Lawsonia Alba and its Endophytic Fungal Metabolites Chemical Constituents

Summary

The work epitomized in the present dissertation is presented in two parts, namely chemical constituents of leaves and fruits of Lawsonia alba (Part-A) and metabolites of Paecilomyces variotii– an endophytic fungus from Lawsonia alba (Part-B).

Part-A

In the current work, leaves and fruits were separated manually from the aerial parts of Lawsonia alba and each part was extracted repetitively (x5) with ethanol at room temperature. Extracts of each part was pooled together separately, freed of the solvent and subjected to solvent separation methods, followed by different chromatographic techniques such as column chromatography, thin layer chromatography and HPLC. Five constituents were obtained and characterized via spectral studies for instance, three known compounds from the leaves luteolin-3′-O–β-D-glucoside (1), luteolin (2), lawsone (3) and two known compounds from the fruits triacontyl (E)-3-(4-hydroxy-3-methoxyphenyl) acrylate (4) and ursolic acid (5). Modern sophisticated and state of the art spectroscopic techniques including UV, MS, IR, ¹³C-NMR (DEPT and BB), ¹H-NMR and 2D techniques (HSQC, HMBC, COSY, NOSY, TOCSY and J-resolved techniques were employed for the structure elucidation of the compounds. In addition to above mentioned techniques, known constituents were identified by comparison with physical and spectral data reported in literature.

Part-B

In this part investigation on metabolites of Paecilomyces variotii- an endophytic fungus from Lawsonia alba was undertaken. Fungal broth and fungal mycelium were separated and repeatedly (x3) extracted with ethyl acetate at room temperature. Each extract was subjected to solvent separation methods, followed by different chromatographic techniques such as column chromatography, thin layer chromatography and HPLC. Eight constituents were obtained and characterized via spectral studies, comprising one new compound lawsozaheer (6) and six hitherto unreported compounds stigmasta-5,7, 22-trien-3 β-ol (7), 4-(2-hydroxyethyl) phenol (8), β-sitosterol (9), stigmasterol (10), viriditoxin (12) and stigmasta-4,6,8(14),22-tetraen-3-one (13) from fungal broth and one unreported compound ergosterol (11) from fungal mycelium. Modern sophisticated and state of the art spectroscopic techniques including UV, MS, IR, ¹³C-NMR (DEPT and BB), ¹H-NMR and 2D techniques (HSQC, HMBC, COSY, NOSY, TOCSY and J-resolved techniques were employed for the structure elucidation of new compound. In addition to above mentioned techniques, known constituents were identified by comparison with physical and spectral data reported in literature.

Bioassays of the plant extracts and the pure compounds obtained in the current research were done including anti-bacterial activity, anti-fungal activity and anticancer activity against three human cancer cell lines, (MCF-7), lung (NCI-H460) and uterine cervix (HeLa). The results are listed in tables (5.2-5.16). The petroleum ether insoluble fractions of both mycelium and broth showed significant growth inhibition/cytotoxicity in all the three cell lines used.

CHAPTER-01———————-

1.0 General Introduction

Allah Almighty has bestowed Pakistan with diversity of medicinal plants. It is known to have highly diversified endemic flora. It is imperative to mention that plants have played a magnificent function in sustaining human health and served humans for instance, seasonings, beverages, cosmetics, dyes and many more as well as valuable component of medicines. Herbal medicines contain as natural substances which are able to promote health and alleviate illness (Lee et al, 2013).

Earlier research reported that therapeutic practice of plants are documented in the Rigveda (4500-1600 BC) and Ayurveda (2500-600 BC). Charaka quoted roughly 50 groups of herbs while Sushruta described 37 sets of 760 herbs. Buddhist era enhanced the use of medicinal plants and provided extensive consideration to foster these plants in a very scientific way (Chopra, Nayar, and Chopra, 1956).

The plant selected for present studies has long been employed in Indo-Pak continent and Middle East countries in coloring hands palm, finger nails and soles of the feet. It has also been employed for hair, eye brows and bread dying, for personal beautification. It is also a good agent for coloring different skins and leathers (D. K. Singh, Luqman, and Mathur, 2015).

Medicinal importance of henna plant cannot be overrated. It has been used from several decades for medicinal purposes. One of the most exciting properties of the plant is its cooling effect. It is believed to be a good cooling agent. This is the reason that it is used to treat scrapes and burns and is frequently employed to treat heat exhaustion and slow down the temperature of a sick person. Different rashes for instance athlete′s ringworm and foot can be treated by exposure of skin to this plant (Kardar, 2005). It also has sun block properties. The plant Lawsonia alba has been stated to have hepatoprotective, analgesic, immunostimulant, hypoglycemic, anti-inflammatory, anti-bacterial, antimicrobial, tuberculostatic and anticancer properties (Goyal, Goyal, and Mehta, 2008).

A great deal of curiosity is found all over the world towards the exploration of medicinal plants. This was the main reason that the foundation of Hussein Ebrahim Jamal (HEJ) Research Institute of Chemistry was laid by the late veteran Scientist Prof. Dr. Salimuzzaman Siddiqui, FRS, in the University of Karachi, which is now believed as one of the best institutions of the world having modern sophisticated facilities for structure elucidation, isolation and different bioassay studies of the medicinal plants.

Owing to its versatile uses, the farming of henna has gained so much prevalence in Pakistan. Punjab and Sindh are the main provinces of Pakistan which are producing henna. The prime intake of henna leaves are mainly for skin and hair drying.

CHAPTER-02

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BIOSYNTHESIS

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CHAPTER-02———————-

2.0 Introduction

The term biosynthesis is employed for the synthesis of natural substances in living cells via sequential enzyme catalyzed reactions from fragments of relatively simple components. Comprehensive investigation and biosynthetic sequences are carried out by multidisciplinary professionals using isotopically labelled precursors, biological amalgamation and general pathways.

Research revealed that in plants which contain chlorophyll for instance, photosynthetic bacteria and algae commence biosynthesis through photosynthesis. Photosynthesis caters chemical energy and substrate material for all resulting biosynthetic reactions. Natural products are divided into two classes on the basis of biosynthetic taxonomy for instance primary and secondary metabolites (Lee et al, 2013).

2.0.1. Primary Metabolites

Primary metabolites are involved directly in plants development, reproduction and normal growth (Nguyen et al, 2016). In plants, photosynthesis is the process which is responsible for conversion of carbon dioxide into carbohydrates which are then converted to adenosine triphosphate (ATP) through citric acid (Krebs cycle). ATP plays a key role in amino acids biosynthesis which are believed to be proteins building blocks. Nucleic acid (DNA and RNA) is the storehouse of genetic information for proteins building plan. Polysaccharides, proteins and nucleic acids are the example of primary metabolites which are essential building blocks of all existing matter (Manitto et al, 1981; Hurtado-Fernández et al, 2011).

2.0.2. Secondary Metabolites

Secondary metabolites are not involved directly in plants development, reproduction and normal growth but do play important role in ecological functions. Primary metabolites are the precursors of secondary metabolites because secondary metabolites are the biosynthetic products of primary metabolites (Von Roepenack-Lahaye et al, 2004). The distribution of secondary metabolites is very limited and is mainly found in microorganisms and plants. There example include antibiotics, alkaloids, flavonoids, phenols, oligosaccharides, terpenes and steroids.

Secondary metabolite building blocks are derived from primary metabolites owing to their pharmacological activity as shown in scheme 3.1 (Dewick, 2002).

Scheme 2.1. Secondary metabolites building block derived from primary metabolites.

2.1 Flavonoids

Flavonoids are broadly disseminated in plant kingdom and are believed to contain a wide range of polyphenolic compounds (Harborne and Williams, 2000). They contain pigments which are responsible for leaves color, particularly in autumn (Saraf, Ashawat, and Saraf, 2007), and are believed to enrich the effect of vitamin ′C′ and act as an antioxidant. Flavonoids are commonly documented to exhibit diverse biological activities such as antitumor, anti-allergies, as liver toxins, anti-inflammation etc. Flavonoids are well-known for protection of blood vessels of human particularly the minute capillaries which are responsible for supply of nutrients and oxygen to cells. It has been shown that flavonoids are crucial phytochemicals believed to lessen aging and mortality (Hertog et al, 1993; Hertog et al, 1995). Some of the recognized and important flavonoids and their roles are listed in table 2.1.

Table 2.1: Types and function of flavonoids

Flavonoids	Functions
Quercetin	Reduces inflammation, prevent growth of head and neck cancer.
Hesperidin	Raises blood level of good cholesterol and decrease bad cholesterol. It also prevents inflammation.
Resveratrol	Decreases the risk of blood cancer, heart disease and stroke.
Anthocyanins	These are potent antioxidants.

Figure. 2.1 Basic Structure of various flavonoids and related compounds.

2.1.1. Biosynthesis of Chalcones

The central C-15 intermediate termed as chalcone is the main precursor for the biosynthesis of all flavonoids. The key precursors employed for the formation of chalcone are 4-coumaroyl-CoA (p-hydroxycinnamic acid CoA ester) and malonyl-CoA. It was investigated that shikimate pathway is embroiled in 4-coumaroyl-CoA formation, which again is the key path to yield phenylalanine, aromatic amino acids and tyrosine in higher plants (Kardar, 2005). Shikimic acid (Scheme 2.2) biosynthesis commences via condensation of phosphoenolpyruvic acid and D-erythrose-4-phosphate. After a number of steps and reaction series shikimic acid is transformed into hydroxycinnamic acid (Scheme 2.3). Chalcone is formed by coupling of 4-coumaroyl-CoA and malonyl-CoA which is then cyclized and catalyzed through chalcone synthase (Scheme 2.4).

Scheme 2.2. Formation of shikimic acid

Scheme 2.3. Biosynthetic conversion of shikimic acid into p-hydroxycinnamic

acid.

Scheme 2.4. Coupling of malonyl-CoA and 4-coumaroyl-CoA to form

chalcone.

2.1.2. Biosynthesis of Flavonones

Chalcones are converted into flavonones via isomerization. Chalcone isomerase is the enzyme involved in the conversion of chalcone into flavonones (Scheme 3.5).

Scheme 2.5. Equilibrium shift of chalcone towards flavanone in aqueous

solution.

CHAPTER-03

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PLANT INTRODUCTION

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Lawsonia alba Lam. (Henna)

Classification of Lawsonia alba Lam.

Kingdom: Plantae – Plants

Subkingdom: Tracheobionta – Vascular plants

Superdivision: Spermatophyta – Seed plants

Division: Magnoliophyta – Flowering plants

Class: Magnoliopsida – Dicotyledons

Subclass: Rosidae

Order: Myrtales

Family: Lythraceae – Loosestrife family

Genus: Lawsonia L. – lawsonia

Species: Lawsonia alba Lam. – henna

CHAPTER-03———————

3.0 Introduction

Lawsonia (family Lythraceae) is a monotypic genus characterized by Lawsonia inermis Linn. (syn. L. alba Lam.). It is known as henna in Arabic, mehndi in Hindi and Urdu. The crop is harvested twice a year (April-May and October-November) from the second year onwards. The plant is cut close to the ground, dried in shade and leaves separated by beating (Kamal and Jawaid, 2010; Rahmat, 2002; Uddin et al, 2011).

3.0.1. Aerial Parts Analysis

Air-dried leaves analysis provided average values of the following: tannins, 10.21%, ash, 14.85%, moisture, 8.97%. Leaves of henna comprise 24-33% water soluble constituents. Lawsone (2-hydroxy-1, 4-naphtoquinone is believed to be the main coloring constituent existing in leaves 1.0-1.4% in concentration (Lal and Dutt, 1933; Singh et al, 2015).

The flowers of henna yield essential oils on steam distillation and has a pleasant fragrance look like tea rose aroma and brown or dark brown colour mignonette (Reseda odorata Linn.). In India henna oil was reported to be extracted on a commercial scale and has been employed for perfumery purposes since antique. α and β-ionones are the main constituents in the flowers of henna; nitrogenous compounds and resins have also been reported. Henna seed analysis shown the following values, protein, 5.0%, moisture, 10.6%, fiber, 33.55%, fatty acids, 10.11%, ash, 4.75% and carbohydrates, 33.62% (Ali and Ansari, 1998; Chaudhary et al, 2010).

3.0.2. Medicinal uses

For medicinal purposes henna plant had been employed over centuries. It is appealed to had been employed as an expectorant, refrigerant, anti-inflammatory, sedative, antipyretic, depurative haematinic, emetic as well as diuretic properties. Research has shown that this plant contains effective anticancer agents (Li et al, 2014).

3.0.2.1. Leaves

The plant leaves have been employed for avoiding graying and falling hair as well as for healthy growth of hair. Henna leaves paste is used for nails, soles, hand palms, hair eyebrow and beard. The leave paste is also employed to safeguard skin form skin, harm and disorder, loss of nail and to protect nail whitlows diseases. It is considerably lucrative in treating bruises, abscesses, skin inflammation, scurvy affections, rheumatism, wounds of circumcision and leprosy. Research reported that in Ayurvedic medicine these are employed to treat fever, cough, strangury, bronchitis, rheumatalgia, inflammations, typhoid, ophthalmia, haemorrhages and haemoptysis (Chaudhary et al, 2010; Li et al, 2014).

3.0.2.2. Flowers

In Ayurvedic medicine the flowers of L. alba are employed as a refrigerant, an antipyretic, a cardio tonic and a soporific. They have also been employed in headache, cardiopathy, anemia, sensation and fever. Its aroma can induce sleep so it is lucrative for treating insomnia (Chaudhary et al, 2010; Kirkland and Marzin, 2003).

3.0.2.3. Seeds

The seeds of henna are used in treating fever, diarrhea, dysentery, abdominal disorders, vaginal discharge, leucorrhoea, insanity, menorrgagia and amentia (Chaudhary et al, 2010; Kirkland and Marzin, 2003). Table 4.1 shows pharmacological activities of different parts of henna plant.

3.1 Chemical Constituents:

A. Harraory was the first person who carried out chemical studies on Lawsonia alba back in 1848. He reported the isolation of a tannin, hennotannic acid which was thought to be the henna′s coloring agent (Lal and Dutt, 1933). Later on, Tommasi in 1920 stated that the main henna leaves coloring agent is lawsone, 2-hydroxy-1,4-napthaquinone melting at 192-195 ºC (decomp.). In 1923 O. A. Oesterle reported crystalline D-mannitol (stable at room temperature) from ethanolic extract (98%).

The main coloring agent of Lawsonia alba ′′lawsone′′ was separated from leaves of henna in 1924 by C. S. Anon. He treated the crushed leaves with a cold or warm alkali earth salt aqueous solution (for instance Mg and Ca salt from the Solvay ammonia-soda process). Another method was published in 1928 by Syed B. Ali who separated lawsone from water extract with benzene. The extract was allowed to treat with Pb acetate and finally H₂S was used to remove Pb (Kardar, 2005).

Lawsone

(2-hydroxy-1,4-napthaquinone)

From Indian specie Lal and Dutt (17) got 2-hydroxy-α-naphthoquinone constituents and its structure was confirmed via direct comparison of its physical data with synthetically made 2-hydroxy-α-naphthoquinone and its derivatives.

In 1950 Kaushal and Anita started research on henna flowers. They focused on essential oils and reported the presence of α-ionone and β-ionone, both having same formula (C₁₃H₂₀O) (Antia and Kaushal, 1950).

α-Ionone β-Ionone

In 1959 S. R. Agarwal et al. analyzed seed oils of L. alba and confirmed the presence of fatty acids for instance behenic, palmitic, arachidic and stearic acid (Aggarwal and Dhingra, 1959).

In 1962 B. N. Sastri separated glucose from the entire plant of L. alba and gallic acid from the leaves (Sastri, 1962).

In 1982 T. Chakrabarry et al. separated two triterpenes (pentacyclic) from the bark of L.alba and separated their structures as 3-β, 30-dihydroxylup-20(29)-en and (20S)-3-β, 30-dihydroxylupane (Chakrabartty, Poddar, and Pyrek, 1982).

(20S) 3 β,30-Dihydroxylupane (3 β,30-Dihydroxylup-20(29)-ene)

Nuraliev et al. in the same year reported three coumarins from the leaves of henna, for instance scopoletin, fraxetin and esculetin (Dzhuraev, Nuraliev, Kurbanov, Akhmedova, and Abyshev, 1982).

In 2001 B. S. Siddiqui et al. separated two new pentacyclic triterpenes, namely lawsonin and lawsonic acid from the aerial portions of henna and their structures were elucidated via spectroscopic studies as 3α-E-ferulyloxy-lup-20(29)-en-28-oic acid and 3α-E-ferulyloxy-urs-11-en-13β-ol respectively (Siddiqui and Kardar, 2001).

In 2003 the same group isolated two more new compounds, namely lawsonadeem and lawsonicin along with a reported compound vomifoliol from the henna plant. Their structures were elucidated with the help of spectroscopic studies as 1-methoxy-13H-dibenzo [b, i] xanthenes-5, 7, 12, 14-tetrone (lawsonadeem), 2,3-dihydro-5-hydroxy-3-(hydroxymethyl)-2-[4-(3-hydroxypropyl-3-methoxyphenyl]-6-methox- benzofuran,4S)-4-hydroxy-4-[(1E,3R)-3-hydroxybut-1-enyl]-3,5,5-trimethylcyclohex-2-1-one (vomifoliol) (Siddiqui, Kardar, Ali, and Khan, 2003). Lawsonicin is structure was later revised by synthesis as dihydrodehydrodiconiferyl alcohol (Meng et al, 2010).

In 2004 B. R. Mikhaeil et al. separated three constituents from the L. alba leaves methanolic extract for instance methoxy-3-methyl-1, 4-napthoquinone, apiin, and p-coumaric acid along with hitherto stated compounds lawsone, apigenin, luteolin and cosmosiin (apigenin-7-glycoside) (Mikhaeil et al, 2004)

In 2005 B. S. Siddiqui et al. further separated two new triterpenoids namely lawsoshamim and lawsowaseem from the L. alba aerial parts. Their structures were defined as 2-acetoxy-3β-hydroxy-olean-12-en-28-oic acid and 3β-hydroxy-24-p–E-coumaroyloxy-olean-12-en-28-oic acid through spectral studies (Siddiqui, Kardar, and Khan, 2005).

In 2005 A. O. Yedeji et al. worked on the leaves of henna through GC and GC/MS and recognized thirty six entities including methyl linolenate, ethyl hexadecanoate, (E)-β-ionone, methyl-(E)-cinnamate and isocaryophyllene (Oyedeji, Ekundayo, and Koenig, 2005).

In 2010 N. X. Cuong separated two new lawsoniasides, A (1) and B (2) along with ten known phenolic constituents from Lawsonia abla leaves methanolic extract. Spectroscopic studies (NMR and FITCRMS) were used to elucidate their structures (Cuong et al, 2010).

In 2011 B. S. Siddiqui et al. separated three new flavonoids, namely lawsonaringenin, lawsochrysin and lawsochrysinin from henna leaves along with four known flavonoids, 3,3′,4′,7-tetrahydroxy flavanone, 3′,4′-dimethoxy flavone, rhoifolin and 7-hydroxy flavone. There structures were interpreted through spectroscopic studies (Uddin et al, 2011). Lawsonaringenin and 3,3′,4′,7-tetrahydroxy flavanone showed good urease inhibition activity and the latter also showed a DDPH radical scavenging activity.

In 2013 B. S. Siddiqui et al. separated seven constituents from stem part of the Lawsonia alba which comprises two new constituents for instance, lawsofructose (1) and lawsorosemarinol, (2), one known compound 2-(β-D-glucopyranosyloxy)-1, 4-naphthoquinone (3) and four unreported compound from Lawsonia alba 7-hydroxy-4-methyl coumarin (7), 3-(4-hydroxy-3-methoxyphenyl)-triacontyl-(Z)-propenoate (6), 3-(4-hyroxyphenyl)-triacontyl-(Z)-propenoate (5) and 4-hydroxy coumarine (4) (Uddin et al, 2013).

2-(β-D-glucopyranosyloxy)-1, 4-naphthoquinone

In 2014 L. Qian et al. isolated three new compounds, biflavonoid A, a bicoumarin A and biquinone besides twelve other reported compounds from the flower of L. alba. Spectroscopic studies were used to elucidate their structures and for further confirmation X-ray diffraction crystallography and chemical electronic circular dichroism (ECD) calculations were also used (Li et al, 2014).

The constituents isolated and reported from various parts of Lawsonia alba are listed in Table 3.2 in order of their publication.

Table 3.1 Constituents of Lawsonia alba Lam. (syn. L. inermis Linn)
S. No.	Name of the Compounds	Plant Material	Molecular Formula	MPC	Ref.
1.	Lawsone	Leaves	C₁₀H₆O₃	195-196	(Fieser, 1948; Tripathi et al, 1978)
2.	D-Mannitol	Whole plant	C₆H₁₄O₆	166	(Fieser, 1948)
3.	α-Ionone	Flowers	C₁₃H₂₀O	137-140	(Datta et al, 1989)
4.	β-Ionone	Flowers	C₁₃H₂₀O	127-128	(Datta et al, 1989; Oyedeji et al, 2005)
5.	Arachidic acid	Seeds	C₂₀H₄₀O₂	75.4	(Aggarwal et al, 1959)
6.	Behenic acid	Seeds	C₂₂H₄₄O₂	75-80	(Aggarwal et al, 1959)
7.	Linoleic acid	Seeds	C₁₈H₃₂O₂	-5	(Aggarwal et al, 1959)
8.	Palmatic acid	Seeds	C₁₆H₃₂O₂	63.1	(Aggarwal and Dhingra, 1959)
9.	Stearic acid	Seeds	C₁₈H₃₆O₂	69.6	(Aggarwal and Dhingra, 1959)
10.	Gallic acid monohydrate	Leaves	C₇H₆O₅. H₂O	225	(Sastri, 1962)
11.	Glucose	Whole plant	C₆H₁₂O₆	147	(Sastri, 1962)
12.	Lacoumarin	Whole Plant	C₁₂H₁₀O₄	162-164	(Bhardwaj et al, 1976)
13.	Laxanthone I	Whole plant	C₁₆H₁₂O₆	286-287	(Bhardwaj et al, 1977)
14.	Laxanthone II	Whole plant	C₁₈H₁₄O₈	180-181	(Bhardwaj et al, 1980)
15.	n-Triacontyl-n-tridecanoate	Whole plant	C₄₃H₈₆O₂	89-90	(Chakrabority et al, 1977)
16.	n-Triacontanol	Whole plant	C₃₀H₆₂O	83-4	(Chakrabority et al, 1977)
17.	Lupeol	Bark	C₃₀H₅₀O	205-206	(Chakrabority et al, 1977)
18.	30-Norlupan-3-ol-20-one	Whole plant	C₂₉H₄₈O₂	233-234	(Chakrabority et al, 1977)
19.	β-Sitosterol	Leaves, bark	C₂₉H₅₀O	136-137	(Muhammad et al, 1984)
20.	Betulin	Bark	C₃₀H₅₀O₂	258-260	(Chakrabority et al, 1977)
21.	Betulinic acid	Bark	C₃₀H₄₈O₃	300-305	(Chakrabority et al, 1977)
22.	Apigenin-7-glycoside	Leaves	C₂₁H₂₀O₁₁	227-230	(Afzal et al, 1980; Mikhaeil et al, 2004)
23.	Apigenin-4′-glycoside	Leaves	C₂₁H₂₀O₁₁	347	(Afzal et al, 1980)
24.	Luteolin-7-O-glucoside	Leaves	C₂₁H₂₀O₁₁	252-254	(Hsouna et al, 2011; Takeda and Fatope, 1988)
25.	Luteolin-3′-O-glucoside	Leaves	C₂₁H₂₀O₁₁	243-245	(Afzal et al, 1980; Takeda and Fatope, 1988)
26.	Acacetin	Leaves	C₁₆H₁₂O₅	260	(Mahmoud et al, 1980)
27.	Acacetin-7-O-glucoside	Leaves	C₂₂H₁₆O₁₀	257	(Mahmoud et al, 1980)
28.	3-O–α-D-Glucoside β-sitosterol	Leaves	C₃₅H₆₀O₆	285	(Mahmoud et al, 1980)
29.	Luteolin	Leaves	C₁₅H₁₀O₆	237	(Mahmoud et al, 1980) (Mikhaeil et al, 2004)
30.	(20S)-3α,30-Dihydroxy lup-20 (29)-ene	Bark	C₃₀H₅₂O₂	266	(Chakrabartty et al, 1982)
31.	3α,30-Dihydroxy lupine-20 (29)-ene (hennadiol)	Bark	C₃₀H₅₀O₂	230-232	(Chakrabartty et al, 1982)
32.	Esculetin	Leaves	C₉H₆O₄	268-272	(Dzhuraev et al, 1982)
33.	Fraxetin	Leaves	C₁₀H₈O₅	227-228	(Dzhuraev et al, 1982)
34.	Scopoletin	Leaves	C₁₀H₈O₄	204-205	(Dzhuraev et al, 1982)
35.	1,2-Dihydroxy-4-glucosyloxy naphthalene	Leaves	C₁₆H₁₈O₈	–	(Muhammad et al, 1984; Takeda and Fatope, 1988)
36.	Stigmasterol	Leaves	C₂₉H₄₈O	170-172	(Muhammad et al, 1984
37.	Lalioside	Leaves	C₁₅H₁₉O₁₀	–	(Takeda and Fatope, 1988; Hsouna et al, 2011)
38.	Lawsoniaside	Leaves	C₂₂H₂₈O₁₃. ½ H₂O	263-264	Takeda and Fatope, 1988; Hsouna et al, 2011)
39.	3-Methylnonacosane-1-ol	Bark	C₃₀H₆₂O	73-74	(Gupta et al, 1992)
40.	Lawsaritol	Roots	C₂₉H₅₀O	124-125	(Alam, Niwa, Sakai, Gupta, and Ali, 1992)
41.	Isoplumbagin	Stem	C₁₁H₈O₃	67-68	(Alam, Niwa, Sakai, Gupta, and Ali, 1992)
42.	Lawsaritol A	Root	C₂₉H₅₀O₂	106-107	(Gupta, Ali, Alam, Niwa, and Sakai, 1994)
43.	2-Phenylethanol	Flowers	C₈H₁₀O	-25.8	(Gupta et al, 1994)
44.	Balanitisin A	Fruits	C₄₅H₇₂O₁₇	274-278	(Khan et al, 1996)
45.	Lawnermis acid	Seed	C₃₀H₄₆O₄	–	(Handa et al, 1997)
46.	Methyl ester of lawnermis acid	Seed	C₃₁H₄₈O₄	–	(Handa et al, 1997)
47.	Lawsonin	Aerial part	C₄₀H₅₆O₄	–	(Siddiqui and Kardar, 2001)
48.	Lawsonic acid	Aerial part	C₄₀H₅₆O₆	–	(Siddiqui and Kardar, 2001)
49.	Lawsonicin	Aerial part	C₂₀H₂₄O₆	–	(Siddiqui et al, 2003) (Meng et al, 2010)
50.	Lawsonadeem	Aerial part	C₂₂H₁₂O₆	–	(Siddiqui et al, 2003)
51.	Vomifoliol	Aerial part	C₁₃H₂₀O₃	–	(Siddiqui et al, 2003)
52.	Apiin	Leaves	C₂₆H₂₈O₁₄	–	(Mikhaeil et al, 2004)
53.	p-Coumaric acid	Leaves	C₉H₈O₃	486-490	(Mikhaeil et al, 2004)
54.	2-Methoxy-3-methyl-1, 4-napthoquinone	Leaves	C₁₂H₁₀O₃	–	(Mikhaeil et al, 2004)
55.	Apigenin	Leaves	C₁₅H₁₀O₅	347.5	(Mikhaeil et al, 2004)
56.	Lawsowasem	Aerial part	C₃₀H₄₆O₃	–	(Siddiqui et al, 2005)
57.	Lawsoshamim	Aerial part	C₃₂H₄₉O₅	–	(Siddiqui et al, 2005)
58.	Ethyl hexadecanoate	Leaves	C₁₈H₃₆O₂	22	(Oyedeji et al, 2005)
59.	Isocaryophyllene	Leaves	C₁₅H₂₄	–	(Oyedeji et al, 2005)
60.	Methyl linolenate	Leaves	C₁₉H₃₂O₂	–	(Oyedeji et al, 2005)
61.	(E)-Methyl cinnamate	Leaves	C₁₀H₁₀O₂	36-38	(Oyedeji et al, 2005)
62.	1,2,4-Trihydroxynaphthalene-1-Ο–β -glucopyranoside	Leaves	C₁₄H₁₈O₁₉	–	(Hsouna et al, 2011)
63.	2,4,6-Trihydroxyac-etophenone-2-Ο–β-D-glucopyranoside	Leaves	C₁₆H₁₈O₈	–	(Hsouna et al, 2011)
64	lawsoniasides, A	Leaves	C₁₈H₂₆O₉	–	(Cuong et al, 2010)
65	lawsoniasides, B	Leaves	C₁₇H₂₄O₉	–	(Cuong et al, 2010)
66	Lawsonaringenin	Leaves	C₂₀H₂₀O₄	–	(Uddin et al, 2011)
67	Awsochrysin	Leaves	C₂₅H₃₀O₄	–	(Uddin et al, 2011)
68	Lawsochrysinin	Leaves	C₂₀H₁₈O₄	–	(Uddin et al, 2011)
69	Lawsofructose	Stem	C₁₀H₂₀O₆	–	(Uddin et al, 2013)
70	Lawsorosemarinol	Stem	C₁₃H₂₀O₅	–	(Uddin et al, 2013)
71	Biflavonoid	Flower	C₃₀H₁₈O₁₂	–	(Li et al, 2014)
72	Bicoumarin	Flower	C₂₁H₁₆O₈	–	(Li et al, 2014)
73	Biquinone	Flower	C₂₃H₁₄O₆	–	(Li et al, 2014)

3.2 Biological activities

Previous investigations revealed that L. alba exhibits a wide range of biological activities. Some important one are listed below.

3.2.1. Antibacterial Activity

Pseudomonas is believed to be gram negative micro-organism hails from family Pseudomonadaceae. This type of pathogens is rampant in nature, impeding water, soil, animals, plants and humans. Pseusedomonas aeruginosa is one of the main source of infection in patients. To explore antibacterial activity dry crude extract of henna was tested employing well-diffusion antibiotic susceptibility method. Henna samples were examined for their antibacterial activity at dissimilar concentrations against extensive range of different micro-organisms containing laboratory standard strain of P. aeruginosa (NCTC 10662) and 11 fresh clinical isolates of P. aeruginosa acquired from patients. Antibacterial activity was shown by all tested isolates but the henna samples collected from Al-sharqyia region showed highest susceptibility against P. aeruginosa as shown in figure (Habbal et al, 2011).

In another study the main coloring agent in leaves of Lawsonia ′′lawsone′′ was stated to have fragile bacterial mutagen against Salmonella typhimurium TA98 strain and was considerably mutagenic against TA 26 strain (Kirkland and Marzin, 2003).

3.2.2. Antifungal Activity

The leaves extract of L. alba was reported to display absolute toxicity against Trichophyton mentagrophytes and Microsporum gypseum. The extract was tested against thirteen ring worm fungi and it displayed broad fungitoxic spectrum. On autoclaving at high temperature the extract′s fungitoxicity stayed unchanged (Chaudhary et al, 2010; V. Singh and Pandey, 1988). L. abla leaves aqueous extract was reported to have antifungal activity against eight main Aspergillus species from maize, paddy and sorghum seed samples (Satish, Mohana, Ranhavendra, and Raveesha, 2007). Essential oils from L. abla grown in Iran were analyzed via GC-MS and they presented significant antifungal activity (Aghel, Ameri, and Ebrahimi, 2005). Leaves ethanol extract of L. alba displayed high antifungal potential against phytopathogenic fungi (Begum, Yusuf, Chowdhury, Khan, and Anwar, 2007). It has also been reported that ethanol and ethyl acetate extracts of henna possess in vitro antidermatophytic properties (Natarajan, Mahendraraja, and Menon, 2000).

3.2.3. Antiviral Activity

It has been reported that L. alba ethanolic fraction show significant activity against Sembiki forest virus (SFV) in chick embryo and Swiss models of mice displaying activities around 100 -65% after the virus challenge of 10 -25 days (Chaudhary et al, 2010; Khan, Jain, Bhakuni, Zaim, and Thakur, 1991).

3.2.4. Antitrypanosomal activity

L.alba leaves methanolic crude extract displayed in-vitro potency against Trypanosoma brucei but not in-vivo. The medication tends to enrich condition of the disease, but this treatment did not affect the pack cell volume and level of parasitaemia (Chaudhary et al, 2010).

3.2.5. Antiparasitic activity

During the survey of medicinal herbs employed in Ivory Coast, 17 plants were assessed in-vitro screening as an antiparasitic drug. Non-polar, polar and alkaloidal fractions of several parts were assessed. The activities were determined included antiscabies, trypanocidal, antimalarial, antihelminthiasis and leishmanicidal etc. Among all these plants L. alba displayed potent trypanocidal activity (Okpekon et al, 2004). Ethyl acetate and pet. ether extract of leaves were evaluated both in vitro and in-vivo for cytotoxicity against Plasmodium falciparum (FcM29-Cameroon strains and FcB1-Columbia) via integrated (3H)-hypoxanthine assay. The leaves extract of L. alba showed significant antiplasmodial activity (El Babili, Bouajila, Valentin, and Chatelain, 2013).

3.2.6. Antimycotic activity

It has been reported that lawsone proved effective against oral Candida albicans isolated from HIV/AIDS patients. The leaves of L. alba were found to display potent fungitoxicity and lawsone was found to be a dynamic factor. The leaves of L. alba displayed ample toxicity against ringworm triggering fungal species for instance Trichophyton mentagrophytes and Microsporum gypseum (Babu and Subhasree, 2009; Prasirst et al, 2004).

3.2.7. Antidermatophytic activity

Hexane, ethyl acetate, and ethanol fractions of L. alba was tested against five strains each of Tinea mentagrophytes and Tinea rubrum. All fractions displayed high antidermatophytic activity in-vitro (Natarajan et al, 2000).

3.2.8. Antioxidant activity

Earlier researches revealed that certain phenolic glycosides present in the leaves butanol fraction of L. alba for instance (lawsoniasides, 2,4,6-trihydroxy-acetophenone2-O-β-D-glucopyranoside,luteolin-7-O-β-D-glucopyranoside, lalioside and 1,2,4-trihydroxynaphthalene-1-O–β-D-glucopyranoside) displayed highly significant antioxidant potential in β-carotene and DDPH assay (Hsouna et al, 2011). Methanolic and aqueous fractions of entire plant also displayed potent ABTS radical and DDPH scavenging activity and prevention of lipid peroxidation, Fe⁺ions and DNA damage (Guha, Rajkumar, Kumar, and Mathew, 2011).

3.2.9. Wound healing activity

The leaves ethanol extract of L. alba was reported to exhibit wound healing properties against Sprague rats employing incision, excision and wound model of dead space (Nayak et al, 2007). Tattoo paste of henna also revealed to be beneficial against wound healing in several skin disorders (Liou et al, 2013).

3.2.10. Anti-inflammatory, anti-diarrheal, analgesic and

antipyretic activity

It is evident from literature survey that L. alba has often been recommended for hand foot syndrome patients due to its analgesic, antipyretic and anti-inflammatory actions (Yucel and Guzin, 2008). L. alba leaves ethanol extract displayed substantial anti-diarrhoeal and moderate analgesic activity in mice models and this study provided scientific origin of its customary use against body pain and loose motion (Sultana and Khosru, 2011).

3.2.11. Enzyme inhibition activity

L. alba leaves ethanol extract and lawsone were tested against trypsin inhibitory activity and displayed IC₅₀ values of 48.6µg/mL and 87 µg/mL respectively (Yogisha, Samiulla, Prashanth, Padmaja, and Amit, 2002).

3.2.12. Diuretic activity

It has been reported that ethanolic and aqueous fractions of L. alba displayed a dose dependent rise in excretion of urine. Ethanolic extract displayed comparatively more diuretic activity than aqueous fraction (Reddy et al, 2011).

3.2.13. Anticancer activity and cytotoxicity

Lawsone a main coloring agent of L. alba was isolated from the leaves and employed as precursor in the synthesis of diverse clinically lucrative anticancer drugs for instance lapachol, atovaquone and dichloroally lawsone (Pradhan et al, 2012; Priya, Ilavenil, Kaleeswaran, Srigopalram, and Ravikumar, 2011; D. K. Singh and Luqman, 2014) . It was reported that leaves chloroform fraction of L. alba displayed cytotoxic effects on MCF-7 and HepG2 having IC₅₀ values of 24.85 µg/ml and 0.3 respectively. Yet, no effect was observed against normal MDA-MB-231 and Caco-2 cell lines (Rahmat, 2002). L. alba leaves essential oil were also found to display cytotoxicity against HepG2 cell (liver cancer) having an IC₅₀value of 24 µg/mL (Rahmat et al, 2006). The roots ethanol extract of L. alba displayed robust antitumor activity against DLA model (Dalton′s Lymphoma Ascites) in Swiss albino model (Priya et al, 2011).

3.2.14. Hepatoprotective activity

L. alba leaves aqueous extract displayed potent hepatoprotective action on CCL₄ –induced liver damage against male wistar albino rats as witnessed by reduced level of serum glutamate pyruvate transaminase, serum bilirubin, serum alkaline phosphatase, serum glutamate oxaloacetate transaminase and a potent anti-lipid per-oxidant influence (Hemalatha, Natraj, and Kiran, 2004; Latha, Suja, Shyamal, and Rajasekharan, 2005).

___________________________________

PART-B

___________________________________

CHAPTER-04

_______________________________________

ENDOPHYTIC

FUNGAL METABOLITES

_______________________________________

CHAPTER-04———————-

4.0 Introduction

Endophytic microorganisms are fungi or bacteria that animate inside of the plant tissues during their life cycle at any moment, without affecting disease symptoms or causing damage to their host. These could be transformed to other generations of plant via vegetative propagules or seeds, letting extensive spreading of these microbes in plants. The distribution and colonization of these microbes could offer a substantial remuneration to their hosts through making excess of metabolites, which helps in protection and existence of the plant. Particularly these secondary metabolites are potent owing to contacts with their host (Souza, Vieira, Rodrigues-Filho, and Braz-Filho, 2011).

Fungi are recognized as plant like organisms without chlorophyll. Endophytic fungi have created curiosity and are center of attention among researchers worldwide due to their appreciation as an unlimited source of biologically structurally novel compounds. Scientific investigations proved that endophytic fungi are vital source of bioactive constituents (Kusari, Pandey, and Spiteller, 2013; Wibowo et al, 2016). Endophytes are significant source of a number of novel bioactive secondary metabolites possibly valuable for human medicines, having antimicrobial, anticancer and several other activities and could serve as a prospective sources of natural products having agrochemical and industrial potentials (Souza et al, 2011). Pathogens and plant pets including bacteria, viruses, insects, fungi and nematodes lessen crops production by 30-50% universally, subsidizing to poverty and malnutrition (Mousa and Raizada, 2013).

4.0.1. Biodiversity and distribution of endophytic fungi

A noteworthy aspect of endophytic fungi is their diverse and widespread occurrence and to inhabit plants in diverse environmental regions for instance Antarctic, Arctic, deserts, geothermal soils, rainforests, oceans, mangrove, coastal forests and swamps. Furthermore, they have been separated from different range of hosts comprising sponges, algae, pteridophytes, angiosperms and gymnosperms (Bills, Menéndez, and Platas, 2012; Kharwar, Mishra, Gond, Stierle, and Stierle, 2011). These fungi are reported to present in all organs for instance (stem, leaf, root, fruit, inflorescence and seed) of plants (Firakova, Sturdikova, and Muckova, 2007). It has been reported that there exist about 420,000 plant species in nature and little have been thoroughly studied regarding to their endophytes origin (Vuorela et al, 2004).

4.0.2. Chemical constituents from endophytic fungi

Research investigations revealed that a number of bioactive compounds have been isolated from endophytic fungi leading to drug discovery as shown below (Mousa and Raizada, 2013; Nicoletti and Fiorentino, 2015). Some important classes include:

4.0.2.1. Terpenoids from endophytic fungi

4.0.2.2. Coumarins

4.0.2.3. Flavonoids

4.0.2.4. Xanthones and quinones

4.0.2.5. Lignans

4.0.2.6. Quinoline

4.0.2.7. Steroids

4.0.2.8. Phenolic compounds

4.0.2.9. Aliphatic Derivatives

4.0.2.10. Polyketide derivatives

CHAPTER-05

_______________________________________

PRESENT WORK

_______________________________________

CHAPTER-05———————-

5.0 Present work

The work epitomized in the present dissertation is presented in two parts, namely chemical constituents of leaves and fruits of Lawsonia alba (Part-A) and metabolites of Paecilomyces variotii-an endophytic fungus from Lawsonia alba (Part-B).

Part-A

5.1 Chemical constituents of leaves and fruits of Lawsonia alba

Considering the biological and other medicinal properties attributed to Lawsonia alba (Henna) as depicted in the aforementioned chapter, investigations on its leaves and fruits were undertaken in the current studies which resulted in isolation and structure elucidation of five known compounds. Leaves and fruits were separated manually from the aerial parts of Lawsonia alba and each part was extracted repeatedly (x5) with ethanol at room temperature and different biological assays were used to determine their biological activities (see 5.4. Biological activities). Each extract was subjected to different chromatographic techniques and solvent separation methods (vide experimental part) which led to isolation and structure interpretation of five constituents for instance luteolin-3′-O–β-D-glucoside (1)), luteolin (2), lawsone (3), triacontyl (E)-3-(4-hydroxy-3-methoxyphenyl) acrylate (4) and ursolic acid (5).

Modern sophisticated and state of the art spectroscopic techniques including UV, MS, IR, ¹³C-NMR (DEPT and BB), ¹H-NMR and 2D techniques (HSQC, HMBC, COSY, NOSY, TOCSY and J-resolved techniques were employed for the structure elucidation of the compounds. In addition to above mentioned techniques, known constituents were recognized by comparison with physical and spectral data reported in literature.

The compounds isolated and characterized via comprehensive spectroscopic techniques in the present study are documented as follows.

5.1.1. Known constituents from the leaves of L. alba

Luteolin-3′-O– β–D-glucoside (1)

(5,7-Dihydroxy-2-(4-hydroxy-3-((4,5,6-trihydroxy-3-(hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) phenyl)-4H-chromen-4-one)

Mol. Formula: C₂₁H₂₀O₁₁

Luteolin (2)

(2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4H-chromen-4-one)

Mol. Formula: C₁₅H₁₀O₆

Lawsone (3)

(2-Hydroxy-1,4-naphthoquinone)

Mol. Formula: C₁₀H₆O₃

5.1.2. Known constituents from the fruits of L. alba

Triacontyl (E)-3-(4-hydroxy-3-methoxyphenyl) acrylate (4)

Mol. Formula: C₄₀H₇₀O₄

Ursolic acid (5)

(1S,2R,4aS,6aR,6aS,6bR,8aR,10S,12aR,14bS)-10-Hydroxy-1,2,6a,6b,9,9,12a-heptamethyl-2,3,4,5,6,6a,7,8,8a,10,11,12,13,14b-tetradecahydro-1H-picene-4a-carboxylic acid)

Mol. Formula: C₃₀H₄₈O₃

Part-B

5.2 Endophytic fungal metabolites of Lawsonia alba

(Paecilomyces variotii)

Endophytic microorganisms are fungi or bacteria that animate inside the plant tissues during their life cycle at any moment, without affecting disease symptoms or causing damage to their host. These could be transformed to other generations of plant via vegetative propagules or seeds, letting extensive spreading of these microbes in plants. The distribution and colonization of these microbes could offer a substantial remuneration to their hosts through making excess of metabolites, which helps in protection and existence of the plant. Particularly these secondary metabolites are potent owing to contacts with their host (Souza, Vieira, Rodrigues-Filho, and Braz-Filho, 2011). Endophytic fungi have created curiosity and are center of attention among researchers worldwide due to their appreciation as an unlimited source of biologically structurally novel compounds. Scientific investigations proved that endophytic fungi are vital source of bioactive constituents (Kusari, Pandey, and Spiteller, 2013; Wibowo et al, 2016).

Investigation on endophytic fungal metabolites of Lawsonia alba (Paecilomyces variotii) were undertaken in the current studies which led to isolation and structure elucidation of one new (novel) and six unreported compounds. Fungus broth and fungus mycelium obtained (vide experimental) were repeatedly (x3) extracted with ethyl acetate at room temperature. Each extract was subjected to different chromatographic techniques and solvent separation methods (vide experimental part) which led to isolation and structure interpretation of one novel compound lawsozaheer (6) and six unreported compounds stigmasta-5,7,22-trien-3β-ol (7), 4-(2-hydroxyethyl) phenol (8), β-sitosterol (9), stigmasterol (10), viriditoxin (12) and stigmasta-4,6,8(14),22-tetraen-3-one (13) from fungus broth. One unreported compound ergosterol (11) was isolated from fungus mycelium.

Modern sophisticated and state of the art spectroscopic techniques including UV, MS, IR, ¹³C-NMR (DEPT and BB), ¹H-NMR and 2D techniques (HSQC, HMBC, COSY, NOSY, TOCSY and J-resolved techniques have been employed for the structure elucidation of new compounds. In addition to above mentioned techniques, known constituents were identified in comparison with physical and spectral data in literature.

The compounds isolated and characterized via comprehensive spectroscopic techniques in the present study are documented as follows.

5.2.1. New constituents from endophytic fungal broth of L. alba

Lawsozaheer (6)

6-Hydroxy-2-(2-hydroxypropyl)-8-methyl-4H-chromen-4-one

Mol. Formula: C₁₃H₁₄O₄

5.2.2. Hitherto unreported constituents from endophytic

fungal broth of L. alba

Stigmasta-5,7, 22-trien-3 β-ol (7)

Mol. Formula: C₂₉H₄₆O

4-(2-Hydroxyethyl) phenol (8)

Mol. Formula: C₈H₁₀O₂

β-Sitosterol (9)

(17-(5-Ethyl-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-ol)

Mol. Formula: C₂₉H₅₀O

. Stigmasterol (10)

(3S,8S,9S,10R,13R,14S,17R)-17-[(E,2R,5S)-5-ethyl-6-methylhept-3-en-2-yl]-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-ol

Mol. Formula: C₂₉H₄₈O

Viriditoxin (12)

Methyl-2-[8-[9,10-dihydroxy-7-methoxy-3-(2-methoxy-2-oxoethyl)-1-oxo-3,4-dihydrobenzo[G]isochromen-8-Yl]-9,10-dihydroxy-7-methoxy-1-oxo-3,4-dihydrobenzo[G]isochromen-3-Yl] acetate

Mol. Formula: C₃₄H₃₀O₁₄

Stigmasta-4,6,8(14),22-tetraen-3-one (13)

Mol. Formula: C₂₉H₄₂O

5.2.3. Hitherto unreported constituents from endophytic

fungal mycelium of L. alba

Ergosterol (11)

Ergosta-5,7,22E-trien-3 β -ol

Mol. Formula: C₂₈H₄₄O

5.3 Analysis of petroleum ether soluble fraction

5.3.1. Gas chromatography (GC)

Gas chromatography was carried out using flame ionization detector (FID) on the less polar capillary column OPTIMA^®-5-Accent (60m x 0.32mm ID with 0.25μm film thickness of 5% phenyl / 95% methyl silicone) installed on a Shimadzu GC -10 for GC-FID analysis. The analysis was performed with an initial temperature 50 ^oC for 2 minutes then ramped at a rate of 7 ^ºC/ min. to a final temperature of 260 ⁰C with holding time of 30 min. Injector with a splitting ratio of 1:8 was set at 235 ^ºC and FID at 400 ^ºC. Carrier and make up gas was nitrogen with a flow of 12.334 ml/min. at a pressure of 90 kpa.

5.3.2. Gas chromatography-mass spectrometry (GC-MS)

For GC-MS experiments Agilent 6890 gas chromatograph, equipped with ZB-5MS (30m x 0.32 ID and 0.25 μm film thickness) was combined with a Jeol, JMS-600H mass Spectrometer operating in EI mode with ion source at250 ^oC and electron energy at 70 eV. Carrier gas was volume was adjusted between 1.0-5.0 µL depending upon the detector response.

5.3.3. Determination of the fatty acids and other non to less

polar constituents

The petroleum ether soluble fractions were analyzed by gas chromatography using flame ionization detector (GC-FID) and GC-EIMS analysis (Masada, 1976). The components were characterized by mass spectral survey (NIST Mass Spectral Search Program, 1998; GC-MS library 1996). Comparison of the mass spectrum of each component obtained by GC-MS, with those reported for the selected compounds permitted to identify various constituents of this fraction.

5.3.4. Characterization of compounds in pet. ether soluble

fraction of endophytic fungal broth (LA-B-PES) of

(Paecilomyces variotii) of L. alba through GC, GC-MS

Twenty compounds were identified in LA-B-PES fraction of fungal filtrate of L. alba (Paecilomyces variotii) through GC/GC-MS (Table-5.1).

Table-5.1. Compounds (> 1%) identified in LA-B-PES fraction of fungal (Paecilomyces variotii) filtrate of L. alba through GC, GC-MS.

No.	Compounds	t_R (min)	Characteristic mass fragments, m/z (rel.% BP)	RI	Area Sum (%)
1	Ethanone, 1-[3-methyl-3-(4-methyl-3-pentenyl) oxiranyl]- (1)	11.522	226 (M⁺not observed), 82(100), 69(86), 55(39), 43(76),	1271	1.75
2	Oxiranemethanol, 3-methyl-3-(4-methly-3-pentenyl)- (2)	11.774	226 (M⁺not observed), 109 (28), 82 (100), 69 (88), 55 (36), 43 (74).	1287	12.26
3	3-Buten-2-ol, 2-methyl-4-(1,3,3-trimethyl-7-oxabicyclo [4.1.0] hept-2-yl)- (3)	12.839	236 (M⁺not observed), 82(48), 43(100).	1364	1.52
4	Benzene, (1-pentylheptyl)- (4)	13.325	246 (M⁺5), 105 (23), 91 (100).	1399	1.13
5	Benzene, (1-propylnonyl)- (5)	13.594	246 (M⁺ 8), 133 (24), 91 (100).	1420	1.16
6	Benzene, (1-ethyldecyl)- (6)	13.938	246 (M⁺4),119 (45), 91 (100).	1448	1.49
7	Benzene, (1-methylundecyl)- (7)	14.608	246 (M⁺ 4), 105 (100).	1499	1.83
8	Benzene, (1-pentyloctyl)- (8)	15.010	260 (M⁺ 8), 105 (22), 91 (100).	1533	2.58
9	Benzene, (1-butylnonyl)- (9)	15.149	260 (M⁺ 4), 147 (22), 91 (100).	1545	1.73
10	1-Penten-3-one, 1-(2,6,6,-trimethl-2-cyclohexen-1yl)- (10)	15.285	206 (M⁺48), 151 (83), 121 (100),	1556	2.92
11	Benzene, (1-propyldecyl)- (11)	15.418	260 (M⁺4), 133 (27), 91 (100).	1568	1.09
12	Phthalic acid, 2,7-dimethyloct-7-en-5-yn-4-yl isobutyl ester (12)	15.915	260 (M⁺ 2), 149 (100), 57 (32).	1608	4.26
13	1-Hexadecanol (13)	16.115	235 (M⁺ not observed), 97 (54), 83 (77), 69 (76), 55 (100), 43 (76).	1624	4.99
14	Benzene, (1-methyldodecyl)- (14)	16.888	260 (M⁺ 4), 105 (100).	1685	1.79
15	1,2-Benzenedicarboxylic acid, butyl 8-methylnonyl ester (15)	18.454	278 (M⁺ not observed), 149 (100).	1784	8.64
16	Oleyl Alcohol (16)	22.083	250 (M⁺ 3), 96 (53), 82 (74), 67 (65), 55 (100), 43 (45).	1933	7.19
17	9-Octadecenoic acid (Z)-, methyl ester (17)	23.529	296(M⁺ not observed), 149(99), 55(100).	1978	1.38
18	Trans-13-Octadecenoic acid (18)	24.605	535 (M⁺ not observed), 55 (100).	2008	1.82
19	1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl) ester (19)	30.161	305 (M⁺ not observed), 167 (54), 149 (100), 71 (26), 57 (42),43 (24).	2146	10.37
20	7, 8-Dimethoxy-3,4-dihydro-2H-dibenzofuran-1-one (20)	25.704	246 (M⁺ 65), 231 (100).	2206	1.15

5.4 Biological Activities

5.4.1. Antimicrobial activities

Antimicrobial activities were determined by disk diffusion method. The ethyl acetate extract of fungal broth (LA-B-EA) and fungal mycelium (LA-M-EA), and then ether soluble and insoluble fractions (LA-B-PES and LA-B-PEIN) and (LA-M-PES and LA-M-PEINS) respectively, were tested against twelve Gram-positive bacteria Staphylococcus aureus, Staphylococcus AB 188, Staphylococcus epidermidis, Streptococcus pyogenes, Streptococcus fecalis, Bacillus cereus, Bacillus subtilis, Bacillus thuringiensis, Micrococcus luteus, Corynebacterium xerosis, Corynebacterium hoffmanii and Streptococcus pneumonia and twelve Gram-negative bacteria Shigella boydii, Salmonella typhi, Salmonella typhi para A, Salmonella typhi para B, Shigella flexneri, Proteus mirabilus, Proteus vulgaris, Escherichia coli, Klebsiella pneumonia, Shigella dysenteriae, Enterobacter and Pseudomonas aeruginosa PA0286 and thirteen fungi Trichopyton rubrum, Microsporum canis, Trichopyton mentegrophyte, Microsporum gypsium, Trichopyton tonsurans, Saccharomyces cerevisiae, Candida albicans, Helementho sporain, Aspergillus flavus, Aspergillus niger, Penicillium sp, Rhizopus sp. and Fusarium sp. Both LA-B-PEINS and LA-M-PEINS fractions showed significant activity against the gram-positive bacteria Staphylococcus aureus. The corresponding LA-B-PES and LA-M-PES fractions did not show any notable activity. Hence LA-B-PEINS and LA-M-PEINS fractions were further evaluated against this bacterium using Microplate Alamar Blue Assay (MABA) (tables-5.5-5.6). In case of Gram-negative bacteria none of these fractions were found active (tables- 5.2-5.3).

On antifungal bioassay, both LA-B-PEINS and LA-M-PEINS fractions showed significant to moderate activity against five fungi. Hence these were further evaluated using Agar Tube Dilution Protocol. LA-B-PEINS showed 75 and 40 % inhibition of Candida albicans and Fusarium lini respectively, while LA-M-PEINS showed 50% inhibition of Candida albicans.

Table 5.2: In vitro antifungal bioassay by Agar Tube Dilution Protocol of LA-B-

PEINS

Name of the Fungus	Linear growth (mm)		% Inhibition by LA-B-PEINS	Std. Drugs	Std. Drugs MIC (µg/mL)
Name of the Fungus	Sample	Control	% Inhibition by LA-B-PEINS	Std. Drugs	Std. Drugs MIC (µg/mL)
Trichophyton rubrum	100	100	0	Miconazole	97.8
Candida albicans	25	100	75	Miconazole	113.1
Aspergillus niger	100	100	0	Amphotericin B	20.70
Microsporum canis	100	100	0	Miconazole	98.1
Fusarium lini	100	100	40	Miconazole	73.50

Remarks: Sample shows significant activity against C. albicans and shows moderate

activity against F. lini

Table 5.3: In vitro antifungal bioassay of LA-M-PEINS (fraction) (Agar Tube

Name of the Fungus	Linear growth (mm)		% Inhibition by NA-B	Std. Drugs	Std. Drugs MIC (µg/mL)
Name of the Fungus	Sample	Control	% Inhibition by NA-B	Std. Drugs	Std. Drugs MIC (µg/mL)
Trichophyton rubrum	100	100	0	Miconazole	97.8
Candida albicans	50	100	50	Miconazole	113.1
Aspergillus niger	100	100	0	Amphotericin B	20.70
Microsporum canis	100	100	0	Miconazole	98.1
Fusarium lini	100	100	0	Miconazole	73.50

Protocol:

Key: Concentration of sample: 400 µg /mL of DMSO

Incubation Temp: 27 (28^o±1^oC)

Incubation period: (7 days)

Remarks: Sample shows moderate activity against Candida albicans

Table 5.4: In vitro antibacterial bioassay by disk diffusion method (zone of inhibition;

mm) of LA-B-PEINS and LA-M- PEINS fractions against Gram-positive bacteria.

S/No

Gram- positive Bacteria

Zone of inhibition

LA-B- PEINS

LA-M- PEINS

Staphylococcus aureus

Staphylococcus AB 188

Staphylococcus epidermidis

Streptococcus pyogenes

Streptococcus fecalis

Bacillus cereus

Bacillus subtilis

Bacillus thuringiensis

Micrococcus luteus

Corynebacterium xerosis

Corynebacterium hoffmanii

Streptococcus pneumoniae

Table 5.5: Microplate Alamar Blue Assay (MABA) of LA-B- PEINS against

Staphylococcus aureus (NCTC 6571)

Name of Bacteria	Percent (%) Inhibition by LA-B- PEINS	Percent (%) Inhibition by ofloxacin

Staphylococcus aureus (NCTC 6571)	84.26%	87.013%

Keys: Concentration of Compound: 150 µg/mL

Remarks: Sample showed highly significant activity against Staphylococcus

aureus with MIC (LA-B-PEINS) = 225µg/mL; (MIC ofloxacin= 125 µg/mL).

Table 5.6: Microplate Alamar Blue Assay (MABA) of LA-M-PEINS fraction

Name of Bacteria	Percent (%) Inhibition by LA-M-PEINS	Percent (%) Inhibition by ofloxacin

Staphylococcus aureus (NCTC 6571)	83.197	87.013%

Keys: Concentration of Compound: 150 µg/mL

Remarks: Sample showed highly significant activity against Staphylococcus

aureus with

MIC (LA-M-PEINS) = 225µg/mL; (MIC ofloxacin= 125 µg/mL).

Table 5.7: In vitro antifungal bioassay by disk diffusion method (zone of

inhibition; mm) of LA-B-PEINS and LA-M-PEINS

S/No	Fungi	LA-B-PEINS	LA-M-PEINS
1	Trichophyton rubrum	10	10
2	Microsporum canis	10	10
3	Trichopyton mentegrophyte	9	–
4	Microsporum gypsium	8	–
5	Trichopyton tonsurans	9	–
6	Saccharomyces cerevisiae	9	–
7	Candida albicans	14	12
8	Helementho sporain	9	–
9	Aspergillus flavus	7	–
10	Aspergillus niger	7	–
11	Penicillium sp.	7	–
12	Rhizopus sp.	7	–
13	Fusarium lini	11	10

5.4.2. Anticancer activity

The ethyl acetate extracts from broth (LA-B-EA) and mycelium (LA-M-EA) and their pet. ether soluble (LA-B-PES and LA-B-PEIN) and insoluble (LA-M-PES and LA-M-PEIN) fractions as well as five pure compounds (7, 8 and 11-13) of endophytic fungus P. variotii from L. alba were tested for their cytotoxic activity in three human cancer cell lines namely breast (MCF-7), lungs (NCI-H460) and uterine cervix (HeLa). The broth extract was found more potent in all three cell lines than mycelium extract (table 5.13, 5.14 and 5.15). The extracts were treated with pet. ether to obtain pet. ether soluble (LA-B-PES, LA-M-PES) and pet. ether insoluble (LA-B-PEINS, LA-M-PEINS) fractions. The LA-M-PEINS was almost equi-potent in all the three cell lines with lower IC₅₀ values. Further work on these fractions led to obtain several pure compounds (vide experimental). Only one pure compound Viriditoxin showed cytotoxicity (tables 5.9, 5.10 and 5.12) with a lesser order than the fractions/extracts. This suggests that some more active constituents may be present and the observation warrants further studies in future.

5.4.2.1. Cytotoxic activity of extracts and fractions of fungal

part of Lawsonia alba.

Table 5.8: Growth inhibition and cytotoxicity induced by extract and fractions

against human breast cancer cell line (MCF-7)

Treatment	Dose (µg/mL)	Growth inhibition	IC₅₀ (µg/mL)	LC₅₀ (µg/mL)
LA-B-PES	10	+23 ± 2.4*	50 ± 3.8	>100
	25	+44 ± 2.8***
	50	+48 ± 3.9***
	75	+61 ± 4.7***
	100	+72 ± 4.0***
LA-B-PEINS	10	+49 ± 2.1***	10 ± 4.8	>100
	25	+77 ± 4.7***
	50	+78 ± 4.4***
	75	+82 ± 2.7***
	100	+87 ± 4.7***

LA-B-EA	10	+50 ± 4.2***	10 ± 4.4	225 ± 3.7
	50	+93 ± 3.6***
	100	-23 ± 2.5***
	200	-39 ± 3.7***
	250	-62 ± 4.4***
LA-M-PES	10	+25 ± 2.1*	20 ± 2.4	78 ± 3.1
	25	+61 ± 3.7***
	50	-44 ± 3.4***
	75	-48 ± 4.7***
	100	-69 ± 3.7***
LA-M-PEINS	10	+48 ± 2.4***	10 ± 3.1	68 ± 4.1
	25	+76 ± 3.5***
	50	-4.0 ± 4.1***
	75	-69 ± 3.4***
	100	-74 ± 2.7***
LA-M-EA extract	10	+34 ± 3.4***	30 ± 4.2	>250
	50	+65 ± 2.7***
	100	+93± 3.9***
	200	-14± 4.8***
	250	-47 ± 3.8***
Reference drug
Doxorubucin µg/mL (µM)	0.01	+4.0 ± 1.0	0.17 ± 0.03 (0.3 ± 0.04)	5.8 ± 1.0 (10 ± 1.2)
	0.1	+37 ± 5.0**
	0.5	+71 ± 2.0***
	5.0	–18 ± 3.0***
	10.0	–50 ± 2.0***

Table 5.9: Growth inhibition and cytotoxicity induced by extracts and fractions

against human lung cancer cell line (NCI-H460)

Treatment	Dose (µg/mL)	Growth inhibition	IC₅₀ (µg/mL)	LC₅₀ (µg/mL)
LA-B-PES	100	+9 ± 3.1	>100	>100
LA-B-PEINS	10	+49 ± 2.1***	10 ± 3.8	>100
	25	+77 ± 3.7***
	50	+78 ± 3.4***
	75	+82 ± 4.7***
	100	+87 ± 3.7***

LA-B-EA	10	+38 ± 2.4***	18 ± 3.2	220 ± 4.0
	50	+87 ± 4.0***
	100	-14 ± 2.8***
	200	-41 ± 2.7***
	250	-60 ± 3.4***
LA-M-PES	10	+25 ± 2.1**	20 ± 3.4	78 ± 2.9
	25	+61 ± 2.7***
	50	-44 ± 3.4***
	75	-48 ± 3.7***
	100	-69 ± 4.7***
LA-M-PEINS	10	+48 ± 2.4***	10 ± 2.7	68 ± 3.8
	25	+76 ± 3.5***
	50	-4 ± 4.1***
	75	-69 ± 3.4***
	100	-74 ± 2.7***
LA-M-EA	10	+8 ± 3.2	60 ± 4.2	240 ± 5.8
	50	+46 ± 4.0***
	100	+99 ± 3.5***
	200	-28 ± 5.7***
	250	-53 ± 4.5***
Reference drug
Doxorubicin µg/mL (µM)	0.01	+4.0 ± 1.0	(0.17± 0.05 (0 (0.2 6 ± 0.08)	5.4 ± 1.2 ( 9.3 ± 1.2)
	0.1	+42 ± 5.0**
	0.5	+76± 2.0***
	5.0	–18 ± 3.0***
	10.0	–53 ± 2.0***

Table 5.10: Growth inhibition and cytotoxicity induced by extracts and fractions

against human uterine cervix cancer cell line (HeLa)

Treatment	Dose (µg/mL)	Growth inhibition	IC₅₀ (µg/mL)	LC₅₀ (µg/mL)
LA-B-PES	10	+40 ± 1.9***	14 ± 2.4	80 ± 3.4
	25	+91 ± 2.5***
	50	-20 ± 3.7***
	75	-44 ± 4.1***
	100	-70 ± 4.5***
LA-B-PEINS	10	+50± 2.9***	10 ± 4.0	>100
	25	+83 ± 3.8***
	50	+98 ± 3.2***
	75	-8 ± 4.0***
	100	-16 ± 4.1***

LA-B-EA	10	+50 ± 2.1***	10 ± 2.5	220 ± 4.6
	50	+93 ± 1.9***
	100	-23 ± 3.7***
	200	-39 ± 5.9***
	250	-62 ± 4.7***
LA-M-PES	10	+36 ± 2.7***	1 16 ± 4.0	74 ± 4.9
	25	+78 ± 3.1***
	50	-25 ± 4.5***
	75	51 ± 4.6***
	100	-60 ± 3.2***
LA-M-PEINS	10	+49± 2.0***	10 ± 3.9	84 ± 5.7
	25	+88 ± 2.9***
	50	-21 ± 3.7***
	75	-44 ± 4.7***
	100	-63± 4.9***
LA-M-EA	10	+45 ± 3.1***	1 15 ± 2.1	180 ± 4.9
	50	+85 ± 4.7***
	100	-23 ± 4.8***
	200	-56 ± 4.0***
	250	-64 ± 3.9***
Reference drug
Doxorubicin µg/mL (µM)	0.0006	+4.0 ± 3.0	0.5±0.02^a 0.88±0.04	5.8 ± 0.1^a (10 ± 0.1)
	0.006	+5.0 ± 3.0
	0.06	+4.0 ± 2.0
	0.6	+60 ± 3.0***
	6.0	–52 ± 7.0***

5.4.2.2. Cytotoxic activity of pure compounds isolated from

leaves and fruits of Lawsonia alba

Table 5.11: Growth inhibition and cytotoxicity induced by pure compounds

against human breast cancer cell line (MCF-7)

Treatment	Dose (µM)	Growth inhibition	IC₅₀ (µM/mL)	LC₅₀ (µM/mL)
Luteolin-3′-O– β-D-glucoside (1)	10	+01 ± 0.6	56 ± 3.4	>100
	25	+19 ± 2.7
	50	+38 ± 2.9***
	75	+84 ± 3.1***
	100	-5 ± 2.5***
Luteolin (2)	100	+1 ± 0.9	>100	>100
Triacontyl (E)-3-(4- hydroxy-3- methoxyphenyl) acrylate (4)	100	+0 ± 1.2	>100	>100
Reference drug
Doxorubicin µg/mL (µM)	0.0006	+4.0 ± 3.0	0.5±0.02^a 0.88±0.04	5.8 ± 0.1^a (10 ± 0.1)
	0.006	+5.0 ± 3.0
	0.06	+4.0 ± 2.0
	0.6	+60 ± 3.0***
	6.0	–52 ± 7.0***

Table 5.12: Growth inhibition and cytotoxicity induced by pure compounds

against human lung cancer cell line (NCI-H460)

Treatment	Dose (µM)	Growth inhibition	IC₅₀ (µM/mL)	LC₅₀ (µM/mL)
Luteolin-3′-O– β-D-glucoside (1)	10	+01 ± 1.9	60 ± 3.2	>100
	25	+08 ± 2.8
	50	+31 ± 3.2**
	75	+85 ± 2.5***
	100	-23 ± 4.9***
Luteolin (2)	100	+0 ± 0.0	>100	>100
Triacontyl (E)-3-(4-hydroxy-3-methoxyphenyl) acrylate (4)	100	+3 ± 1.5	>100	>100

Reference drug
Doxorubicin µg/mL (µM)	0.0006	+4.0 ± 3.0	0.5±0.02^a 0.88±0.04	5.8 ± 0.1^a (10 ± 0.1)
	0.006	+5.0 ± 3.0
	0.06	+4.0 ± 2.0
	0.6	+60 ± 3.0***
	6.0	–52 ± 7.0***

Table 5.13: Growth inhibition and cytotoxicity induced by pure compounds against

human uterine cervix cancer cell line (HeLa)

Treatment	Dose (µM)	Growth inhibition	IC₅₀ (µM/mL)	LC₅₀ (µM/mL)
Luteolin-3′-O– β-D-glucoside (1)	10	+01 ± 1.4	54 ± 2.8	>100
	25	+21 ± 2.1*
	50	+45 ± 2.9***
	75	+82 ± 3.9***
	100	+99 ± 4.2***
Luteolin (2)	100	+1 ± 1.0	>100	>100
Triacontyl (E)-3-(4-hydroxymethoxyphenyl) acrylate (4)	100	+1 ± 1.1	>100	>100

Reference drug
Doxorubicin µg/mL (µM)	0.0006	+4.0 ± 3.0	0.5±0.02^a 0.88±0.04	5.8 ± 0.1^a (10 ± 0.1)
	0.006	+5.0 ± 3.0
	0.06	+4.0 ± 2.0
	0.6	+60 ± 3.0***
	6.0	–52 ± 7.0***

5.4.2.3. Cytotoxic activity of pure compounds isolated from

fungal part of Lawsonia alba.

Table 5.14: Growth inhibition and cytotoxicity induced by pure compounds

against human breast cancer cell line (MCF-7)

Treatment	Dose (µM)	Growth inhibition	IC₅₀ (µM/mL)	LC₅₀ (µM/mL)
Viriditoxin (12)	10	+11 ± 2.4	26 ± 2.4	>100
	25	+32 ± 3.1***
	50	+73 ± 2.6***
	75	+85 ± 4.9***
	100	-19 ± 4.1***
Stigmasta-5,7,22-trien-3 β-ol (7)	100	+0 ± 0	>100	>100
Stigmasta- 4,6,8(14),22- tetraen-3-one (13)	10	+11 ± 1.3	62 ± 4.9	>100
	25	+30 ± 3.0**
	50	+40 ± 4.2***
	75	+60 ± 3.2***
	100	+78 ± 3.3***
4-(2-Hydroxyethyl) phenol (8)	100	+0 ± 0	>100	>100
Ergosterol (11)	100	17 ± 1.9	>100	>100

Control absorbance (515 nm): MCF-7 (2.6 ± 0.9)

Table 5.15: Growth inhibition and cytotoxicity induced by compounds against

human lung cancer cell line (NCI-H460)

Treatment	Dose (µM)	Growth inhibition	IC₅₀ ( (µM/L)	LC₅₀ ( (µM/mL)
Viriditoxin (12)	10	+14 ± 2.7	20 ± 2.2	60 ± 5.2
	25	+63 ± 3.1***
	50	-43 ± 3.4***
	75	-61 ± 5.1***
	100	-71 ± 5.1***
Stigmasta-5,7,22-trien- 3- β-ol (7)	100	+0 ± 0	>100	>100
Stigmasta-4,6,8(14), 22- tetraen-3-one (13)	10	+3 ± 0.5	80 ± 3.9	>100
	25	+15 ± 2.3
	50	+28 ± 1.7**
	75	+45 ± 3.2***
	100	+61 ± 4.5***
4-(2-Hydroxyethyl) phenol (8)	100	+2 ± 1.0	>100	>100
Ergosterol (11)	100	4 ± 1.9	>100	>100

Table 5.16: Growth inhibition and cytotoxicity induced by pure compounds

against human uterine cervix cancer cell line (HeLa)

Treatment	Dose (µM)	Growth inhibition	IC₅₀ (µM/mL)	LC₅₀ ( (µM/mL)
Viriditoxin (12)	10	+44 ± 1.9***	12 ± 2.9	56 ± 5.0
	25	-4 ± 5.7***
	50	-44 ± 4.7***
	75	-67 ± 4.1***
	100	-72 ± 3.4***
Stigmasta-5,7,22-trien-3- β-ol (7)	100	+0 ± 0	>100	>100
Stigmasta-4,6,8(14),22- tetraen-3-one (13)	10	+1 ± 0.8	82 ± 3.9	>100
	25	+2 ± 0.9
	50	+10 ± 0.7
	75	+42 ± 1.2***
	100	+61 ± 2.6***
4-(2-Hydroxyethyl) phenol (8)	100	+2 ± 1.4	>100	>100
Ergosterol (11)	100	0 ± 0	>100	>100

Reference drug
Doxorubicin µg/mL (µM)	0.0006	+4.0 ± 3.0	0.5±0.02^a 0.88±0.04	5.8 ± 0.1^a (10 ± 0.1)
	0.006	+5.0 ± 3.0
	0.06	+4.0 ± 2.0
	0.6	+60 ± 3.0***
	6.0	–52 ± 7.0***

Part-A

5.5 Results and Discussion

5.5.1. Luteolin-3′-O–β–D-glucoside (1)

5,7-Dihydroxy-2-(4-hydroxy-3-((4,5,6-trihydroxy-3-(hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) phenyl)-4H-chromen-4-one (1)

Compound 1 was found as yellowish gummy solid. Its HREI-MS displayed molecular ion peak at m/z 448.0892 corresponding to the molecular formula C₂₁H₂₀O_11.The IR absorption bands were noted at 3589 for hydroxyl and at 1598 for α, β– unsaturated carbonyl and C-O stretching vibration occurred at 1120 cm^-1. The absorbance in the UV spectrum were noted at λ_maxat 213 and 269 nm. Analysis of the ¹H-NMR, ¹³C- NMR, DEPT, HSQC and HMBC (Table 6.1 vide experimental) showed luteolin type structure with sugar moiety at 3′ position. The ¹H-NMR displayed a series of signals ranging from δ_H 3.40-3.90 pertaining to sugar moiety. The anomeric proton and carbon of sugar moiety were observed at δ_H 4.92 (1H, d, J = 7.2 Hz), and δ_C 103.2 (CH, 1′′). Two meta-coupled protons (doublets, J = 2.0 Hz) at δ_H 6.35 and 6.13 were attributable to H-8 and H-6 respectively. The presence of another phenyl unit with three substituents were indicated by signals at δ_H 7.41 (dd, J = 9.0 Hz, 3.0 Hz H-6′), 7.40 (d J = 3.0 Hz H-2′) and 7.30 (d J = 9.0 Hz H-5′) respectively. One singlet for one hydrogen was observed at δ_H 6.54, owing to H-3. The ¹³C-NMR data showed a ketone carbonyl at δ_C 183.4 (C-4), two δ_H olefinic carbons at δ 165.1 (C-2) and 104.7 (C-3), three oxygen bearing carbons at δ 149.0 (C-5), 163.0, (C-7) and 149.0 (C-4′). The ¹H-NMR and ¹³C-NMR signals of sugar moiety δ_H 4.92, (1H, d, J = 7.2 Hz, δ_C 103.2 (CH, 5′′), 3.42, (1H, m), 71.2, CH, 4′′), 3.51, (1H, s), 77.5 (CH, 3′′), 3.47, (1H, s), 69.7, CH, 2′′), 3.55, (1H, s), 74.7, (CH, 1′′) revealed the presence of O-glycoside unit. In addition to this, the NMR data (Table 6.1 vide experimental) also revealed diastereotopic′′ protons CH₂ directly attached at C-2′′ [H-6a, 3.91, (1H, dd, J = 12.0 Hz, 1.8 Hz), [H-6b, 3.72 (1H, dd J = 12.0 Hz, 5.4 Hz, CH₂ C-6′′). These protons are diastereotropic owing to presence of chiral center at position 2′′. The type of carbons were identified from broad band (BB), DEPT 90 and DEPT 135 respectively. In the light of above data, the compound was identified as luteolin-3′-O-glucoside.

HMBC correlation of compound 1

Figure 5.1

5.5.2. Luteolin (2)

3-(3,4-Dimethylphenyl)-6,8-dimethyl-1-methylene-1,4-dihydronaphthalene (2)

Compound 2 was found as yellowish powdered solid. Its HREI-MS displayed molecular ion peak at m/z 286 corresponding to the molecular formula C₁₅H₁₀O₆_.The IR absorption bands were noted at 3307 for hydroxyl and intensive band at 1608 cm^-1 showed the presence of α, β– unsaturated carbonyl. The absorbance in the UV spectrum were noted at λ_max at 208 and 253 nm which indicates that the compound hails from the flavonoid group. Analysis of the ¹H-NMR, ¹³C-NMR, DEPT, HSQC and HMBC (Table 6.2 vide experimental) showed luteolin type structure without sugar moiety at any position. The ¹H-NMR did not display any series of signal ranging from δ_H 3.40-3.90 pertaining to sugar moiety. The ¹H-NMR showed two meta-coupled protons (doublets, J = 2.0 Hz) at 6.19 and 6.44 were attributed to H-6 and H-8 respectively. The presence of di-substituted phenyl unit was confirmed by the signals at 7.36 (d, J = 7.8 Hz H-2′), 7.36 (dd, J = 8.5 Hz, 2.0 Hz H-6′) and 6.88 (d, J = 8.5 Hz, H-5′). One singlet was observed at δ 6.52, integrating for one hydrogen, due to H-3. The ¹³C-NMR data showed a ketone carbonyl at δ_C 181.7 (C-4), two olefinic carbons 164.2 (C-2) and 113.9 (C-3), four oxygen bearing carbons at 151.6 (C-5), 164.3 (C-7), 141.0 (C3′) and 149.7 (C-4′) (Dordevic, Cakic, and Amr, 2000; Xu et al, 2009). The type of carbons were identified from broad band (BB), DEPT 90 and DEPT 135. Finally, through all the spectroscopic techniques the compound was identified as luteolin.

HMBC correlation of compound 2

Figure 5.2

5.5.3. Lawsone (3)

2-Hydroxynaphthalene-1,4-dione (3)

Compound 3 was obtained as red dye. Its HREI-MS displayed molecular ion peak at m/z 174 corresponding to the molecular formula C₁₀H₆O₃_.The IR absorption bands were noted at 3373 for hydroxyl (O-H stretching) and the carbonyl vibration band appeared at (C=O) 1641 cm^-1. Carbon carbon double bond (C=C) vibrational band of naphthalene ring appeared at 1578 and 1592 cm^-1. The absorbance in the UV spectrum were noted at λ_max 296, 339 and 416 nm. Analysis of the ¹H-NMR, ¹³C-NMR, DEPT, HSQC and HMBC (Table 6.3 vide experimental) showed naphthoquinone type structure. The ¹H-NMR showed signal at δ_H 8.10 (2 H, d, J = 7.8 Hz, H-5, H-8) and at 7.77 (2 H, d, J = 7.8 Hz, 7.2 Hz, H-6, H-7). One singlet was observed at δ_H 6.30 (1 H, d, H-3). The ¹³C-NMR showed signals at δ_C 184.9 (C-1) and 181.1 (C-4) which indicated the presence of two carbonyl groups. Other signals appeared at δ_C 156.2 (C-2), 110.1 (C-3), 126.6 (C-5), 133.1 (C-6), 135.2 (C-7), 126.4 (C-8), 132.8 (C-9) and 129.3 (C-10). The HREIMS spectrum exhibited the molecular ion at m/z 174 [M⁺] and other fragmented ions as m/z 146, 118, 105, 89 and 77. The type of carbons were identified from broad band (BB), DEPT 90 and DEPT 135 respectively. Through all the spectroscopic techniques the compound was identified as 2-hydroxy-1,4-naphthoquinone, commonly known as lawsone or hennotannic acid (Mahkam, Nabati, and Rahbar Kafshboran, 2014).

HMBC correlation of compound 3

Figure 5.3

5.5.4. Triacontyl (E)-3-(4-hydroxy-3-methoxyphenyl) acrylate

(4)

Compound 4 was obtained as white solid. Its HREI-MS displayed molecular ion peak at m/z 614 corresponding to the molecular formula C₄₀H₇₀O_.The IR absorption bands were noted at 3579 (OH stretching) and 1729 cm^-1 (C=O). The absorbance observed in the UV spectrum were at λ_max 230, 292 and 317 nm. Analysis of the ¹H-NMR, ¹³C-NMR, DEPT, HSQC and HMBC (Table 6.4 vide experimental) showed substituted cinnamate type structure with long chain alkane. The ¹H-NMR showed aromatic signals at δ_H 7.01 (1H, d, H-2), 6.90 (1 H, d, H-5) and 7.06 (1H, dd, H-6). The ¹H-NMR also had olefinic signals at δ_H 7.60 (1H, d, H-7) and at 6.29 (1H, d, H-8). Ester signals were observed in the ¹H-NMR spectrum at δ_H 4.16 (2H, t, H-10). The ¹H-NMR further showed three protons singlet at δ_H 3.91 (3H, s, H-40) which indicated the presence of methoxy group attached to benzene ring. The ¹³C-NMR showed a signal for carbonyl group at δ_C 167.3 (C-9) and aromatic carbon signals at 127.0 (C-1), 109.2 (C-2), 146.6 (C-3), 147.8 (C-4), 114.6 (C-5) and 123.0 (C-6). Olefinic signals were observed at δ_C 144.6 (C-7) and 114.6 (C-8). The ¹³C-NMR spectrum also showed signal at δ_C 64.6 (C-10) attached to oxygen. The ¹³C-NMR further showed a methoxy signal at δ_C 55.9 (C-40). Other signals for long chain alkyl groups (-CH₂-) were also observed between δ_C 22.6-31.9 for total 28 CH2 groups. The terminal methyl group was observed in the ¹³C-NMR spectrum at δ_C 14.1 (C-39). The HREI-MS spectrum showed the molecular ion at m/z 614 [M⁺] and other fragment ions as m/z 599, 586, 558, 194, 177, 137 and 57. The type of carbons were identified from broad band (BB), DEPT 90 and DEPT 135 respectively. In light of these data, the compound was identified as triacontyl (E)-3-(4-hydroxy-3-methoxyphenyl) acrylate.

HMBC correlation of compound 4

Figure 5.4

Part-B

5.5.5. 6-Hydroxy-2-(2-hydroxypropyl)-8-methyl-4H-chromen-

4-one (6)

Compound 6 was a white powdered. Its HREI-MS displayed molecular ion peak at m/z 234.0892 corresponding to the molecular formula C₁₃H₁₄O₄. The IR absorption bands were noted at 3474 (hydroxyl) and 1662 (α, β– unsaturated carbonyl) cm^-1. The UV spectrum showed λ_max at 242 and 291 nm. Analysis of the ¹H-NMR, ¹³C-NMR, DEPT, HSQC and HMBC (Table 6.5 vide experimental) showed a benzopyrone type ring. The ¹H-NMR showed signals at δ_H4.19 (1H, m, J = 6 Hz, 6.6 Hz, 6.6 Hz, H-2 and 1.27 (3H, d, J = 6 Hz, H-3′). The ¹H-NMR further showed two diastereotropic protons at δ_H 2.64 (2 H, dd, J = 14.4 Hz, 7.8 Hz, H-1′a) and 2.70 (2H, dd, J = 14.4 Hz, 4.8 Hz). Two meta coupled aromatic signals at 6.65 (1H, d, J = 2.4 Hz, H-5) and 6.63 (1H, d, J = 13.8 Hz, H-7), one singlet at 6.05 (1H, s, H-3). The ¹³C-NMR showed carbonyl signal at δ_C 180.0 (C-4), and olefinic singals at 167 (C-2) and 112 (C-3). This very downfield shift of olefinic carbon δ_C 167 (C-2) witnessed the direct attachment of oxygen with double bond. The ¹³C-NMR showed aromatic singals at δ_C 101.7 (C-5), 161.5 (C-6), 118.0 (C-7), 143.6 (C-8), 163.2 (C-9) and 118.1 (C-10) respectively. Its HREI-MS spectrum showed the molecular ion at m/z 234.0892 [M⁺] and m/z 190 showing the loss of ethyl alcohol. The type of carbons were identified from broad band (BB), DEPT 90 and DEPT 135 respectively. In the light of above data, the compound was identified as 6-hydroxy-2-(2-hydroxypropyl)-8-methyl-4H-chromen-4-one.

HMBC correlation of compound 6

Figure 5.5

5.5.6. Ergosterol (11)

Ergosta-5,7,22 E-trien-3 β -ol

Compound 11 was a white crystalline solid. Its HREI-MS displayed molecular ion peak at m/z 396.3548 corresponding to the molecular formula C₂₈H₄₄O_.The IR absorption bands were noted at 3373 (hydroxyl) and 1641 (olefinic C=C), 2940.7 (aliphatic C-H) and 2867.9 cm¹. The UV spectrum had λ_max at 240 and 270 nm. Analysis of the ¹H-NMR, ¹³C-NMR, DEPT, HSQC and HMBC (Table 6.8 vide experimental) showed a sterol type structure with extended conjugation at C-6 and C-7. The ¹H-NMR spectrum showed signals at δ_H 3.63 (1H, m, H-3), two diastereotropic at 2.46 (1H, ddd, J = 14.5 Hz, 2.5 Hz, H₄α) and 2.86 (1H, t, J = 12.0 Hz, H_4β). It further extended conjugation by protons signals at δ_H5.55 (1H, d, J = 3.0 Hz, H-6) and 5.38 (1H, d, J = 3.0 Hz, H-7). An olefinic (C=C) was also present at C-22 and C-23 as indicated by protons at δ_H5.14 (1H, dd, H-22) and 5.25 (1H, dd, H-23). Other ¹H-NMR signals were present at δ_H 0.63 (3 H, s, H-18) 0.94 (3 H, s, H-19), 1.02 (3 H, s, J = 6.5 Hz, H-21), 1.88 (1 H, m, H-24), 0.82 (3 H, d, J = 6.6 Hz, H-26), 0.84 (3 H, d, J = 6.6 Hz, H-27) and 0.91 (3 H, d, J = 6.8 Hz, H-28). The ¹³C-NMR spectrum showed six signals for olefinic carbons at δ_C 131.9 (C-5), 119.5 (C-6), 116.2 (C7), 135.5 (C-8), 135.3 (C-22) and 132.1 (C-23) respectively and a CH signal at (δ_C 70 for C-3) (Kamboj and Saluja, 2011). The type of carbons were identified from broad band (BB), DEPT 90 and DEPT 135 respectively. These data led to identify the compound 11 as ergosterol.

5.5.7. Stigmasta-5,7,22-trien-3 β-ol (7)

Compound 7 was a white crystalline solid. Its HREI-MS displayed molecular ion peak at m/z 410.3548 corresponding to the molecular formula C₂₉H₄₆O_. The IR absorption bands were noted at 3373 (hydroxyl), 1641 (olefinic C=C) cm^-1, 2940, 2867 (C-H) cm¹. The UV spectrum had λ_max at 240 and 270 nm. Analysis of the ¹H-NMR, ¹³C-NMR, DEPT, HSQC and HMBC (Table 6.6 vide experimental) showed a sterol type structure with extended conjugation at C-6 and C-7. By comparison of this compound with ergosterol only little difference was noted with one additional CH₂ group in compound 7. Thus in ergosterol CH₃ group was present at C-24 (C-28 CH₃) while in this compound ethyl group was present at C-24 (C-28 CH₂ and C-29 CH₃). The ¹H-NMR spectrum showed two diastereotropic protons at δ_H 1.49 and 1.59 each (H, m, H-28a and H-28b) while this was absent in ergosterol. It further showed a signal at δ_H 3.63 (1H, m, H-3) and two additional diastereotropic protons at δ_H2.46 (1H, ddd, J = 14.5 Hz, 2.5 Hz, H₄α) and 2.86 (1H, t, J = 12.0 Hz, H_4β). The extended conjugation at C-6 was demonstrated by protons at δ_H 5.55 (1H, d, J = 3.0 Hz, H-6) and 5.38 (1H, d, J = 3.0 Hz, 2.6 Hz, H-7). An olefinic double bond at C-22 was exhibited by protons at δ_H5.16 (1H, dd, H-22) and 5.35 (1H, dd, H-23). Other ¹H-NMR signals included δ_H0.63 (3H, s, H-18), 0.94 (3H, s, H-19), 1.02 (3H, s, J = 6.5 Hz, H-21), 1.88 (1H, m, H-24), 0.82 (3H, d, J = 6.6 Hz, H-26), 0.84 (3H, d, J = 6.6 Hz, H-27) and 0.81 (3H, t, J = 6 Hz, 7.5 Hz, H-29). The ¹³C-NMR spectrum showed signal for six olefinic carbons at δ_C131.9 (C-5), 119.5 (C-6), 116.2 (C7), 135.5 (C-8), 135.3 (C-22) and 132.1 (C-23) respectively (Kamboj and Saluja, 2011). The type of carbons were identified from broad band (BB), DEPT 90 and DEPT 135 respectively. These data led to identify the compound 7 as stigmasta-5,7,22-trien-3 β-ol.

5.5.8. 4-(2-Hydroxyethyl) phenol (8)

Compound 8 was obtained as white gummy solid. Its HREI-MS displayed molecular ion peak at m/z 138.0680 corresponding to the molecular formula C₈H₁₀O₂_.The IR absorption bands were noted at 3393.0 (hydroxyl) and 1501 -1510 (aromatic band) cm^-1. The UV spectrum had λ_max at 208, 254 and 347 nm. Analysis of the ¹H-NMR, ¹³C-NMR, DEPT, HSQC and HMBC (Table 6.7 vide experimental) showed a phenol type structure. The ¹H-NMR spectrum showed signals at δ_H2.78 (2H, t, J = 6.6 Hz, 13.2 Hz, H-1′), 3.81 (2H, t, J = 3 Hz, 3.6 Hz, H-2′), 6.77 (2H, d, J = 8.4 Hz, H-2, H-6) and 7.07 (2H, d, J = 8.4 Hz, H-3, H-5). The ¹³C-NMR spectrum showed aromatic carbons at δ_H 154.1 (C-1), 129.0 (C-4), 130.1 (C-3/5), and 115.5 (C-2/6) respectively. Its HREIMS spectrum showed molecular ion at m/z 138 [M⁺] and other fragment ions at m/z 107, and 77. It was observed that the mass spectrum of 4-(2-hydroxyethyl) phenol had fragment m/z 107 as base peak owing to the stability of tropylium ion formation. At the end by going through all the spectroscopic techniques the compound was identified as 4-(2-hydroxyethyl) phenol, which is commonly known as tyrosol (Linstrom and Mallard, 2001). The type of carbons were identified from broad band (BB), DEPT 90 and DEPT 135 respectively. In the light of above data, the compound was identified as 4-(2-hydroxyethyl) phenol.

HMBC correlation of compound 8

Figure 5.8

CHAPTER-06———————-

6.0 Experimental

6.1. General experimental procedure

The ultraviolet (UV) spectra were recorded on Thermo Scientific UV-Visible Spectrophotometer. Infrared (IR) spectra were measured on VECTOR₂₂ spectrophotometer. P-2000 Polarimeter was used to measure optical rotations. The Electron Impact Mass spectra were taken on Finnigan Mat 312 mass spectrometer and the Msroute JMS 600H. HREIMS spectra were recorded on MAT 95 XP Thermo Finnigan mass spectrometer. FAB spectra were measured on JEOL JMS-HX110 mass spectrometer, employing methanol as solvent and glycerol as matrix. NMR spectra were taken on Bruker AV 600, 500, 400, 300 spectrometer at 600 MHz, 500, 400, and 300 MHz for proton NMR (¹HNMR) spectroscopy. ¹³C NMR spectra were taken on Bruker avance AV-600 CRYO PROBE (600 MHz), and Bruker avance 500 spectrometer (500 MHz) employing TMS as internal reference. Two dimensional techniques (2D) for NOESY, COSY, HSQC, HMBC, TOCSY and J-resolved spectra were also taken on Bruker avance AV-600 CRYO PROBE (600 MHz), and Bruker avance 500 spectrometer (500 MHz) employing TMS as an internal reference.

Vacuum liquid chromatography (VLC) was executed employing TLC grade silica gel (E. Merck) 60 GF ₂₅₄. For column chromatography (CC), Silica gel 60 (mesh size 72-235, Machery-Nagel), Sephadex lipophilic LH-20, (Sigma-Aldrich) and reverse phase RP-18 (Merck) were employed. UV ranging from 254-366 nm were used for the detection of chemical constituents and ceric sulphate (1% Ce(SO)₂in 10 % H₂SO₄) was used as spraying agent. Recycling preparative HPLC was accomplished via employing HPLC (LC-908W Recycling Preparative HPLC) and solvent system water-methanol in the ratio of 1:1 was employed as mobile phase.

Part-A

6.2. Plant Material

The aerial parts of the plant Lawsonia alba (stem, leaves and fruits) were collected from the area of Karachi University in March 2013 and identified by Dr. Afsheen Athar Department of Botany, University of Karachi. A voucher (specimen G. H. No. 7292) has been deposited in the Herbarium of Karachi university. Leaves and fruits were separated manually from the aerial parts of Lawsonia alba.

6.2.1. Extraction of leaves and isolation of compounds

Dried Leaves (1.5 kg) were repeatedly (x5) extracted with ethanol at room temperature. A syrupy ethanolic concentrate (60.12 g) was obtained on removing solvent at reduced pressure. This concentrated syrup was partitioned between ethyl acetate and water. This was divided into pet. ether soluble (PES; 7.23 g; solvent removed) and petroleum ether insoluble fractions (PEI; 30.0 g).

The PEI fraction was subjected to normal column chromatography (silica gel; pet ether and ethyl acetate; (100:0 0:100). 50 fractions were obtained and combined on the basis of thin layer chromatography (TLC) to furnish 8 fractions (Fr-1, Fr-2, Fr-3, Fr-4, Fr-5, Fr-6, Fr-7 and Fr-8). The fraction Fr-2 was further subjected to pencil column chromatography (silica gel; pet ether and ethyl acetate (100:0- 0:100). 9 fractions were obtained and combined on the basis of thin layer chromatography (TLC) to furnish 1 major fraction, which was subjected to prep. TLC to obtained lawsone (3) (6 mg).

The PES fraction was subjected to normal column chromatography (silica gel; pet ether and ethyl acetate (100:0 0:100). 35 fractions were obtained and combined on the basis of thin layer chromatography (TLC) to furnish 4 fractions (PES-Fr-1, PES-Fr-2, PES-Fr-3 and PES-Fr-4). PES-Fr-1 was subjected to pencil column chromatography (silica gel; pet ether and ethyl acetate (100:0 0:100). 12 fractions were obtained and combined on the basis of thin layer chromatography (TLC) to furnish 3 fractions (Fr-1-1, Fr-1-2 and Fr-1-3). Fraction Fr-1-2 was again subjected to pencil column chromatography silica gel; pet ether and ethyl acetate (100:0 0:100) to obtained one major fraction, which was subjected to prep. TLC to obtained luteolin (2) (6.5 mg).

The main aqueous phase of leaves was extracted with n-butanol (Scheme 6.3). The butanolic phase was dried over anhydrous sodium sulphate and concentrated under reduced pressure to obtain a gummy residue (21.0 g). This was subjected to lipophilic sephadex (LH20) column chromatography (methanol-chloroform, 1:1). 40 fractions were obtained which were combined on the basis of thin layer chromatography (TLC) to furnish 6 fractions (Fr-1, Fr-2, Fr-3, Fr-4, Fr-5 and Fr-6). The fraction Fr-2 was subjected to RP-18 (reverse phase chromatography; water-methanol, (100:0 0:100). 8 fractions were obtained and these fractions were combined on the basis of reverse phase thin layer chromatography to furnish 3 fractions (Fr-2-1, Fr-2-2 and Fr-2-3). The fraction (Fr-2-1) was further subjected to prep TLC to obtain luteolin-3′-O–β-D-glucoside (1) (7 mg).

6.2.2. Extraction of fruits and isolation of compounds

Dried fruits (1.4 kg) were repeatedly (x5) extracted with ethanol at room temperature. A syrupy concentrate (50.15 g) was obtained on removing solvent at reduced pressure. This was partitioned between ethyl acetate (EA) and water. The EA phase was dried (anhyd. Na₂SO₄) and freed of solvent. The residue obtained was divided into pet. ether soluble (PES; 5 g; solvent removed) and petroleum ether insoluble fractions (PEI; 10. 0 g).

The PES fraction was subjected to normal column chromatography (silica gel; pet ether and ethyl acetate (100:0 0:100). 7 fractions were obtained and combined on the basis of thin layer chromatography (TLC) to furnish 3 fractions (Fr-1, Fr-2 and Fr-3).

The fraction Fr-2 was further subjected to pencil column chromatography (silica gel; pet ether- ethyl acetate; (100:0 0:100). Fractions were combined on the basis of (TLC) to furnish 2 major fractions (Fr-2-1 and Fr-2-2). Fr-2-1 was subjected to prep. TLC to obtain triacontyl (E)-3-(4-hydroxy-3-methoxyphenyl) acrylate (4). Fr-2-2 was subjected to prep. TLC to obtain ursolic acid (5).

Part-A

6.3 Characterization of Constituents

6.3.1. Luteolin-3′-O–β–D-glucoside (1)

(5,7-Dihydroxy-2-(4-hydroxy-3-((4,5,6-trihydroxy-3-(hydroxymethyl) tetrahydro-2H-pyran 2-yl) oxy) phenyl)-4H-chromen-4-one)

–

Luteolin-3′-O– β-D-glucoside was isolated as yellow gummy solid (7.0 mg) from the n-butanol soluble fraction of leaves methanolic extract. The molecular formula was derived as C₂₁H₂₀O₁₁ by exact mass measurement via HREI-MS.

Spectral Data

UV λ_max(log ɛ) in MeOH: 213 nm and 269 nm.

IR ^ʋ_max (KBR): 3589 (OH), 1598 (C=O), 1120 (C-O), cm^-1.

HREI-MS m/z: 448.10084 [M⁺] (calculated for C₂₁H₂₀O₁₁ 448.10054)

¹H- and ¹³C-NMR: Table 6.1

Table: 6.1: ¹³C-NMR (150 MHz), ¹H-NMR (600 MHz) Spectroscopic

Data^a forLuteolin-3′-O–β-D-glucoside (1). Solvent (MeOH)

Position ^δC_, mult ^δH_,mult (J in Hz) HMBC^b

2 165.1, C

3 104.7, CH 6.54, s 2, 4, 9

4 183.4, C

5 149.0, C

6 101.3, CH 6.13, d (2.0) 7, 9

7 163.0, C

8 96.0, CH 6.35, d (2.0) 4, 6, 9, 10

9 159.7, C

10 104.4, C

1′ 123.3, C

2′ 114.9, CH 7.40, d (3.0) 6′

3′ 150.0, C

4′ 149.0, C

5′ 117.8, CH 7.30, d (9.0) 4′, 6′

6′ 119.4, CH 7.41, dd (9.0, 3.0) 2′, 4′, 2

1′′ 103.2, CH 4.92, d (7.2) 3′

2′′ 69.7, CH 3.47

3′′ 77.5, CH 3.51 1′′

4′′ 71.2, CH 3.42, m 3′′, 6′′

5′′ 74.7, CH 3.55 d 3′

6′′ 62.5, CH₂ H-6a, 3.91, 1H, dd, (12.0, 1.8) 3′′

H-6b, 3.72, 1H, m (12.0, 5.4)

^αChemical shift values are in ppm and assignments are based on DEPT, COSY, HSQC and HMBC experiments. ^b Carbon atoms correlated to proton resonance in the ¹H-NMR column.

6.3.2. Luteolin (2)

2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4H-chromen-4-one)

Luteolin was isolated as yellowish powdered solid (7.0 mg) from the ethyl acetate soluble fraction of leaves methanol extract. The molecular formula was derived as C₁₅H₁₀O₆ by exact mass measurement via HREI-MS (Lin, Pai, and Tsai, 2015).

Spectral Data

UV λ_max(log ɛ) in CHCl₃: 208 and 253 nm.

IR ʋ_max (KBR): 3307 (OH), 1608 (C=O), cm^-1.

HREI-MS m/z: 286.0497 [M⁺] (calculated for C₁₅H₁₀O₆ 286.0477)

¹H- and ¹³C-NMR: Table 6.2

Table: 6.2: ¹³C-NMR (150 MHz), ¹H-NMR (600 MHz) Spectroscopic Data^a

Luteolin (2). Solvent (DMSO)

Position ^δC_, mult ^δH_,mult (J in Hz) HMBC^b

2 164.2, C

3 102.9, CH 6.44, s 10

4 181.7, C

5 151.6, C

6 100.5, CH 6.19, d (2.0) 8

7 164.0, C

8 94.0, CH 6.44, d (2.0) 6, 10

9 162.1, C

10 103.8, C

1′ 121.5, C

2′ 161, CH 7.40, d (2.0) 1′, 4′

3′ 141.0, C

4′ 149.7, C

5′ 116.1, CH 6.88, dd (8.5)

6′ 119.0, CH 7.36, d (2.0)

^αChemical shift values are in ppm and assignments are based on DEPT, COSY, HSQC and HMBC experiments. ^b Carbon atoms correlated to proton resonance in the ¹H-NMR column.

6.3.3. Lawsone (3)

(2-Hydroxy-1,4-naphthoquinone)

Lawsone was isolated as red-orange dye (7.0 mg) from the ethyl acetate soluble fraction of leaves methanol extract. The molecular formula was derived as C₁₀H₆O₃ by exact mass measurement via HREI-MS (Mahkam et al, 2014).

Spectral Data

UV λ_max(log ɛ) in DMSO: 296, 339, 416 and 448 nm.

IR ʋ_max (KBR): 3170 (OH), 1680 and 1648 (C=O), 1578 and 1592

(C=C), 1215 (C-O) cm^-1

HREI-MS m/z: 174.0323 [M⁺] (calculated for C₁₀H₆O₃ 174.0317)

¹H- and ¹³C-NMR: Table 6.3

Table: 6.3 ¹³C-NMR (150 MHz), ¹H-NMR (600 MHz) Spectroscopic Data^a

Lawsone (3). Solvent (MeOH)

Position ^δC_, mult ^δH_,mult (J in Hz) HMBC^b

1 184.9, C

2 156.2, C

3 110.2, CH 6.3, s 2, 4, 9

4 181.9, C

5 126.6, CH 8.10, d (7.8) 7

6 133.1, CH 7.77, dd (7.8, 7.2) 5, 7

7 135.2, CH 7.77, dd (7.8, 7.2) 8

8 126.4, CH 8.10, d (7.8) 1, 6, 7, 10

9 132.8, C

10 129.3, C

^α Chemical shift values are in ppm and assignments are based on DEPT, COSY, HSQC and HMBC experiments. ^b Carbon atoms correlated to proton resonance in the ¹H-NMR column.

6.3.4. Triacontyl (E)-3-(4-hydroxy-3-methoxyphenyl)

acrylate (4)

Triacontyl (E)-3-(4-hydroxy-3-methoxyphenyl) acrylate was obtained as white solid from petroleum ether soluble fraction of fruits methanol extract. The molecular formula was derived as C₄₀H₇₀O₄ by exact mass measurement via HREI-MS.

Spectral Data

UV λ_max(log ɛ) in DMSO: 296 nm, 339 nm, 416 nm

IR ʋ_max (KBR): 3373 (O-H), 1641 (C=O), 1578

and 1592 (C=C), cm^-1.

HREI-MS m/z: 614.5298 [M⁺] (calculated for C₄₀H₇₀O₄614.5274)

¹H- and ¹³C-NMR: Table 6.4

Table: 6.4 ¹³C-NMR (150 MHz), ¹H-NMR (600 MHz) Spectroscopic Data^a

Triacontyl (E)- 3-(4-hydroxy-3-methoxyphenyl) acrylate (4).

Solvent: CDCL₃

Position ^δC_, mult ^δH_,mult (J in Hz) HMBC^b

1 127.0, C

2 109.2, CH 7.01, d (2.0) 5, 6

3 146.6, C

4 147.8, C

5 114.6, CH 6.90, d (8.0) 1, 2, 6

6 123.0, CH 7.06, dd (8.0, 2.0) 2, 4, 5

7 144.6, CH 7.60, d (16.0) 2, 6, 9

8 115.6, CH 6.29, d (16.0) 1

9 167.3, C

10 64.6, CH₂4.16, t (6.4, 6.8) 11, 12

11 31.9, CH₂

12-18 29.0-29.7, CH₂ Seven signal

19 28.7, CH₂

20 25.9, CH₂

21-38 22.6-22.62

39 14.1, CH₃0.85,

40 55.9, CH₃3.91, s 2

^αChemical shift values are in ppm and assignments are based on DEPT, COSY, HSQC and HMBC experiments. ^bCarbon atoms correlated to proton resonance in the ¹H-NMR column.

Part-B

6.4 Lawsonia alba fungul part

6.4.1. Isolation of endophytic fungi from leaves

Apparently healthy looking leaf samples of Lawsonia alba were collected in sterile plastic bags from children garden and HEJ Research Institute of Chemistry, University of Karachi at 15 days interval throughout the reporting period. Fifty randomly selected leaves were used for isolation of endophytes each time. The samples were processed within 24 hours of collection. The leaves were washed thoroughly with running tap water to remove dirt from the surface, and cut into approximately 1cm² pieces with the help of a sterile pair of scissors. For surface sterilization, the leaf segments were dipped in 70% ethanol for 5 seconds then immersed for 90 seconds in 4% sodium hypochlorite and finally rinsed in sterile distilled water (Dobranic, J.K. et al, 1995). After blotter drying, three sterilized segments of a leaf were placed in each sterile Petri-dish containing agar media. The samples were incubated for 2-5 weeks at room temperature. The plates were checked every day after inoculation and any fungus that grew out from the cut ends of the leaf segments was isolated, purified, and maintained at 4 ^ºC on Potato Dextrose Agar for further identification. The whole procedure was repeated at 15 days intervals on three different media viz, malt agar, Czapecks Dox agar, and potato dextrose agar. The fungi were identified after reference to Raper and Fennel (1965), Domsch et al, (1980) and Ellis (1976, 1978).

6.4.2. Development of protocol for mass multiplication of the

fungi for isolation of secondary metabolites

Flasks (250mL) containing 100ml of Potato Dextrose, malt extract, or Czapecks Dox Broth were autoclaved at 121 ^ºC for 15 minutes. After cooling down, the broths were inoculated with a 5 mm diameter inoculum disc of a fungus using an actively growing culture of the selected endophyte. The flasks were incubated for 4 weeks at 28 ºC in a cool incubator and also in a shaking incubator. The fermentation broths were then filtered through two-folds of sterile cheese cloths to get the fungal biomass.

6.4.3. Extraction of fungal broth and isolation of

compounds

Fungus broth (LA-B-PES) was repeatedly (x3) extracted with pet. ether at room temperature to obtain pet. ether fraction. The pet. ether fraction was dried over anhydrous Na₂SO₄, filtered and freed of solvent under reduced pressure to obtained the pet. ether soluble (PES) fraction (200.0 mg). After extracting with pet. ether, fungus broth was repeatedly (x3) extracted with ethyl acetate (EtOAc) at room temperature to obtain ethyl acetate soluble fraction (LA-B-EA). The ethyl acetate fraction was dried over anhydrous Na₂SO₄, filtered and freed of solvent under reduced pressure to obtained the ethyl acetate fraction (6.0 g).

The pet. ether soluble (PES) fraction (200.0 mg) was freed of the solvent and taken in methanol. The concentrated methanol solution was kept in fridge overnight when a white crystalline mass deposited which was filtered and identified as stigmasta-5,7,22-trien-3 β-ol (7). The mother liquor further crops of stigmasta-5,7,22-trien-3 β-ol (43 mg).

The ethyl acetate (EtOAc) fraction (6 g) was subjected to column chromatography (silica gel; pet ether- EtOAc, (100:0 0:100). 60 fractions (Fr-1-60) were obtained and combined on the basis of thin layer chromatography (TLC) to furnish 5 fraction (Fr-1 through Fr-5). The fraction Fr-1 was subjected to pencil column chromatography silica gel; pet ether- EtOAc, (100:0 0:100). 3 fractions were obtained (Fr-1-1, Fr-1-2 and Fr-1-3) on mixing same practices. The fraction Fr-1 and Fr-2 were white powder with some impurity. The impurity was removed by washing it with methanol. Fr-1 and Fr-2 furnished pure β-sitosterol (9) and stigmasterol (10) respectively.

Fr-2 was also subjected to pencil column chromatography (silica gel; pet ether- EtOAc, (100:0 0:100). 2 major fractions Fr-2-1 and Fr-2-2 were obtained on combining on the basis of TLC. The fraction Fr-2-2 was subjected to prep. TLC and afforded 4-(2-hydroxyethyl) phenol (8).

The fraction Fr-4 was subjected to pencil column chromatography (silica gel; pet ether- EtOAc, (100:0 0:100). Fractions obtained were combined on the basis of TLC to furnish 3 fractions (Fr-4-1-Fr-4-3). The fraction Fr-4-3 was subjected to prep. TLC and furnished a novel compound lawsozaheer (6).

6.4.4. Extraction of fungal mycelium and isolation of

compounds

Fungus mycelium was repeatedly (x3) extracted with pet. ether at room temperature to obtain pet. ether soluble fraction (LA-M-PES), which was dried over anhydrous Na₂SO₄, filtered and freed of the solvent under reduced pressure to obtain the pet. ether fraction (300.0 mg). After extracting PES fraction, fungul mycelium was repeatedly (x3) extracted with ethyl acetate at room temperature to obtain ethyl acetate soluble fraction (LA-M-EA), which was dried over anhydrous Na₂SO₄, filtered and freed of the solvent under reduced pressure to obtain the ethyl acetate fraction (EAS) (18.0 g).

The pet. ether soluble (PES) fraction (300.0 mg) was taken in small amount of dichloromethane and kept in fridge overnight. A white crystalline residue settled down which was separated and characterized as ergosterol. The mother liquor furnished further crops of ergosterol (11). Final mother liquor was mixture of several compounds in very small quantities and was not proceeded further.

Part-B

6.5 Characterization of constituents

6.5.1. Lawsozaheer (7)

6-Hydroxy-2-(2-hydroxypropyl)-8-methyl-4H-chromen-4-one

Lawsozaheer was isolated in the form of white powder (6.5 mg) from the ethyl acetate fraction of fungal broth. The molecular formula was derived as C₁₃H₁₄O₄by exact mass measurement via HREI-MS.

Spectral Data

UV λ_max(log ɛ) in MeOH: 242 nm, 291 nm

IR ʋ_max (KBR): 3474 (O-H), 1662 (C=O) cm^-1.

[α]_D²⁷: – 0.016 (MeOH, c 0.09)

HREI-MS m/z: 234.2527 [M⁺] (calculated for C₁₃H₁₄O₄234. 2503)

¹H- and ¹³C-NMR: Table 6.5

Table: 6.5 ¹³C-NMR (150 MHz), ¹H-NMR (600 MHz) Spectroscopic Data^a for

Lawsozaheer (6). Solvent, MeOH

Position ^δC_, mult ^δH_,mult (J in Hz) HMBC^b

2 167.1, C

3 112, CH 6.05, s 2, 10, 1′

4 182.0, C

5 101.7, CH 6.65, d (2.4) 6, 7, 9, 10

6 161.5, C

7 118.0, CH 6.63, d (13.8) 5

8 143.6, C

9 163.2, C

10 118.1, C

1′ 43.4, CH₂2.64, dd (14.4, 7.8) 2, 3, 2′, 3′

2.70, dd, (14.4, 4.8) 2, 3, 2′, 3′

2′ 66.3, CH 4.19, m 2

3′ 23.5, CH₃1.27, d (6) 1′, 2′

4′ 23.2, CH₃1.93_,s 9

^α Chemical shift values are in ppm and assignments are based on DEPT, COSY, HSQC and HMBC experiments. ^b Carbon atoms correlated to proton resonance in the ¹H-NMR column.

6.5.2. Stigmasta-5,7,22-trien-3 β-ol (7)

Stigmasta-5,7,22-trien-3 β-ol was isolated as white crystals (43 mg) from petroleum ether soluble fraction of fungal broth. The molecular formula was derived as C₂₉H₄₆O by exact mass measurement via HREI-MS.

Spectral Data

UV λ_max(log ɛ) in MeOH: 240 nm and 270 nm.

IR ʋ_max (KBR): 3373.6 (O-H), 1641.6 (C=C), 2940.7 and

2867.9 (C-H) cm¹.

[α]_D²⁷: -0.001 (CDCl₃, c 0.08)

HREI-MS m/z: 410.3597 [M⁺] (calculated for C₂₉H₄₆O 410.3548)

¹H- and ¹³C-NMR: Table 6.6

Table: 6.6 ¹³C-NMR (150 MHz), ¹H-NMR (600 MHz) Spectroscopic Data^a

Stigmasta- 5,7,22-trien-3 β-ol (7). Solvent, CDCl₃

Position ^δC_, mult ^δH_,mult (J in Hz) HMBC^b

1 36.3, CH₂ 1.83,0, m

2 35.1, CH₂1.75, m

3 70.4, CH 3.63, m

4 36.6, CH₂H₄α 2.46, ddd (14.5, 2.5) 2, 3, 6

H₄β 2.86, t (12)

5 131.9, C

6 119.5, CH 5.55, d 4

7 116.2, CH 5.38, d

8 135.5, C

9 48.1, CH 1.92, t

10 38.3, C

11 24.9, CH₂2.33, m

12 36.6, CH₂1.25, m

13 43.5, C

14 48.1, CH 1.92, m

15 24.9, CH₂2.33, m

16 27.8, CH₂1.71, m

17 56.0, CH 1.25, m

18 11.3, CH₃0.63,s 12, 13, 14

19 17.6, CH₃0.94, s 1, 9, 10

20 39.4, CH 2.09, m 17, 22, 23

21 21.1, CH₃1.02, d (6.5) 17, 20, 22

22 135.3, CH 5.16, dd, (4.0, 4.0) 20, 23, 24

23 132.1, CH 5.35, dd, (3.0, 3.0)

24 42.8, CH 1.88, m 22, 23, 25, 26, 27, 28, 29

25 33.1, CH 1.43, m 24, 26, 27

26 19.6, CH₃0.82, d (6.5) 24, 25, 27

27 19.4, CH₃0.84, d (6.5) 24, 25, 26

28 19.7, CH₂1.49, 1.59, m 24, 29

29 19.9, CH₃0.81, t (7.5) 24, 25, 28

^α Chemical shift values are in ppm and assignments are based on DEPT, COSY, HSQC and HMBC experiments. ^b Carbon atoms correlated to proton resonance in the ¹H-NMR column.

6.5.3. 4-(2-Hydroxyethyl) phenol (8)

4-(2-Hydroxyethyl) phenol was obtained as white gummy solid from ethyl acetate fraction fungal broth. The molecular formula was derived as C₈H₁₀O₂ by exact mass measurement via HREI-MS.

Spectral Data

UV λ_max(log ɛ) in DMSO: 208 nm, 254 nm and 347 nm.

IR ʋ_max (KBR): 3393.0 (O-H) and 1501 -1510 cm^-1 (aromatic band)

HREI-MS m/z: 138.0697 [M⁺] (calculated for C₈H₁₀O₂ 138.0680)

¹H- and ¹³C-NMR: Table 6.8

Table 6.7 ¹³C-NMR (150 MHz), ¹H-NMR (600 MHz) Spectroscopic Data^a4-(2-

Hydroxyethyl) phenol (8). Solvent, MeOH

Position ^δC_, mult ^δH_,mult (J in Hz) HMBC^b

1, C
5, CH 6.77, d, (8.4) 1, 3
1, CH 7.07, d, (8.4) 4, 1, 1′
0, C
1, CH 7.07, d, (8.4) 1, 4
5, CH 6.77, d, (8.4) 1, 3

1′ 38.2, CH₂ 2.78, t, (6.6) 1, 2, 2′

2′ 63.8, CH₂ 3.81, t, (6.6) 2, 1′

^αChemical shift values are in ppm and assignments are based on DEPT, COSY, HSQC and HMBC experiments. ^bCarbon atoms correlated to proton resonance in the ¹H-NMR column.

6.5.4. Ergosterol (11)

Ergosta-5,7,22E-trien-3 β -ol

Ergosterol was isolated as white crystals (8 mg) from petroleum ether soluble fraction of fungal mycelium. The molecular formula was derived as C₂₈H₄₄O by exact mass measurement via HREI-MS.

Spectral Data

UV λ_max(log ɛ) in CHCl₃: 240 nm and 270 nm.

IR ʋ_max (KBR): 3373.6 (O-H), 1641.6 (C=C), 2940.7 ¹ and

2867.9 (C-H), cm¹.

[α]_D²⁷: -0.001 (CDCl₃, c 0.08)

HREI-MS m/z: 396.3597 [M⁺] (calculated for C₂₈H₄₄O 396.3548)

¹H- and ¹³C-NMR: Table 6.8

Table: 6.8 ¹³C-NMR (150 MHz), ¹H-NMR (600 MHz) Spectroscopic Data

^aErgosterol (11). Solvent CDCl₃

Position ^δC_, mult ^δH_,mult (J in Hz) HMBC^b

1 36.3, CH₂ 1.83,0, m

2 35.1, CH₂1.75, m

3 70.4, CH 3.63, m

4 36.6, CH₂H₄α 2.46, ddd (14.5, 2.5) 2, 3, 6

H₄β 2.86, t (12)

5 131.9, C

6 119.5, CH 5.55, d 4

7 116.2, CH 5.38, d 10, 13

8 135.5, C

9 48.1, CH 1.92, m

10 38.3, C

11 24.9, CH₂2.33, m

12 36.6, CH₂1.25, m

13 43.5, C

14 48.1, CH 1.92, m

15 24.9, CH₂2.33, m

16 27.8, CH₂1.75, m

17 56.0, CH 1.25, m

18 11.3, CH₃0.63, s12, 13, 14

19 17.6, CH₃0.94, s 1, 9, 10

20 39.4, CH 2.09, m 17, 22, 23

21 21.1, CH₃1.02, d (6.5) 17, 20, 22

22 135.3, CH 5.16, dd, (4.0, 4.0) 20, 23, 24

23 132.1, CH 5.35, dd, 3.0, 3.0)

24 42.8, CH 1.88, m 22, 23, 25, 26, 27

25 33.1, CH 1.43, m 24, 26, 27

26 19.9, CH₃0.82, d (6.6) 24, 25, 27

27 19.7, CH₃0.84, d (6.6) 24, 25, 26

28 19.6, CH₃0.91, d (6.8) 24, 25

^α Chemical shift values are in ppm and assignments are based on DEPT, COSY, HSQC and HMBC experiments. ^b Carbon atoms correlated to proton resonance in the ¹H-NMR column.

CHAPTER-08———————-

Aging

The process of rising aged or maturing.

Antioxidant

Those chemicals, which are required to hinder oxidation of other molecules and help to slow down oxidative stress, are called antioxidants.

Antimicrobial

Such type of metabolites or chemicals that are capable to kill microbes are said to be antimicrobs.

Antibacterial

Such type of phytochemicals or drudge which having property to destroy bacteria are called antibacterial.

Anticancer

Chemicals substances responsible to slow the growth of cancer causing cells are called anticancer.

Antifungal

A chemical which having ability to kill the fungi are called antifungal.

Antiviral

Photochemical which are responsible to murder viruses which cause to illness are called antiviral.

Analgesic

Any substance having ability to relieve pain.

Anti-inflammatory

A substance or a chemical that Controls inflammation.

Antioxidant potential

A chemical compound or substance that inhibits damaging effects of oxidation.

Arthrosclerosis

Atherosclerosis is a disease of the arterial blood vessels (arteries), in which the walls of the blood vessels become thickened and hardened by the deposition of fats.

Biosynthesis

The way through which organic molecules are forming fundamental building blocks in living things.

Carcinogenic

The substance that causes cancer.

Catalyst

Substances which required increasing or decreasing chemical reactions are called catalyst.

Cytotoxicity

Chemical substances which are capable to make toxic to specific cells are called

cytotoxicity.

Chromatography

The analytic technique which is use to isolate and separated different chemicals from mixtures

Esterification

A chemical reaction resulting in the formation of at least one ester product.

HPLC

High performance liquid chromatography is power full analytic technique which are used to separate a mixture of compounds those molecules which disturbed to immune system are said immunodulators.

Infra-red spectroscopy (IR)

Spectroscopic technique used to determine functional groups in a molecule is called IR spectroscopy.

In-vitro

The experiments which carried out external side of living thing are called IN-vitro.

In-vivo

The tests which carried out in side of living things are called In-vivo.

Metabolites

Metabolites are chemicals which are involves in metabolism in various type of living system.

Obesity

An excessive accumulation of fat in the body.

Phytochemical

Such type of plant extracted chemicals which are essential for biological activities are called phytochemicals.

Primary metabolites

Primary metabolites are those chemicals which are directly involved metabolisms and ordinary growth.

Secondary metabolites

Those metabolites which are not directly involved in metabolism but indirectly work in process of metabolism are said secondary metabolites.

Spectroscopy

Interaction between matter and radiation energy for elucidation of molecule is called spectroscopy.

Scavenging ability

Ability to remove unwanted substances.

Terpenoids

Any of a class of hydrocarbons that consist of terpenes attached to an oxygen-containing group. Terpenoids are widely found in plants, and can form cyclic structures such as sterols.

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