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′-Oβ-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:

  1. 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)

 

  1. 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-pE-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-Oα-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-Οβ -glucopyranoside Leaves C14H18O19 (Hsouna et al, 2011)
63. 2,4,6-Trihydroxyac-etophenone-2-Οβ-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-Oβ-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, Feions 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

  1. 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 IC50  value 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

  1. 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′-Oβ-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

  1. Luteolin-3′-O βD-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

 

  1. Luteolin (2)

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

Mol. Formula: C15H10O6

 

  1. Lawsone (3)

(2-Hydroxy-1,4-naphthoquinone)

Mol. Formula: C10H6O3    

 

5.1.2. Known constituents from the fruits of  L. alba

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

Mol. Formula: C40H70O4

  1. 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

  1. 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

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

 

Mol. Formula: C29H46O

 

  1. 4-(2-Hydroxyethyl) phenol (8)

Mol. Formula: C8H10O2

 

  1. 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

 

  1. 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                  

 

  1. 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

  1. 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 tR (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)

Sample Control
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)

Sample Control
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 (28o±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;

  1. 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
1

2

3

4

5

6

7

8

9

10

11

12

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

20

11

8

10

8

10

9

9

8

9

8

9

20

10

8

10

8

8

9

9

8

9

8

9

 

 

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±0.02a

0.88±0.04

5.8 ± 0.1a

(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±0.02a

0.88±0.04

5.8 ± 0.1a

(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±0.02a

0.88±0.04

5.8 ± 0.1a

(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±0.02a

0.88±0.04

5.8 ± 0.1a

(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±0.02a

0.88±0.04

5.8 ± 0.1a

(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′-OβD-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 C21H20O11. 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 λmax at 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 δH 3.40-3.90 pertaining to sugar moiety. The anomeric proton and carbon of sugar moiety were observed at δH 4.92 (1H, d, J = 7.2 Hz), and δC 103.2 (CH, 1′′). Two meta-coupled protons (doublets, J = 2.0 Hz) at δH 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 δH 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 δH 6.54, owing to H-3. The 13C-NMR data showed a ketone carbonyl at δC 183.4 (C-4), two δH 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 δH 4.92, (1H, d, J = 7.2 Hz, δC 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 λmax 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 δH 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 δC 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 C10H6O3The 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 λmax 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 δH 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 δH 6.30 (1 H, d, H-3). The 13C-NMR showed signals at δC 184.9 (C-1) and 181.1 (C-4) which indicated the presence of two carbonyl groups. Other signals appeared at δC 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 λmax 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 δH 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 δH 7.60 (1H, d, H-7) and at 6.29 (1H, d, H-8). Ester signals were observed in the 1H-NMR spectrum at δH 4.16 (2H, t, H-10). The 1H-NMR further showed three protons singlet at δH 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 δC 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 δC 144.6 (C-7) and 114.6 (C-8). The 13C-NMR spectrum also showed signal at δC 64.6 (C-10) attached to oxygen. The 13C-NMR further showed a methoxy signal at δC 55.9 (C-40). Other signals for long chain alkyl groups (-CH2-) were also observed between δC 22.6-31.9 for total 28 CH2 groups. The terminal methyl group was observed in the 13C-NMR spectrum at δC 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 λmax 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 δH  4.19 (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 δH 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 δC 180.0 (C-4), and olefinic singals at 167 (C-2) and 112 (C-3). This very downfield shift of olefinic carbon δC 167 (C-2) witnessed the direct attachment of oxygen with double bond. The 13C-NMR showed aromatic singals at δC 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 λmax 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 δH 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, H4β). It further extended conjugation by protons signals at δH 5.55 (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 δH 5.14 (1H, dd, H-22) and 5.25 (1H, dd, H-23). Other 1H-NMR signals were present at δH 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 δC 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 (δC 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 λmax 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 δH 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 δH 3.63 (1H, m, H-3) and two additional diastereotropic protons at δH 2.46 (1H, ddd, J = 14.5 Hz, 2.5 Hz, H4α) and 2.86 (1H, t, J = 12.0 Hz, H4β). The extended conjugation at C-6 was demonstrated by protons at δH 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 δH 5.16 (1H, dd, H-22) and 5.35 (1H, dd, H-23). Other 1H-NMR signals included δH 0.63 (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 δC 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 (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 C8H10O2The IR absorption bands were noted at 3393.0 (hydroxyl) and 1501 -1510 (aromatic band) cm-1. The UV spectrum had λmax 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 δH 2.78 (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 δH 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)2 in 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′-Oβ-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′-OβD-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 λmax (log ɛ) in MeOH:      213 nm and 269 nm.

IR ʋmax (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

Dataa for  Luteolin-3′-Oβ-D-glucoside (1). Solvent (MeOH)

Position             δC, mult                δH, mult (J in Hz)                             HMBCb

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)

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.

 

6.3.2. 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 λmax (log ɛ) in CHCl3:           208 and 253 nm.

IR ʋmax (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 Dataa    

                              Luteolin (2). Solvent (DMSO)

Position             δC, mult               δH, mult (J in Hz)                           HMBCb

 

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)

 

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.

 

6.3.3. 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 λmax (log ɛ) in DMSO:     296, 339, 416 and 448 nm.

IR ʋmax (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 Dataa   

                         Lawsone (3). Solvent (MeOH)

Position             δC, mult                δH, mult (J in Hz)                               HMBCb

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

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

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

HREI-MS m/z:                          614.5298 [M+] (calculated for C40H70O4 614.5274)

1H- and 13C-NMR:                     Table 6.4

 

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

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

Solvent: CDCL3

     Position             δC, mult                δH, mult (J in Hz)                             HMBCb

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,   CH2                           4.16, 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, CH

21-38                   22.6-22.62                        

39                        14.1, CH3                               0.85,

40                        55.9, CH3                                3.91, s                                            2

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.

 

 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 C13H14O4 by exact mass measurement via HREI-MS.

Spectral Data

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

IR ʋmax (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 C13H14O4 234. 2503)

1H- and 13C-NMR:                      Table 6.5

 

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

Lawsozaheer (6). Solvent, MeOH

Position             δC, mult                δH, mult (J in Hz)                               HMBCb

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,    CH2                     2.64, 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,    CH3                     1.27, d (6)                                  1′, 2′

4′                    23.2,    CH3                      1.93, 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 λmax (log ɛ) in MeOH:         240 nm and 270 nm.

IR ʋmax (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 Dataa    

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

Position             δC, mult                δH, mult (J in Hz)                               HMBCb

1                         36.3, CH2                         1.83,0, m

2                         35.1, CH2                          1.75, m

3                         70.4, CH                   3.63, m

4                         36.6, CH2                          H4α   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, CH2                         2.33, m

12                       36.6, CH2                         1.25, m

13                       43.5, C

14                       48.1, CH                  1.92, m

15                       24.9, CH2                         2.33, m

16                       27.8, CH2                         1.71, m

17                       56.0, CH                  1.25, m

18                       11.3, CH3                         0.63,  s                                                      12, 13, 14

19                       17.6, CH3                        0.94, s                                      1, 9, 10

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

21                       21.1, CH3                       1.02, 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, CH3                       0.82, d (6.5)                                24, 25, 27

27                       19.4, CH3                       0.84, d (6.5)                                24, 25, 26

28                       19.7, CH2                       1.49, 1.59, m                               24, 29

29                       19.9, CH3                       0.81, 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 λmax (log ɛ) in DMSO:     208 nm, 254 nm and 347 nm.

IR ʋmax (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 Dataa 4-(2-

Hydroxyethyl) phenol (8). Solvent, MeOH

Position             δC, mult                δH, mult (J in Hz)                              HMBCb

  • 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′

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.

 

6.5.4. 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 λmax (log ɛ) in CHCl3:         240 nm and 270 nm.

IR ʋmax (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)                               HMBCb

1                         36.3, CH2                         1.83,0, m

2                         35.1, CH2                          1.75, m

3                         70.4, CH                   3.63, m

4                         36.6, CH2                          H4α   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, CH2                         2.33, m

12                       36.6, CH2                         1.25, m

13                       43.5, C

14                       48.1, CH                  1.92, m

15                       24.9, CH2                         2.33, m

16                       27.8, CH2                         1.75, m

17                       56.0, CH                  1.25, m

18                       11.3, CH3                        0.63, s                                                         12, 13, 14

19                       17.6, CH3                       0.94, s                                       1, 9, 10

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

21                       21.1, CH3                       1.02, 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, CH3                       0.82, d (6.6)                              24, 25, 27

27                       19.7, CH3                       0.84, d (6.6)                              24, 25, 26

28                       19.6, CH3                       0.91, 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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