Nutritional and medicinal applications of Moringa oleifera Lam.—Review of current status and future possibilities

Accepted Manuscript Title: Nutritional and medicinal applications of Moringa Oleifera Lam.—Review of current status and future possibilities Authors: Swati Gupta, Rohit Jain, Sumita Kachhwaha, S.L. Kothari PII: DOI: Reference:

S2210-8033(17)30053-2 http://dx.doi.org/doi:10.1016/j.hermed.2017.07.003 HERMED 189

To appear in: Received date: Revised date: Accepted date:

1-11-2016 17-3-2017 31-7-2017

Please cite this article as: Gupta, Swati, Jain, Rohit, Kachhwaha, Sumita, Kothari, S.L., Nutritional and medicinal applications of Moringa Oleifera Lam.—Review of current status and future possibilities.Journal of Herbal Medicine http://dx.doi.org/10.1016/j.hermed.2017.07.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Nutritional and medicinal applications of Moringa Oleifera Lam. – Review of current status and future possibilities

Swati Gupta1, Rohit Jain1, Sumita Kachhwaha2, S L Kothari3* Affiliations: 1-Department of Biosciences, Manipal University Jaipur, India 2-Department of Botany, Bioinformatics Infrastructure Facility, University of Rajasthan, Jaipur, India 3-Amity Institute of Biotechnology, Amity University Rajasthan, Jaipur, India *Corresponding author: [email protected], Telefax No.: +91 141 2703439

Graphical abstract

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Abstract Moringa oleifera Lam. (vern. Drumstick) is a mineral rich, medicinally important tree species of family Moringaceae. It has a wide range of culinary applications, and has bioremediation, nutritional and medicinal properties. Conventionally, seeds of the tree have been used as natural coagulants and flocculents in waste water treatment. Its leaves and bark act as biosorbent for remediation of heavy metals and dyes. The tree provides remedies for a range of diseases and disorders by dint of its unique combination of numerous phytochemicals. Gum exudates of the tree are of immense medicinal importance due to their applications in biodegradable drug delivery systems and in treatment of asthma, dysentery and intestinal cancer. The multidimensional utilities of M. oleifera may cause overexploitation of this tree, posing danger to the existing natural variability in the near future. Therefore, there is a need for conservation of the species, for ethnobotanical, pharmacological, nutraceutical and biodiversity purposes. Development of tissue culture propagation methods will assist in preserving some of the germplasm of the species. In the present review, consolidated analysis of the role of biotechnology in conservation and genetic enhancement of nutritional medicinal and commercial value of the tree has been placed in perspective, together with an up to date review on phytochemical analysis of the plant, and its utility has been discussed to invite the attention of the scientific community to further consider the study of this miracle tree species and its nutritional and pharmaceutical properties. Abbreviations:

Kn: Kinetin BAP: 6-Benzylaminopurine IBA: Indole-3-butyric acid IAA: Indole-3-acetic acid

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NAA: 1-Naphthalene acetic acid TDZ: Thidiazuron 2,4-D: 2,4-Dichlorophenoxyacetic acid TRIA: Triacontanol PGR(s): Plant growth regulator(s) MS: Murashige and Skoog Keywords: Moringa oleifera, Alzheimer’s, cancer, Moringa gum, antioxidant

1. Introduction Plants provide the richest resource of natural compounds, which are used directly or indirectly for a wide range of applications for the wellbeing of human populations and domestic animals (Khanuja, 2012). These compounds are primarily phytomolecules synthesized through primary and secondary metabolic pathways in plants (Arora et al., 2013). The dependence of humans on plants for their basic requirements like food, medicine, clothes, shelter etc is as old as mankind itself (Goyal et al., 2007), and still in the modern age the majority of commercial products including pharmaceutical and healthcare, food and beverages, textiles, cosmetics and aromas are obtained from plants (Khanuja, 2012). Therefore, plants are and will remain economically, industrially, environmentally, spiritually, historically, and aesthetically important for survival, sustenance and prosperity of life on the Earth (Arora et al., 2013). Moringa oleifera Lam. (syn. M. pterygosperma Gaertn.) is a minerals and vitamin rich, nutritious and medicinally important tree species of the Hindustan center of crop origin, belonging to the family Moringaceae, and commonly called Drumstick, Ben Oil or Horseradish tree (Arora et al., 2013; Ramachandran et al., 1980) 3

Moringaceae is a monogeneric family including the single genus Moringa with 10-12 species. The plant M. oleifera is characterized by tripinnate leaves, yellow or white petioles, hanging 3sided pods and whitish grey corky bark (Fig. 1a-c). Other characteristics include bisexual, stalked, white or creamy axillary flowers, globular winged seeds, pendulous ribbed capsules and soft, fissured tuberous taproots (Bhandari, 1995; Ramachandran et al., 1980; Seshadri and Nambiar, 2003). The tree also exudes gum through lysigenous traumatic gum ducts developed in the bark (Bhandari, 1995; Subrahmanyam and Shah, 1988). The shape and color of the gum exudates varies from stalactite pieces to tears and yellow to reddish brown or black (fig 1d) in color (Bhandari, 1995; Morton, 1991; Panda et al., 2006; Ramachandran et al., 1980). These gum exudates are water insoluble due to their mucilaginous texture and belong to the tragacanth or hog gum series (Ramachandran et al., 1980) It is a fast growing, medium sized deciduous tree, propagated as a perennial plant from cuttings and seeds (Ramachandran et al., 1980; Seshadri and Nambiar, 2003). It mainly grows in semiarid, tropical and subtropical regions, although the drought resistance properties of the plant makes it more suitable for drier regions (Farooq et al., 2012; Leone et al., 2015; Seshadri and Nambiar, 2003; Thurber and Fahey, 2009). The roots are susceptible to water-logging and tend to rot in such conditions. It is also able to tolerate a wide range of soil types, the optimum being a well-drained sandy or loamy soil (pH 5-9) (Patel, S. et al., 2010; Ramachandran et al., 1980). M. oleifera is valued for its multiple economic, medicinal and neutraceutical properties worldwide. This plant has been honored as “Botanical of the Year – 2007” by the National Institute of Health (NIH). The Africans used to call it “Never Die” or “Miracle Tree” for its ability to treat more than 300 diseases.

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All the plant parts including immature pods, leaves, mature seeds, flowers and roots have long been consumed by humans for a variety of applications. The immature pods, leaves, flowers and mature seeds are used as vegetables (Farooq et al., 2012; Ramachandran et al., 1980; Stevens et al., 2013). Culinary practices are affected by varying traditions and taste preferences (Seshadri and Nambiar, 2003). Traditional dishes across the world include the use of tender young plants, young and mature leaflets and flowers in soup and sauce preparations (Morton, 1991; Stevens et al., 2013). Leaves are also used in salad preparation with groundnut (Fahey, 2005; Morton, 1991; Stevens et al., 2013), herbal tea preparations, porridge, complementary baby foods, spice and as garnish. The fresh leaves serve as a good snack when chewed raw (Ramachandran et al., 1980; Stevens et al., 2013). In addition, seeds obtained from tender and mature pods are popularly used in pickling (Ramachandran et al., 1980). Seeds obtained from young pods are eaten green, and mature seeds are either roasted or fried before consumption and taste like peanuts (Fahey, 2005; Morton, 1991; Ramachandran et al., 1980). The extremely pungent roots are mixed with vinegar and salt and serve as a substitute to the popular condiment “horseradish” (Morton, 1991). This review focuses on the important medicinal and neutraceutical properties and also the biotechnology of this high value plant. M. oleifera.

2. Methodology A thorough and critical survey of the literature related to Moringa research was conducted up to March 2017. Various online and offline resources were taken into consideration. The primary source of data collection for the review included research papers and review articles published by reputed publishers such as Springer, Elsevier, Routledge and Taylor & Francis imprints, BMC

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and PLOS One. Online databases including NCBI, Scopus and Science Direct were also referred to for collection of data on Moringa oleifera. The paid articles were accessed through the Bioinformatics Infrastructure Facility Center sponsored by DBT, New Delhi located at the University of Rajasthan campus. Some other ‘grey literature’ sources such as webpages, conference proceedings, book chapters and theses were also reviewed to access the maximum possible information about the potential utilities, extent of research carried out and current bottlenecks in the Moringa oleifera research. The Flora of the Indian Desert (Bhandari, 1995), a comprehensive book based on practical survey of the Desert zone of India, was referred to for collection of information about the geographical distribution, taxonomy and morphology of the tree. Herbarium “RUBL”, located at University of Rajasthan Campus was also visited to collect data regarding the morphological and identification characteristics of the tree. The importance of this plant in Ayurvedic, Unani, Siddha and folk medicinal formulations was further validated through the Indian Medicinal Plant Database, NMPB (http://www.medicinalplants.in). Literature searching was conducted using Google Scholar and PubMed using the following keywords: Moringa oleifera, drumstick, sajina, Moringa pterygosperma, metabolite profiling of Moringa, metabolite profiling of drumstick, phytochemical analysis of Moringa, in vitro propagation, micropropagation, uses of drumstick tree, and importance of Moringa oleifera. The commercial importance of the tree was reviewed by surveying different market places and online stores for the availability of Moringa oleifera products. In this review the challenges associated with commercialization of the products of drumstick tree, along with the scope of biotechnological interventions as a remedy, are discussed. The review thus highlights the need for future research pertaining to the application of biotechnology for enhancement of commercial value of the tree.

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3. Advances in M. oleifera Research/ Multidimensional potential or utilities of M. oleifera 3.1 Bioremediation potential Use of M. oleifera as a water purifier has been a common practice for decades, and research in this direction revealed the significant bioremediation potential of different components of the tree. During the early 1990’s, the efficiency and properties of the drumstick tree as a natural coagulant were assessed and it was found that M. oleifera seeds, while being biodegradable and non-toxic, can act as a viable substitute to industrial coagulants such as alum (Sutherland et al., 1994). This coagulating potential is attributed to dimeric cationic proteins (13 kDa) and flocculating proteins (about 6.5 kDa) as isolated and purified from aqueous extracts of M. oleifera plant parts (Gassenschmidt et al., 1995; Ndabigengesere et al., 1995). In fact, the purified proteins were reported to be more effective as the coagulation process is mediated through adsorption and neutralization of colloidal charges (Ndabigengesere et al., 1995). These promising results led to extensive research on the isolation and purification of efficient coagulants from M. oleifera extracts, and as a result a variety of coagulating and flocculating components were isolated (for instance Okuda et al. (2001) successfully purified an active coagulant from the salt solution of M. oleifera seeds crude extract, while hemaggulatinating proteins and a lectin coagulant were isolated and purified from M. oleifera tissue extracts and seeds, respectively (Santos et al., 2009). The coagulant activity of different extracts and purified proteins was reported to be comparable to that of the most commonly used coagulant, aluminium sulphate and as a consequence use of M. oleifera as a substitute to these chemical coagulants is encouraged for turbid water treatments (Pritchard et al., 2010), along with the use of its flower extracts for disinfection of the waste

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water (Moura et al., 2011). Further, Broin et al. (2002) cloned cDNA encoding one of the flocculating proteins present in M. oleifera seeds and the resulting recombinant proteins thus obtained were used against clay particles, as well as both Gram positive and Gram negative bacteria. Besides being used as a natural coagulant, flocculent and microbicidal, the tree has also been explored for its potential to remediate toxic compounds usually found in industrial soil and waste water. The presence of toxins in effluents, due to improper disposal practices, leads to soil and water pollution and disturbance in the food chain associated with a particular ecosystem. Hence, strong emphasis has been made over the past few years for the development of efficient methods for removal of toxicants. Use of M. oleifera as a natural biosorbent for remediation of toxicants is one novel approach, in which researchers have explored different parts of the tree for their remedial efficiency using different parameters. Biosorbents developed by chemical modification of leaves serve as an excellent substitute to the conventionally used adsorbents for removal of heavy metals, particularly Cd(II), Cu(III), Ni(II) and Pb(II) from the aqueous solution (Reddy et al., 2010a; Reddy et al., 2012). In a similar study, Reddy et al. (2010b) demonstrated that M. oleifera bark can also serve as an efficient biosorbent for biosorption of Pb2+, and concluded that such biosorbents can serve as efficient substitutes to the existing synthetic adsorbents such as activated carbon, ion exchange resins, etc. Efficient removal of azo and anthroquinonic dyes using seed extracts and organic compounds such as benzene, toluene, ethylbenzene and cumene using pods of the tree has also been illustrated (Akhtar et al., 2007; Beltrán‐Heredia and Sánchez Martín, 2008). These studies not only highlight the potential of M. oleifera as an efficient and environmentally friendly coagulant and biosorbent for bioremediation of polluted water and land, but open new

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gateways for developing commercially viable coagulants, flocculants and biosorbents for remediation of xenobiotics.

3.2 Nutritional value M. oleifera has been used as a dietary supplement due to its rich nutritional content. It is an outstanding indigenous source of vitamins, proteins and minerals (Fahey, 2005). The tree contains digestible proteins, iron, magnesium, calcium, vitamins (B6, B2 and C), and carotenoids. M. oleifera is one of the richest natural sources of provitamin A (Maheshwari et al., 2014; Mehta et al., 2011; Seshadri and Nambiar, 2003). Every part of this tree has been found to possess many nutrients. Flowers, leaves, young shoots and immature pods are good sources of methionine and are also rich in phosphorus, calcium and iron. The refined seed oil is an acceptable substitute to olive oil, due to the presence of all the essential fatty acids as in olive oil (Morton, 1991). The lipid composition of seeds is even greater than that of soyabean, making it nutritionally important. The high content of essential amino acids in the seeds makes it an excellent substitute to legumes, which are poor in sulphur containing amino acids (Ferreira et al., 2008). The leaves of the plant are also significant in terms of their use as a nutritional supplement to combat malnutrition (Fahey, 2005; Seshadri and Nambiar, 2003; Thurber and Fahey, 2009). According to Botany of the Plant Industry as well as Trees of Life (an NGO), “leaves of M. oleifera possess: 4 times more calcium and two times more protein than milk, 7 times more Vit. C than oranges, 3 times more potassium and iron than banana and spinach respectively and 4 times more Vit. A than carrots” (Thurber and Fahey, 2009) - and therefore, this plant is unique as

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it is very rare to find such a diversified nutrient profile in a single plant species. (Razis et al., 2014). This tree serves as a sustainable and economically sound nutrient rich food supplement, for those suffering from malnutrition, especially in developing countries. In addition, it is also being used to boost food security, foster rural development and support sustainable land.

3.3 Pharmaceutical value The medicinal virtues of M. oleifera are well known through its use as a traditional medicine for a number of diseases and disorders since ancient times (Ramachandran et al., 1980). This tree has been popularly used as a folk medicine for anaemia, arthritis and rheumatism, asthma, constipation, diarrhea, stomach pain, ulcers, intestinal spasms, headache and sore gums (Mehta et al., 2011; Pandey et al., 2012; Razis et al., 2014). The tree possesses abortifacient, antirheumatic, anti-inflammatory, bactericidal and diuretic activities and also serves as antidote, emetic, purgative, stimulant, tonic, vermifuge (anthelmintic medicine) and vesicant (Fatima et al., 2014; Mehta et al., 2011; Patel, J.P. et al., 2010). The gum exudates are not only reported to be used in treating various chronic disorders, but are also being used as a potential substitute to synthetic binders and suspending agents in drug delivery systems (Jarald et al., 2012; Panda and Ansari, 2013; Panda et al., 2008; Varma et al., 2014). Various performulation studies ascertained the suitability of M. oleifera gum as a mucoadhesive polymer, disintegrant, binder (Patel et al., 2012) and as a sustained release polymer (Varma et al., 2014), while the drug dissolution and disintegration studies showed that the rate/proportion of drug release can be adequately monitored by varying the Moringa gum concentration in the tablet formulation (Basawaraj et al., 2010; Panda et al., 2008). Later, in vitro toxicity assays of Moringa gum confirmed its non-toxic

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nature, thus rendering it safe for both topical and tablet/non-topical formulations (Jarald et al., 2012; Panda et al., 2006). Shah et al. (2011), evaluated the efficiency of Moringa gum as a pharmaceutical excipient based on various physicochemical parameters and concluded that the gum obtained from M. oleifera serves as an excellent natural source of pharmaceutical excipient. Therefore, Moringa gum exudates can efficiently serve as a non-toxic, natural substitute to the toxic, synthetic excipients, leading to enhanced therapeutic efficiency of the drug by improvising the stability, dissolution rate and disintegration rate of the drug. Similarly, Moringa coagulant can be used as an efficient amorphous state stabilizer in many drug formulations such as ibuprofen, meloxicam and felodipine (Bhende and Jadhav, 2012). The salt extract of Moringa seeds has also been particularly used with Povidone (polyvinylpyrrolidone, PVP) to improve solubility of the partially soluble NSAIDS (Noolkar et al., 2013). Recent studies indicate that the tree gains its exceptional ability to treat such a wide range of medical conditions from its rich phytochemical composition. An overview of some of the important medicinal properties and associated phytochemicals follows:

2.3.1 Antioxidant activity: The antioxidant activity of different parts of M. oleifera is mainly attributed to the presence of ascorbic acid (Vit. C), β-carotene (Kumar et al., 2012; Mahajan and Mehta, 2007), quercetin, kaempferol (Gupta et al., 2012), and phenolic acids (Anwar et al., 2007; Mahajan and Mehta, 2007; Sreelatha et al., 2011). Also, isothiocyanates, polyphenols and rutin from leaves (Bajpai et al., 2005; Tumer et al., 2015); tocopherols, myricetin and lectins from seeds (Mahajan and Mehta, 2007; Santos et al., 2005; Singh et al., 2013); procyanidins from bark (Atawodi et al., 2010) and palmitic acid, phytosterols 9-octadecenamide from the flowers (Inbathamizh and

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Padmini, 2012) have been regarded as potent antioxidants. Monopalmitic acid, oleic acid, trioleic triglycerides present in seed oil are also reported to exhibit antioxidant activity (Lalas and Tsaknis, 2002; Mahajan and Mehta, 2007). Antioxidant activities of different extracts obtained from different plant parts have been reported in several studies. Extracts of leaves in organic solvents such as methanol and acetone have been found to exhibit antioxidant activities (Atawodi et al., 2010; Charoensin, 2014; Moyo et al., 2012b; Ogbunugafor et al., 2012; Siddhuraju and Becker, 2003; Vongsak et al., 2013). Similarly, the antioxidant activities of aqueous extracts of roots (Satish et al., 2013) and methanolic extracts of stems and pods (Gupta et al., 2012; Kumbhare et al., 2012) have also been ascertained using both in vitro as well as in vivo assays. Sreelatha and Padma (2010), reported that the diverse antioxidant profile of leaves also corresponds to the cryoprotective nature of the plant. The plant parts can also be used as natural preservative for fat (Patel, S. et al., 2010).

2.3.2 Antitumor activity: Extracts of the stem and seeds are reported to exhibit cytotoxic, anticancer and antitumor activities (Araújo et al., 2013; Onsare and Arora, 2015; Shaban et al., 2012). Similarly, leaf extracts prepared in different solvents have also been found to exhibit anticancer, cytotoxic (Berkovich et al., 2013; Nair and Varalakshmi, 2011), antiproliferative (Pamok et al., 2012), antimyelomic (Parvathy and Umamaheshwari, 2007), antileukemia, antihepatocarcinoma (Khalafalla et al., 2010) and chemoprotective (Anwar et al., 2007; Murakami et al., 1998) activities.

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Phytochemical analysis revealed that these activities are credited to 3-O-6x-oleoyl-β-Dglucopyranosyl-β-sitosterol, β-sitosterol-3-O-β-D-glucopyranoside, 4(α-L-rhamnosyloxy) phenylacetonitrile, 4-hydroxyphenylacetonitrile and 4-hydroxyphenyl-acetamide in seeds (Guevara et al., 1999; Villasenor et al., 1989); cis-9-hexadecenal, quinic acid, 3,5-dihydroxy-6methyl-2,3-dihydro-4H-pyran-4-1, 9-octadecenamide, methyl octadecenoate in flowers (Inbathamizh and Padmini, 2012) and quercetin, kaempferol (Krishnamurthy et al., 2015; Sreelatha et al., 2011), (4-[(4′-O-acetyl-α-L-rhamnosyloxy) benzyl]isothiocyanate, O-ethyl-4-(αL-rhamnosyloxy) benzyl carbamate, 4-(L-rhamnosyloxy) benzyl isothiocyanate niaziminin and niazimicin (Anwar et al., 2007; Murakami et al., 1998) in leaves. Recently, it was found that the antitumor/anticancer activities of different leaf extracts is mediated through the antioxidant and apoptosis inducing activities of the plant (Jung, 2014; Tiloke et al., 2013).

2.3.3 Antimicrobial activity: Antibacterial activities of different components of the plant including leaves (Abalaka et al., 2012; Doughari et al., 2007; Kekuda et al., 2010; Mandal et al., 2014; Moyo et al., 2012a; Oluduro, 2012; Rahman et al., 2009; Thilza et al., 2010; Valarmathy et al., 2010), seeds (Auwal et al., 2013; Jabeen et al., 2008; Oluduro et al., 2010; Viera et al., 2010) and pods (Arora and Onsare, 2014) have been reported against E. coli, S. typhi, P. aeruginosa, E. cloace, P. vulgaris, S. aureus, M. kristinae, E. aerogenes, Shigella, B. cereus, Streptococcus-B-haemolytica, B. subtilis, K. pneumonia, B. megaterium, S. lutea, B. sterothermophilus, S. pyogenes, V. cholera, S. entridis, enteropathogens and wound bacteria.

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Extracts of seeds (Chuang et al., 2007; Jabeen et al., 2008; Oluduro et al., 2010) and leaves (Chuang et al., 2007; Kekuda et al., 2010; Oluduro, 2012) are also reported to exhibit significant anti-fungal activity against T. mentagrophyte, Pullarium sp., A. flavus, Penicillium sp., A. niger, A. oryzae, A. terreus, A. nidulans, F. solani, R. solani, C. cladosporioides, P. sclerotigenum and Dermatophytes (T. rubrum, E. xoccosum, M. canis). Significant antimicrobial activity of root extracts against E. coli, S. aureus, P. aeruginosa, P. mirabilis, Penicillium sp., Mucor sp., A. niger and C. albicans have also been reported. In addition, the antiviral activities of seeds against HSV-1 (Ali et al., 2004) and that of leaves against Epstin Barr Virus (EBV), HIV and HSV-1 (Lipipun et al., 2003; Nworu et al., 2013; Sudha et al., 2010) have also been tested successfully. Further investigations showed that these antimicrobial activities are possibly attributed to the presence of 4(α-L-rhamnopyranosyloxy)benzyl isothiocyanate, methyl N-4-( α-Lrhamnopyranosyloxy)benzyl carbamate, 4-(α-D-glucopyranosyl-1-4 α-L-rhamnopyranosyloxy)benzyl thiocarboxamide (Oluduro et al., 2010; Pandey et al., 2012), 4-( α-Lrhamnopyranosyloxy) benzyl glucosinolate, (Goyal et al., 2007), together with proanthocyanidins and glucomoringine (Maldini et al., 2014; Singh et al., 2013). The presence of cardiac glycosides in pods (Arora and Onsare, 2014), kaempferol, rhamnetin, kaempferitin, isoquercitrin and pterygospermin in flowers, spirochin and anthonine in roots (Farooq et al., 2012; Mehta et al., 2011; Raj et al., 2011) and aglycon of deoxy-niazimicine (N-benzyl,S-ethyl thioformate) in bark (Nikkon et al., 2003) also contribute to their antimicrobial nature.

2.3.4 Anti-inflammatory activity:

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The presence of potent anti-inflammatory agents including 4-[(α-L-rhamnosyloxy)benzyl] isothiocyanate, 4-[(4′-O-acetyl-α-L-rhamnosyloxy)benzyl]isothiocyanate (Morton, 1991; Stohs and Hartman, 2015; Waterman et al., 2014), quercetin, kaempferol glucosides, (Coppin et al., 2013), 4-[2-O-acetyl- α-L-rhamnosyloxy)benzyl]isothiocyanate, 4-[(3-O-acetyl-αrhamnosyloxy)benzyl]isothiocyanate (Cheenpracha et al., 2010), 3,5-dihydroxy-6-methyl-2,3dihydro-4H-pyran-4-1, 9-octadecenamide (Inbathamizh and Padmini, 2012), aurantiamide acetate and 1,3-dibenzyl urea (Maheshwari et al., 2014; Pandey et al., 2012; Sashidhara et al., 2009) in different parts of M. oleifera have been reported. The reported anti-inflammatory and anti-arthritic activities of seed extracts are ascribed to the presence of glycosides (Gupta et al., 2005; Hamza, 2010; Mahajan et al., 2007). Further, Sulaiman et al. (2008), demonstrated that anti-inflammatory activity of leaves is mediated through inhibition of inflammation signaling pathways. The role of leaf and root extracts in treatment of inflammation (Ezeamuzie et al., 1996; Pal et al., 1995) and that of pod extracts in amelioration of inflammation associated disorders such as cancer, asthma, allergic rhinitis, atopic dermatitis and rheumatoid arthritis (Lee et al., 2013; Muangnoi et al., 2012) has also been studied.

2.3.5 Cardio-protective activity: The presence of O-[2′-hydroxy-3′-(2″-heptenyloxy)]-propyl undecanoate, O-ethyl-4-[( α-L(rhamnosyloxy)-benzyl] carbamate, methyl p-hydroxybenzoate, (Faizi et al., 1998; Pandey et al., 2012; Stohs and Hartman, 2015) and 4-(α -L-rhamnosyloxy benzyl)-O-methyl thiocarbamate (Farooq et al., 2012; Pandey et al., 2012) correspond to the hypotensive nature of the plant as a

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whole, while N- α-L-rhamnopyranosyl vincosamide from the bark is reported to exhibit cardioprotective effects (Morton, 1991). The hypotensive and bradycardiac activities of leaves are attributed to niazinin, niazimicin, niaziminin, niazimin, niazirin, niazicin, niazirinin, niazirin 4-[(4′-O-acetyl-α-rhamnosyloxy) benzyl]isothiocyanate (Bose, 2007; Faizi et al., 1994a; Faizi et al., 1994b), and glucomoringine is reported to have a hypertensive effect (Dangi et al., 2002; Maheshwari et al., 2014). Leaves are also reported to exert a positive effect on the circulatory system/capillaries and reduce mortality and morbidity as a consequence of coronary heart diseases, due to the presence of gossypetin, quercetagenin and proanthocyanidins (Kumar et al., 2012; Seshadri and Nambiar, 2003). Nandave et al. (2009), demonstrated that the cardioprotective activity of leaves is mediated through its antioxidant, antiperoxidative and myocardial preservative effects. Later, Abdulazeez et al. (2016) reported that the blood pressure effect of alkaloids and flavonoids present in seeds and leaves is due to their inhibitory effect on angiotensin converting enzymes.

2.3.6 Immunomodulatory activity: Leaf extracts have been found to exhibit both immunomodulatory and immunostimulatory activities (Rachmawati and Rifa’i, 2014). The immunomodulatory effect of leaves is mediated through a reduction in cyclophosphamide induced immunosuppression by stimulating both cellular and humoral immunity (Gupta et al., 2010), which is attributed to the presence of compounds like isothiocyanates and glycoside cyanides (Sudha et al., 2010).

2.3.7 Neuroprotective Effect:

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Hannan et al. (2014), revealed that leaves exert a neuroprotective effect by promoting neuronal survival and neurite outgrowth. Leaf extracts are also reported to exhibit a protective effect against Alzheimer’s disease by altering brain monoamine levels and electrical activity (Ganguly and Guha, 2008). Further, it was found that the central inhibitory/CNS depressant action of leaves is mediated through its analgesic and anticonvulsive activities (Bakre et al., 2013; Gupta et al., 1999; Ray et al., 2003).

2.3.8 Treatment of Metabolic disorders: Leaf extracts have been reported to possess hypocholesterolemic (Ghasi et al., 2000), antihyperglycemic, antihyperlipidemic and hepatoprotective activities (Divi et al., 2012; Jain et al., 2010; Jaiswal et al., 2009; Kumar and Pari, 2003; Ndong et al., 2007; Pari and Kumar, 2002). Later, Tahiliani and Kar (2000) and Chumark et al. (2008) affirmed the presence of antihyperthyroidism and anti-atherosclerotic activities in leaves. Also, extracts of roots have been found to possess estrogenic, anti-estrogenic, progestational and anti-progestational activities (Bose, 2007; Shukla et al., 1988) along with liver and kidney protective effects (Mazumder et al., 1999). Phytochemical analysis showed that β-sitosterol, 4-[α-(L-rhamnosyloxy) benzyl]-o-methyl thiocarbamate (trans) are potent cholesterol lowering, anti-diarrheal, antispasmodic, hypolipidemic agents (Anwar et al., 2007; Jain et al., 2010; Maheshwari et al., 2014), while quercetin-3-glycoside is responsible for the antidiabetic (Farooq et al., 2012), antidyslipidemic (Mbikay, 2012) and hypoglycemic (Maheshwari et al., 2014) effects. Also, moringine and moringinine are reported to have a role in antihypoglycemic properties of the plant (Kar et al.,

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1999; Maheshwari et al., 2014; Mehta et al., 2011). The presence of insulin like plant proteins in leaf extracts has been recently reported for treatment of diabetes (Paula et al., 2016). Stohs and Hartman (2015), reported the presence of a novel compound, niaziridin, which enhances the gastrointestinal absorption of vitamins and nutrients. The presence of quercetin and kaempferol in flowers and pods has been reported to be associated with their antidiabetic and hepatoprotective activities (Farooq et al., 2012; Gupta et al., 2012).

2.3.9 Other activities: M. oleifera has also been reported to have wound healing activities. Potent wound healing agents such as vicenin-2 (Muhammad et al., 2013), quercetin, kaempferol and phytosterols (Hukkeri et al., 2006) have been reported in ethyl acetate extract of the leaves. Recently, Lambole and Kumar (2012) reported dexamethasone present in bark as an efficient wound healer. It mediates the activity by suppressing anti-healing agents. The wound healing activity has also been reported in the seeds of M. oleifera (Bhatnagar et al., 2013; Parwani et al., 2016). Leaves, seeds and roots of the plant also possess various other activities, such as schizonticidal, antiulcerogenic (Dahiru et al., 2006; Patel, J.P. et al., 2010), larvicidal (De Oliveira et al., 2011; Prabhu et al., 2011), antiurolithiatic and anti-ulcerative colitis (Gholap et al., 2012; Karadi et al., 2006), whereas the gum exudates of plant are reported to exhibit macrofilaricidal activity (Kushwaha et al., 2011). Extracts of the leaves contribute significant radioprotective effects as estimated by Rao et al. (2001) using comparative scoring of aberrations in bone marrow metaphase chromosomes between mice pre-treated with leaf extracts and those without any pre-treatment prior to irradiation. Seeds are reported to exhibit antipyretic (Hukkeri et al., 2006), aphrodisiac (Zade et

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al., 2013), antihypersentitivity and anti-anaphylactic (Mahajan and Mehta, 2007) activities along with being a potent antitoxidant to fluoride toxicity (Ranjan et al., 2009). The anti-asthamatic and anti-rhinitis activities of seed extracts were attributed to the presence of moringine and moringinine, which play a significant role in bronchiole relaxation and treatment of respiratory tract infections (Agrawal and Mehta, 2008; Mahajan and Mehta, 2008). Mehta et al. (2003), reported the presence of anti-atherosclerotic activity in pods, while the protective effect of the phenolic compounds of the tree against oxidative stress was recently reported by Ramabulana et al. (2016). Evidently, drumstick tree serves as a good source of neutraceuticals which are very rare to find altogether in a single plant in sufficient quantities. Thus, the tree can be regarded as medicinally important for prevention as well as cure.

3.4 Other economic utilities Apart from the above discussed properties, the tree has also been studied for some other economic utilities such as feed value and a source of natural gas. M. oleifera has emerged as an important cattle feed that can be used as a substitute to conventional animal feeds such as sunflower seed cake and alfalfa (Babiker et al., 2016; Sarwatt et al., 2002) due to its high nutritional value. Use of Moringa silage as a feed has been reported to exhibit a significantly positive effect on milk quality and quantity in dairy cows (Mendieta-Araica et al., 2011), and milk yield and growth performance in goats and lambs (Babiker et al., 2016). Further, Aregheore (2002) suggested that use of the tree foliage as a part of dairy forage for goats serves as an economical source of protein supplementation. The cost of beef production and nutritional

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quality of roughages was estimated to be optimum when M. oleifera was used as a feed, thereby serving as an economical forage for cattle (Foidl et al., 2001; Roy et al., 2016). The tree has also been reported to be an efficient feedstock for biofuels such as biogas (Foidl et al., 2001) and biodiesel (Kafuku and Mbarawa, 2010; Rashid et al., 2008). The vegetable oil obtained from the seeds of tree has been reported to be of superior quality than that of Jatropha curcas (Kibazohi and Sangwan, 2011). These findings provide new horizons that could be investigated through sustainable development and green technology. In addition to culinary uses, the oil obtained from the seeds has also been used for a variety of purposes such as lighting, hair dressing, lubrication in watches, soap making and perfume production (Morton, 1991). The seed cake remaining after oil extraction is commonly used as a biofertilizer due to its rich nutrient content. Pontual et al. (2012) reported the caseinolytic and milk coagulating activities of proteases present in flowers, while in a recent similar study, milk clotting enzymes were purified and characterized from seeds and were successfully employed in cheese making, thus suggesting an alternative to conventional animal rennets (Ahmed, 2016). Gum exudates obtained from excisions on the bark have been used in leather tanning and calico printing (Morton, 1991). Lee et al. (2016), recently developed an antimicrobial, antioxidative and biodegradable packaging material from puffer fish skin gelatin containing M. oleifera leaf extract. Therefore, the tree is not only important from medicinal and nutritional perspectives, but also has a vast range of other potential utilities.

4. Commercial value of the plant

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The considerable medicinal potential has led to the development of various commercial formulations, such as Shrigu, Rumalaya and Septilin (The Himalaya Drug Company, Bangalore India), Livospin (Herbals APS Pvt. Ltd., Patna, India), Orthoherb (Walter Bushnell Ltd. Mumbai, India), while Ratnagiri Rasa, Sarasvata Ghrta, and Sudarsana churna are some of its ayurvedic formulations. The gum exudates of the tree are valued in the Indian as well as global markets for its dark colored texture and rich medicinal value. The increasing global demand and limitations in the availability of the gum has also resulted in its increasing costs.

5. Need for M. oleifera biotechnology In the past few years, Moringa has gained the attention of both scientific and business communities due to its economic importance in terms of food, medicines, waste management and sustainable agriculture practices. The extensive research and attempt for commercialization of drumstick tree and its products has led to an urgent need for its conservation - from dietary, pharmacological, ethnobotanical and biotechnological perspectives. One solution to the increasing demand for M. oleifera based products is biotechnological approaches, which include in vitro propagation for mass multiplication, enhanced biosynthesis of secondary metabolites through metabolite profiling and pathway engineering and recombinant technology.

5.1 Problems associated with M. oleifera propagation The Drumstick tree is easily propagated by stem cuttings as well as seeds, but propagation by cutting reduces the growth of the mother plant and also affects the yield and sometimes can 21

cause death of the mother plant (Islam et al., 2005). Plants obtained by seed propagation vary in genotypes leading to variations in phenotypes and nutritional values (Riyathong et al., 2010). The tree also has low seed viability and low seed germination rates, which in turn results in slow population growth rates (Stephenson and Fahey, 2004). The need for large areas of agricultrural land for mass cultivation of M. oleifera, to meet the increasing demand of industries for production of finished goods is somewhat impractical in a society/place where scarcity of cultivable land is a major concern due to increasing population and industrialization. M. oleifera is highly susceptible to infestations with flies, aphids, thrips and other insects, leading to foliar damage which in turn results in decline in total biomass and thereby making it unfit for consumption/use as food and medicine. Additionally, the frost sensitive nature of the tree makes it almost impossible to grow it in open lands of temperate zones (Förster et al., 2013). Being the sole cultivable species of family Moringaceae, the tree has to meet the growing demands of local population for its use as food, medicine and water clarifiers which may lead to gradual decline in its biodiversity due to reducing natural tree resources (Steinitz et al., 2009; Stephenson and Fahey, 2004). Apart from these issues, one of the major drawbacks that usually holds back the commercialization of such medicinal plant species is fluctuations in metabolic constituents due to inconsistent environmental conditions, harvesting conditions and other variations which in turn causes difficulties in standardization of commercial formulations. Moreover, the geographical distribution of the tree also results in variations in morphological, metabolic and genetic characteristics as a consequence of different climatic conditions prevailing in different zones (Bhatia et al., 2013; Chatterjee et al., 2010).

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5.2 Biotechnological strategies: potential solution Biotechnology is the only field of science that not only aids in gaining insights into the actual mechanisms of living bodies but also provides numerous techniques which can be employed for their further improvisation. Plant biotechnology is one such specialized field of biotechnology in which biotechnological tools and techniques enable one to explore plant systems and to understand mechanisms associated with different activities followed by their improvisation such as enhanced growth and yield, rapid mass multiplication, increased metabolite production and secretion. Plant tissue culture, metabolite profiling, genetic and pathway engineering are some of the common tools of plant biotechnology which are being used extensively to achieve different objectives of crop improvement in terms of yield, disease resistance and enhanced nutritional value. Plant tissue culture via in vitro propagation helps in the conservation of endangered, medicinally and economically important plant species. It also provides scope for improvisation by generation of disease/virus free plants, maintaining supply of seasonal crops throughout the year and rapid bulk propagation. Plants possess a variety of phytochemicals which exhibit numerous medicinal and non-medicinal properties, but lack of knowledge about the phytocomposition and pathways involved makes it difficult to exploit their properties at commercial scale. Metabolite profiling aids in key enzymes involved in biosynthesis of commercially important phytochemicals. Genetic engineering leads to generation of new varieties possessing the desired characteristics and are commonly referred to as genetically modified (GM) plants. In addition, the analysis of morphological, biochemical and genetic variability using suitable molecular markers not only helps in the selection of superior varieties of interest for future

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breeding programs and mass multiplication for commercial purposes, but also provides insights into the effects of different biotic and abiotic factors on the morphological, phytochemical and genetic characteristics (Goyal et al., 2015). Such analysis facilitates the selection and identification of superior varieties of plants with particular desired characteristics, which can be further subjected to mass multiplication and breeding programs.

5.2.1

Micropropagation

In recent years, in vitro micropropagation based techniques have proved a fruitful approach for mass multiplication and germplasm conservation of endangered as well as economically and medicinally important plants (Goyal et al., 2015; Jain et al., 2009; Steinitz et al., 2009; Stephenson and Fahey, 2004). Plant tissue culture technologies serve as an excellent alternative to conventional propagation methods, resulting in year round availability of the tree which involves rapid clonal mass multiplication in a stipulated time and space. This approach enables meeting the huge industrial demands for drumstick tree as raw materials in variety of drug and dietary formulations. Moreover, in vitro regeneration and multiplication techniques are not only being used as a onestop solution to all the major concerns of issues such as overexploitation, enhancement of nutritional value and disease resistance but can also be used for commercial scale production of plant products (as reported in a study by Jain et al. (2011), where they have optimized in vitro propagation protocol for biosynthesis of steroidal lactones in an endangered medicinal herb Withania coagulans).

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Therefore, current biotechnology research is focused on optimization of in vitro propagation methods using different parts of M. oleifera in order to meet the commercial demand and overcome the formerly discussed issues. In vitro propagation of M. oleifera was reported by Stephenson and Fahey (2004), in which they used meristematic stem nodes and immature seeds as explants and reported that the explants showed best response in full and half strength Murashige and Skoog (MS) medium. Multiplication of shoots was dramatically increased when the nodal segments were cultured on membrane raft. The use of isothiazolones for improved survival rate of the rooted plantlets was also reported in the study. In a similar report by Förster et al. (2013), nodes taken from the sterile seedlings of two different ecotypes were cultured on MS medium supplemented with different concentrations of 6Benzylaminopurine (BAP). Results revealed that MS medium without BAP and with 0.5mg/L BAP showed best response in terms of shoot length (3.81 - 4.10 cm) and number of offshoots (4.34 – 4.40), respectively. In experiments conducted using nodal segments (with axillary buds) as an explant, Islam et al. (2005) reported that at BAP (1mg/L and 1.5mg/L) all the explants produced shoots and maximum number of shoots (4±0.29) were obtained with 1mg/L BAP. On the other hand, Mathur and Kamal (2012) reported the best response on MS medium supplemented with BAP:1-Naphthalene acetic acid (NAA) at 3:3mg/L. Further, Islam et al. (2005) found that repeated subcultures on same BAP concentrations resulted in increased number of shoots (25) and concluded that BAP is the most effective plant growth regulator (PGR) for shoot induction and multiplication. They also recorded 100% rooting in MS medium without any PGRs, but maximum number of roots (2.2 - 3.7 and 2.8) was reported on MS

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medium containing Indole-3-acetic acid (IAA) at 0.05, 1 & 2mg/L and Indole-3-butyric acid (IBA) at 2mg/L, respectively (Islam et al., 2005). Studies based on establishment of an in vitro propagation method using seeds are also reported. In a study carried out by Marfori (2010), nodes from seedlings in a sterile environment were subjected to MS medium supplemented with varying concentration regimes of different growth hormones and the highest number of axillary shoots per explants (4.6) was obtained in medium containing 2.5µM BAP, 5µM Kinetin (Kn) and 2.5µM Thidiazuron (TDZ). Other cytokinins, i.e. Kn (10µM) and TDZ (5-10µM) showed inhibitory effect. Saini et al. (2012) also reported that nodal segments obtained from aseptically grown seedlings, when inoculated on MS medium containing 4.44µM BAP showed best response (9±1.0) and highest number of roots (8±0.8) were observed on IAA (2.85µM) and IBA (4.29µM). Devendra et al. (2012) carried out tissue culture study using zygotic embryos as explants. Zygotic embryos grown on MS medium containing 10.75µM NAA resulted in white organogenic callus, which was further subjected to induction of somatic embryos using MS media supplemented with 4.52µM 2,4-Dichlorophenoxyacetic acid (2,4 D) and 11.09µM BAP. They concluded that MS medium with 13.31µM BAP showed highest somatic embryo induction frequencies with germination efficiency of 77.2%. Further, maximum rooting efficiency (78.4%) was reported in MS medium supplemented with 10.75µM NAA. Similar studies of callus cultures were also carried out using leaves and cotyledons as explants. Highest callus growth was obtained using leaf explants in MS medium supplemented with 0.1mg/l 2,4-D (Khalafalla et al., 2011). Nieves and Aspuria (2011) established callus culture for cotyledons and observed increased callus formation on MS medium containing 2.5mg/L BAP.

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Also, enrichment of media with TDZ results in compact, nodular and loose calli (Nieves and Aspuria, 2011). Riyathong et al. (2010) used stem explants obtained from in vitro raised seedlings and 100% shoot formation was obtained on MS medium supplemented with BAP (1 - 4 mg/L), and the highest number of shoots per explant (10.2) was achieved with BAP (2mg/L). In a recent report by Avila-Treviño et al. (2017), successful micropropagation protocol was established using buds and cotyledonary nodes of the tree without using any plant growth regulators and they further illustrated the genetic similarity among the regenerated plantlets using RAMP markers. The tissue culture studies are summarized in Table1. Thus, it can be concluded that the in vitro propagation studies carried out so far provide a platform for further/future interventions in order to obtain superior varieties with enhanced medicinal and nutritional value. In one such attempt, Mathur and Kamal (2012) studied the biosynthesis of “trigonelline – a metabolically active pyridine alkaloid” in different components of M. oleifera including the callus cultures and reported that the trigonelline production in in vitro grown cultures can be significantly enhanced using nicotinic acid as precursor in the medium. Additionally, use of plant cell suspension cultures and hairy root cultures for large scale production of commercially important secondary metabolites have been suggested by Rao and Ravishankar (2002), whereas Förster et al. (2013) concluded that in vitro cultures are successfully being used in medicine and food industries as a source for important metabolites. The importance of comprehensive metabolite profiling and engineering was highlighted by Zhou and Wu (2006). They illustrated the role of plant tissue culture techniques along with plant

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genomics, transcriptomics, proteomics and metabolomics for production of important plant pharmaceuticals and neutraceuticals on a commercial scale. Therefore, it is inferred that comprehensive knowledge of the metabolites present and their biosynthesis pathway is the key behind converting plant cells and tissues into large bioreactors which are often referred to as “plant cell factories” for commercial production of phytomolecules/phytochemicals.

6. Conclusion and Future Prospects In recent years, great emphasis has been laid on the utilization of natural resources as a source of food, medicine and waste management due to the harmful effects posed by the synthetic resources, in addition to their non-renewable nature. Moringa oleifera Lam. is a tree with multidimensional utilities which serves as an excellent reservoir for pharmaceuticals and neutraceuticals. The rich phytochemical profile and advances in biotechnological techniques has led to generation of new avenues aimed towards enhancement of overall commercial value of the tree. In vitro propagation techniques have emerged as a life-line for ex situ conservation of endangered and elite germplasm and also lay the foundation for the concept of “plant cell factories” i.e. the natural bioreactors for production of plant products. This concept provides new insights into development of more effective, eco-friendly and biodegradable products using mass multiplication and production techniques. Establishment of plant cell factories demands thorough knowledge of the metabolomics of the plant as it facilitates in identification and alteration/modulation of the key components for enhanced production of targeted plant product(s).

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Although efficient in vitro propagation protocols for M. oleifera have been established, comprehensive studies involving the use of metabolite profiling and engineering for enhanced metabolite production in in vitro cultures are still lacking. Knowledge of biosynthetic pathways of the desired compounds in plants and in culture systems will facilitate the modulation and fabrication of such compounds in in vitro cultures. Therefore, a comprehensive metabolite profiling followed by biosynthetic pathway identification using high throughput techniques such as GC-MS and NMR is essential for identification and characterization of novel neutraceuticals and pharmaceuticals. The use of biotechnological approaches thereby not only facilitates selection and conservation of superior varieties but is also a stepping stone to commercialization of important/targeted plant products. There is no doubt that the shortcomings in M. oleifera commercialization can be overcome by the use of biotechnological tools, and it is likely that the future research in this context will make M. oleifera an important solution for the various health and environmental issues of the present era/generation.

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Figure Legends: Fig. 1 Moringa oleifera Lam. a. Tree growing in natural habitat b. Tree in flowering stage c. Tree in fruiting stage d. Gum secretion from the cut marks on the stem

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Table 1: Table 1: In vitro propagation studies in M. oleifera Explant Used

PGRs Used

Meristematic Nodes,

BAP, Ancymidol,

Immature Seeds

NAA, Kinetin

Nodes

BAP, IBA, IAA

Method of Propagation Reference(s) Stephenson and Direct Shoot Induction Fahey (2004) Direct Shoot Induction

Islam et al. (2005)

Direct Shoot Induction

Marfori (2010)

BAP, Kinetin, Seeds TDZ, NAA Khalafalla et al. Leaves

2,4-D

Callus (2011) Nieves and

Cotyledons

BAP, 2,4-D, TDZ

Callus Aspuria (2011)

Leaves, Zygotic

2,4-D, NAA,

embryos

Kinetin, BAP

Nodes

IBA, BAP

Devendra et al. Somatic embryogenesis (2012) Mathur and Direct Shoot Induction Kamal (2012)

Triacontanol Nodes

(TRIA), BAP, IAA,

Direct Shoot Induction

Saini et al. (2012)

IBA Förster et al. Nodes

BAP, IAA

Direct Shoot Induction (2013)

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Indirect Avila-Treviño et Nodes, Leaf

No PGR(s)

regeneration/callus al. (2017) induction

55