INTRODUCTION AND REVIEW OF LITERATURE

1.1 Background

The kingdom fungi (singular: fungus) are a distinct, humongous and diverse group of eukaryotic organisms distributed worldwide favoring varied habitats. This includes yeast, rusts, smuts, molds, mildews and mushrooms (Hawksworth. 2001; Kirk et al., 2008). These are placed distinctly apart from the animal and plant kingdom because of unique biochemical, morphological and molecular characteristics (Burns. 2006). Globally, about 1.5 to 5.1 million species of fungi has been reported, among which only 5% have been classified. According to early taxonomic studies in the 18th and 19th century, fungi were classified broadly on the basis of morphological and physiological characters (Zhou et al., 2010; Petersen and Knudsen, 2015). Recently, molecular genetics have paved new avenues for modern classification of fungi and surpassed the conventional classification based on phenotypic characters or used together to resolve the taxonomic issues. Phylogenetic analysis has placed these organisms in a distinct kingdom known as fungi (Hawksworth 2006; Ruggiero et al., 2015). The kingdom is further classified in single subkingdom which is comprised of seven phyla (Hawksworth 2006; Ruggiero et al., 2015). Among all the groups, Ascomycetes (Morchellaceae) was focused in the current project due to its nutritional, myco-chemical, nutraceutical, socio economical and complexity of their molecular characteristic.

1.2 Ascomycetes

Ascomycetes constitute a major taxonomic group within the true fungi (Eumycota) where the majority of the Morchella species collected from the northern and lower plain irrigated areas viz., Morchella crassipes, Morchella elata, M. pulchella, M. eohespera, M. galilaea and M. eximia. Ascomycetes constitute largest group of sac fungi having 64000 recognized species that were reported from various eco-geographical and ecosystem origin (Kirk et al., 2001). They were divided into various orders and classes. According to classification of Schoch et al. (2009), these classes were: Sordariomycetes, Laboulbeniomycetes, Dothidiomycetes, Leottiiomycetes, lecanoromycetes, eurotiomycetes, Orbiliomycetes, Schizosaccharomycetes, Arthoniomycetes, Pezizomycetes, Saccharomycetes, noelectomycetes and Pneumocystidiomycets. The basic classification of a subclass of ascomycetes that’s divided into three order Gymnomycota, Oomycota and Eamycota. The Oomycota further divide in to two c

lasses, Phycomycetous and Zygomycetes. Similarly, Emycota divided into two classes, Deuteromycetes (ascomycetes) and Mycophycophata (Hawksworth. 2006).

The basic classification of a subclass of ascomycetes that’s euascomycetes has been much censured due to its complex nature. As in the case of classification systems, a single morphological character was used to separate the euascomycetes (Lumbsch et al., 2005). Numerous of these characters, however, have been found less helpful in phylogeny than originally perceived (Ariyawansa et al., 2015). Moreover, besides these, molecular and phylogenetic research based on 18SrDNA sequences phase shown the three major groups in

Ascomycota: a). Euascomycetes, a class of filamentous fungi (FM) which are characterized by the formation of asci in a fruiting body (ascoma); b). Hemiascomycetes (HM), encompassing best of the yeasts characterized by the lack of mutually ascogenous hyphae (AH) and fruiting body formation; c). Archiascomycetes, with Taphrina, fission yeast Pneumocystis and related taxa, which are vastly capricious in biochemical and morphological characters (Berbee and Taylor, 1992; Bruns et al., 1992; Tehler et al., 2000).

1.3 Distribution, Occurrence and Socio-economic aspects

Ascomycetes species thrive in various habitats and ecosystems with high distribution in aquatic, moist and terrestrial land. Although, these fungi are typically saprobes but also live as parasitic, mutualistic and pathogenic mode of life. Moreover, about 40 % of ascomycota are found in association with algae in lichens contributing around 8 % of our land Earth. These fungi are found enormously in industrial, natural and agricultural environments and have been extracted from harsh climate habitats such as from deep-sea wood (Kohlmeyer. 1977), earth

24 | P a g e sediments and from depths of many rocks in the Antarctica continent (Raghukumar et al., 2004; Selbmann et al., 2005). Globally, wild Morchella species (spp) are greatly valued for being edible mushrooms (Ascomycetes). Morchella spp. are known with different indigenous names for instance Morchel (Germany), Morielje (Netherlands), Morilla (Mexico and Spain), Morille (France), Murkla (Sweden), Olote (Mexico), Pallohuhtasieni (Finland), Pique (Chile), Pumpalka, (Bulgaria), Rundmorkel (Norway), Smardz (Poland), Smrž obecný (Cheez), Spongioli (ancient Grace), Yangdujun (China), Zbírciog (Romania) and Guchi (urdu/Pakistani) (Rolfe and Rolfe 1925; Korhonen. 1986; Chandra. 1989; Kreisel. 2005; Hamayun et al., 2006). Morchella spp. are widely distributed in different countries for instance USA, Pakistan, India, China, Israel and Turkey (Goldway et al., 2000; Pilz et al., 2007; Barseghyan and Wasser, 2008: Masaphy et al., 2009; Michael et al., 2016; Taskin et al., 2015). Various species of Morchella have shown their varied natural habitats viz., Coniferous, tropics, subtropics, temperate, temperate rain and alpine forest. These species are also found in Rocky Mountains, orchid, hardwood, sugar cane and grass fields (Table 1.1) (Korhonen 1986; Guzman. 1998; Hamayun et al., 2003; Kellner et al., 2004, Liu, 2005; Negi. 2006; Pilz et al., 2007; Kuo. 2008; Kuo et al., 2013; Richard et al., 2015; Badshah et al., 2015; Badshah et al., 2018). M. elata, M. conica, M. crassipes M. esculenta has a great socio economic importance globally (Pilz et al., 2007; Matocec et al., 2016). The huge trader countries i.e., Republic of China, United States of America, Israel, sub-continent, and Turkey (Mediterranean and Aegean) export Morchella to various parts of Europe (Pilz et al., 2007). Morchella (morels) are a key source of income for people living near forests and in villages. They are collected for beneficial subsistence, recreational and commercial harvests purposes (Anderson et al., 2002; Roman and Boa, 2004; Farlane et al., 2005). Morchella is among the world’s most valuable and highly prized edible fungi due to their impressively gratifying flavor and sporadic fruiting season (Lakhanpal et al., 2010; Taşkın et al., 2011; Enshasy et al., 2013; Badshah et al., 2015). Commercially export of Morchella species is common in US (Ower et al., 1986), the rising demand has integrated cultivation in a particular indoor facility using advanced technology. Although, initial efforts have been made at growing wild edible Morchella in the different conduction in Yunnan Province (Zhao et al., 2009). In Pakistan, the Morchella species collection comprises over 5000 families in the region (Saqib et al., 2011). A sum of 14,000-28000kg Morchella species from the Swat (Pakistan)

25 | P a g e was founded to export annually to other area of Pakistan as well as in international markets. It raised significant revenue (US 643000$/year) in Swat valley. These collectors use to obtain the lowest money in trade chain of mushrooms due to incremental addition value by traders and sometimes by discrimination of the middle suppliers and merchants (Sher and Shah, 2014).

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Table.1.1. Global distribution of Morchella species in natural habitat STATES No.# of Common species Habitat Citation species Alaska 1 Morchella tomentosa Temperate rainforest Kuo. 2008 Chez 1 M. semilibera Hardwood, Forest Pilz et al., slope 2007 China 1 M. bicostata Deciduous tress Liu. 2005 Finland 2 M. esculenta. M. elata Forest, orchid, Korhonen. 1986 Germany 3 M.esculenta, M. Rocky mountain range Kellner et crassipes. M.spongiola al., 2004 India 5 M. semilibera, M. Subtropical forest, Negi. 2006 crassipes, M. conica, Himalayans M. esculenta, M. elata Israel 4 M.esculenta, M. Tropic subtropics vulgaris M. conica, M. Barseghyan elata, and Wasser, 2008. Mexico 2 M. rufobrunnea, Subtropical forest Guzman. M. guatemalensis 1998 Newfoundland 2 M. eohespera, Mountains National Voitk et al., M. laurentiana Park 2016 North 3 M. americana, M. Coniferous forests Kuo et al., America prava , M. importuna boreal, cold temperate 2013; Richard et al., 2015. Norway 2 M. elata Norway spruce forest Pilz et al., 2007

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Pakistan 3 M. esculenta, Alpine forest, Hamayun et M. crassipes, sugarcane and grasses al.,2003; M. pulchella field Badshah et al., 2015; Badshah et al., 2018. Sweden 3 Morchella species Burned soil Pilz et al., 2007 Turkey 5- 49 M. mediterraneensis, Pine forest Taşkın et al., M.fakeness,M. 2015. conifericola M. magnispora M. galilaea USA 3 M. prava, M. temperate or boreal Richard et americana, and M. forests, al., 2015 importuna 1.4 Morchella: Taxonomy and Systematics

Morchella belongs to ascomycetes. Members of this genus have 8-ascospores enclosed in a special sac-like structure called ascus. There are as many as 60 Morchella species/taxa reported globally (Loizides et al., 2016). Additionally, due to a complex taxonomy and lack of sequence data at NCBI (molecular data), the identification of Morchella has been conflicting (Richard et al., 2015). The molecular analyses none the less have suggested that Morchella has its place to family Morchellaceae and order Pezizales, as a separate genus. However, the number of species within the genus remains unclear. The European mycologists have identified three species viz. M. elata, M. esculenta and M. conica (Cai et al., 2006). The systematic and taxonomic studies have shown a series of multi-loci phylogenetic lineages, consisting of three separate lineages namely: ancestral M. rufobrunnea (M. anatolica), late diverging M. elata (Distantes sensu) (Clowez 2012) and Morchella esculenta (Morchella sensu Clowez 2012). These clades were dated back to beginning of cretaceous period about 125 MY (Taskin et al., 2010; O’Donnell et al., 2011; Du et al., 2012) as summarized below (Table 1.2).

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The above-mentioned clades contained a minimum of 27 and 36 phylogenetically separate species correspondingly (Voitk et al., 2014). Moreover, fungal taxonomic names (binomial system) have been confined only to four species and the species identified using phylo-species within these two clades were designated by typical abbreviated form of genus name and species followed by Arabic numbering such as Mel (Elata Clade) codes, Mes (Esculenta Clade). The taxonomy of Asian and North American Morchella species are based on epithets (Arora. 2008; Imazeki et al., 1988). Phylogeny is not in agreement with the names applied to Morchella species which suggested that many Morchella species demonstrates a high continental endemism and parochialism in the northern hemisphere which is steady with their phylogenetic derivation in Laurasia (O’Donnell et al., 2011). Generally, mycologist rely on phenotypic characteristics, however due to the polymorphic nature of Morchella species, the reported number of species could not be determined with confidence and ranged between 3 to 50 (Bunyard et al., 1994). The Morchella hunters, on the other side, recognized morels in three groups: the yellow morels, black morels the half-free morel. Although various scientific names are used, including Morchella conica, M. rotunda, M. angusticeps, M. vulgaris, M. elata, M. esculenta, M. deliciosa, and M. crassipes (Kellner et al., 2005;), the use of emerging and advance molecular phylogenetic skills and comprehensive taxonomic approaches have made convenient and feasible towards the identification of Morchella species resolving several of the old arguments but also led to new ones (Pilez et al., 2007).

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Table. 1.2 Morchella Species and their major clade

Clade Macro-Morphological characters Citation Elata Ridges (R) at stage of maturity black / dark brown O’Donnell et al., 2011; (DB), Partially longitudinally parallel or, arranged Taskin et al., 2010; sinus is present, ascocarps may be conical as well as Du et al., 2012 cylindrical. Almost never rufescent, usually facultatively(F),endophytic(E),and obligate pyrophilic (OP). Esculenta Ochraceous or buff ridges, maturity not dark, O’Donnell et al., 2011; sporadically arranged, ascocarps usually ovoid, sinus Taskin et al.,2012; (S) present / absent sometimes rufescent. Probably Du et al.,2012 endophytic.

Rufobrunnea Ridges buff or silvery white, at minimum O’Donnell et al., 2011; incompletely longitudinally arranged, ascocarps (Fb) Taskin et al., 2010; usually acutely conical, often rufescen, sinus absent Du et al., 2012 (SB), Stalk through dark pruinescence (DP). Might be endophytic or facultatively saprotrophic.

1.5 Molecular phylogenetic help to resolve the issue of Morchella species The taxonomical, binomial, arabic names of Morchella species were not reliable for mycologist, due to lack of morphological characters. The current rapid progresses in phylogenetic analysis and DNA sequencing techniques have permitted mycologists to overcome problems in chemosystematics, mycotaxonomy and interpret the evolution of fungi (Koufopanou et al., 1997; Geiser et al., 1998; Yang. 2011). The concept of GCPSR (Cracraft. 1983; Taylor et al., 2000), presented a consensus scheme for the identification of Morchella species (Revankar and Sutton 2010; Zeng et al., 2013; Elliott et al., 2014; Pildain et al., 2014). With the aid of GCPSR method based on the LSU-EFL-A-RPBL-RPB2 combined-gene dataset of genus Morchella, three (3) new species in the Elata Clade were recognized viz., Mel-36 from

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Canada, Mel-37 from Argentina; and Mel-35 from Australia (Pildain et al., 2014). However, about 71 distinctive species have been documented in Morchellaceae on phylogenetic basis.

1.6 The phylogenetic and morphological taxonomy of Morchella

Morchella species classification shifted to a polythetic scheme, where higher characters are carefully assessed and compared but none of them important for classification. Therefore, taxonomic ranking is commonly done on a ‘best match’ criteria, rather than on constantly dichotomous variable characters (Loizides et al., 2015). The new recent information on the Index Fungorum three hundred and fifteen (315) names (http://www.indexfungorum.org /names/names.asp), in Morchella has been described at species (S), sub-species and variety level. Majority of the species were described from Europe and merely reported from Asia. Further, inadequate microscopic and macroscopic specific distinctions and high levels of variations in color and fruiting bodies at various stages (Du et al., 2014) affected by climate and ecological parameters. So, the Morchella species varies from three to 50 leading to puzzling produced by due to of synonyms and homonyms (Bresinsky et al., 1972; Gessner et al., 1987; Volk and Leonard, 1989; Jung et al., 1993; Bunyard et al., 1994, 1995; Kanwal et al., 2011; Kuo et al., 2012; Clowez 2014; Richard et al., 2014). The morphological variation in various species of Morchella were further examined and confirmed with the help of molecular techniques, therefore numerous phylogenetic species were recognized including Mes-1, Mel-1, Mel-4, Mel-3, Mel-36, Mel-15, M. rufobrunnea, M. steppicola Zerova, M. tomentosa M. Kuo, M. anatolica, (Stefani et al., 2010), M. semilibera DC. M. punctipes Peck M. angusticeps Peck (Peck. 1903), M. Australiana, M. anatolica (Işiloğlu et al., 2010), and M. rufobrunnea (Guzman and Tapia 1998; O’Donnell et al., 2011; Kuo et al., 2012; Richard et al., 2014; Elliott et al., 2014). The numerous specimens studied as result twenty-one new species have been classified (Kuo et al., 2012; Clowez. 2012; Richard et al., 2015). Therefore, the application of emerging studies such as multilocus sequences may make it easy to identify Morchella species.

1.7 Health allied effect of Morchella species Mushrooms are cogitated as a better source of drugs in modern medicine. Mushrooms are much valued due to their vast role in remedial of many diseases for instance cancer and diabetes. This is largely due to their biochemical composition and nutritional value. In recent

31 | P a g e years their pharmaceutical and cosmeceutical value have expanded vigorously (Samorini. 2001; Alam et al., 2009; Bodha et al., 2010; Chunwei et al., 2013; Ma et al., 2015). Different Morchella species used in traditional drug were reported for quick healing and as a disinfectant, for digestive system, an immune-stimulant (Prasad et al., 2002; Mahmood et al., 2011; Lone et al., 2012; Mala et al., 2012), and as a general tonic for cough and cold (Nautical et al., 2001; Rokaya et al., 2010; Sher et al., 2014). In traditional Chines Medicine (TCM) mushrooms were used for dyspepsia, excessive chest mucus and breathing troubles (Jianzhe and Xiaolan. 1987; Duncan et al., 2002). Nutraceutical and clinical trials were previously reported by Mau et al. (2004) and Tsai et al. (2006). According to Nitha and Janardhanan, (2008), the ethanolic (EtOH) extract of M. esculenta mycelia against gentamicin and cisplatin the nephron-protective effect of induced nephrotoxicity in mice. M. esculenta against Carbon tetrachloride (CCl4), induced chronic hepatotoxicity toxic in liver disease (Nitha et al., 2013). The anti-inflammatory properties of Morchella species were studied highlighting the significance of dose reliant curing of both critical and chronic inflammation (Nitha et al., 2007; Kim et al., 2011). According (Stojkovic et al., 2013) Morchella esculenta revealed that crude methanolic (MeOH) extractions exhibited anti-mutagenic capability (AM) against Salmonella typhimurium. The mushroom (Basidiomycetes) were also historical background in the folk remedy (Wasser and Weis, 1999; Lindquist et al., 2005; Sullivan et al., 2006). Therefore, numerous edible morels species are now commercially grown and about 200 wild species are being utilized in medicines (Aida et al., 2009). A.bisporus (AB), Lentinus edodes (LE) and Pleurotus species contain bioactive compounds (Greve et al., 2010; Willared et al., 2013; Stajic et al., 2013; Beulh et al., 2013; Beekman and Barrow, 2014; Evidente et al., 2014; Huang and Nie, 2015; Zhang et al., 2016). Recent investigations have focused on introducing novel bioactive compounds that can trigger biological responses of immune cells as well as antitumor, antidiabetic, and hepatoprotective capacity positively (Lindequist et al., 2005; Kalogeropoulos et al., 2013). The pervious literature articulated that Morchella esculenta (yellow), have many bioactive compounds, mineral contents, fat, protein, carbohydrates, ash, and amino-acid and fibers (Litchfield et al., 2006; Dursun et al., 2006). The Agarics cordyceps extract was currently tested in asthmatic babies in remission stage Aman et al., (2000). Amauroderma camerarium has been

32 | P a g e used against the Trichomonasvaginalis (TV), Lentinus edodes, Grifola frondosa, Schizophyllum commune, and Ganoderma lucidum Cooi and Liu, (2000) are essential natural forest resources of immunomodulatory agents, Zhang et al., (2005). C. sinensis supplement could affect spermatogenesis by cordycepin (3’deoxyadenosine) as elevated serum cordycepin levels increased the improvement of spermogenesis and more testosterone levels (Yang et al., 2006; Duarte et al., 2007; Sun et al., 2010).

1.8 Antioxidant potential of Morchella Species The high antioxidant activity potential was reported for M. esculanta, M. vulgaris, M. elata, M. conica, M. crassipes and M. rotanda (Mahfouz et al., 2006; Badshah et al., 2015).

The methylene chlorides (CH2Cl2), aqueous extract of M. esculenta in ethanol were reported to have antioxidant potential, with various assays (NF-Kb activation, and prevention of severe and chronic redness in vive) (Nitha et al., 2007; Kim et al., 2011). M. esculenta, M. anguisticeps, and M. conica were used in various assay of Scavenging effect, DPPH, inhibition of superoxide generation, lipid peroxidation and inhibition Chelating effects on ferrous ions (Mau et al., 2004; Puttaraju et al., 2006; Turkoglu et al., 2006; Elmastas et al., 2006; Ozturk et al., 2010; Nitha et al., 2010; Heleno et al., 2013). The isolated polysaccharides from the fruiting bodies of M. esculenta have shown antioxidant properties (Rotaoll et al., 2005; Elmastas et al., 2006; Nitha and Janardhanan, 2008). These are containing a huge amount as active constituents with great antioxidant potential, attributed commonly to polyphenolic compounds and shield against the superior possessions of dangerous free radicals (FR) in our body (Lin and Mau, 2002; Lin and Chen, 2002; Barja. 2004). Therefore, stopping oxidative damage (OD) diseases such as cardiovascular, cancer disease, neurological disorders, diabetes, arthritis leukemia and other degenerative ailments linked with special aging, (Shah and Channon, 2004; Cheung. 2005; Barros et al., 2007; Kim et al., 2008; Smolskait et al., 2015). Several researchers noted that antioxidant content and bio-chemicals of wild mushrooms in many parts of the world (Barros et al., 2007; Elmastas et al., 2007; Kim et al., 2008; Chen et al., 2010; Puttaraju et al., 2016). The methanolic extracts of Ganoderma sinensis were studied for their antioxidant activities. It was observed that extract exhibited elevated antioxidant activities 70 – 99 percent 1 at 20 mg/ml and stumpy IC50 values of extract 0.95–10.00 mg /ml (Wachtel et al., 2010; Hu et al., 2010). Phellinus baumii, Pleurotus abalones, Agaricus bisporus possess stout antioxidant enzymatic activities (Siu and Chen, 2014). A recent study from Nepal revealed the antioxidant

33 | P a g e potential in the polyphenolic constituents of mushrooms (Tamarakar et al., 2016). 1,1-diphenyl, 2-picrylhydrazyl (DPPH assay); ABTS 2, 2-azino-bis 3- ethylbenzothiazoline-6-sulphonic acid;

H2O2 and O2), RP (FRAP ferric reducing antioxidant power) ,(HDP, Heme degradation of peroxides and FOX ferrous oxidation-xylenol) and lipid peroxidation inhibitors (TBA) reactive substances (RS), assays to determine the great antioxidant potential (Table 1.3) (Mau et al., 2001; Fui et al., 2002; Yang et al., 2002; Cheung et al., 2003; Turkoglu et al., 2006; Sarikurkcu et al., 2008 ; Carocho and Ferreira. 2013). Moreover, no studied was reported the antioxidant activities of isolated compound, lipids, polyphenols, and crude polysaccharides from Morchella species in Pakistan. Although ROS (reactive oxygen species) are over formed in one site, enzymatic and non-enzymatic antioxidants maintain the balance in another site. When the system is unbalance, the cumulative damage of amino acid, lipids, proteins, DNA and membrane damage caused by excess Reactive oxygen species (ROS) produced in the physique leads to the occurrence of what is called oxidative stress, and this is believed to cause ageing and many health diseases (Droge. 2002; Ou et al., 2003; Hanson. 2005). Therefore, mushrooms protect our body from free oxidative radicals and oxidative stress.

1.9 Antitumor properties of Morchella against Breast cancer Malignancy is the primary origin of death worldwide, and human breast cancer is the utmost one. According Jemal et al. (2003) the occurrence and mortality rates of breast cancer in female still rank high in global epidemiologic research over the earlier few years. Improvements in cytotoxic and hormonal treatments have not led to sustained cure in breast cancer. Actually in 1970, the estrogen (ER)-positive MCF-7 cell line was plagiaristic from a patient with metastatic breast cancer (BC) (Levenson and Jordan, 1997). Therefore, the MCF- 7 cell has become an ideal model system for the research of breast cancer as it narrates to the vulnerability of the cells to apoptosis. In spite of the fact that numerous tumors initially treatment by chemotherapy, breast cancer cells can subsequently subsist and gain resistance to the cure (Campbell et al., 2001; Rao, et al., 2005). M. esculenta has been proven to have antitumor activities and anti-inflammatory (Nitha et al.2007; Nitha et al., 2013), which were attributed to the possession of polysaccharides (Yang et al., 2014). Further, it has become gradually significant in the treatment of a number of main solid tumors, predominantly metastatic and drug-resistant BC (breast cancers) (Spencer et al., 1994). The death ratio due to breast cancer in the U.S is about one in fourth, which is the most common malignancy in women

34 | P a g e globally, which made a total number of 18% of female cancer. Although, globally 600,000 annual deaths reported due to breast cancer (Kumar et al., 2011; Siegel et al., 2013). Moreover, current emerging research, the discovering of natural compounds is the demand of the new era, because of precise function of natural compounds and high effectiveness (Kohen et al., 2005: Vollmar et al., 2013). Commonly, Ganoderma applanatum is preferred for treatment of human breast cancer, especially terpenes derived resin (Lukmanual et al., 2015). The Song Gen (Phellinus linteus) extract can prepare innovative prostate cancer cells (PC) to apoptosis in athymic bare rates (Tsuji et al., 2010). Another common anticancer mushroom Hericium erinaceus (lions Mane) produced aromatics compound (isohericenone J and hericerin A) laterally with various recognized active compounds, 4- (3′,7′-dimethyl-2′,6′- octadienyl)-2-formyl-3-hydroxy-5-methoxybenzylalcohol) and (isoericerin, hericerin, N- dephenylethyl isohericerin, hericenone), D-arabinol ester , diterpene , resorcinols, hericenols and erinacerins, from a extract of the H. erinaceous whole body, (Miyazawa et al., 2012; Kobayashi, et al., 2014 ; Zhang et al., 2015; Le et al., 2015).

Although most affective anticancer compound isolated from A. mellea that armillarikin, armillaridin, melleolides, and arnamial (Bohnert et al., 2011; Chen et al., 2014; Chen., 2013; Chang et al., 2015).

Furthermore, a numerous bioactive component, such as polyphenols, polysaccharides and triterpenoids, have been isolated from wild mushrooms. Many analgesic active compounds, in medicinal mushrooms, have antitumor activities (Wasser and Weis, 1999; Ikekawa, 2000; Feng et al., 2001). Although at minimum 30 medicinal mushrooms has produced bioactive compounds with distinct anticancer potential in xerographs, only a few amount has used in the ensuing step, viz. aims clinical valuation for anticancer effect in human patient. Furthermore, antiestrogens have provided the strongest endocrine rehabilitation for breast cancers in the first phase, therapeutic choices are limited to estrogen receptor (ER) negative tumors, which are often more antagonistic. In fact, approximately two-thirds of estrogen receptor ER-positive breast cancers react to estrogen ablation (Elstne et al., 2002). Pleurotus eryngii is another medicinal mushroom to have anticancer potential against a wide range of cancers, including MCF-7 breast cell line, Various kinds of biological compounds have been isolated from this medicinal mushroom e.g., 2, 3, 6, 23-tetrahydroxy-urs-12-en-28-oic acid which possess

35 | P a g e promising MCF-7 (Breast cancer) effect in, in vitro study (Xu et al., 2015). Therefore, they wild mushroom Pleurotus tuberregium has possessed anti-breast bioactive compounds in its fruiting bodies e.g. carboxymethylated P-glucan (CMPTR). CMPTR possess promising anti-breast cancer potential in breast cancer cell line (MCF- 7), possessing anti-proliferative activity, arrest cell cycle at G1 phase, inducing apoptosis, down-regulate the mien of Bcl-2 protein, up-regulate the expression of Bcl-2 or Bax proteins (Zhang, et al., 2006). Moreover, everyday foods, such as coffee cruciferous, fish, vegetables tea and soy products, have been specified to be correlated with the risk of human breast cancer by emerging studies (Sun. 2006; Wuah. 2008; Tang et al., 2009; Zheng et al., 2013). Therefor edible mushroom, as a common food supplied in daily diet globally, contains a plenty of nutraceutical active compounds. Although Polysaccharide Are the most active compound isolated from wild mushroom, which has immune modulating and antitumor properties (Wasser et al., 2011). Further the emerging laboratory research has proven the antitumor activity of medicinal mushrooms, in equally in vivo and in vitro (Sliva 2008; Suarez et al., 2013), as well as adjuvant treatments with wild medicinal mushroom different extracts were shown to be capable of improving the prognosis of human breast cancer (Eliza et al., 2012). Currently no literature is available on antitumor activity of M. eximia and M. galilaea. Another common anticancer mushroom Hericium erinaceus (lions Mane) produced aromatics compound (isohericenone J and hericerin A) along with different recognized compounds, 4- (3′,7′-dimethyl-2′,6′-octadienyl)-2- formyl-3-hydroxy-5-methoxybenzylalcohol) and (isoericerin, hericerin, N-dephenylethyl isohericerin, hericenone ), D-arabinol ester , diterpene , resorcinols, hericenols and erinacerins, subsequently an extract of H. erinaceous ascocarps bodies, (Miyazawa et al., 2012; Kobayashi, et al., 2014 ; Zhang et al., 2015; Le et al., 2015). Although most affective anticancer compound isolated from A. mellea that armillarikin, armillaridin, melleolides and arnamial (Bohnert et al., 2011; Chi and Chen. 2013; Chen et al., 2014; Chang et al., 2015).

1.10 Morchella species against gram-positive and gram-negative bacteria

Moreover, the antibacterial effects of many medicinal mushroom species and bioactive constituents on both G-negative and G-positive bacteria have been confirmed through different

36 | P a g e studies. The antimicrobial activities of M. esculenta and Verpa bohemica from Kashmir valley reported against E. coli by (Shameem et al., 2017). First study reported Badshah et al., (2012) that M. esculenta in different solvent extract showed the strong antibacterial activity against viz. Bacillus subtilis (ATCC6633), Staphylococcus aureus (ATCC6538), Vibrio cholerae (ATCC6643), Klebsiella pneumoniae (MTCC618), Escherichia coli (ATCC15224), Enterobacter aerogenes (ATCC13048). The fruiting body of M. esculenta possesses antibacterial properties (Kalyoncu et al., 2010; Alvas et al., 2012). Earlier research showed that the antimicrobial activity against Salmonella typhimurium, Staphylococcus aureus, E. coli, Listeria monocytogenes and Enteobacter cloacae (Haleno et al., 2013). The pervious literature indicated that significance of Agaricus blazei in various extract to stabilizing liver function of hepatitis B patients, moreover authentic research is required to confirm such effects. It has been reported that antibacterial potential of wild mushroom of different extracts may be secondary, with a pure polysaccharide main fraction of A. brasiliensis has been revealed to growth host tolerance against some contagious agents through stimulation of the microbial activity of macrophages (Batterbury et al., 2002; Martins et al., 2008).

1.11 Myco-chemicals Wild and edible mushrooms have a tremendous assortment of bioactive compounds with nutraceutical as well as medicinal properties (Lindquist et al 2005; Poucheret et al., 2006; Kalac et al., 2009). Most of those complex constituents present in the polyphenolic, polysaccharide, and degreased lipid fractions of wild species. Moreover, mushrooms are not an important part of our regular food; though, their uses ratio increase globally due to the best taste and the occurrence of bioactive compounds with pharma properties. Many of those compounds can be found in the polyphenolic, polysaccharide, and lipid atoms (Heleno et al., 2012).

1.12 Polyphenolic compounds Phenolic compounds are formed from the main class of secondary metabolites that are derivatives of the pentose phosphate, shikimate pathway and phenylpropanoid metabolism (Fig. 1). Polyphenolic compound possesses an aromatic/ arenes ring deportment one / more hydroxyl groups, and their structures can array from that of a simple phenolic molecule to that of a complex high molecular weight polymer (Balasundram et al., 2006). Although structural

37 | P a g e plasticity, hydroxylation, conjugation, and methoxylation contribute to a wide range of naturally occurring polyphenolic molecule, total numbers of these compounds are estimated more than 8000. The most common groups of phenolic compounds are flavonoids and phenolic acid (Harborne, 1986; Harborne and Williams, 2000). The polyphenolics compound of different solvents activates were revealed in M. conica, M. anguisticeps, M. esculenta, reported by (Mau et al., 2004; Puttaraju et al., 2006; Turkoglu et al., 2006; Anguiano et al., 2007; Ramirez et al., 2007; Ozturk et al., 2010; Heleno et al., 2013; Vieira et al., 2016). Additionally various research work displayed that phenolic acid profile of Morchella species viz, Morchella conica aqueous extract have 4.05 mg/g, 12.85 mg/g gallic acid and tannic, 4.96 mg/kg protocatechuic acid, 2.48 mg/kg include 20.8 mg/kg protocatechuic acid, 2.15 mg/kg p-coumaric acid, 12.9 mg/kg cinnamic acid 55.2 mg/kg (phydroxybenzoic acid), and 1.83 mg/ kg gallic acid (Vieira et al., 2016) while in Morchella anguisticeps 0.15 mg/g syringic acid and 0.94 mg/g protocatechuic acid , 3.2 mg/g gallic acid and 8.63 mg/g tannic acid (Puttaraju et al., 2006). The polyphenolic acid profile of Morchella. esculenta range 0.24 mg/g, 0.06 mg/g protocatechuic acid 0.1 mg/g (p-hydroxybenzoic acid), and 0.01 mg/g p- coumaric acid (Heleno et al., 2013). Moreover, the amino acid, sugar, and mineral profiles of Morchella species have been described earlier, (Beluhan and Ranogajec, 2011) as has the fatty acid (FA) profile, with linoleic acid (LA) being the key compound (Vieira et al., 2016). Yildiz et al. (2014) likewise noted the extraction of total phenolic compound from M. esculenta. The previous studies M. esculenta collected from South Waziristan shown the presence of health benefit phenolic compounds and antioxidant activity (Badshah et al., 2015). The present scientific knowledge of isolation, identification, antioxidant, and antitumor activity of polyphenolic is limited. The different phenolic compounds were detected in Ganoderma lucidum that’s benzoic acid, Caffeic acid, catechin, chlorogenic acid, Protocatechuic acid, p-hydroxybenzoic acid, vanillic acid, syringic acid, p-coumaric acid, rutin and t-cinnamic acid, L. edodes, Gallic acid, p-hydroxybenzoic acid, vanillic acid, syringic acid, p-coumaric acid, rutin, abscisic acid and t-cinnamic acid, and Hericium erinaceus also have gallic acid, vanillic acid, protocatechuic acid, p-coumaric acid, syringic acid, ferulic acid, rutin, and quercetin (Yildiz et al., 2014). Polyphenolic compounds were reported for their antimicrobial, antithrombotic, anti- inflammatory, cardio protective and anti-allergic effects (Manach et al., 2005). The isolated

38 | P a g e phenolic compound plays a key role in the skin color, besides contributing towards development and reproduction, providing fortification against microbes and predators (Benavente,1997; Bravo, 1998; Samman et al., 1998; Middleton et al., 2000; Alasalvar et al., 2001; Puupponen et al., 2001; Manach and Scalbert, 2005). Moreover, the numerous mushrooms contain health benefit polyphenolic compounds (Hertog, et al., 1993; Parr et al., 2000). Some isolated phytochemical compound from wild mushroom of Ophiocordyceps sinensis are in class of Proteins; (C5H14N2), (C11H8N2), (C17H12N204), (C16H12N2O2), C7H19N3), (Wei et al., 2014; Jiraungkoorskul and Wannee, 2016). The Fatty acids (FA): Docosanoic acid, oleic acid, lauric acid, linoleic acid, lignoceric acid, myristic acid, palmitic acid, palmitoleic acid, pentadecanoic acid, stearic acid, succinic acid (Wu et al., 2014). Some of Phenolic acids(PA): syringic acid, Acetovanillone, protocatechuic acid, hydroxybenzoic acid, salicylic acid, Isoflavones: Daidzein, genistein, glycitein, orobol (Max et al., 2009), Vitamins, inorganic and volatile compounds (Yu et al., 2012). 1.13 Polysaccharides Morchella esculenta is conventionally utilized as nutritional supplements and also as cancer therapy because of the possession of many bioactive compounds, including polysaccharides, trace elements, proteins, dietary fibers and vitamins (Litchfield et al., 1963). Many Morchella species have strength endorsing effects and bio-activities are ascribed to water soluble polysaccharides, numerous studies were reported polysaccharides potential, comprising their isolation, purification and structure characterization of their activities, for both water soluble exo and endopolysaccharides (EP). Precisely, endopolysaccharide (EP) of Morchella esculenta sunken fermentation was described to persuade important anti atherosclerosis activities and anti hyperlipidemic; anti-hyperlipidemic (lipid lowering) potent was gritty by identifying body weights (BW) and serum lipid index of hyperlipidemic mice. Therefore, the endo-polysaccharide (EP) caused a decline in body mass in a dose-dependent range (DDR), and a non-dose-dependent (NDD) alteration in the serum lipid index (Liu et al., 2016). Moreover, extraction, isloation characterization and purification of water soluble polysaccharides from fruiting parts of Morchella esculenta were newly reported by Yang et al., (2015). These crude water soluble polysaccharides were mainly polymers of, galactose arabinose, glucose and mannose, with a usual molecular mass of 43.625 Da (Yang et al., 2015). Further two other crude water soluble polysaccharides from Morchella esculenta (MEP I and

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II) were extracted and purified by (Cui et al., 2016) with mean molecular mass of 192 and 53.3 kDa, showing immune-stimulatory and immunomodulatory activities (Cui et al., 2011). Further, various types of crude water soluble polysaccharides and polysaccharide conjugates were reported in mushrooms. They accepted in few countries for clinical practices to cancer ailment, containing “Lentinan” from Lentinus edodes, Sonifilan” from Schizophyllum commune, “Krestin” from Trametes versicolor, “Grifolan” from Grifola frondosa, and “Pleuran” from Pleurotus ostreatus (Zaidman et al., 2005). Their mycological activity has been associated to their difference immunomodulation effects. Pure polysaccharide was obtained from higher basidiomycetes, that is β-glucans, have been isolated from 100 of various wild mushroom species (Ferreira, 2010). The tocopherols are abundant in the biolipidic dilution, that are vital nature antioxidants potent and stope free radical, reacting with peroxyl radicals formed from polyunsaturated fatty acids (PUFA) in lipoproteins or membrane phospholipids or to produced stable lipid hydroperoxides (Eldin et al., 1996). Pure polysaccharides extracted from wild mushroom have been globally studied in the pharma due to their antimicrobial potent and functions, that’s as anti-carcinogenic, antiviral, antioxidant, anti-inflammatory, anti-hypertensive, antidiabetic, immune-stimulatory and immune-regulation abilities. (Lee et al., 2010; Liu et al., 2012; Cao and Wang., 2012; Wang et al., 2013; Zhao et al., 2015). Water soluble polysaccharides can summon the immune system, as it growths thymus and spleen indices (Bao et al., 2013), endorse lymphocyte proliferation / maturation (Engelet al., 2013; Naeem et al., 2014), stimulate cytokine secretion (Neimert et al., 2011; Honda et al., 2012). Medicinal mushroom derived polysaccharides now had been regular used in clinical treatments (Tanget et al., 2012), the king mushrooms (Grifola Frondosa) is one of the supreme valued folk pharma, and has been used as body tonic for a long time in Japan, China, and other Asian countries, it has freshly concerned and considerable application for its health (Shih et al., 2008). Fruiting bodies and cultured liquid vegetative from the mushroom had been noted to comprise various biologically active constituents (Cui et al., 2007). Grifola frondosa has common mushroom, that have contained active isolated compound in the previous literature (Yang et al., 2007; Lee et al., 2010). Pure polysaccharides are globally recognized for its several activities as well as antiviral, antioxidant (Zhao et al., 2015), anti-carcinogenic (Wang et al., 2013), anti-inflammation (Lee et al., 2010), anti-hypertensive, immune-stimulatory (Talpur et

40 | P a g e al., 2002; Kodama et al., 2005) and antidiabetic (Manohar et al., 2002). Also detail studied were reported in Table (1.4).

Polyphenols

Phenolic acid Flavonoids Non-phenolic Flavonoids

Anthocyanin Antioxidants

Flavonols Flavanols

Flavones Flavanones

Isoflavones

Fig. 1: Classification of natural polyphenols by Weinreb et al. (2004)

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Table 1.4. Anti-carcinogenic activates of polysaccharides isolated from different mushroom species.

Anti-carcinogenic Mushroom species Polysaccharide Reference type sources Melanoma Cordyceps militaris Fruiting body Lee and Hong, 2011 Lung cancer Cordyceps militaris Fruiting body Park et al., 2009 Breast cancer Ganoderma lucidum Fruiting body Zhao, and Hu, 2010 Prostate cancer Ganoderma lucidum Fruiting body Slivova, and Sliva,2005 Cervical cancer Ganoderma lucidum Fruiting body Chen et al., 2009 Cervical cancer Angelica sinensis Mycelia Cao et al., 2010 Prostate cancer Agaricus blazei Fruiting body Yu et al., 2009 Breast cancer Pleurotus geesteranus Fruiting body Zhang et al., 2011 Breast cancer Pleurotus tuberregium Sclerotia Zhang et al., 2006 Lung cancer Clitocybe alexandri Fruiting body Vaz et al., 2010 MCF-7 human Suillus luteus Fruiting body Santos et al., 2013 breast cell lung cancer, colon cancer

1.14 Nutritional value of Morchella Species

Non-essential (NE) and essential amino acids (ES) are present in many edible mushrooms (Chang and Miles, 1989). The composition of amino acid amount varies in different wild mushroom species (Ferreira et al. 2016). The quality of mushroom depends on the amount of amino-acid (Sudheep and Sridhar, 2014, Teklit. 2015). The flavor of a mushroom is different due presence of the concentrations of glutamic and aspartic acids; glycine, alanine and serine- threonine (sweet amino-acids); bitter amino acids (histidine, arginine, leucine, isoleucine, phenylalanine, methionine, and Valine) and tyrosine and lysine (tasteless amino acids) (Pomeranz. 2012, Kalac 2016). P. sajor-caju and P. ostreatus contained the huge amount of arginine, glu acid, asp acid, leucine, threonine and alanine reported by (Mendez et al., 2005;

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Chirinang et al., 2009; Ogundele et al., 2016). M. esculenta have a high composition of protein 1.62, carbohydrate 4.25 and lipids 1.96 g /100 DW (Sarbhoy et al., 1996; Boda et al., 2012). According to pervious studied the nutritional value were recorded in various Morchella species viz, Morchella crassipes, Morchella conica, Morchella hortensis, and Morchella elata (Beluhan and Ranogajec., 2011; Heleno et al., 2013; Vieira et al., 2016). The present scientific knowledge of nutrition potential of Morchella species is limited, where still no baseline data are available in Pakistan. Currently, due to high amount of protein and best flavor, and nutritional aspects of wild edible mushrooms is increasing (Kalac. 2009). In the advanced countries, low cholesterol and energetic currency has been esteemed (Demirbap. 2001). The fruiting bodies are rich in polysaccharides, proteins, amino acid minerals and nitrogen, they have precise low-fat calories (Mau et al., 2004; Heleno et al., 2013; Vieira et al., 2016). The fruiting bodies (Fb) also contained the cellulose, natural polymeric substances hemicellulose, lignin and crude fiber in the range of (28.5–41.0, 13.0–39.3%, 14.0–20.20% and 14.1–20.2%), (Ragunathan et al., 1996; Manzi et al., 2001; Mattila et al., 2001). Further (Manzi et al., 2001; Ouzouni, et al., 2009) reported that wild mushroom contains 90% water, minerals (phosphorous, zinc, and magnesium), 20-30 % Proteins, high in fibers (F), carbohydrate and low calories foods owed to their low ratio in fat. Globally, extensive research has been carried out on past and current trend of distribution and medicinal use of Morchella species as well as associated health concerns as a results of consumption of wild edible species (Alonso et al., 2000 , Carvalho et al., 2005 ; Cocchi et al., 2006; Svoboda et al., 2006; Ouzouni et al., 2009; Li et al., 2011; García et al., 2013; Petkovšek and Pokorny, 2013; Schlecht and Säumel., 2015). In this way consumption of Morchella Species for treatment purposes is potentially associated with toxicological hazards. Beside many other chemicals ingredients, uptake, translocation and subsequent bioaccumulation of toxic trace metals such as Cd, Mn, Co, Cr, Pb, As and Zn, may pose potential hazards to the consumers (Rath et al., 2009). These Morchella Species grows in natural, colder mountainous habitats, geochemically inconsistent areas and anthropogenic ally polluted soil of Pakistan, which may enhance the probability of metal uptake (Kucera. 2004). The intake of heavy metals (Pb, Zn, Cd, Cu, Ni) can critically cause exhaustion of approximately necessary nutrients in the human, which in turn sources a decline in intrauterine growth retardation (IGR), immunological defenses, (caused by Pb, Cd, and Mn), psychosocial

43 | P a g e and erectile dysfunctions, disabilities associated with low food and a high occurrence of upper gastrointestinal cancer (Iyengar and Nair, 2000 ; Turkdogan et al., 2003). Similarly Cd causes bone fracture, cancer, kidney dysfunction and hypertension (Satarug et al., 2000).The High concentration of Pb has chronic difficult health effects including dermatogenic, respiratory issue, elevated bloochronic, difficult, improper effects, including, tumor infection, and infertility (Oliver, 1997 and Wang et al., 2006).

1.15 Phosphodiesterase inhibitory effects of Mushroom

Phosphodiesterase are the important enzyme in the regulation of different smooth muscle and play a key important physiological role by regulating the interior cell level of cyclic nucleotides (CN) They are divided into different isoenzymes, PDE2, PDE1, PDE4, PDE3, and PDE5(Beavo et al., 1990; Nicholeson et l., 1991). The phosphodiesterase isoenzyme, (PDE5) plays a very key role in male sexual diseases “erectile dysfunction” by preventing the hydrolysis of cGMP. PDEs catalyze the hydrolysis of (cGMP), (cAMP) (Andersson, and Wagner,1995). Erectile dysfunction is a common disease (Jeffecoate, 1997), affecting about 40 % of adult men over the age of 35- 40, 70 % male over the age of 60-70. As estimated by (Goldstein. 2000) the number of patients will be around 330 million next 25 years. Problem statement Scientific information with respect to various species of Mushroom (Morchella spp.) of Asian region, found in the natural habitat of Pakistan in different season is scanty. Through this study, it would be the first base line information pool and to further classify through various identification tools (molecular and taxonomic) and by molecular phylogeny, extraction of polysaccharides, polyphenolics and different solvent extract, antibacterial, anti-tumor and antioxidant activity, and evaluation against PDE5 and PDE6 of Morchella species. Morchella species have great value and play important role to develop socioeconomic and livelihood of the less development region of Pakistan. The local people used for different remedies through by traditional knowledge, without any scientific information. The Morchella species were identified by hunter from different colours. In current studies the identification problem was resolved through by molecular markers. Further in our study confirmed that have a potential of nutraceutical, as well as therapeutically.

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1.16 Aims and objectives The wild edible and medicinal mushrooms are widely consumed throughout the world for their nutraceutical and health aspects. Pakistan is home of many edible mushrooms of which Morchella species are highly valuable. However, little systematic studies are available on the identification; nutritional and medicinal value of Morchella species found at higher altitudes and selected lower plain area of Pakistan. Therefore, the present studies were conducted to achieve the following objectives:

 To identify selected Morchella species by using morphological and molecular markers.  To extract and isolate polysaccharides, polyphenols and lipids from fruiting bodies of selected Morchella species  To determine antibacterial, antioxidant, anti-cancerous and phosphodiesterase activity of the selected Morchella species  To evaluate nutritional value of the selected Morchella species

MATERIALS AND METHODS The summary of the research work was carried out is as follows: 1. Collection/sampling was brought to Plant Genetics Laboratory, where morphological assessment and molecular analysis were performed. The DNA sequencing were done commercially from Alva Laboratory, Spain. 2. The Biological activities to include antibacterial, antioxidant and anticancer activity were performed at Irma Lerma Rangel college of Pharmacy, Health science center Texas A & M University, USA, supported by IRSIP, HEC, Pakistan during (2017). Furthermore, the Phosphodiesterase’s PDE5 &PDE6 was done commercially from Kinexus, Canada. 3. The Amino acid analyses were performed at N.A.R.C, Islamabad, Pakistan. 4. The NMR spectroscopic analyses were carried out at Irma Lerma Rangel College of Pharmacy, Health science center Texas A & M University, USA. Section A: Identification

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2.1. The collection/sampling sites/study area

The sampling sites were visited based on previous knowledge gained from local pickers and native market traders of Morchella species. Morchella species are commonly found in the higher mountainous regions and lower plainsin the irrigated (Charsadda) areas of Pakistan. These areas were targeted because of their wide geographical, physical variations, high moistureand snowfall, cool and moderate temperature. In South Asia, Pakistan is famous for its different seasons: a dry and cool winter, pleasant spring; hot temperature in summer with rainy season; and autumn (October to December). The rich diversity of Morchella species in the higher mountainous region of Pakistan was due to prolong season of spring, from March through June. Whereas, the plain irrigated areas are enriched with the huge population of light to pale colour Morchella species. The study area was primarily divided into two sections the lower plain irrigated field area i.e. Charsadda, a district with hot summers and typically cool winter; and the high mountainous region having diverse flora and fauna, featured with freezing (sub-zero) temperatures. Higher altitudinal regions selected in Pakistan were: Skardu, Murree, Swat and Azad Jammu Kashmir (AJK). Details are shown in Table 2.1.

Azad Kashmir is in Northeast of Pakistan and fall within Himalayan mountain belt. It lies between 34°35'17.99"N and 73°54'39.59"E. It holds sin hilly and mountainous geography, characterized by deep ravines, rough, and rolling terrain. The north is covered with moist temperate, while south has sub-tropical climates. Moreover, average rain falls between 100 to 150mm, in the northern district with 30-60% precipitation is in the shape of snow. The average temperature varies between 04°C to 20°C and 13296 km2area cover approximately (Singh et al., 2004).

Skardu is situated within the capital administration of Gilgit Baltistan, Northern Pakistan. Skardu is located at 35°18 '21.55"N and 75°36'51.14"E. It has about 15000km2area covers and total population of 214,848. The climate of this site is moderate, ranges between 39.5 to 16.1°C.

Swat is located at 34º 51′ to 34º 55′ N and 72°26'36.84"E with area of about 5,337 km2, and have population of about 3.3 million (Census Report, 2000). Murree is bounded in the east

46 | P a g e by river Jhelum, in the north-west by the Khyber Pakhtunkhwa and in the south by the sub- mountainous area of Rawalpindi. Murree lies between 33°54'27.98"N and 73°23'29.40"E with total area of about 697.5 km2. The total annual rain fall is recorded as 1789mm.

Charsadda (34°23'2N,71°48'18E) is bounded by the Malakand district on the North side, Mardan district on the East, the Nowshera and Peshawar districts on the South and the Mohmand agency (FATA) on the West. The maximum rise in temperature is recorded in the month of June, whereas a sudden decrease in temperature is recorded from October and onwards. The coldest month here is December and January. The annual rainfall is about 400 to 600 mm. The average winter rainfall is higher than that of the summer rainfall. Major tree species of Charsadda are Morus alba, Dalbergia latifolia (Shawa) and Eucalyptus globules (Lachi).

Table. 2.1: Field areas and distribution of Morchella species in Pakistan

Sampling sites Latitude N Longitude E Number of species Skardu GB 35°18' 21.55" 75°36'51.14 30 Swat 34º 51′ 72°26'36.84 250 Murree 33°54'27.98 73°23'29.40" 100 Azad Kashmir 34°35'17.99 73°54'39.59 200 Charsadda 34°23'2 71°48'18 350

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Fig. 2.1. A Map illustrates sampling sites in Pakistan.

2.2 Sample Processing and Preservation The specimens of Morchella and other related species were collected during several field surveys conducted in different geographical areas such as at higher altitudes, lower altitudes and irrigated areas of Pakistan during 2014 to 2016. The field information regarding habitat, distribution, altitude, surrounding flora and soil characteristics were noted along with in situ photography of the specimens. After collection, samples were shade dried, labeled and keep at 4°C. A specimen of each site has also been succumbed to the Herbarium of Department of Plant Sciences, Quaid-i-Azam University, Islamabad, Pakistan (ISL). The stored samples were further studied for macroscopic characters. Furthermore, the maturity process was extensively studied in the field from repeated collections of fresh ascocarp at various stages of development. For microscopic studies, slides were prepared using potassium hydroxide (KOH 0.05) as a growing medium, with Congo-Red (0.02) and Melzer’s reagent, (amyloid) which was sporadically used as obligatory to stain various microscopic characters. All microscopic

48 | P a g e characters were carefully studied under HM-Lux Letiz Compound Microscope (German) at 40X magnification, mounted in Glycerin and distilled water.

Identification was carried out through studying taxonomically important characters enlisted from the previous literature (Taskin et al., 2015; Richard et al., 2015; Michael et al., 2017). Important characters studied were: paraphyses shape, size, apices, numeral and alignment of septa, the sterile ridges (T-elements), stipe cortex, (T- elements), spore (Length and width), and shape character, surface, asci size and ascus base.The fresh and dried Morchella spacemen used for some microscopic and macroscopic characters and obtained their maximum values. Spore measurements were taken, by used of simply naturally evicted spores directly found from a spore print. The Morchella spacemen were kept in normal tap water for 30 mints. At least 30 fully mature spores from each ascocarps were measured. The scale bar used for measure of various spore.

2.3.1. Molecular study Molecular study was carried out for six Morchella species, as follows:

2.3.2. DNA extraction and amplification

Total genomic DNA was extracted from dried specimens using a modified extraction protocol following Murray and Thompson (1980). A small part of each specimen was ground with the help of a micro pestle in 600 µl of CTAB buffer (CTAB 2%, NaCl 1.4 M, EDTA pH 8.0, 20 mM, Tris-HCl pH 8.0 100 mM). The resultant paste was incubated for 15 min at 65ºC. An equal volume of chloroform:isoamylalcohol (24:1) was added to the sample and cautiously agitated to prepare an emulsion. It was then centrifuged for 10 min at 13000 g and the supernatant with DNA was taken. The supernatant was allowed to precipitate by adding an equal volume of isopropanol. The precipitating sample was centrifuged again for 15 min under same conditions to obtain a pellet. The pellet was washed in cold 70% ethanol, centrifuged again for 5 min and dried at room temperature. It was finally re-suspended in 200 µl dd.H2O.

PCR amplification was performed with rDNA ITS (ITS1-5.8S-ITS2) region based primer pairs: ITS4 and ITS5 (White et al. 1990); EF1-983F and EF1-1567R (Rehner and Buckley, 2005); EF1-α gene, while CRPB1A (Castlebury et al. 2004) and RPB1-Cr (Hall 2003)

49 | P a g e were employed for RPB1. The 9F/3R was used for RNA polymerase II (RPB2) (Liu et al. 1999). PCR reactions were performed with following conditions: 95 ºC for 5 min, trailed by 35 cycles at 94 ºC, 54 ºC and 72 ºC for 45s, 30s and 45s respectively with a final 72 ºC step for 10 min. PCR products were electrophoresed in 1.2% agarose gel and positively amplified reactions were successively sequenced with single reverse primer. Chromatograms were assessed for putative reading errors and corrections were made accordingly.

2.4. The phylogenetic analysis BLAST algorithm search (NCBI) was carried out for the obtained sequences. The initial comparison of the obtained sequences and those held in the GenBank were made using Bio- edit software and sequence alignments were done in MEGA (v 7.0) software. The phylogenetic analyses were also performed in MEGA (v 7.0) with default parameters. Neighbor-Joining (NJ) trees were constructed and bootstrap consensus trees were generated using p-distance method. The bootstrap analysis with 1000 permutations were performed for all samples and the obtained values were shown on tree branches. Section B: The Biological Activities 2.5. The biological activities and Myco-chemical analysis

These and the subsequent analyses were carried out on two most frequently found species: M. galilaea and M. eximia.

2.5.1. Sample preparation

The fresh fruiting bodies of M. galilaea and M. eximia were cleaned with double sterile distilled water, cut into pieces and shade dried. The dried spacemens were ground and homogenized finely and keep at 40C for production of crude extracts in solvents of different polarity.

2.5.2. Extraction

For the crude methanolic extracts, 100g powder each of M. galilaea and M. eximia were soaked in a flask for 60 hours with regular shaking to obtained extraction. The crude extract mixture was filtered using Whatman filter paper no.1 and the filtrate was obtained in a Buchner funnel (50mm). The residue in the flask was treated with equal amounts of same solvent for the second time. Both extracts were collected, concentrated and dried using rotary evaporator at

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40˚C (WG-EV311-USA). The MeOH and EtOH extracts thus obtained were stored in a desiccator. The final extracts were weighed for both the species: M. galilaea and M. eximia. The extracts were keep at 4˚C for further analyses.

2.6. Extraction of Polyphenolic Compounds

Extracts preparation of Morchella galilaea and M. eximia were carried out using nonpolar and polar solvents (Markham, 1982). The M. eximia and M. galilaea (10-50 gm) of each fine-dried powder were isolated by stirring with 70 ml of methanol (Me OH) 80% (v/v) at 60˚C for 3 hours, then centrifuged (Sorvall RC6Germany) at 4000 rpm for 15min at 4˚C and sifted over whatman filter paper no 1. The remaining methanolic extract was evaporated using a rotary evaporator at 45˚C (WG-EV311 USA) (Can et al., 2014; Kim et al., 2006) rapidly. The remaining residues were liquefied in 10 ml acidic water at (pH 2) and extracted with Ethyl acetate (15 ml), and Diethyl ether (15 ml), consecutively. The Organic phases (OP) were keep and evaporated until dry under condensed pressure in a rotary evaporator at 45˚C. The obtained total phenolic 1.53 and 1.35 gm, respectively. The isolated polyphenolics were stored at -20 ˚C for further assays. A schematic overview for the extraction and purification of polyphenols are shown in (Fig 2.2).

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Fig. 2.2. Graphical presentation, Extraction and isolation of Polyphenolics

2.7. Isolation of pure polysaccharides

The dried samples of M. galilaea and M. eximia (60 gm) were pulverized to powder and acquiesced to eradicate degreasing lipids by adding of 650 ml of absolute ethanol (80% EtOH) under reflux at 110◦C for 3 h. The residue found was then extracted treated three times with 500 ml of boiling water (BW) under reflux 90-80◦C for 3h each time. The extract was filtered under vacuum pressure and centrifuge at 3500 rpm at 45 min, the supernatant treated with a rotary evaporator at 80◦C and then added 750 ml absolute ethanol (95%) and keep over night at 4˚C. Extract was centrifuged at 6000 rpm up to 30 min, the resultant precipitous (crude polysaccharides) was clean serially with ethanol, acetone, ether. Additional vacuum dried. The weight of the obtained polysaccharides was 3.03 gm, and 2.88gm. Overall schematic overview for the extraction of crude polysaccharide compound processes are shown in (fig 2.3).

Fig. 2.3. Graphical representation of Extraction and isolation of polysaccharides

2.8. Isolation of Polyphenols by NMR

The obtained polyphenolics of M. galilaea and M. eximia (10mg), as mentioned in previous section, were freeze dried over P2O5 in vacuum for one week, treated with deuterium. After

52 | P a g e lyophilizing (-75°C, pressure 10 bars for 30 mints). The extracted polyphenolics of M. galilaea and M. eximia were added with deuterium oxide (D20) (Chase et al.,1997). NMR spectra were measured at 27°C on Bruker advance DPX-300 Ultra spectrometer (Leeuwen et al., 2008).

2.9. Cell proliferation or MTT Assay

This experiment focused on the cell viability was carried out to determine cellular proliferation, inhibitory activity and cytotoxicity of methanolic extracts of both M. galilaea and M. eximia. The healthy and mycoplasma-free cells of 70-80% of confluence were used. About 5×103 cells per each well were seeded in 96-well plates in 100 μl of complete culture medium (2,5-diphenyltetrazolium bromide).Although cell line were incubated at 37°C for one night to allow cells’ attachment. Cells were serum starved for 4h before conducting any further experiments.

The final concentration of each methanolic extract and polysaccharide in each well were 0.0, 0.005, 0.05, 0.5, 5, 25, and 50μg/ml in 100μl of culture medium and DMSO was used as control. Moreover, standard Nilotinib (commercial drug) were compared with isolated extract (M. galilaea and M. eximia). Following incubation for 72 or 96 hrs, 10μl of cell counting kit-8 (Dojindo molecular Tech, Inc., Rockville, MS, USA) was added into each well. Plates were then incubated for 2-4 hrs shaken for 5 min and the absorbance was read on ELISA reader at

450 nm. The 50% of cell inhibition (IC50) was calculated.

2.10. Antioxidant activity: The DPPH free radical scavenging Assay

The hydrogen donating or radical scavenging ability M. galilaea and M. eximia were measured in terms of stable radical 1,1-diphenyl-2-picrylahydrazyl (DPPH), equipped by dissolving 5.2 mg DPPH in 200ml of methanol. The methanolic extract and various isolates of M. eximia and M. galilaea were dissolved in HPLC methanol grade (Sigma-Aldrich) in different concentrations. A sample of 1ml was assorted with 1 ml DPPH solution and incubated in completely dark room at room temperature for 30 mints. After completion of incubation interval, optical density of the samples was noted at 517nm using a spectrophotometer (Du 800 UV-VIS spectrophotometer). Ascorbic acid was used as standard in 5mg/50ml prepared different dilution. All analyses were carried out in triplicate (Brand-Williams et al., 1995;

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Sharma & Bath, 2009). In order to decipher the active dose of extract sufficient for 50% inhibition of DPPH (IC50), following procedure was adopted.

The radical scavenging activity was deliberated as % of DPPH discoloration using the following formula:

% scavenging DPPH free radical = 100 × (1 – Abs control – Abs sample / Abs control)

AbsSample is the absorbance of the extract of test (M. eximia and M. galilaea), where Abscontrol is the absorbance of control reaction (DPPH solution, blank without extract). The schematic overview for DPPH assay has been shown in (fig 2.3).

Fig.2.4: Graphical representation of Antioxidant activity (DPPH assay)

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2.11. Antibacterial Assay

2.11.1. Preparation of methanolic and ethanolic samples

Samples prepared for assay of antibacterial activity were of two types. The methanolic and ethanolic extracts of M. galilaea and M. eximia of fruiting bodies were treated with pure dimethylsulphoxide (DMSO) to make final stock solutions of concentration 100, 50 and 30mg per ml (Bbosaet al., 2007). These were then keep at 4˚C. The final stock solution was used for further fractionation with dimethylsulphoxide by serialized dilution using standard formula. The various concentrations of 30, 50, 100 mg/ml were finally used for analyses. The antibiotic streptomycin (2mg/ml) dissolved in DMSO were used as positive control (PC) while Dimethylsulphoxide (DMSO) was used as negative control.

2.11.2. Preparation of Nutrient Agar medium

The agar medium was made by dissolving 20g of nutrient agar in 1000ml distilled water. The mixing was facilitated by boiling. The conical flask (1000ml) having medium was plugged with cotton (CP) and autoclaved at 121°C for 30mints and 15psi pressure (sterilization). The medium was cooled and placed at room temperature and was allowed to attain 45-50˚C. The medium was then decanted in 30ml amount in sterile petri dishes (150 -15mm) and were allowed to get solidified for 30-35 minutes. Subsequently, all petri dishes were incubated at 37˚C for 40 minutes to assess sterility. All the petri dishes were sited in reversed position to keep the moisture over the medium layer. The plates were tightly wrapped and stored at 4˚C until further use.

2.11.3. Preparation of broth cultures

The nutrient broth was prepared for each bacterium type by dissolving 0.13g of nutrient broth fine powder in 10ml of sterilized distilled water. The test tube with broth medium were plugged with cotton (CP) and sterilized through autoclaving for 20 mins at 121˚C. The broth was permitted to cool down under aseptic conditions. Then the revived strains (microorganism) were inoculated to the Broth culture (BC). This assortment in test tube was kept in a shaker for 24 hours to obtain good growth.

2.11.4. McFarland 0.5 BaSO4 turbidity standard

The standard was prepared by adding 0.5 ml of 0.048M BaCl2 to 99.5 ml 0.36N H2SO4. The (5-6 ml) Barium Sulphate turbidity standard was stored in a screw cap test tube and was used to assess the turbidity (Koneman, 1988).

2.11.5. Test Organisms

Four strains of bacteria were used which were: Bacillus-subtilis (ATCC6633), Staphylococcus-aureus (ATCC6538), Klebsiella-pneumoniae (MTCC618) and Enterobactor- aerogenes (ATCC13048). The initial two was G- positive and the latter two were G- negative. The bacterial fresh strains were obtained from the Department of Microbiology, Quaid-i-Azam University, Islamabad.

2.11.6. Preparation of Inoculum

The pallets of bacteria obtained from 24 hours old culture in nutrient broth were assorted with physiological saline. The turbidity of the medium was adjusted by tallying sterile 8 physiological saline in comparison to the McFarland 0.5 BaSO4 turbidity standard (10 colony forming unit (CFU per ml). These inocula were used in training of the lawn for bacterial rate growth on Nutrient Agar plates.

2.11.7. Preparation of Bacterial Plates

After 24 hours of growth the broth cultures of each test organism were inoculated on the nutrient agar plates with the help of sterilized cotton swab to make a lawn of test microorganisms. Distinct sterilized petri dishes and cotton swabs were used for each test microorganism under the sterilized/aseptic conditions.

2.12. Agar Diffusion Method

Agar well diffusion assay was used to analyse the antibacterial properties of M. galilaea and M. eximia. As a first step the sterile cork borer (3mm) were used to make holes in the lawns of media cultures. Three different concentrations of each extract (30mg/ml, 50mg/ml and 100 mg/ml) were dispensed into separate wells. The 2mg/ml solutions of the positive control (Streptomycin) were also applied on each test organism following the same method. The petri

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plates were incubated at 37oC for 24 hours. Plates were prepared in triplicate for each extract. The sensitivity of test organism was determined by measuring ‘the zone of inhibition’ surrounding the wells. Mean diameter of pure zones in each plate was calculated. All the materials used were subsequently inactivated and sterilized through autoclaving.

2.13. Evaluation against PDE5 & PDE6

The cyclic-nucleotide phosphodiesterases placed in the compound profiling method were cloned, expressed and purified using standard procedures. Quality control (QC) testing was adopted to ensure compliance to the accepted standards. The PDE-GloTM Phosphodiesterase assay kit was purchased from PerkinElmer and the procedures were followed as prescribed in the supplier instructions. 5mg each of the M. galilaea and M. eximia were used in the present case. A stock solution was prepared with a final volume to 100μl, off which 5μl was incorporated into each assay.

Section C: Nutritional Analysis

2.14. Amino acid Analysis

Samples were prepared for amino acids (AA) according to the assay of Tkachuk and Irvine (1969). The Morchella eximia and M. galilaea (50mg) was hydrolyzed with 4.5ml of 6N HCl at 100 °C for 720 min. The assortment of NaOH pellets employed in a desiccator for 10- 12h to eliminate the HCl. The HCl was removed by placing the mixture in a desiccator containing NaOH pellets for 10-12hrs. Citrate buffer 27ml (pH 2.2) having Brij-35 Octonic acid and detergent was well mixed to the removal of HCl through Whatman filter paper 125mm and amino acids analysis. Automatic amino acid analyzer Spackman et al., (1958) using cation exchange chromatography (CEC).

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Section D: Data Analysis

2.15. The Biological Activities

The detailed statistical analyses were performed on observed data for both species. The data were analyzed using descriptive statistics tools following Steel and Torrie, (1980). Biological activities for DPPH, Anticancer/anti-tumor potentials of M. galilaea and M. eximia were analyzed by statistical software GraphPad Prism version 5.0 (San Diego, CA, USA).

2.16. Antibacterial Assay In the antibacterial assay, observations based on M. galilaea and M. eximia were analyzed by tabulating the mean values of the three replicates. The data has been presented in graphical form with standard error of the mean (GraphPad Prism version 5.0 San Diego, CA, USA).

2.17. Amino acid Analysis Data were recorded from respective species for amino acid profiles and tabulated in MS Excel 2007 program. Means values were computed from three independent replicates and their descriptive statistics were generated for each profile. The data was analyzed by t-test interpretation using statistical Statistix 8.1 (Analytical Software, 2005).

RESULTS

Summary of results

Analysis of all collected samples revealed as many as six morpho-types of (Morchella) in Pakistan. In section A the morphological descriptions have been presented following the taxonomic literature. The details in description are meant to highlight the morphological variation within these taxa. Furthermore, fresh molecular sequences have been generated for these morpho-types to confirm their identification, comparable to the global material. In the subsequent sections the biological activities have been performed such as: antibacterial

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analysis, antioxidant activities, anticancer activities and Phosphodiesterase inhibitory activity. Followed by the assessment of nutritive value by assessing the amino acid profile and finally the spectroscopic analysis was carried out. The results pertaining to these analyses have been summarized as follows:

Section A: Identification of Morchella species

Morphological and molecular identification

3.1 Morchella galilaea Masaphy & Clowez in Clowez, Bull Soc Mycol France 126 (3-4): 238. 2012.

ASCOMATA 55–70 mm tall (15–25 dry length). Hymenophore 30–40 (5–15 dry length) mm tall, 20–25 mm wide, sub cylindrical, conical, primary ridges 20–35µm, pitted, two types: long elliptical ridges and narrow polygonal ridges). Sterile ribs 15.5–26.14 mm tall and longitudinal 6–10.1 mm wide, elastic, soft, thin, to form elongated hymenial pits, rounded and white when young, often compressed, ridges glabrous. Hymenial pits yellow gray, whitish gray, light gray, silvery gray and blackish gray when young and turn in color to light olive brown, greyish-light brown, yellow brown at maturity. Fruiting body or Ascocarp margin (AM) simple, connecting straight into the stipe.

STALK 22–36× 6–8 mm, thickening laterally towards the base, cylindrical; surface white when young, yellowish white at maturity. Context whitish, 0.7–1.2 mm thick in the hollow hymenophore; whitish and glabrous with sterile inner surface.

ASCOSPORE smooth, elliptic, contains homogeneous contents. Asci 8-spored 14.5 × 11.7 μm, (length /wide ratio=1.23), hyaline.

PARAPHYSES sub-clavate or cylindrical, 52–130 × 8–12 µm, apices round or subacute, 2–3- septate, hyaline or with brownish homogeneous contents (Figure 3.1).

Material examined: Pakistan, Province: Khyber Pakhtunkhwa, District Charsadda, 31°43'51E, 34°8'43N, Alt. 906ft. under Saccharum officinarum and Triticum aestivum, Hussain Badshah, 15 December 2015.

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Ecology and Distribution: This species occurs in the Saccharum officinarum and Triticum aestivum crops in the lower plain irrigated sandy areas of District Charsadda, Province Khyber Pakhtunkhwa, Pakistan.

Distinguishing features: An autumn species, lower plain irrigated area, hyaline (170- 226) µm, spore 14.5 × 11.7μm, associated with Saccharum officinarum (sugarcane) and Tritium aestivum (wheat).

Fig. 3.1: Morchella galilaea. A: Field photograph of ascocarp; B–C: Spore within asci, Paraphyses respectively

Nucleotide sequences and phylogeny

The translation elongation factor (TEF) was amplified for two isolates (13066 Chd20 and 13067 Chd30). The generated sequences length was 563bp. After alignment of the query sequences with closely related reference sequences, the whole alignment set revealed indels at 2, 61, 482, 483, 484, 560, 561, 563 nucleotide sites, respectively. The stretches also showed 15 single nucleotide polymorphism sites (SNPs). Similarly, the RPB1 sequenced for two isolates (Isolates Chd20 and Chd3) and aligned with closely related sequences. These two sequences

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showed indels on 2-9 and 767-799 nucleotides sites respectively, while detected 33 SNPs in the stretches suggesting that RPB1 is more polymorphic as compared to TEF.

TEF sequences (Isolates 13066 Chd20 and 13067 Chd30) matched with top four Morchella sp. Mes-16 (M. galilaea) GenBank sequences (accession nos: JN085288, GU551531, GU551151, GU551154 and GU551147) with 100% identities and 100% Query cover.

Similarly, RPB1 sequences (Isolates Chd20 and Chd3) also matched with top four Morchella sp. Mes-16 (M. galilaea) GenBank sequences (Accession no: JN085335, GU551259, GU551629, JU551266) with 99% identities and 100% Query cover. The NCBI – blast analysis revealed sequence matches TEF -RPB-1. Separate phylograms have been generated for TEF and RPB-1 (Figs 3.2 & 3.3 respectively) sequences showing the identity of M. galilaea.

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Fig. 3.2: Phylogram of M. galilaea obtained from TEF sequences and is based on Neighbor-Joining method. Chd20 & Chd30 represented the Pakistani accessions

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Fig. 3.3: Phylogram of M. galilaea obtained from RPB1 sequences and is based on Neighbor-Joining method. The sequences labeled as Pakistan in parenthesis represent M. galilaea from collected material

3.2 Morchella pulchella Clowez & Franc. Petit in Clowez, Bull. Soc. Mycol. Fr. 126:314. 2012.

ASCOMATA 60–70 × 52–56 in size, brown, conical. Hymenophore 50 × 40 mm, sub-conical, pitted, pits short and elongated, ridged (both regular and irregular in shape, primary ridges 26 per ascoma, 2–5 mm long, pitted 55–60 per pit, 3 mm, over groove character is present at base of fruiting body. sharply attached to stipe.

STALK, 15–25 mm long, hollow, yellowish, convex, rays are present in base with middle depression.

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ASCOSPORE 20.4 × 15.6 µm, ellipsoid, smooth, with homogenous contents. ASCI 8-spored, 11.5 × 10.2 µm, cylindrical, hyaline.

PARAPHYSES shorter than asci, 78–150 × 10–14 µm, cylindrical, subclavate, apices rounded, narrow, mostly subacute, rarely acute, septate, hyaline with homogenous contents, yellow hyphal elements on sterile ridges 77–181 × 7.9–26 µm (Figure 3.4).

Material examined: Pakistan, Province Khyber Pakhtunkhwa, District Swat, Malam Jabba,

34°47'54.44"N 72°34'33.37"E, altitude 1800 m, under Pinus wallichiana and in grasses, 15 April 2015, Collector: Hussain Badshah B10, (ST-QAU38.4).

Ecology and Distribution: Morchella pulchella grows naturally on calcareous soil and in mountains and higher altitudinal regions, in the highly snow fall area under Pinus wallichiiana trees in Pakistan. The growing period is March to early June.

Distinguishing features: Paraphyses shorter than asci, yellow hyphal elements on sterile ridges. Spore cylindrical with length to width ratio of 1.12, hyaline length/width (Qm) ratio 1.5.

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Fig. 3.4: Morchella pulchella (ST-QAU 38.4) A: Field photo of Ascocarp. B–D: asci with ascospores. C: Paraphyses

Sequence characterization and phylogenetic analysis

The TEF locus amplification generated 636 bp long sequence. The sequence alignment with other similar sequence stretches revealed indels at position 152, 188, 352, 378, 454, 554, 578 nucleotide sites, respectively. The stretches also showed 4 SNPs. Similarly, the RPB1 isolates sequences (M. pulchella) were 784 bp long. The alignment with sequences of the closely related species showed indels at position 152, 98, 150, 154 and 272, 345, 464, 598, 674, 784 a nucleotides sites respectively. Moreover, 5 SNPs were found in the stretches which suggests RPB1 is highly polymorphic as compared to TEF and recommended for DNA-based molecular identification of Morchella species at species level. BLASTn analysis using the TEF sequence as the query matched with sequences of M. pulchella (accession no. KM491180 with 100% identity) and two sequences of Morchella sp. Mel-23 (Accession nos. GU551381 &

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GU551390, 100% and 99% identity, 636, 636 and 635, 636 bp). Similarly, the RPB1 sequence matched with M. septentrionalis (accession nos. KM588048 and JX173414 with 100% identity, 785, 785 bp) and M. pulchella (accession no. HM056439 with 100% identity, 785, 785 bp). Also, the RPB2 sequence matched with M. pulchella (accession no. KM588041, 100% identity, 809, 809 bp) and two sequences of M. septentrionalis (accession nos. KM588048 and JX173414 with 100% identity, 805,805 bp). This analysis showed a highly polymorphic nature for the present collection. The NCBI –blast analysis revealed sequence matches TEF and RPB1. Identity with 98% Query cover), Mr.3 (KP670926) matched with GQ228470 (717, 724 (99%) identity with 98% Query cover). This analysis showed a highly polymorphic nature for the present collection. The NCBI-blast analysis revealed sequence matches ITS. The results of the phylogenetic analysis have been given in the figure 3.5.

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Fig. 3.5: Phylogenetic tree based on partial TEF sequences. Posterior probabilities and bootstrap values are given on each node of the tree; the bootstrap values enclosed in the parentheses show low bootstrap values. The Pakistani specimen in bold was identified as Morchella pulchella.

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3.3 Morchella crassipes Clowez 2012, (Vent.: Fr.) Pers., Syn Meth Fung:621. 1801.

ASCOMATA 30–50 mm tall, 42 mm wide, Hymenophore 20 ×45 mm, round-conical or sub conical, pitted, deep pits short and narrow elongated, ridged (irregular and regular shape, primary ridges (No,29–35), 3–6 mm long, pitted (No, = 59–64) with 3.4 mm. over groove character is present at base of fruiting body. Sterile Ribs (SR) 12.5–22.1 mm tall, fruiting body or Ascocarp margin (AM) Simple connecting straight into the stalk with no a sharp bend.

STALK hollow, yellowish, rays and middle depression are present in base, Stalk 12–30 mm length, 8–10 mm wide, cylindrical, bulky marginally towards the base surface, white glabrous and yellowish glabrous with sterile inner surface. Context whitish, 0.5–1.1 mm thick in the hollow.

ASCOSPORES smooth, elliptical with homogeneous contents. ASCOSPORE 55- 130 µm, Asci 8-spored, smooth, ca. 20 µm × 14.82 µm (L/W= 1.35), ellipsoid.

PARAPHYSES 95–150 µm long, subclavate or cylindrical, apices rounded or ovoid, 1–2- septate, hyaline or with blackish homogeneous (Figure 3.6).

Material examined: Pakistan, Province Panjab, Murree Tehsil, 33°57'42.76"N, 73°23'29.88"E, Altitude 2100 m, under Pinus wallichiana April 2014, Coll. Hussain Badshah (Mr53, Mr54 and Mr.57).

Ecology and Distribution: M. crassipes occurs on organic rich, moist soil under shade of old trees such as Pinus wallichiana.

Distinguishing features: Fruiting body hard with soft stalk, conical or sub conical, pitted, deep pits short and narrow elongated, over groove, spore L/W=1.35. Naturally grows in the end of March and middle of April especially in lesser Himalaya Mountains.

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Fig. 3.6: Morchella crassipes. A. Ascocarp in field. B. Spore with in ascospore. C. Paraphyses.

Sequence characterization and phylogenetic analysis of Morchella crassipes

The ITS locus amplification revealed a sequence of 651 bp in length. The sequence alignment with other related similar sequences stretches revealed indels on 59, 69, 170, 193, 294, 458, 651 nucleotide sites, respectively. The stretches also showed 110 single nucleotide polymorphism sites (SNPs), which shows high polymorphism. The sequences generated using ITS rDNA marker were closely matched with GenBank sequences of ITS (accession nos. KP670933, KP670934 and KP670932) of Morchella crassipes GenBank sequences (Accession no: KY402197 100% or 877/877bp identity with 100% Query cover, GQ228463 and GQ228461 99% or 876/877bp with 100% Query cover respectively). The results were also confirmed with phylogenetic analysis (3.7). The analysis involved total of 24 ITS rDNA sequences which were retrieved from GenBank.

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Fig. 3.7: Phylogram of M. crassipes obtained from ITS sequences and is based on Neighbor- Joining method. The bootstrap values are shown on the branch of each node.

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3.4 Morchella elata Fr.: Fr. in Fries, Syst Mycol 2:8. 1822.

ASCOMATA 25–55 mm tall, 13–18 mm wide, hymenophore 15×43 mm, ovoid, sub- conical, primary ridges (30-55) and narrow pits are present, i.e. these are dark brown to black with verticals elongated ridges and narrow polygonal ridges. Sterile ribs 11.5–20.14 mm tall, longitudinally arranged, 5–9.1 mm wide, tough, thin, anastomosing to form long hymenial rounded pits network. Hymenial Pits, yellow gray, light gray, blackish gray, silvery gray while at primary stage when young becoming light yellow brown, blackish-light blackish at maturity. Fruiting body or Ascocarp margin Sample connecting straight into the stalk (Stipe) with no sharp bend.

STALK 10–50 mm length, 6–8 mm wide, soft and hollow thin and straight towards the base, surface light yellowish white, glabrous. Context present, 0.5–1.2 mm thin in the hollow hymenophore, creamy, with sterile inner surface.

ASCOSPORES (90-130μm) smooth, elliptical with homogeneous contents. Asci 8-spored, 26 μm length and 15.3μm wide (L/W=1.69) and 15.31–225 × 17–24 μm, hyaline, cylindrical.

PARAPHYSES 90 -260μm sub-clavate or cylindrical, apices rounded or subacute, with 2–3- septate, hyaline or with brownish homogeneous content (Figure 3.8).

Material observed: Pakistan, Azad Jammu Kashmir, Athmuqam Tehsil, 34°35'17.99"N and 73°54'39.59"E, Altitude 4720ft, under Pinus wallichiana 21 April 2014, Coll. Mr. Hussain Badshah (Mr4and KM3).

Ecology and Distribution: M. elata occurs on organic rich well exhausted moist soil under shade of forest trees like Pinus wallichiana.

Distinguishing features: Ascomata longer than stalk, stalk soft thin straight towards the base, surface light yellowish white, glabrous. Elongated paraphyses, 90 -260 μm, spore L/W= 1.69, mostly grows in the lesser Himalayan range in late April.

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Fig. 3.8: Morchella elata A. filed photo, B. Asci with spore, Paraphyses

Molecular characterization and phylogenetic analysis of Morchella elata

The ITS marker was amplified for isolate Mr.8 (accession no. KP670928), The ITS locus amplification revealed 727 bp read. After alignment of the query sequences with closely related reference sequences the whole alignment set revealed indels on 54, 73, 455, 473, 478, 55, 573, 577, 671, 683, 727 nucleotide sites respectively. The stretches also showed 15 single nucleotide polymorphism sites (SNPs). Similarly, the sequence comparison revealed single nucleotide polymorphism which is evident in the phylogram (Fig 3.9) in which all species have been clustered in distinct clades. The sequences generated using ITS rDNA marker were closely matched with top GenBank sequences such as our ITS sequence Mr.8 (accession no. KP670928) matched with top two Morchella elata GenBank sequences (Accession no: GQ228470, 718, 725(99%) bp identity with 99% Query cover and with EF080996 718,726 (99%) bp identity with 99% Query cover respectively), Mr.4 (accession no. KP670930) matched with GQ228470658,716 (92%) bp identity and EF080996 658,716 (92%) bp identity with 98% Query cover respectively), Mr.22 (KP670929) matched with (accession nos. GQ228470718,723 (99%) bp identity with 99% Query cover and with EF080996 718,723 (99%) bp identity with 99% Query cover respectively), Mr.36 (accession no. KP670931)

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matched with GQ228470 (691, 697 (99%) bp identity with 91% Query cover), Mr.24 (accession no. KP670930) matched with GQ228470 (541, 551 (98%) bp

Fig. 3.9: Phylogram of Morchella elata obtained from ITS sequences and is based on Neighbor-Joining method. The bootstrap values are shown on the branch of each node.

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3.5 Morchella eximia Boud., Icon Mycol, expl pl. 6, pl. 208. 1909.

ASCOMATA 55–60 mm tall, 45 mm wide, hymenophore 45× 60 mm, conical, pitted, pits short and elongated, ridged conical and ovoid pitted, pits elongated, ridged (regular and irregular shape), primary ridges (n = 36) 2.2–5.1 mm long, pitted (n = 50–65) with 3.1 mm.

STALK 25–30 mm hollow, yellowish, no sharp bends, and regular rays are present in base with middle depression.

ASCOSPORE 110-170 µm, smooth, ASCI8-spored, elliptical 17.4 µm long and 9.2 µm wide, the average length (Me) and length to width (Qm) ratio is 1.92 µm.

PARAPHYSES 90-226 µm, cylindrical, apices ovoid, mostly subacute, rarely acute, hyphal elements on sterile ridges 73–170 × 7–24 µm. Context are present, 0.7–1.3 µm, hollow hymenophore (Figure 3.10).

Material observed: Pakistan, Province, Khyber Pakhtunkhwa, District Swat, Malam Jabba, 34°47'54.44"N 72°34'33.37"E, altitude 1800 m, under Pinus wallichii and Abies pindrow. April 2014, Coll. Hussain Badshah, Maqsood afridi & S.A Shah

Ecology and distribution:

Higher altitudinal regions characterized by sandy soil or clay and covered by dead organic matter.

Distinguishing features: Polymorphic ascomata, brown and blackish, Stalk absent of sharp bends, spore L/W= 1.92, Paraphyses cylindrical, apices ovoid, higher mountain range in burnt ground.

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Fig 3.10: A. fruiting body of Morchella eximia B. Asci with spore and Paraphyses

Molecular characterization and phylogenetic analysis of Morchella eximia

The ITS locus amplification revealed 427 bp, the sequence alignment with other related similar sequences were aligned which demonstrated indels on 187, 258, 280, 362, 390, 427 nucleotide sites respectively. The stretches also showed 75 single nucleotide polymorphism sites (SNPs). Phylogram was clearly bifurcated into two main clades (Clade I and Clade II). In clade I, seven Morchella species were clustered together while four were grouped in clade II (Fig 3.11) The studied Morchella eximia (H13) showed higher homology with already reported Morchella eximia (KM587970.1) with 49 bootstrap values. The clustering of Morchella eximia together supports that ITS. Likewise, two Morchella septimelata species also showed higher resemblance with each other by 93 bootstrap values. In clade II, three Morchella species (KM587986.1, KM587991.1 and JQ723045.1) grouped together with bootstrap values of 52. Morchella septimelata (JQ723041.1) act as outgroup in this tree. This analysis showed a highly polymorphic nature for the present collection.

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49 H13_ITS Sample_NameH13 23 KM587970.1 Morchella eximia 12 AJ698475.1 Morchella esculenta JQ618567.1 Morchella septimelata Clade I 14 93 JQ618755.1 Morchella septimelata KM485946.1 Morchella importuna JQ723040.1 Morchella septimelata KM587986.1 Morchella eximia Clade II KM587991.1 Morchella sextelata 52 JQ723045.1 Morchella sp. JQ723041.1 Morchella septimelata

1

Fig. 3.11: Phylogram of Morchella eximia obtained from ITS sequences and is based on Neighbor-Joining method. The bootstrap values are shown on the branch of each node.

3.6 Morchella eohespera Beug. Beug, Voitk & O’Donnell, sp. nov. FIG A-D

ASCOMATA 40–95 mm tall, 32–65 mm wide hymenophore 29 × 55 mm, ovate to conical, longitudinal ridged with narrow pitted attached to stipe sulcus size 2.2 to 4.1mm deep and 3.1 to 4mm wide, primary regular ridges 10-22 some shorter, less horizontal secondary ridges are black to yellowish. A small transecting ridge and elongated primary vertical pits are present. Although in some ascomata, pits are broader than tall; marvelously tomentose; “pale olive buff (POB)” to “orange citrine (OC)” primarily, in stage “snuff brown (SB)”, after dried “pale pinkish buff (PPB).

STALK hollow with white granules. 20–44 mm tall, 17–32 mm wide, cylindrical to tapering downward, sometimes sub-clavate with a few folds at the base, hollow, upper portion 3–4mm thick.

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ASCOSPORE 10–14 µm, ridges approximately sub-capitate to capitate elements. ASI-8 spores, 18.2 µm length, 13 µm wide, with L/W=. 1.4, ellipsoidal, with ascospore 80 µm, hyaline, content homogeneous.

PARAPHYSIS 90- 162 µm long and 4 to 6 µm wide at base position, typically expanding to 7.5 to 11.5 µm apex position, Elements on the sterile ridges are observed, 120 to 170 µm, straight to marginally sinuous, clavate, lanceolate, capitate or sub capitate, septate with a 60– 90 µm long terminal. Cell filled with rough material (Figure 3.12).

MATERIAL EXAMINED: PAKISTAN, Gilgit Baltistan Province: District Skardu: 35°18'21.55"N 75°36'51.14 E, Coll. Hussain Badshah June 2014.

Ecology and Distribution: Engelmann spruce, P. mariana, Amelanchier, Corylus, Salix, and calcareous, moistly sandy soil, is observed to support highly the growth of M. eohespera.

Distinguishing feature: fruiting body: large in size, blackish and yellow, spore ellipsoidal, length and wide ratio 1.4, cell filled with rough material, pale pinkish, stalk cylindrical to tapering downward and few folds at the base.

Fig.3.12: Morchella eohespera. A. Field photo, Paraphyses. C. Spore, D. Ascus base

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Molecular characterization and phylogenetic analysis of Morchella eohespera

The translation elongation factor (TEF) was amplified for isolate (Mr1) with sequence length 558 bp, the sequence alignment with other related similar sequences showed on indels at 178, 254, 352, 374, 388, 456, 472, 492, 558. The stretches also showed 5 single nucleotide polymorphism sites (SNPs). The present collection thus shows highly polymorphism. The sequences generated using TEF marker were closely matched with top GenBank sequences such as our TEF sequence Mr.1 matched with GenBank sequences (KT819379,557,558 (99%) bp identity with 100% Query cover, with JQ321846 (Mel-19), 557,558 (99%) bp identity, with JN085083, 557,558 (99%) identity, with GU551566, 557,558 (99%) bp identity) (Fig 3.13). This analysis showed a highly polymorphic nature for the present collection. The NCBI blast analysis revealed sequence matches TEF.

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Fig. 3.13: Phylogram of M. eohespera obtained from TEF sequences and is based on Neighbor-Joining method. The bootstrap values are shown on the branch of each node. Morchella elata as chosen out-group.

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Table 3.1: Morphological characters of Morchella species and their composition

Species Associated Ascomata Primary Sterile Ascocarp Stipe Hollow Stipe Spore Spore Paraphyses Altitudes Location name species size Mm ridges Ribs margin size L/W shape size .Mm Mm µm Shape- Ft / m Colour , Qm µm ratio

M. S. 55–70 20-35 15.5– Straight 22– Elliptical 52–130 × 906 Charsadda galilaea officinarum, 26.14 36 8–12 Conic Cylindrical- 1.4 /cylindrical Yellowish

T.aestivum M. Pinus 50-40 60- 70× 10.5- Over 18- ellipsoid 1800 Malam pulchella wallichiana 52- 56 18 groove 30 Jabba Brown nature All are in hollow Convex- 1.5 200-260 yellow

M. Pinus 30 - 50 29-59 12.5– straight 8 – 1.35 ellipsoid 90- 150 2100 Murree crassipes wallichiana 20.24 10 Conical Cylindrical/ gloubrios, white M. elata Pinus 25–55 30-55 11.5– straight 10– 1.62 cylindrical 90- 260 4720 Azad roxburghii, 20.14 50 Kashmir Ovoid Black

M. Pinus 55-60 36-65. 73- Simple 25- 1.92 elliptical 90 - 226 1800 Malam eximia pinaster, 170× 30 Jabba 7- 24 PinusNigra Ovoid Yellowish /.conical M. Engelmann 40 -95 20-35 60- Simple 20 – 1.4 ellipsoid 90-162× 6- 2226 Skardu eohsepra spruce, P. 187 44 4 mariana Ovate / white conical

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Section B: Biological Activities 3.7 Antibacterial activity

The results revealed that at 100 mg/ml concentration of methanolic extract, both Morchella species exhibited strong antibacterial activity against Bacillus subtilis (Figures 3.14-3.15, 3.16 and 3.17). Nevertheless, maximum (36.4mm) inhibitory activity against Enterobactor aerogenes was possessed by methanolic extract of Morchella galilaea followed by Morchella eximia (29.16mm). Further at (100mg/ml) concentration, the methanolic extracts prepared from fruiting bodies of M. eximia exhibited the strong inhibitory effects against Klebsiella pneumoniae (MTCC618). The M. galilaea showed the maximum antibacterial activity against the Staphylococcus aureus (32.2 mm). The ranking of M. eximia and M. galilaea for their antibacterial activity against Bacillus subtilis were as follow: M. eximia > M. galilaea. Maximum antibacterial activity against Enterobacter aerogenosa was exhibited by the fruiting body of M. galilaea.

Moreover, the inhibitory effects of M. eximia against Enterobacter aerogenes were less effective as compare other extracts. The sequence of selected Morchella species for antibacterial against was as: M. galilaea > M. eximia.

Similarly, at 50mg/ml concentration, the methanolic extracts prepared from fruiting bodies of M. galilaea exhibited the strong inhibitory effects (27.8mm) against Staphylococcus aureus. Whereas, M. eximia revealed the prevailing inhibitory activity against Enterobacter aerogenes (27.3mm) and Staphylococcus aureus (26.2mm). While M. galilaea revealed (25.2mm) zone of inhibition against Staphylococcus aureus Klebsiella pneumoniae. Maximum antibacterial activity (23.4 mm) against Bacillus subtilis was exhibited by M. galilaea followed by M. eximia with (23.0 mm) respectively. M. galilaea possessed 22.3mm zone of inhibition against Enterobacter aerogenes. Similarly, the same concentration (30mg/ml) of methanolic extract prepared from fruiting body of M. eximia exhibited 24.3mm zone of inhibition against Staphylococcus aureus. The M. galilaea showed (24mm) zone of inhibition against Klebsiella pneumoniae. Further that it was found that at 30mg/ml the methanolic extract of M. galilaea and M. eximia showed the similar result against both Enterobacter aerogenes and Klebsiella pneumoniae respectively. Similarly, the ethanolic extract of M. galilaea and M. eximia at 100mg/ml concentration shown similar antibacterial activity against Klebsiella pneumoniae and Staphylococcus aureus. The M. eximia revealed strong antibacterial effects (27.4mm) against Staphylococcus aureus and maximum antibacterial activity

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is shown against Klebsiella pneumoniae by M. galilaea (30.4mm). Similarly, the maximum (24.8 and 24.2mm) inhibitory activity against Enterobacter aerogenes and Bacillus subtilis was possessed by ethanolic extract (100mg/ml) of M. eximia. The results further revealed that maximum antibacterial activity against Enterobacter aerogenes and Bacillus subtilis was exhibited by ethanolic extracts prepared from fruiting body of M. galilaea.

The ranking of M. eximia and M. galilaea for their antibacterial activity against Klebsiella pneumoniae and Staphylococcus aureus were as follow: M. galilaea > M. eximia respectively.

Further the antibacterial activity of ethanolic extract concentration at 100 mg /ml isolated from fruiting body of M. eximia and M. galilaea showed (30.1mm and 29.2 mm) zone of inhibitions against Klebsiella pneumoniae respectively. The maximum zone of inhibitions was (29.2 and 27.2mm) against Staphylococcus aureus by M. eximia and M. galilaea. Over all the ranking of M. galilaea for their four antibacterial activity strains were Klebsiella pneumoniae > Staphylococcus aureus >Bacillus subtilis > Enterobacter aerogenes respectively.

The results further revealed maximum antibacterial activity (24.8mm and 24.2 mm) against Enterobacter aerogenes and Bacillus subtilis was exhibited by ethanolic extracts prepared from fruiting body of M. eximia. The antibacterial activity of ethanolic extract at concentration 50 mg /ml isolated from fruiting body of M. eximia and M. galilaea showed (24.1 mm) zone of inhibitions against Staphylococcus aureus. Maximum (23.2mm) growth inhibitory activity against Klebsiella pneumoniae was exhibited by M. galilaea. Both M. eximia and M. galilaea produced (21.5mm and 21.2 mm) zone of inhibition against Enterobactor aerogenes respectively. The M. eximia (19.1mm) indicated less growth inhibitory effect against Staphylococcus aureus.

Similarly, the ethanolic extract (30mg/ml) of M. eximia and M. galilaea showed highly effective (19.4 mm) against Staphylococcus aureus and Bacillus subtilis respectively. M. galilaea produced (20.1 mm) zone of inhibition against Klebsiella pneumoniae. The results revealed that maximum inhibitory zone (18.7mm and 18.6mm) against Staphylococcus aureus and Bacillus subtilis was produced by M. eximia as compared to M. galilaea.

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M. galilaea 40 Staphylococcus aureus Bacillus subtilis 30 Klebsiella pneumoniae Enterobactor aerogenes 20

(mm) ZOI 10

0

Fig 3.14: Antibacterial activity for methanol and ethanol extracts (100, 50, 30mg/ml) of M. galilaea collected from the higher mountainous region and plain irrigated lower area. Antibiotic streptomycin was used as positive control while pure DMSO was used as negative control. The data has been presented as mean+/- SE (standard error bars).

M. eximia

40 Staphylococcus aureus Bacillus subtilis 30 Klebsiella pneumoniae Enterobactor aerogenes 20

ZOI (mm) ZOI 10

0

Fig. 3.15: Antibacterial activity for methanol and ethanol extracts (100, 50, 30mg/ml) of M. eximia collected from the higher mountainous region and plain irrigated lower area. Antibiotic streptomycin was used as positive control while pure DMSO was used as negative control. The data has been presented as mean+/- SE (standard error bars).

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Fig 3.16: (1-4) The antibacterial activity of M. galilaea, methanolic extract at (100 mg/ml) against K. pnumonieae, enterobactor aerogenes, Staphylococcus aureus, Bacillus subtilis.

Fig. 3.17 (5-6). The antibacterial activity of M. galilaea and M. eximia, methanol extract at (100 mg/ml) against Bacillus subtilis, Enterobactor aerogenes (7-8) Staphylococcus aureus, K. pnumonieae. (9-10) Staphylococcus aureus, K. pnumonieae (ethanolic extract)

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3.8 Antioxidant activity (DPPH free radical scavenging Assay) of M. eximia and M. galilaea

Results presented in (Table 3.2) shown that polyphenols isolated from M. galilaea exhibited dose dependent activity at 1 mg/ml (76.7 %) and 28.5 % at 0.01 mg/ml. For lipids maximum scavenging activity was recorded at 3 mg/ml (58.40%) while minimal activity (20.16%) at 0.01mg/ml. Polysaccharides of M. galilaea scavenged 34.92% DPPH radical at 1.7 mg/ml and 11. 48% at 0.01 mg/ml. The methanol extract of M. galilaea showed maximum scavenging effect (70.79 %) at 1 mg/ml and minimum scavenging effect at 0.01 mg/ml (5.98%).

Similarly, polyphenols of M. eximia exhibited 75.3% scavenging potential at 1 mg/ml while lipids showed 78.5% at 2 mg/ml. The crude polysaccharides of M. eximia showed maximum scavenging effect at 1.7 mg/ml (31.6%) and low at 0.03 mg/ml (13.6%). The maximal free radical scavenging effect of methanol extract of M. eximia was 72.64% at 1 mg/ml. It was observed that polyphenols of M. galilaea exhibited higher antioxidant capacity as compared to lipids and crude polysaccharides. However, scavenging effect of lipids was higher in M. eximia than M. galilaea.

The IC50 value was summarized in the (Figur3.19). The DPPH assay can be assessed by the resolve of the IC50 values, which correspond to the quantity of different extract required to scavenge 50% of the DPPH Free radicals. High IC50 values revealed the low antioxidant activity, whereas the low IC50 values showed the high antioxidant activity.

The low IC50 value was recorded for polyphenols of the M. eximia (0.2738 mg/ml) and M. galilaea (0.2958 mg/ml) respectively. The IC50 value of lipids from M. eximia was 0.4657 mg/ml and from M. galilaea 5.413 mg/ml. The IC50 value of crude polysaccharides of M. eximia (24.73 mg/ml) was higher than that of M. galilaea (11.96 mg/ml). Similarly, methanol extract of M. eximia and M. galilaea has IC50 values of 0.5157 mg/ml and 0.5899 mg/ml respectively. The commercial standard ascorbic acid showed highest scavenging potential at 15 mg /ml (83.2 %) and lowest (5.77%) with IC50 (10.22 mg /ml) (Fig 3.19).

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IC50 Values of DPPH assay

24.73

11.96 10.22

5.413

0.5157 0.2738 0.4657 0.5899 0.2958

Methanolic Polyphenol Lipids Crude Methanolic polyphenol Lipids Crude Ascorbic acid extract ployscahride extract ployscahride M.eximia M.galilaea

Fig. 3.18: Antioxidant activity of fruiting bodies of M. eximia and M. galilaea. The half maximal inhibitory concentration (IC50) for both the species were compared.

DPPH radical scavenging activity tests

Fig.3.19: Standard ascorbic acid measured as DPPH radical scavenging activity

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Table 3.2: Antioxidant activity of M. galilaea and M. eximia

Concentration

mg/ml DPPH % inhibition of free radicals M. galilaea M. eximia

Polyphenols Lipids Crude methanolic Crude Lipids Polyphenols methanolic polysacch extract polysaccha extract aride ride

3 - 58.40 ------2 - 49.03 - - - 78.5 - - 1.7 - 41.67 34.92 - 31.6 73.1 - - 1.5 - 39.20 28.38 - 30.3 68.5 - - 1.3 - 40.75 29.90 - 24.5 61.7 - - 1.1 - 37.59 35.62 - 24.5 56.9 - - 1 76.7 36.68 28.14 70.79 23.1 59.4 75.3 72.64 0.8 64.4 34.24 26.09 57.08 26.0 52.1 72.2 62.74 0.7 58.5 29.55 24.60 55.44 17.0 48.1 56.8 45.26 0.5 53.0 27.25 22.67 39.87 21.5 41.9 48.6 54.39 0. 3 42.6 20.60 23.58 27.86 -* 36.3 45.1 32.72 0.2 37.2 -* 16.92 22.8 9.1 30.7 39.3 24.49 0.1 34.9 24.02 14.72 20.1 10.5 - 37.1 25.1 0.08 33.8 18.34 12.92 16.7 12.3 31.0 35.6 17.89 0.07 32.7 25.70 11.33 13.06 11.4 26.1 32.8 16.47 0.05 31.6 22.25 9.56 10.24 14.1 25.7 31.7 13.53 0.03 29.4 25.26 10.89 7.89 13.6 26.9 29.7 12.35 0.02 28.8 20.60 9.07 6.7 -* 24.6 30.1 10.03 0.01 28.5 20.16 11.48 5.98 -* 23.2 24.7 8.82

(-*) No results at this concentration

(-) activity was not performed at this concentration as required prime results remained signify beforehand range.

The antioxidant activities of both species have been summarized (Table 3.8.1) to observe and profoundly examine the optimum results, a wide range of extract concentration were utilized i.e. 0.01mg/ml to 3mg/ml. Polyphenols and methanolic extract concentration ranging from 0.01mg/mg to 1mg/ml were observed for M. galilaea and M. eximia respectively and both

demonstrated virtuous activity in ascending order. As we got the required results at 1mg/ml concentration that’s why no further concentration range were checked for them.

The Crude polysaccharide were observed with concentration grades from 10mg/ml to 1.7mg/ml for both specimen and further examination was not performed due to obtain required results with the concentration range of 1.7mg/ml. In case of M. eximia, activity was started at 3mg/ml concentration and also noticed no results at 0.03mg/ml concentration. The concentration range were 0.01mg/ml to 3mg/ml for Lipids using M. galilaea and 0.01mg/ml to 2mg/ml for M. eximia

3.9 Anticancer/anti-tumor potentials of M. galilaea and M. eximia assessed on MCF-7

The results summarized in (Fig 3.20, 3.21) revealed anticancer effects of methanolic extract and crude polysaccharides of M. galilaea and M. eximia on MCF-7 (human breast cancer cell line). A study was first performed to find their potentials on concentration (0-100) ug/ml. Such result showed that the methanolic extract of M. galilaea and M. eximia against MCF-7 cell line revealed the most promising anti proliferative effects at 50-100 ug/ml. The Fig. 3.22, 3.23 additionally, summarized the commercial anticancer drugs Nilotinib revealed superior anticancer activity (3.87 g/ml), as compared to extract of M. galilaea and M. eximia have the IC50% (25.69 and 39.97g/ml) respectively. Furthermore, the methanolic extract of M. eximia and M. galilaea at (100µg/ml) have effective potential to inducing (77.1-78.9 %) growth reduction in MCF-7 cancer cell line respectively. Whereas, methanol extract of M. eximia and M. galilaea (50) µg/ml have strong potential to inducing (62.1-64 %) growth reduction in MCF-7 respectively. Moreover, crude polysaccharides of M. galilaea and M. eximia (0-100) µg/ml was used against cancer cell line MCF-7, this revealed adverse result. The crude polysaccharide had no serious effects on inhibiting the growth rate of MCF-7 cancer cell line. Therefore, the result concluded that the methanolic extract of M. eximia, and M. galilaea have good anticancer potential.

Anti-tumor activity of extract of M. galilaea against human breast cancer cell line MCF-7. 150

100

50

Cellviabililty (%)

0 CTR 0.1 0.5 1 5 10 50 100 concentration ug/ml

Fig. 3.20: Effect of methanolic extract of M. galilaea against MCF-7 human breast cancer cell line. Standard error and mean values were analyzed through GraphPad Prism 5.

Anti-tumor activity of crude polysaccharide of M. galilaea against human breast cancer cell line MCF-7. 200

150

100

50

Cellviabililty (%)

0 CTR 0.1 0.5 1 5 10 50 100 concentration ug/ml

Fig. 3.21: Effect of crude polysaccharide of M. galilaea against MCF-7 human breast cancer cell line. Data were analyzed by using GraphPad Prism 5.

Anti-tumor activity of extract of M. eximia against human breast cancer cell line MCF-7. 200

150

100

50

Cellviabililty (%)

0 CTR 0.1 0.5 1 5 10 50 100 concentration ug/ml

Fig. 3.22: Effect of M. eximia methanolic extract against MCF-7 human breast cancer cell line. Standard error and mean values were analyzed through GraphPad Prism 5.

Anti-tumor activity of crude polysaccharide of M. eximia against human breast cancer cell line MCF-7. 200

150

100

50

Cellviabililty (%)

0 CTR 0.1 0.5 1 5 10 50 100 concentration ug/ml

Fig. 3.23: Effect of crude polysaccharide of M. eximia against MCF-7 human breast cancer cell line. Standard error and mean values were analyzed through GraphPad Prism 5.

Fig. 3.24: IC50 methanolic extract of M. galilaea and M. eximia (25.69, 39.97 ug/ml) respectively

Nilotinib against MCF-7 cancer cell line

1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3

CellViability % 0.2 0.1 0.0 0 5 10 25 50 100 Concentration (ug/ml)

Fig 3.25: Nilotinib the commercial cancer drugs (use as standard) against MCF-7 cancer cell line Standard error and mean values were analyzed through GraphPad Prism 5.

3.10 Phosphodiesterase inhibitory activity of M. galilaea and M. eximia

The results observed as % activity change compared to control are presented in (Table.3.3). During present investigation the values obtained for this assay showed an activation of the Morchella galilaea and Morchella eximia ranging from 996,162 CPM up to 1,872,947 CPM and with a % change of 346% to 650% respectively above the control values.

3.10.1 Drug Sildenafil (Viagra)

Sildenafil (also sold as the brand name Viagra) is a drug used to treat erectile dysfunction and pulmonary arterial hypertension. Results that’s are summarized in (Table 3.4) shown us that this drug was tested at 10µM against the two phosphodiesterases, with the results showing an inhibition of PDE5 at -64% and a nominal activation (essentially no change) of +3% with PDE6.

Table 3.3: Effect of methanolic extract Morchella galilaea on PDE5 and PDE6 phosphodiesterase activity

Phosphodiesterase Control (CPM) Morchella galilaea (CPM) Difference % CRC

PDE5 288,204 1,284,366 996,162 346%

PDE6 241,445 1,364,244 1,122,799 465% Morchella eximia (CPM) PDE5 288,204 2,161,151 1,872,947 650%

PDE6 241,445 1,717,169 1,475,724 611%

Table 3.4: Effect of the drug Sildenafil on PDE5 and PDE6 phosphodiesterase activity

Phosphodiesterase Control (CPM) Sildenafil (CPM) Difference % CRC

PDE5 288,204 103,730 (184,475) -64% PDE6 241,445 248,513 7,068 3%

Section C: Nutritional Aspects in Morchella

3.11 Amino acid analysis of M. galilaea and M. eximia

Amino acid composition of the dried fruiting body of M. eximia and M. galilaea is summarized in (Table: 3.5). The amount of amino acids in M. galilee was (43.55 g/100g). Whereas the total amount of amino acids in M. eximia was (41.25 g/100g). The results showed that a total seventeen amino acids were reported in M. galilaea and M. eximia. It was found that M. galilaea possessed amino acids leucine (9.05g/100g), lysine (7.176g/100g), valine, (6.2g/100g), phenylalanine (5.24g/100g), threonine (3.74g/100g), isoleucine (5.6g /100g, arginine 5.15 g/100g), tyrosine (3.41g/100g) in high amounts whereas methionine (1.88g/100g) and histidine (2.56 g/100g) were present in low amounts. The non-essential amino acid glutamic acid (21.74 g/100g), aspartic acid (9.7 g/100g), Proline, (5.91g/100g), glycine (3.22g/100 g), serine (4.3 g/100g) and alanine (3.93g/100g), were found in high quantity. The content of cysteine (0.51g/100g) was low

The amino acid composition of the dried fruiting body of M. eximia was leucine (9.04 g/100g), lysine (7.08 g /100g), valine, (6.32 g/100g), phenylalanine (5.18 g/100g), threonine (3.87 g/100g), isoleucine (5.66 g/100g) and arginine (5.46 g/100g). Histadine (2.55 g/100g), tyrosine (2.59g/100g), methionine (2.01 g/100g) were present in low amounts. The non-essential amino acid glutamic acid (22.45g/100g), aspartic acid (9.78 g/100g), Proline, (6.07 g/100g), glycine (3.15 g/100g), serine (4.35 g/100g) and alanine (3.93g/100g), were found in high quantity. The content of cysteine (0.50g/100g) was low. The determination of biological value of amino-acid by Kuhnau (1949) procedure of rating of protein using over all sum of the percentage of essential amino acid. The table (Table: 3.6). summarized the amino acid score of M. galilaea that was found to be (83.36 and 94.70 g/100g). The comparison of nutritive value of M. galilaea with human milk was calculated. Using human milk as a reference standard, the M. galilaea contained total value of (81.41 g/100g), pure value (84.61 g/100g) and supplementary value of the amino acid as (3.28 g/100g) respectively. The amino acid score of M. eximia was found to be (58.63 and 94.70 g/100g). The M. eximia contained the total value of amino acid (87.92 g/100g), pure value (94.59 g/100g) and supplemental value of the amino acid (3.72 g/100g) respectively (Table: 3.7). The

supplementary data of amino acid profiling such as minimum, maximum, means, Standard error and their relative figure were summarized in Appendix 1.

Table 3.5: Amino acid profile of Morchella galilaea and Morchella eximia. The t-test description showed non-significant values between Amino acid profile of both species.

Amino acid gAA/100g Percentage composition gAA/100g Percentage composition

M.galilaea M. eximia Glutamic acid 9.47062 21.74 9.261 22.45 Serine 1.87324 4.30 1.79561 4.35 Histidine 1.11799 2.57 1.05011 2.55 Glycine 1.40436 3.22 1.30045 3.15 Threonine 1.64984 3.79 1.59748 3.87 Arginine 2.40152 5.51 2.25267 5.46 Alanine 1.71359 3.93 1.6203 3.93 Tyrosine 1.48611 3.41 1.0677 2.59 Cysteine 2.23 0.51 0.208207 0.50 Valine 2.73642 6.28 2.60731 6.32 Methionine 8.23 1.89 0.830833 2.01 Phenylalanine 2.28239 5.24 2.13695 5.18 Isuleucine 2.4665 5.66 2.33447 5.66 Leucine 3.94447 9.06 3.72977 9.04 Lysine 3.12584 7.18 2.92114 7.08 Proline 2.57553 5.91 2.50536 6.07 Aspartic acid 4.26065 9.78 4.03303 9.78 Total amino-acid 43.55507 - 41.25239 - aEAA7(Total AA)*100 39.09632794 - 39.16851626 - EAA10(Total AA)*100 60.90373176 - 60.8314595 -

aEAA10; lysine, threonine, tryptophan, valine, histidine, methionine, arginine, isoleucine, leucine, phenylalanitie.

EAA7; non -essential amino acids; Aspartic acid, Proline, tyrosine, alanine, Glycine, Glutamic acid, serine

A

B

Fig. 3.26: Typical chromatograms of amino acids determined by high performance Amino Acid Analyzer. A. Morchella galilaea, B Morchella eximia

Table 3.6: in vitro calculation of amino acids score of M. galilaea and M. eximia based on composition of essential amino acids

Amino acid FAO references M. galilaea M. eximia Amino-acid Amino-acids protein score* score*

M. galilaea M. eximia Leucine 7.04 9.04 9.041 128.37 128.43 Lysine 5.44 7.18 7.081 131.93 130.17 Threonine 4 3.79 3.788 94.70 94.70 Methionine 3.52 2.93 2.064 83.36 58.64

+Cysteine Phenylalanine 6.08 8.65 7.666 142.30 126.09

+tyrosine Valine 4.96 6.28 6.320 126.67 127.43 Isuleucine 4 5.66 5.659 141.57 141.47

*The ratio of the amount of each (EAA) in food protein to the amount of the respective amino acid in the same quantity of FAO reference protein.

Table 3.7: Nutritive value calculation of M. galilaea and M. eximia by the methods of Kuhnau, (1949)

Amino acids Human Milk M.galilaea Reference protein Reference reference M.eximia of M.galilaea protein M.eximia protein Leucine + 14.60 14.70 14.7 0.0 0.0 isoleucine Lysine 6.60 7.2 7.1 3.5 0.0 Threonine 1.60 3.8 3.8 0.0 0.0 Methionine +cyc-s 4.00 2.4 2.1 3.0 1.9 Phenylalanine 9.50 8.6 7.7 8.6 1.8 +tyrosine Valine 6.30 6.3 6.3 6.3 0.0 sum 42.60 42.9 41.60 - -

Pure value [(43.55-8.6)/42.93)] *100 = 81.41

Supplementary value (84.69- 81.40945) = 3.28

Pure value [(41.25-1.9)/41.60)]*100 = 94.51

Supplementary value (87.92- 84.2) =3.72

Section D: Nuclear Magnetic Resonance spectroscopy (NMR) of Morchella

3.12 Nuclear Magnetic Resonance spectrum of analysis of Morchella galilaea

Nuclear Magnetic Resonance spectroscopy (300 Htz, NMR) spectroscopy was used for structural elucidation tool to identify the protons in the isolated compounds. 1HNMR can differentiate easily between the aliphatic and aromatic protons according to the chemical shift () for the assigned protons. That was initial screening to assess the medicinal potential of selected Morchella species. Therefore, the determination of different functional group was studied, which is the preliminary step in medicine isolation and characterization, we have performed this assay and used this information for further evaluation.

The 1H NMR, 13C NMR, and DEPT spectra of the purified polyphenolic derivative (1) isolated from Morchella galilaea are represented in (Fig 3.28, A-C1).

Compound 1 (polyphenolic derivative) was isolated as white amorphous powder whose aliphatic region of the 1H-NMR (Figure 3.28 A) showed two multiplets at δ 0.83-0.87 and 1.95-2.05, pentet at δ 1.47, two triplets at δ 2.17 and 2.73, and a singlet peak at δ 2.08. A characteristic multiplet was identified at the vinylic or aromatic region at δ 5.25-5.39.

The assigned aliphatic and vinylic protons were confirmed through 13C NMR, and DEPT spectra as shown in (Figure 3.28. B and C, respectively). The carbon atoms carrying aliphatic protons were detected in the up field region of 13C NMR at δ 14.37, 22.42, 22.55, 24.95, 25.67, 27.06, 29.00, 29.18, 29.45, 31.14, 31.36, 31.74, and 34.14. These carbon atoms were further 13 attested by C DEPT spectrum as CH3 for the peak at δ 14.37, and as CH2 for the peaks at δ 22.43, 22.56, 24.95, 25.67, 27.07, 29.01, 29.19, 29.46, 31.36, 31.76, and 34.14, respectively). The two characteristic 13C NMR peaks at δ 128.20 and 130.16 were attested for a p-substituted benzene ring which were confirmed by 13C DEPT at δ 128.21 and 130.09 in the downfield region. Furthermore, a carbonyl group was detected by 13C NMR peak at δ 174.93.

Further analysis by 1HNMR (300 MHz), 1H NMR (300 MHz, DMSO) δ 5.25-5.39 (m, 3H), 2.73 (t, 1H, J = 6.0 Hz), 2.17 (t, 2H, J = 7.5Hz), 2.08 (s, 1H), 1.95-2.05 (m, 3H), 1.47 (p, 2H, J = 6.0 Hz), 1.10-1.40 (m, 15H), 0.85 (t, 3H), 13C NMR (300 MHz, DMSO) δ 174.93 (1C, C=O), 130.16

(1C, Ar-CH), 128.20 (1C, Ar-CH), 34.14 (1C, Aliphatic-C, CH2), 31.74 (1C, Aliphatic-C, CH2),

31.36 (1C, Aliphatic-C, CH2), 31.14 (1C, Aliphatic-C, CH2), 29.45 (1C, Aliphatic-C, CH2), 29.18

(1C, Aliphatic-C, CH2), 29.00 (1C, Aliphatic-C, CH2), 27.06 (1C, Aliphatic-C, CH2), 25.67 (1C,

Aliphatic-C, CH2), 24.95 (1C, Aliphatic-C, CH2), 22.55 (1C, Aliphatic-C, CH2), 22.42 (1C, 135 Aliphatic-C, CH2), 14.37 (1C, Aliphatic-C, CH3). DEPT C (125 MHz, DMSO) δ 130.16 (1C,

Ar-CH), 128.20 (1C, Ar-CH), 34.14 (1C, aliphatic-C, CH2), 31.76 (1C, aliphatic-C, CH2), 31.36

(1C, aliphatic-C, CH2), 29.46 (1C, aliphatic-C, CH2), 29.19 (1C, aliphatic-C, CH2), 29.01 (1C, aliphatic-C, CH2), 27.07 (1C, aliphatic-C, CH2), 25.67 (1C, aliphatic-C, CH2), 24.95 (1C, aliphatic-C, CH2), 22.56 (1C, aliphatic-C, CH2), 22.43 (1C, aliphatic-C, CH2), 14.37 (1C, aliphatic-C, CH3).

A. 1HNMR

B. 13CNMR

C. 13C DEPT

B.

Fig. 3.27: (A) 1H NMR (300 MHz), (B) 13C NMR, (C) C135 DEPT Spectra of the purified polyphenolic compound (1) of Morchella galilaea dissolved in DMSO.

3.13 Nuclear Magnetic Resonance spectrum of analysis of Morchella eximia

The 1H NMR, 13C NMR, and DEPT spectra of the purified polyphenolic derivative (2) isolated from Morchella eximia are represented in Fig 3.29. A-C). Compound 2 (polyphenolic derivative) was isolated from Morchella eximia. The spectral analysis of Morchella eximia were recorded in DMSO.

The chemicals shift of (Fig 3.29), A- B- C. 1H NMR, 13C NMR, DEPT C135 of Purified polyphenolics of M. eximia reported in ppm.1H NMR (300 MHz, DMSO-d) δ 5.41-5.20 (3H, m), 2.73(1H, t), 2.17 (2H, t), 2.02 (1H, s), 2.03-1.98 (3H, m), 1.47 (2H, m), 1.4-1.1 (11H, m), 0.85 (3H, t).13C NMR (125 MHz, DMSO) δ 174.93 (1C, C=O), 130.16 (2C, Ar-CH), 128.19 (2C, Ar-

CH), 34.11 (1C, aliphatic CH2), 31.36 (1C, aliphatic CH2), 29.45 (1C, aliphatic CH2), 29.46 (2C, aliphatic CH2), 29.07 (2C, aliphatic CH2), 29.01(1C, aliphatic CH2), 27.06 (1C, aliphatic CH2), 135 25.66 (1C, aliphatic CH3), 24.94 (1C, aliphatic CH2), 14.36 (1C, aliphatic CH3).DEPT C (125

MHz, DMSO) δ 130.16 (1C, Ar-CH), 128.21 (1C, Ar-CH), 34.11(2C, aliphatic CH2), 31.36 (1C, aliphatic CH2), 29.46 (2C, aliphatic CH2), 29.19 (1C, aliphatic CH2), 29.07 (1C, aliphatic CH2),

29.01(1C, aliphatic CH2), 27.06 (2C, aliphatic CH2), 25.66 (1C, aliphatic CH3), 24.94 (1C, aliphatic CH2), 22.44(1C, aliphatic CH2), 14.37 (1C, aliphatic CH3).

A. 1HNMR

B. 13CNMR

C. 13C DEPT

Fig. 3.28: (A) 1H NMR (300 MHz), (B) 13C NMR, (C) C135 DEPT Spectra of the purified polyphenolic compound (2) of Morchella eximia dissolved in DMSO.

.

DISCUSSION

The present study encompasses several aspects: a) the morphological and molecular data based identification of Morchella species with the view to find the most reliable morphological characters that could be used in species identification particularly the Pakistani taxa; b) Biological activity analyses on two frequently found and promising taxa (the biological activities included: anti-bacterial, anti-oxidant, anti-tumor and phosphadase activity; c) the nutritional value (based on their amino acid profile); and d) the spectroscopic analysis (the NMR based characterization for bioactive compound identification). The present study is one of the first detailed analysis on any

Pakistani Morchella species, therefore most of the data presented here is of novel in nature and provides a baseline study for future detailed analysis.

Choosing Morchella for the present study has its own merits, i.e. Morchella is found naturally and has been reported to be picked by locals traditionally from a wide area mostly in the northern mountain ranges of Pakistan. The food and culinary uses of Morchella cannot be over emphasized however, this commodity here in Pakistan has been used rarely since most of the collection (picked from the wild) is exported raw. Therefore, the local morpho-genetic variation largely remained undetermined and unexplored. There are very few scientific studies available in the country that have highlighted the proper identification of these morphotypes and very few have reported their potential nutraceutical value. Therefore, it is the real need of hour to explore the hidden diversity of Morchella in this region and study their nutritional and myco-chemical potentials. This has been precisely the objective of the present study. The study revealed quite intriguing results which have been discussed here as follows:

4.1 The morphological diversity of Morchella in Pakistan Morphologically Morchella is notoriously diverse. The global taxonomic and systematic studies on Morchella especially the morphology-based identification is poor and confusing due to tremendous variability in form and colour. The field-based experience suggests that this variability was natural and deemed so due to the impacts of ecological and climatic changes (Du et al. 2014). This has led to describe this genus, generally, as an assemblage of widespread cryptic species and a major impediment for the morphological identification of species (Taylor et al. 2000).

Availability of DNA-based techniques and the phylogenetic analysis could greatly improve the fungal taxonomy and systematics and has been utilized vastly in understanding evolution of fungi (Koufopanou et al. 1997, Geiser et al. 1998, Yang 2011, 2013). The specific markers warrant mentioning here for instance: Internal transcribed spacer (ITS), Translation elongation factor (TEF1) Large subunit (LSU) and RNA polymerase II largest subunit (RPB1 and RPB2) (Dettman et al. 2003, Revankar & Sutton 2010, Taşkın et al. 2010, 2012, O’Donnell et al. 2011, Du et al. 2012a, Zeng et al. 2013, Elliott et al. 2014, Pildain et al. 2014, Voitk et al. 2014). These have frequently been used in delimitation of fungi at species level including Morchella (Du et al. 2012, Schoch et al. 2012, Richard et al. 2015, Ali et al. 2016).

In the present study, similar combined taxonomic approach (based on morphological and molecular data) has been followed achieving species level identification in Morchella. Though, this has never been easy, yet the previous experience with ITS and LSU markers with rust fungi (Ali et al. 2016; 2017), proved useful in species recognition. Furthermore, several morphological characters proved stable and thus were useful in combinations for species identification. This included: ascocarp primary ridges, their shape, hymenal pit colour, ascocarp attachment to stipe, stalk colour at maturity, asci size, paraphyses shape and size, septate or aseptate. Similar studies based on morphological descriptions have been provided by Clowez (2012) and more recently by Taskin et al. (2015). However, there were few clear challenges too as described below:

4.1.1 Morchella crassipes vs M. esculenta Identification of M. crassipes was confusing and problematic when comparison was made with M. esculenta. Morphologically these two taxa were parallel in color and differed slightly in few other characters, for example: in the size of ascomata (150–180 mm), Hymonopore pileus (100–120 or 50–55 mm), ascospore dimensions (21–26×12–15.5 µm), ascus size (255–325 or 18– 25 µm), Stalk size (100–120.9 mm). The dimension for M. esculenta were: the size of ascomata 150–190 mm, hymonophore pileus (70–80.6 or 90–60.2 mm), ascus (215–355 9 or 16.5–25 µm), ascospores (17–25–9 11–13.5 µm), Stalk (80–100 or 30.2–50.4 mm) (Waraitch 1976; Weber 1988). Therefore, DNA based analysis could provide more diagnostic information for determination of species. However, intriguingly the differences between M. crassipes and M. esculenta at the molecular level were even less evident from the reported NCBI databases (Kellner et al. 2005). The morphological characters of Pakistani specimens were found convergent in addition with natural habitat as compared to the North American species (Richard et al., 2015). However, based on phylogenetic analysis the Pakistani specimens (Accession nos. KP670933 and KP670934) showed stronger affinity with M. crassipes (GenBank Accession no. KY402197) with well supported (75% bootstrap value) topology.

4.1.2 Several newly recorded Morchella species from Pakistan

Morchella pulchella

The confirmation of M. pulchella from an apparently undisturbed mountainous site in Pakistan, also suggests that M. pulchella is a species with a Eurasian distribution, unlikely to have been

anthropogenically introduced to this region. M. pulchella has been previously reported only from China (Du et al. 2012a, 2012b), Turkey (Taşkin et al. 2010) France (Clowez 2012, Richard et al. 2015) and lately from Spain (Alonso et al. 2016). We report here M. pulchella based on rDNA sequence data from Pakistan. It is highly likely that M. puchella may represent species complex and therefore further study is needed to understand the taxonomy and biology of this important fungus.

Morchella elata Morphologically M. elata has been described on the basis of ascomata characteristics with straight longitudinal and typically parallel (or sometimes vertical) crests with transverse anastomoses. In literature the only iconographic reference was given by Fries (1822), however, Fries (loc. cit.) also described M. elata based on living material from Sweden. Since the present material from Pakistan showed close morphological affinities with M. elata with with several morphological exceptions, hence phylogenetic analysis based on ITS rDNA data deemed necessay. The molecular analysis on these isolates: Mr.4, Mr. 8, Mr. 22, Mr. 36, Mr. 3 and Mr. 24) clustered (with M. elata and M. pulchella) (Mel-31), Mel-23 (unknown species in elata clade), and M. sp. complex (Mel-23/24/31/32) (Loizides et al.,2016; Taskin et al., 2016). Since no previous report has been made on this species, suggest this is the first record of this species in Pakistan. Morchella eohespera Ascomata of North American specimen were 45–100 mm long, ovate and conical longitudinal ridges are present, Stalk, 20–40 mm, cylindrical, spore ellipsoidal, hyaline. The M. eohesphera, micro-morphological characters for instance the spore size was small. These grow in small groups in late spring especially on grassy slopes, calcareous soil near conifers sites as also reported from North America and Eurasia (Richard et al., 2015; Voitk et al., 2016). The morphological characters of Morchella eohespera collected from Skardu district, Pakistan is revealed similarity with the north American species. In the phylogenetic analysis, the presently collected samples (Mr.1) gained strong bootstrap support (99%) and appeared in the clade with Mel-19 (Morchella eohespera) thus recognized as M. eohespera which is again the first record of species from Pakistan.

Morchella eximia M. eximia (Mel-7) described from Europe, Asia, Australia and South America (Du et al. 2015). The first taxonomic study was performed by Solak et al. (2002) in Turkey, Solak et al. collected M. eximia from Turkey near conifers and regenerating post-fired forest. M. eximia has been reported fire-adapted species (Du et al. 2015). The comparative analysis of the present specimens showed many similarities with Turkish specimen, i.e. spore elliptical, asci 8, stalk length (20–30 mm), fruiting body 50–60 mm length and conical (Richard et al. 2015). In our phylogenetic analysis the ITS sequence (isolate H13) clustered with M. eximia (accession no. KM857970) from GenBank which corroborates the morphological identification of these specimens as M. eximia. Though it has already been reported from Asia, but this is the first report of the species to be recorded from Pakistan.

4.2 The biological activities in Morchella

Antibacterial activity Pathogenic bacterial and fungal resistance to synthetic antibiotics has pushed interest towards exploring and use of natural products as antimicrobial agents, and this trend has increased in recent decades (Aziz et al., 2007; Badshah et al., 2015). During present investigation the M. galilaea and M. eximia exhibited antibacterial activities against pathogenic bacterial strains. The methanolic extract was more effective against Enterobacter aerogensis, Klebsella pnumoneae and Staphylococcus aureus. These results confirmed the pervious findings of Gbolagade et al., (2006) and Lima et al., (2016) who have reported antibacterial activity of Agricus blazi mushroom against pathogenic bacteria. Similarly, Badshah et al., (2012) have reported the methanolic extract of M. esculenta as highly effective in inhibiting growth of pathogenic bacteria. M. esculenta is used in health care in different parts of the world and possess antimicrobial activities (Tietel et al., 2017). Researches have shown that several antibiotics like penicillin, streptomycin, chloramphenicol and vancomycin have been derived from fungi (Hardman et al., 2001; Davies and Davies.2010; Furusawa et al., 2018). Previously, the methanolic extracts of Morchella esculenta revealed antibacterial activity against Staphylococcus aureus, Escherichia coli, Listeria monocytogenes and Enterobacter cloacae (Heleno et al., 2013).

Thus, mushroom are factories for the synthetics of different biologically active compounds with potential therapeutics properties (Shameem et al., 2017) and therefore can be exploited in the formulation of novel antibacterial drugs. In current studies the antibacterial activity of M. galilaea and M. eximia may be due to the presence of phenolic and other bioactive compounds such as polysaccharides, lipids and flavonoids, which are abundantly reported in Morchella. In the present studies the methanolic extracts showed promising inhibitory activity against bacterial growth in contrast to the ethanolic extracts. Estrela et al. (2000) have reported that the size of the microbial inhibition zone depends up on the solubility and ability of the bioactive substance to diffuse, which was better achieved in methanol.

Antioxidant and anti-tumor efficacy of Morchella Generally mushroom are an excellent source of natural antioxidants found in their mycelia and fruiting bodies (Laith. 2010; Ziaja et al., 2015; Sanchez. 2017). The presence of polyphenols, polyketides, steroids and terpenes confer such activity in mushrooms (Turkoglu et al., 2007; Barros et al., 2008; Ramesh et al., 2010). Major groups of polyphenolics are phenolic acid such as the hydroxyinnamic acids, hydroxybenzoic and cinnamic acid (Choi et al., 2014; Helen et al., 2015). Wild mushrooms also contain other bioactive components such as flavonoids, terpenoids, lipids, polysaccharides and glycosides (Franziska et al., 2010; Lee et al., 2013). These active components found in the studied mushrooms are responsible for their higher antioxidant activity. The antioxidant activity of methanolic extract of both the Morchella species showed a strong and wide range similarity with previously reported data Kosanic et al. (2016). Previous studies have shown that Morchella conica, Morchella esculanta and Morchella angusticeps were a rich source of natural antioxidants and exhibited higher radical scavenging effects (Barros et al., 2008; Gursoy et al., 2009; Heleno et al., 2013; Badshah et al., 2015; Boonsong et al., 2016).

In the pursuit to find the best candidate, all macro constituents have been tested. For instance, the polysaccharides of wild mushrooms have potential antioxidant activity (Zhang et al., 2008; Wasser et al., 2010; Klaus et al., 2011; Chen et al., 2012; Kozarski et al., 2014; Siu et al., 2014). The isolated lipids showed relatively higher antioxidant capacity as compared to polysaccharides. Morchella eximia and M. galilaea having all such constituents showed promising antioxidant and anticancer activity in addition to their usefulness in curing of cardiovascular diseases and blood pressure (Shah et al., 2016; Rosselló et al., 2016). During present studies the

methanol extract of M. eximia and M. galilaea also showed the anticancer potential (as revealed against MCF-7 cancer cell lines). For this purpose, several fractions such as crude polysaccharide, polyphenols and lipids were tested independently. It was intriguing to find that the inhibition efficacy of polyphenols was maximum. In contrast to this the inhibitory potential of polysaccharides was much less. Results further showed that application of crude polysaccharides to the cell line exhibit the inhibitory activity only partially. Therefore, polyphenolic efficacy in Morchella species helped cure cancer development. Hence, morels with such potential may be a preferred choice as they have no side effects being edible too.

Phosphodiesterase enhancing activity of Morchella Compound profiling was undertaken to evaluate the impact of methanolic extract of Morchella galilaea and Morchella eximia and a reference drug on the two cGMP phosphodiesterases (PDE5 and PDE6) using the commercial PDE-GloTM phosphodiesterase assay kit. The drug Sildenafil, when tested at 10 µM showed a reduction in signal compared to the control (-64%) with the PDE5, whereas there was essentially no change (+3% activation) with PDE6. In contrast, the extract of Morchella galilaea and Morchella eximia significantly increased the enzymatic activity of both PDE5 and PDE6 although the activation appeared to be more pronounced with Morchella eximia than with Morchella galilaea. As expected, Sildenafil produced inhibition of PDE5, which is at least 10-fold more sensitive to this drug than PDE6 according to the scientific literature. It has been reported that the Ki for inhibition of PDE5 by Sildenafil is usually in the 1-10 nM range, whereas the Ki for inhibition of PDE6 by the drug is closer to 50 nM. It might be expected that complete inhibition of both PDE5 and PDE6 would have occurred with the 10 µM Sildenafil used in the current study. In the present analysis the extracts of the Morchella galilaea and Morchella eximia showed and enhancing potential that increased the concentration of PDE5 from 996,162 CPM up to 1,872,947 CPM. This was assessed as a 346% to 650% increase respectively above the control values. 4.3 The nutritional value of Morchella

4.3.1 The amino acid profile Wild mushrooms have a great nutritional value due to presence of protein content (FAO, 1991). However, the amount and type of amino acid present is deemed more important. This situation differs among organisms besides its dependence on different ecological factor as well as

fruiting body size and maturity level (Faiz and Sesli 2009). Non-essential amino acids play a key role in communication of gene expression, cell signaling, flowing of blood and transportation of food system, maturation of different tissue, microbial growth in intestine and cell immune response (Wang et al., 2013).

Amino acid profiling of Morchella revealed presence of seventeen amino acids each in M. galilaea and M. eximia, the most abundant amino acid being glutamic acid. Glutamic acid plays an essential role in regulating the amino acid metabolism, glutathione which are necessary to inhibit the poison peroxidase and the polyglutamate folate cofactors (Bimal et al., 2014). The M. galilaea and M. eximia contain high amount of glutamic acid as compared to other mushroom species such as: Pleurotus ostreatus (9.01g/100g,) P. sajorcaju (10.3g /100g), Boleuts eduils (2.56 g/100g), Genoderma lacedium, (0.575 g/100g), and G. adspersum (0.1962 g/100g) (Hadar and Arazi.1986; Bander. 2004; Zaho et al., 2004; Dundar et al., 2008; Kvrak. 2015; Bakır et al., 2018).

Glycine is an important amino acid which regulate different metabolic activity, prevent free radicals, tissue damages, wound injury, production of protein synthesis and rising immunity, diabetes, heart disease, cancer, inflammatory diseases and ischemia reperfusion damaging (Wang et al., 2013). The Glycine was found in M. eximia and M galilaea. Previous studies have reported the presence of this amino acid in P. ostreatus (17.1 g/100g), P. florida (0.0029g/100g), Boleuts eduils (0.82 g/100g), Genoderma lacedium, (0.828g/100g), and G. adspersum (0.627 g/100g) (Wang et al., 2001; Shashirekha et al., 2005; Dundar et al., 2008; Bakır et al., 2018). Aspartic acid plays a key role in secretion of different hormones. They are well known precursor of amino acid. Aspartic acid was reported very low amount in different mushrooms i.e. Pleurotus ostreatus 2.061g/100g, Boleuts eduils 1.45 g/100g, Genoderma lacedium,0.508 g/100g, and G. adspersum 0.864 g/100g, P. florida 0.0003 g/100g as compare to current study (Zaho et al., 2004; Shashirekha et al., 2005; Kvrak. 2015; Bakır et al., 2018).

Among essential amino acids, threonine and methionine were present in M. eximia and M. galilaea. Threonine is known to inhibit nervous disorders (Hyland. 2007). Methionine plays an important role for treating various liver diseases, alcoholism, depression problem, allergies, asthma, radiation side effects (RSE), schizophrenia, and Parkinson disease (Mischoulon and Fava, 2002). Furthermore, Tryptophan served as a precursor for serotonin which is reported to increase tolerance to pain (Segura and Ventura, 1998). The second highest amount of amino acid leucine

and lysine were found in M. eximia and M galilaea as compared to commonly reported mushroom species like Pleurotus ostreatus, Pleurotus florida, Boleuts eduils, Genoderma lacedium, and Genoderma adspersum (Zaho et al., 2004; Kvrak. 2015; Bakır et al., 2018). Lysine, an essential amino acid, is necessary for optimal growth (Chen et al., 2003). Similarly, Leucine was found in slightly higher quantities while the Methionine and Cysteine were lower in concentration compared to the reference protein (FAO, 1991). Hence, when compared to the human milk proteins (as a reference), the amino acid constituents in M. eximia and M. galilaea could be rated as of high nutritional value besides their nutraceutical potential due to the abundance of different amino acids.

4.4 The preliminary spectroscopic analysis based on 1H NMR, 13C NMR and C135 DEPT

4.4.1 Purified polyphenolics of M. galilaea The signals of purified polyphenolics of Morchella galilaea, δ 0.85 (t, 3H, 3J [1H-1H] = 6 Hz), δ 1.23 (m, 15H), probably due to CH - Alkyl attached with electron donating group. The peaks of δ 2.02 (d, 3H, 3J [1H-1H] = 6 Hz), δ 2.08 (s, 1H), δ 2.17 (t, 2H), may be due to alkyl group attached with electron with drawing group. The signals appeared at 7.5 Hz - δ 5.32 ppm would be due to benzene -CH and OH respectively. The 13C NMR (300 MHz, DMSO); whereas the peaks at δ 174.93 indicated the presence of carboxylic (1C, C=O) functional groups. The peaks appeared in the range (34.14- 14.34) showed the existence of alkyl group such as R-CH3, R-CH2- R, R-CH-R, R-C-R. These peaks (at 130.15-128.20) depicted presence of either vinyl or aromatic group. DEPT, contain the CH2-CH2 attached with electric negative elements, while CH-CH3 attached with positive elements. The 13C NMR the single peaks at δ 170 indicated carbonyl functional group derivate (C = O), whereas the peaks at 130 -128 showed the presence of aromatic moieties. The peaks appeared in the range of 14.36- 34.11 revealed the existence of different alkyl groups such as R-CH3, either R-CH2-R. Further the various alkyl chain was reported (Morcombe and Zilm. 2003) in different stage of isolation of polyphenolics from mushroom.

4.4.2 Purified polyphenolics of M. eximia The group present CH2- CH2 - CH2-CH2, CH2, CH2 attached with electro negative atoms, whereas CH3-CH - CH3- CH3 have showed with positive atoms. The signals of purified polyphenolics 1H NMR δ 5.41-5.20 (3H, m), probably R2C=C(R)H, due to vinyl functionality, the

peaks appeared at 2.73 (1H, t), 2.17 (2H, t), 2.02 (1H, s), 2.03-1.98 (3H, m) may be due to the presence of acetyl or alkyl attached with electronegative atoms while the peaks at 1.47 (2H, m), 1.4-1.1 (11H, m) showed the existence of alkyl group. The small peaks at 0.85 (3H, t) due to shielded alkyl proton. Jamie Marie. (2007) and Robert (2014) reported the various vinyl functional groups in various mushroom.

CONCLUSIONS  This study reports new records of Morchella galilaea, M. elata, M. eximia, M. pulchella, M. eohsepera and M. crassipes from Pakistan. The phylogenetic analysis based on ITS, RPB1, RPB2 and TEF sequences aided in the identification of these species on the basis of morphological data.  Higher antibacterial activity was recorded for methanolic extract (100 mg/ml) of both the Morchella species than ethanolic extracts against pathogenic bacterial strains. It was found that methanolic extract was more effective as compared to the ethanolic extract. Moreover, a higher zone of inhibition was recorded against Staphylococcus aureus (ATCC6538) and Enterobacter aerogenes (ATCC13048) with methanolic extracts of M. galilaea at 100 mg/ml. Maximum zone of inhibition against Bacillus subtilis (ATCC6633) and Klebsella pneumoniae (MTCC618) was recorded for methanolic extract of M. eximia at 100 mg/ml.  In the anti-proliferative activities, the methanolic extract of M. galilaea and M. eximia showed the most promising antiproliferative impact.  The methanolic extracts of M. galilaea and M. eximia showed no inhibition instead showed high activations of PDE5 and PDE6 enzymes.  Both the M. galilaea and M. eximia demonstrated the presence of 17 amino acids in different quantities. The most abundant amino acid was glutamic acid. The essential amino acid of both the Morchella species showed great similarity with human milk reference protein.

 The spectroscopy 1H NMR, 13C NMR, C135 DEPT of isolated polyphenolic of M. galilaea and M. eximia, of M. galilaea M. eximia showed the presence of aromatic and vinylic functional groups. However, further studies are required to fully characterize the biologically

Future recommendations

The present study was one of the unique study of its kind in Pakistan with objective to identify Morchella species found in natural habitats of Mountain regions of Pakistan by using Morphological and Molecular markers and to investigate bionchemica, nutritional and biological activities. With new records of Morchella species (Morchella galilaea, M. elata, M. eximia, M. pulchella, M. eohsepera and M. crassipes) from these three regions (Khyber Pakhtunkhwa, A. J & K and Gilgit-Baltistan) of Pakistan revealed great importance for conservation and further multiplication in the natural habitat. Extensive field studies are needed to explore untapped diversity of morels from the unexplored areas such as Baluchistan and several districts of Khyber Pakhtunkhwa especially located in the Hindukush and lesser Himalayan Mountain ranges in Pakistan. Moreover, proper taxonomic revision, descriptions based on microscopic characters especially through scanning electron microscopy and comparison of taxa may be carried out to elucidate taxonomic and biochemical attributes. Sustainable harvest also needed to be addressed while linking such resources for commercialization. International standards would be attained after short to long term interventions through academia and R & D organizations of Pakistan in collaboration with international organizations keeping all treaties and conventions into account, where CBD stands top most priority in this connection. Furthermore, the phylogenetic study based on ITS, RPB1, RPB2 and TEF sequences for identification of these new records provided new insights for nutritional importance adding significance to explore metabolomics by NMR based investigations, genetics as well as ecological based studies under changing climatic scenarios. Exploiting these species would add its role for isolation, purification and characterization of important compounds and can be used for natural food preservatives and culinary treatments. Moreover, clinical trials of newly identified compounds may also be helpful to address nutritional

insecurity faced in rural populations of Pakistan achieving Sustainable Development Goals (SDGs). Use of newly identified compounds may also compliment in the synthesis of drugs is being recommended. Method development for isolation of new compounds and cultivation techniques can also be disseminated through print media (research journals, scientific magazines, brochures and monographs) and social media for scientific community. Vibrant and pragmatic steps are suggested for conservation, multiplication and commercialization in developing countries like Pakistan which is striving for socio-economic development at large and prosperity.