigenin and Chrysoeriol. High pressure liquid chromatography (HPLC) analysis was carried out to analyze the major compound, Carvacrol, present in the aqueous extract of P. amboinicus plants obtained from Taichung of Taiwan [50]. The results showed an abundance of Carvacrol occurrence in the aqueous extract, with 1.88 mg/g of the extract. Eight compounds from the ethyl acetate fraction of P. amboinicus leaves collected from Egypt were isolated and identified [48]. Based on the UV, NMR spectra and their physical data, these isolated compounds were identified as 5,41 -Dihydroxy-3,7- dimethoxy flavone (3-methoxy genkwanin), 5,41 -Dihydroxy-6,7-dimethoxy flavone (Crisimaritin), p-Coumaric acid (Hydroxy cinnamic acid), Caffeic acid, 3,5,7,31 ,41 -Pentahydroxy flavanone (Taxifolin), Rosmarinic acid, Apigenin and 5-O-Methyl-luteolin. For the first time, they reported the occurrence of 3-Methoxy genkwanin, p-Coumaric acid and 5-O-Methyl-luteolin in this plant. In the same study, total phenolic content was higher in the stem extract (9.6 mg/g) compared to the leaf extracts (8.4 mg/g) and the root extracts (5.4 mg/g). Molecules 2016, 21, 369 11 of 26 Table 3. The known non-volatile constituents of P. amboinicus. Compound Name Formula Plant Origin/Part Analytical Method References UV/NMR/UPLC/MS/HPLC HPLC UV/NMR/UPLC/MS/HPLC UV/NMR/UPLC/MS/HPLC UV/NMR/MS/HPLC UV/NMR/MS/HPLC [48,51] [51] [48,51] [48,51,52] [52] [52] UV/NMR/UPLC/MS UV/NMR UV/NMR/UPLC/MS UV/NMR/UPLC-MS HPLC UV/NMR UV/NMR/MS/HPLC UV/NMR/UPLC/MS/HPLC UV/NMR/UPLC/MS UV/NMR/UPLC/MS UV/NMR/UPLC/MS UV/NMR/UPLC/MS UV/NMR/UPLC/MS [48,49] [49] [48] [48] [51] [49] [52] [48,51] [48] [48] [48] [48] [48] Phenolic acids Caffeic acid Gallic acid p-Coumaric acid Rosmarinic acid Salvianolic acid A Shimobashiric acid C9 H8 O4 C7 H6 O5 C9 H8 O3 C18 H16 O8 C26 H22 O10 C36 H32 O16 India, Egypt/Leaf, stem, root (Methanol extract) India/Stem (Methanol extract) India, Egypt/Leaf, stem, root (Methanol and ethyl acetate fraction) India, Egypt, Thailand/Leaf, stem, root (Methanol and ethyl acetate fraction) Thailand/Aerial parts (Water extract) Thailand/Aerial parts (Water extract) Chrysoeriol Cirsimaritin Eriodictyol Luteolin Rutin Salvigenin Thymoquinone Quercetin 5,41 -Dihydroxy-6,7-dimethoxy flavone 1 5,4 -Dihydroxy-3,7-dimethoxy flavone 5-O-Methyl-luteolin 3,5,7,31 ,41 -Pentahydroxy flavanone 1 4 ,5,7-Trihydroxyflavone (apigenin) C16 H12 O6 C17 H14 O6 C15 H12 O6 C15 H10 O6 C27 H30 O16 C18 H16 O6 C10 H12 O2 C15 H10 O7 C17 H14 O6 C17 H14 O6 C16 H12 O6 C15 H12 O7 C15 H10 O5 Philippines, Egypt/Leaf, stem, root (Chloroform extract; Ethyl acetate fraction) Philippines/Leaf (Chloroform extract) Egypt/Leaf, stem, root (Ethyl acetate fraction) Egypt/Leaf, stem, root (Ethyl acetate fraction) India/Stem (Methanol extract) Philippines/Leaf (Chloroform extract) Thailand/Aerial parts (Water extract) Egypt/Leaf, stem, root (Ethyl acetate fraction) Egypt/Leaf, stem, root (Ethyl acetate fractions) Egypt/Leaf, stem, root (Ethyl acetate fractions) Egypt/Leaf, stem, root (Ethyl acetate fractions) Egypt/Leaf, stem, root (Ethyl acetate fractions) Egypt/Leaf, stem, root (Ethyl acetate fractions) Flavonoids Molecules 2016, 21, 369 12 of 26 Figure 2. The structures of some of the non-volatile chemical constituents. Figure 2. The structures of some of the non-volatile chemical constituents. μ μ content was found to μ be highest in the root extracts (126 µg/g), followed by the The tannin leaf (90 µg/g) and stem extracts (81 µg/g). The use of high resolution Ultra Performance Liquid Chromatography (UPLC)-MS analysis identified the presence of Rosmarinic acid, Chrysoeriol, Caffeic acid and p-Coumaric acid in the ethyl acetate fractions of stems and roots. However, the compounds, Eriodyctiol, Luteolin and Quercetin were found only in the ethyl acetate fractions of stems [48]. Bhatt et al. [51] have analyzed the phytochemicals present in the methanolic stem extracts of P. amboinicus. Their study revealed the existence of total phenolic content (49.9 mg gallic acid equivalent (GAE)/g extract), condensed tannins (0.7 mg tannic acid equivalent (TAE)/g extract) and total flavonoids (26.6 mg rutin equivalent (RE)/g extract). Further, HPLC analysis of the extract confirmed the occurrence of bioactive polyphenols such as Rosmarinic acid (6.16 mg/g extract), Rutin (0.32 mg/g extract), Caffeic acid (0.77 mg/g extract), Gallic acid (0.26 mg/g extract), p-Coumaric acid (0.10 mg/g extract) and Quercetin (0.15 mg/g extract). The chemical composition of aqueous leaf extracts of P. amboinicus was reported to contain tannins, flavonoids, saponins, polyuronides and Molecules 2016, 21, 369 13 of 26 steroid glycosides [28]. In the same study, about 11 phytocompounds constituting 97.6% of the total extract were confirmed using GC-MS analysis. The major components included Linalool (50.3%), Carvacrol (14.3%), Geranyl acetate (11.7%) and Nerol acetate (11.6%). Chen et al. [52] have isolated and identified four bioactive compounds with transcription factor inhibition activity from aerial parts (stem and leaves) of P. ambonicus collected from Thailand. They separated the compounds from water extracts using HPLC column-based fractionation to detect the compounds using mass spectrometry and NMR analysis. The identified non-volatiles included Rosmarinic acid, Thymoquinone, Shimobashiric acid and Salvianolic acid. 4. Bioactivities of P. amboinicus 4.1. Antimicrobial Activities P. amboinicus extract, from crude extract to an essential oil, contains innumerable biological constituents owing to its chemical diversity. Plant phytochemicals possess antimicrobial activity against a wide range of bacteria, yeast and mold, but vary in quantity and quality depending on the bioactive constituents [3,4,53]. Its wide range of chemical diversity containing phytochemicals such as terpenes, alcohols, acetones, phenols, acids, aldehydes and esters is often used as components in the pharmaceutical industry. Table 4 provides the detailed antimicrobial activities of different parts of P. amboinicus and its major compounds. 4.1.1. Antibacterial Activities Bacteria are prokaryotic microorganisms usually found on the surface of the skin, mucosal layer and intestinal tract of humans and animals. The genus Staphylococcus is one example of common bacteria found to reside on the skin and in mucous membrane and is mostly harmless. Yet, there are dangerous bacteria classified as human pathogens, causing contagious diseases with a fatal prognosis [54]. These bacteria are usually inhibited by taking antibiotics. However, recently drug resistance developed by microbes is increasingly observed and is a global phenomenon. Therefore, continuous exploration of medicinal plants for effective drugs is an ongoing process. From early years, P. amboinicus has been used as folk medicine to fight pathogenic bacterial activity. In Cuba, a decoction of the leaves was given to patients suffering from chronic cough or tuberculosis and later scientific studies revealed P. amboinicus having anti-Mycobacterium tuberculosis activity [55]. Hot water extract of P. amboinicus leaves inhibited growth of pathogens, Escherichia coli and Salmonella typhimurium while stimulating the growth of Lactobacillus plantarum [56]. This antibacterial activity of plant extracts is most likely due to the combined effect of adsorption of polyphenols to bacterial membranes with membrane disruption and subsequent leakage of cellular contents [57,58], and the generation of hydro peroxide from polyphenols [59]. Further, it was shown that unsterilized ethanolic leaf extract of P. amboinicus exhibits antibacterial activity against diabetic wound pathogens, E. coli, S. aureus, P. mirabilis, P. aeruginosa and K. pneumonia [60]. Current attention is focused on inhibition of antibiotic-resistant bacteria as it is gradually becoming a major problem in the medical industry. Essential oil of P. amboinicus is reported to have a synergistic effect on the antibiotic toxicity toward resistant bacterial strains when combined with aminoglycosides. This makes P. amboinicus essential oil a possible source of a natural product with bacterial resistance-modifying activity [61]. In another study, Vijayakumar et al. [62] used leaf extract of P. amboinicus to biologically synthesize zinc oxide nanoparticles (Pam-ZnO NPs). These Pam-ZnO NPs successfully controlled the growth of methicillin-resistant Staphylococcus aureus biofilms (MRSA ATCC 33591) at the concentration of 8–10 g¨ mL´1 . Molecules 2016, 21, 369 14 of 26 Table 4. Pharmacological properties of P. amboinicus different parts. Pharmacological Activity Plant Part Used Bioactive Compound Potential Effect Antibacterial activity Leaf extract/Essential oil/Decoction Biogenic zinc oxide nanoparticles Pam-ZnO NPs control the growth of methicillin-resistant Staphylococcus aureus biofilm; inhibits growth of Escherichia coli, Salmonella typhimurium & Mycobacterium tuberculosis. Antifungal activity Leaf extract/Essential oil Carvacrol, p-Cymene, α-Terpinolene & β-caryophyllene Fungitoxic properties against Aspergillus flavus, Aspergillus niger, Aspergillus ochraceus, Aspergillus oryzae, Candida versatilis, Fusarium sp. GF-1019, Penicillium sp., Saccharomyces cerevisiae, Candida albicans, C. tropicalis, C. krusei & C. stellatoidea. Antiviral activity Leaf/Ethanolic extract - Exhibited antiviral activity against viruses (VSV, HSV1 & HIV). Activity against Respiratory diseases Leaf extract/Decoction or juice/Essential oil - Used as folk medicine in brazil for influenza, cough, expectorant, bronchitis and throat problems; given orally to control asthma & catarrh; used as bronchodilator. Lavicidal potential Leaf extract/Essential oil Pam-ZnO NPs (zinc oxide nanoparticles) Exhibited up to 100% mortality in Anopheles stephensi, Culex quinquefasciatus & Culex tritaeniorhynchus. Oral Diseases Essential oil Carvacrol Antagonistic effect when used with mouthwash. References [54–63] [13,63] [28,64–66] [11,12,67–72] [14,47,62,73–75] [76] Digestive diseases (Diarrhea, Constipation, dyspepsia, indigestion & as carminative) Leaf extract/Juice - Stimulates growth of Lactobacillus plantarum and inhibits growth of selected food-borne pathogens (Escherichia coli & Salmonella typhimurium); relieves constipation troubles; prevents formation of gas in the gastrointestinal tract & facilitates expulsion of gas Antitumor activity Leaf extract/Crude hydro alcoholic extracts Flavone (Luteolin), flavonols Inhibited the growth of sarcoma 180 & Ehrlich ascite carcinoma tumors in mice; showed significant anticancer activity through inducing apoptosis in A549 (human lung cancer) cell line. Antiinflammatory activity Aerial part/Ethanol, methanol & hexane extract Rosmarinic acid, Shimobashiric acid, alvianolic acid L, Rutin, Thymoquinone, Quercetin Concentration of 0.1 mg/mL inhibited 10%–50% DNA binding activities; inhibited the binding of AP-1 to its consensus DNA sequence; decreased carrageenan-induced paw edema up to 40%; significantly increased IgG, IgM & lysozyme activity in rats. Analgesic activity Leaf extract - Provides remedy for headache, backache & musculo-skeletal problems. [52,81–83] Wound healing activities Leaf & Root Aqueous extract - Increased wound healing activity in experimentally induced diabetic mice & againt murrels. [60,84–87] [30,56,67,68,77] [78,79] [34,50,52,78,80] Cardiovascular disorders Leaf aqueous extract - Positive inotropic activity in the isolated frog heart; effective for treating congestive heart failure. Skin disease (Anti-dandruff, Cuts, Skin Allergy; Burns) Leaf extract/Essential oil/Leaf juice/Paste Thymol, 1,8-Cineole, β-Pinene, α-pinene, phenolic compounds Inhibits the growth of Malassezia furfur; applied on cut as antiseptic promoted better healing; paste was effective against skin allergies, skin burns. Insect bites Leaf aqueous extracts - Potency as antidote for scorpion (Heterometrus laoticus) venom with >50% efficiency. Lactogenic properties Leaf Nutrient content (iron & carotene) Increased breast milk in new mothers. Anti-epileptic activity Leaf, stem, root Extract (aqueous & alcoholic) Alkaloids, flavonoids & saponins Effective as an anticonvulsive and/or antiepileptic medicine. Activity against Genitourinary diseases Leaf Decoction/Ethanolic & aqueous leaf extract - Effective against urinary diseases in the Amazon & India; to relieve kidney troubles, treat vaginal discharges; used after childbirth; increased urine volume & electrolyte concentration in male albino rats. [67,69,95–98] Antioxidant activity Leaf extracts/Essential oil Carvocrol & Thymol Exhibited significant inhibition in DPPH free radical & hydroxyl radical formation. [16,34,99,100] Other diseases Leaf - Fevers, meningitis, eye diseases. [67,101–103] [67,88] [68,89–92] [68,93] [80] [11,68,94] Molecules 2016, 21, 369 15 of 26 4.1.2. Antifungal Activities There is also vast evidence that P. amboinicus plays a crucial role in hindering the growth of disease causing fungus. However, little is known of the derivatives and its effectiveness when used together with industrial drugs. In evaluating the interference of P. amboinicus essential oil on the anti-Candida activity of some clinically used antifungals (itraconazole, ketoconazole and amphotericin B), it showed a diverse level of interference. Essential oil exhibited prominent interference on the activity of itraconazole, providing a synergic effect on C. albicans, C. tropicalis, C. krusei and C. stellatoidea. Whereas, interference on the anti-yeast activity of ketoconazole was antagonic and synergic when interacting with C. albicans, C. guilliermondii and C. stellatoidea. Amphotericin B, on the other hand, showed a small interference on the anti-yeast activity [63]. In another research, antifungal activity of the volatile oil was studied against various fungi by an agar well diffusion susceptibility test. In that, growth of Aspergillus ochraceus, Aspergillus niger and Penicillium sp. was inhibited by 60%, 64% and 60%, respectively, with 10 µL of volatile oil [13]. 4.1.3. Antiviral Activities A large number of active agents are available for the symptomatic treatment of sexually transmitted diseases (STDs) and acquired immune deficiency syndrome (AIDS). Nevertheless, the emergence of drug-resistant strains and dose-limiting toxic effects has complicated the treatment of these diseases and necessitated the search for new antimicrobial substances from various sources. In the last decade, major advancements have been reported in the field of “microbicides”, i.e., compounds or formulations which, when applied topically can prevent the transmission of STDs including AIDS [28]. These include a few from plant sources such as gossypol derivatives, praneem polyherbal preparations and plantibodies. Likewise, extracts of P. amboinicus were tested and reported to have antiviral activity against Herpes Simplex Virus-1 (HSV1) [64] and anti-HIV inhibition activity [65]. Besides that, ethanolic extract of P. amboinicus was reported to have selective antiviral activity on Vero cell lines at 0.1 mg/mL minimum inhibitory concentration when tested against HSV1 and Vesicular Stomatitis (VSV) viruses [66]. 4.2. Respiratory Disorders P. amboinicus is frequently cited in the treatment of chronic coughs, asthma, bronchitis and sore throat in India and the Caribbean Islands [67–69]. In accordance with that, leaves of P. amboinicus had positive bronchodilator activity when tested on guinea pigs [70]. In Eastern Cuba, essential oil from aerial parts of P. amboinicus is used to treat asthma [11]. Decoction or juice made from leaves together with other herbs is also taken orally to control asthma. This decoction is also used to treat catarrhal infections where it clears the excessive build-up of thick phlegm or mucus in an airway or cavity of the body [71]. Collectively, the reason behind this could be high amounts of Carvacrol [11] and Thymol [12] found in the essential oil of the plant. Carvacrol and Thymol are an excellent expectorant and used to treat various respiratory disorders. It is suggested that a drink or a bath of P. amboinicus juice/decoction can be a worthy treatment for influenza, cough, bronchitis and throat problems [72]. 4.3. Activity against Digestive Diseases P. amboinicus is a popular treatment for dyspepsia, indigestion and diarrhea, and a carminative in India and Africa [30,67,68,77]. In India, the leaves of P. amboinicus are consumed along with buttermilk, yogurt, or any other probiotic sources during pathogen-induced diarrhea. The leaves are known to have a prebiotic effect on the probiotic bacteria Lactobacillus plantarum. They utilize the phytoconstituents of the leaves by producing necessary metabolic enzymes. A detailed examination by Shuba and Bhatt [56], describes the mode of hot water extract (HWE) of P. amboinicus leaves on growth inhibition of Escherichia coli and Salmonella typhimurium (pathogens) while stimulating the growth of Lactobacillus plantarum. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) Molecules 2016, 21, 369 16 of 26 gel showed the presence of phenolic acid decarboxylase enzyme induced in the presence of HWE, which indicated the utilization of polyphenols by the bacteria. Cells grown on HWE also showed β-galactosidase activity, indicating their ability to utilize sugars present in HWE. This provides evidence in the traditional use of the leaves in the alleviation of diarrhea by accelerating microbial gut balance during infection. In addition, P. amboinicus juice obtained from pounded leaves is used as a drink to cure constipation in Indonesia and Malaysia [77]. 4.4. Antiepileptic Activity Various literatures have reported the use of P. amboinicus in the treatment of nervous disorders, including epilepsy and convulsions [68]. In Cuba, it is used as an anticonvulsive and antiepileptic drug [11]. Bhattacharjee and Manjumder [94], tested the anticonvulsant activity of the leaf, stem and root alcoholic extract separately on Swiss albino mouse models by maximal electric shock-induced seizures and pentylenetetrazole-induced seizures. They found significant anticonvulsant activity in both the models with alcoholic leaf extract recording the highest activity. They also predicted that the presence of alkaloids, flavonoids and saponins in these extracts may be responsible for this activity. 4.5. Antitumorigenic Activities The antitumor activity of hexane extracts of P. amboinicus has been reported [78]. The results showed a significant inhibition on the growth of Sarcoma-180 tumor in mice treated with the hexane extracts of P. amboinicus. A dose of 350 mg/kg of hexane extracts of P. amboinicus significantly reduced the growth of S-180 tumor with 66% inhibition, while doses of 100, 150 and 250 mg/kg reduced the inhibition to 44%, 45% and 47%, respectively. There were no significant differences in body weight before and after the treatments. This was comparable to metrotexat, a cancer-treating drug which may cause very serious, life-threatening side effects but reduces 100% of tumoral growth. Nevertheless, hexane extracts of P. amboinicus is plant-based, therefore, the severity of side effects can be reduced while the tumoral growth is being destroyed. P. amboinicus ethanolic extract showed significant anticancer activity through inducing apoptosis in the A549 (human lung cancer) cell line [79]. 4.6. Anti-Inflammatory Activities The hexane extract (HE) of P. amboinicus was also shown to exhibit anti-inflammatory activity [78]. A significant reduction of the paw edema was observed at doses of 150, 250 and 350 mg/kg of the HE of P. amboinicus. The highest percentages of reduction of the paw edema were observed in the groups treated with 250 (41%) and 350 mg/kg (33%) of the HE of P. amboinicus. Interestingly, the lowest percentage of inhibition of paw edema was observed in the groups that were treated with 10 mg/kg of the indomethacin, a non-steroidal antiinflammatory drug. Rheumatoid arthritis is a chronic inflammatory disease. The activator protein-1 (AP-1) controls the expression of inflammatory cytokines, whereas tumor necrosis factor (TNF-α) plays a key role in the pathogenesis of inflammatory bone resorption. The active constituents of P. amboinicus were shown to possess AP-1 and TNF-α inhibitory activities [52]. However, they further suggested validating the AP-1 and TNF-α inhibitory potential of P. amboinicus for the treatment of rheumatoid arthritis. Treatment of leaf methanolic extracts of P. amboinicus resulted with moderate to high anti-inflammatory activity in experimental mice [34]. In vitro and in vivo studies have revealed the potent anti-inflammatory activity of aqueous extract of P. amboinicus [50]. The anti-inflammatory activity was related to modulation of antioxidative enzymes in the liver with a decreased malondialdehyde level. Also, they observed the production of TNF-α and cyclooxygenase 2 (COX-2) in the tissue of edema paw induced in mice. In vitro studies revealed the production of proinflammatory mediators in RAW 264.7 cells. Most recently, Silitonga et al. [80] reported the significant improvement of immunoglobulin levels (IgG, IgM) and lysozyme activity in rats when treated with ethanolic leaf extract of P. amboinicus. Molecules 2016, 21, 369 17 of 26 4.7. Wound Healing Activities Few studies have investigated the ability of P. amboinicus to reduce blood sugar levels. Some of the phytochemicals found in P. amboinicus have been proven to play an important role towards blood sugar level lowering mechanisms (Table 4). This herb has the ability to prevent or decrease the risk of infection and its complications in diabetic patients [84]. Application of a paste prepared using P. amboinicus showed an enhanced wound healing ability by immune-stimulation in diseased giant murrels [85]. Likewise, P. amboinicus leaves and root derived paste (10%) has been shown to exhibit thorough epithelialization on the excision wound in albino rats after 12 days of application [86]. The use of polyherbal suspension prepared from P. amboinicus and Punica granatum was shown to exhibit good wound healing properties in laboratory mice [87]. Further, ethanolic extract of P. amboinicus reduced the wound area by up to 76.6% in diabetic mice induced by monosodium glutamate. It was observed that the plant extract promoted wound healing by increased wound contraction, enhancing collagen deposition and reducing the wound epithelialization period [60]. 4.8. Effects against Skin Diseases P. amboinicus has been used in Brazil since the early days for the treatment of skin ulcerations caused by Leishmania braziliensis [89]. In India, the juice of the leaves is used to treat skin allergies [90]. It is also used to treat burns in Asian regions [68]. When the leaf paste is baked on a flame and applied to cuts or burns, it acts as an antiseptic and promotes healing [91]. Essential oil of P. amboinicus also inhibits the growth of dandruff-causing fungus Malassezia furfur, and was tested using the agar diffusion method and compared against Ketaconazole-based shampoo as the standard [92]. 4.9. Effects against Animal and Insect Bites Leaves of P. amboinicus are also used as a poultice for centipede and scorpion bites in Asian regions, including Malaysia [68]. It is reported that aqueous extracts (0.706 mg/mL and 0.406 mg/mL) of P. amboinicus to be more than 70% efficient when tested against fibroblast cell lysis [93]. This implies the aqueous extracts to have a tendency to be scorpion (Heterometrus laoticus) venom antidotes. However, the same paper also reported its cytotoxicity to be questionable. 4.10. Lactogenic Activity In Indonesia, P. amboinicus is used as a traditional food in soup to stimulate lactation for the month or so following childbirth. The leaves are commonly consumed by mothers who have given birth in North Sumatra, in particular the Batak tribe. The leaves of this herb are believed to increase the production of breast milk due to the high content of nutrients, especially iron and carotene. Consumption of leaves significantly increases minerals such as iron, potassium, zinc and magnesium in milk, thus, improving the infant’s weight and health holistically [80]. 4.11. Antioxidant Activities The essential oil P. amboinicus possesses a significant antioxidant property against stress-created in cell line-induced lung cancer in both (in vitro and in vivo) models which could be due to the presence of phytochemical compounds such as Carvocrol and Thymol. Non-enzymatic antioxidant-reduced glutathione was found to be increased in the P. amboinicus essential oil treated mice. The presence of important bioactive compounds confirms the possibility of its use in pharmaceutical drug formulations. The use of essential oil of P. amboinicus is cheaper than natural drug formulation and also without any side effects on the animal model reported [34]. For the first time, the aqueous leaf extract of P. amboinicus was reported to possess higher superoxide- scavenging, nitric oxide-scavenging and ferrous ion-chelating capacity [99]. A report by Bhatt and Negi [16] showed the highest polyphenolic content with appreciable total antioxidant and 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical-scavenging properties in the solvent extract of P. amboinicus leaves. Similarly, Molecules 2016, 21, 369 18 of 26 Khanum et al. [100] found lower content of total flavonoids and total phenolics and antioxidant activity in an ethanolic leaf extract of P. amboinicus. 4.12. Oral Diseases Caries and periodontal disease are especially of concern to public health, where they affect a large part of the population. P. amboinicus, rich in carvacrol, has shown an antagonistic effect when used with mouthwash to avoid bacterial growth in the oral cavity [76]. This could be a potential alternative treatment for diseases related to oral cavities. 4.13. Larvicidal Potential Mosquitoes have the ability of carrying and transmitting human and animal diseases across countries causing hundreds of millions of clinical cases and millions of deaths annually. Senthilkumar and Venkatesalu [14] reported the possible use of P. amboinicus essential oil as a low cost eco-friendly resource for inhibiting the malarial vector mosquito population. The LC50 values of the oil were found to be 33.5 and 28.3 ppm after 12 and 24 h, respectively. Likewise, Lima et al. [73] reported larvicidal activity (LC50 value: 58.9 ˘ 0.4 µg/mL) of the essential oil of P. ambonicus against the mosquito (Aedes aegypti) which is a chief vector of dengue, yellow fever and dengue hemorrhagic fever. In another study, the essential oil of P. amboinicus was shown to act as a good larvicidal agent against the mosquito, Anopheles gambiae after 48 h [47]. In an investigation by Baranitharan et al. [74], the highest larvicidal activity against Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus was found in the ethyl acetate leaf extracts of P. amboinicus. More recently, Jayaraman et al. [75] have reported the larvicidal potential of different solvent extracts of P. amboinicus leaves against Aedes aegypti, Culex quinquefasciatus, and Anopheles stephensi. P. amboinicus zinc oxide nanoparticles (Pam-ZnO NPs) showed 100% mortality of fourth instar mosquito larvae of Anopheles stephensi, Culex quinquefasciatus and Culex tritaeniorhynchus at the concentration of 8 and 10 g/mL. The histopathological studies of Pam-ZnO NPs-treated A. stephensi and C. quinquefasciatus larvae revealed the presence of damaged cells and tissues in the mid-gut. The damaged tissues suffered major changes, including rupture and disintegration of the epithelial layer and cellular vacuolization [62]. This biological control could be slow, but a long-lasting, inexpensive alternative and harmless to the ecosystem. 4.14. Activity against Cardiovascular Disorders P. amboinicus is also used in the Caribbean, to treat congestive heart failure [67]. The aqueous extracts of the fresh leaves of P. amboinicus exhibited dose-dependent positive inotropic activity in the isolated frog heart without affecting the heart rate [88]. This may be attributed to the increase in sodium influx thereby causing greater intracellular availability of calcium. In this report the bioactivity of the tissue-cultured extracts of P. amboinicus to the parent plant was also described. Both extracts from tissue-cultured and parent plant produced a comparable significant effect indicating that they both can be used as a source of biochemical production. 4.15. Activity against Genitourinary Diseases The leaves of P. amboinicus are frequently utilized in the treatment of urinary diseases in the Amazon and India [69,95]. This species is also reported to relieve kidney troubles and treat vaginal discharges, and is taken as a drink after childbirth [67]. Urolithiasis is a condition when stony concretions form in the bladder or urinary tract. Many remedies have been employed during the treatment of urinary stones. The juice of P. amboinicus has been used as a natural remedy to dilute the crystals in the urinary tract in India from ancient times [96]. The antilithiotic activity of the concentrated fresh juice of the leaves of P. amboinicus is proved by Jose et al. [97]. The said study on urine analysis revealed significant reduction in calcium, oxalates and total protein level compared to the control. Further histopathological results showed an absence of crystal and normal-sized tubules with a single epithelial lining. He suggested this antilithiotic activity could be associated with calcium oxalate Molecules 2016, 21, 369 19 of 26 origin. The diuretic properties of ethanolic and aqueous extracts of P. amboinicus were evaluated by determination of urine volume and electrolyte concentration in male albino rats. Furosemide (10 mg/kg) was used as a standard, while normal saline (0.9%) was used as a control. Both ethanolic and aqueous extracts (500 mg/kg) have shown a significant increase in the volume of urine and urinary concentration of Na, K and Cl ions and were comparable to furosemide. This study concludes that the leaves of P. amboinicus possess diuretic activities [98]. 4.16. Analgesic Activity In Africa, P. amboinicus is used as a remedy for headaches [81]. The aqueous extract of P. amboinicus leaves showed an analgesic and anti-inflammatory property, mainly modulated by controlling inhibition of proinflammatory mediators [52]. It is also used to treat musculo-skeletal conditions such as a stiff neck and backache [82,83]. 4.17. Activity against Other Diseases P. amboinicus is an important herb in Asia and South America for the treatment of infectious diseases such as fevers [67,101], cholera and meningitis [102]. It also used to treat sensory disorders associated with ear and eye problems. For example, P. amboinicus seed oil is a treatment for acute edematous otitis acuta in Polynesia [103], whereas in India its leaves are rubbed into the eyes to alleviate conjunctivitis [67]. 5. Culinary Uses 5.1. Nutritional Values Herbs have been used extensively in culinary purposes since ancient times. Many delicious cuisines we enjoy contain various dietary herbs to increase the taste and flavor of the food. Herbal plants also have lots of health benefits attributed to their nutritional content [17,27]. Hence, P. aromaticus can be a good source of nutritive compounds which help to enhance the taste and also prolong the shelf life of food products. A study validates the presence of high minerals, precisely calcium and potassium, at 0.158% and 0.138%, respectively [17]. The detailed nutritional components are presented in Table 5 [17,19,27]. These minerals are necessary to build and maintain strong bones and to retain normal function of heart, kidneys, muscles and nerves. P. amboinicus also has a significant content of iron at 0.262%. Iron is an important component of hemoglobin aids red blood cells to carry oxygen throughout the body. Hemoglobin represents about two-thirds of the body’s iron and its deficiency causes anemia. Adding to that, this plant also contains total Xanthophylls (0.356 mg/g of dry weight of the plant) which consist of Neoxanthin, Violaxanthin, Leutin, Zeaxanthinics. It also has α-Carotene (0.157 mg/g of dry weight) and β-Carotene (0.0035 mg/g of dry weight) [104]. All this makes P. amboinicus a unique dietary supplement. Table 5. Nutritional content of P. amboinicus. No. Principles 1. 2. Proteins Vitamins + Ascorbic acid + Thiamine Minerals + Calcium + Phosphorous + Potassium + Sodium + Magnesium Trace metals + Iron + Zinc + Copper + Chromium Soluble dietary fibers Insoluble dietary fibers Phytic acid Soluble oxalate 3. 4. 5. 6. 7. 8. Nutrient Content 0.6% 0.003% 0.00008% 0.158% 0.016% 0.138% 0.0047% 0.088% 0.262% 0.0003% 0.00012% 0.000022% 0.31% 1.56% 0.00092% 0.02% Molecules 2016, 21, 369 20 of 26 5.2. Use as a Food Additive The highly aromatic leaves of P. amboinicus are used in cooking to enhance the taste and aroma of food. The herb is often used as a substitute for oregano in the food trade. The leaves of P. amboinicus are added when marinating food, for food stuffings [104] and for flavoring meat dishes, e.g., beef, lamb and chicken [105,106]. The strong oregano-like flavor of this herb is an excellent choice to disguise the smell of meat while enhancing the taste. It is also used for the same reason in seafood cuisines such as fish and shellfish [67] to mask the smell of fish, which may prevent someone from enjoying their meal. In addition to this, P. amboinicus is used to spice dishes containing tomato sauces [10]. In India, the leaves are sometimes eaten raw with bread and butter or added to fritters. The tender and soft leaves are crunchy and have an astringent taste when chewed raw. They also may be added to beer and wine for flavoring [67]. 6. Ornamental and Other Social Uses P. amboinicus is grown as an ornamental in home gardens and hanging baskets for its attractive heart-shaped foliage and expression of fresh aroma when touched [10]. The variegated version with white-edged leaves, Plectranthus amboinicus “Variegata”, looks particularly attractive as an ornamental plant, especially when planted in hanging baskets or grown as a garden border. This plant is resistant to diseases and is able to survive drought due to its succulent leaves which can retain water. They can grow beautifully with minimum maintenance. Added to their culinary and medicinal value, P. amboinicus makes a preferred potted plant in many home gardens. P. amboinicus has scented leaves and these are often rubbed into the hair and body after bathing to replace body odor with a refreshing scent [67]. Many health and wellness spas around the world use essential oils of this plant to induce a calming sensation for aromatherapy. In the Amazon, the leaves are mixed with sugar and used as an intoxicant [68]. Whereas, in Tonga and Martinique, the leaves are used for the cleaning of textiles and to perfume them [10,46]. P. amboinicus is also used for spiritual and religious purposes and offered to the spirits when a house is being built in some community [67]. 7. Conclusions and Recommendations P. amboinicus is an important aromatic medicinal herb packed with many bioactive constituents and nutrients, which are important for maintaining good health. The plant has shown a wide range of biological properties and proved to be effective in curing respiratory, cardiovascular, oral, skin, digestive and urinary diseases. The biological properties are attributed to the occurrence of a wide range of bioactive compounds in the plant extracts as well as an essential oil. Thus, it can be stated that P. amboinicus has huge future prospects in meeting the global demand for natural, cost-effective and safer bioactive molecules in pharmaceutical and nutraceutical industries. However, additional research efforts are required to isolate, identify and interpret or authenticate the effectiveness of bioactive compounds from P. amboinicus. Though several classes of phytocompounds are isolated and authenticated from this herb, their bioactivity and toxicity studies under in vivo conditions using animal models are limited to only a few compounds. Till now, no scientific evidence is available on the human safety aspects of P. amboinicus even though it is used widely in folk medicine. Further, some detailed investigations should be aimed at understanding the effectiveness of these isolated compounds in treating other human illnesses. Acknowledgments: The authors are grateful to the Department of Crop Science, Faculty of Agriculture, University Putra Malaysia for supporting the research work. Author Contributions: G.A., M.K.S. and U.R.S. together initiated and designed the work. G.A. and M.K.S. contributed to literatures collection and drawn chemical compound structures. M.K.S. and U.R.S. drafted the manuscript. All authors have read the manuscript before submission. Conflicts of Interest: The authors declare no conflict of interest. Molecules 2016, 21, 369 21 of 26 Abbreviations The following abbreviations are used in this manuscript: SPME: GC: LSC: GLC: GC-MS: GC-FID: NMR: UV: HPLC: UPLC: GAE: TAE: RE: Pam-ZnO NPs: TNF-α: COX-2: AP-1: HWE: HE: SDS-PAGE: HSV 1: STDs: AIDS: HIV: VSV: Solid phase micro extraction Gas chromatography Liquid-solid chromatography Gas-liquid chromatography Gas chromatography-mass spectroscopy Gas chromatography outfitted with flame ionization detector (GC-FID) Nuclear magnetic resonance Ultra violet High pressure liquid chromatography Ultra performance liquid chromatography gallic acid equivalent Tannic acid equivalent Rutin equivalent P. amboinicus Zinc oxide nanoparticles Tumor necrosis factor Cyclooxygenase 2 Activator protein-1 Hot water extract (HWE) Hexane extract Sodium dodecyl sulfate polyacrylamide gel electrophoresis Herpes simplex virus-1 Sexually-transmitted diseases Acquired immune deficiency syndrome (AIDS) Human immunodeficiency virus Vesicular stomatitis virus References 1. 2. 3. 4. 5. 6. 7. 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