Alkaloids in S. Buxifolia Root and Stem Bark of S

Introduction

The natural products and natural medicine are nowadays withdrawing attention than the allopathic system. They remain the best sources of drugs and drug leads, and this remains true today despite the fact that many pharmaceutical companies have deemphasized natural products research in favor of HTP screening of combinatorial libraries during the past 2 decades. Moreover, this system of medicine is pollution free and less toxic with minor side effects. Out of the 6000 plants mentioned in the traditional systems of medicine, only 350 species are under use (Malviya et al., 2012).

Family Rutaceae comprises about 158 genera and 1900 species widely distributed in both temperate and tropical zones of the new, as well as, the old world. It is represented by 23 genera and over 80 species occurring mostly in the tropical and subtropical Himalayas and the Western Peninsular of India. (Mabberley, 2008).

The family consists mainly of trees or shrubs and a very small proportion of herbs, all of which flourish in the tropics and sub- tropics. It belongs to the order Sapindales and has an ancient name known as Rue family (Bailey, 1958). Rutaceae has been used in the folk medicine for the treatment of gastrointestinal

Introduction disorder such as anorexia, constipation and nausea, extract of Rue family has been used as a vermifuge, several species are used as tonic and diuretic (Metcalf and Chalk, 1950).

Rutaceae is of great economic importance with the most valuable members yielding Citrus fruits such as oranges, lemons, mandarins, limes, tangerines and grapefruit. Numerous species are cultivated for their precious essential oils such as Neroli and Bergamot that are used in the natural healing and perfume industries. Citrus genus is the most important source of essential oil as Citrus oils have been widely used in both perfumery and food industries.

The constituents of such major Citrus fruits as lemon (Clark and Bernhard, 1960) orange (Shaw and Coleman, 1974) and grapefruit (Umano et al., 1994) are well characterized.

Rutaceae is best known by the exotic genus Citrus, because of its commercially consumed fruits. Other groups of the same subfamily of Citrus are commonly cultivated as ornamentals in America, including species of Atalantia, Clausena, Murraya, and Swinglea.

Genus Atalantia includes approximately 20 speciecs (Roskov et al., 2016). Amongst Atalantia species, Atalantia buxifolia or Severinia buxifolia (Poir.) Tenore is the plant of our interest.

Introduction

Severinia buxifolia (Atalantia buxifolia) which is commonly known as the thorny, evergreen shrub. In Hanian province of China, the roots of Atalantia buxifolia are used in folk medicine for the treatment of influenza, cough, malaria, and stomachache by the people of Li nationality. Acridone alkaloids were the major alkaloids reported in Atalantia buxifolia, these alkaloids nucleus possess insect antifeedant and cytotoxic activities, respectively (YangTao, 2012). Atalantia buxifolia has been used in chinese folk medicine for treatment of chronic rheumatism, paralysis, snake bite and malaria, Atalantia buxifolia is also used for foundation and as a hedge plant (Wu and Chen, 2000). Moreover, leaves are used as yeast cake (Gilman, 1999).

This study hightlights the phytochemistry and activity of the leaves. Other organs were not studied as there is only one tree in the Orman garden. The only available organ is the leaf.

The present study includes the following: 1. Literature survey and reference books about Severinia buxifolia (Poir.)Tenore. (Atalantia buxifolia) 2. A botanical study of the different organs as well as DNA fingerprinting. 3. Preliminary phytochemical screening of the leaves.

Introduction

4. Investigation and identification of essential oil of the leaves by GC ̸ MS. 5. Investigation and identification of lipoidal matter of leaves by GC ̸ MS. 6. HPLC analysis of butanol, ethyl acetate and methylene chloride fractions of leaves of Severinia buxifolia (Poir.) Tenore. 7. Isolation, purification and characterization of the major chemical constituents from different fractions using different chromatographic and spectral methods. 8. Biological study of the ethanolic extract as well as its fractions prepared from the leaves together with the major isolated compounds.

Introduction

Review of literature This literature survey was performed on Severinia buxifolia (Poir.) Ten. as an attempt to shed light on the data reported concerning their major constituents and biological activities as well as to serve as a helpful guide during the foregoing study.

Major Constituents It was reported that S.buxifolia (Poir.) plant, family Rutaceae characterized by presence of the most important constituents. Examples are volatile oil, flavonoids, coumarins, alkaloids, triterpenes, sesquiterpenes and other miscellaneous compounds. In the leaf extract, the major compounds found are coumarins and volatile oil. No acridone alkaloids or limonoids were detected except in the root bark only (Bacher et al.,2010) as the following:

1- Flavonoids Flavonoids represent one of the largest and most studied classes of phenylpropanoid-derived plant specialized metabolites, with an estimated 10,000 different members. Structurally, they consist of two main groups, the 2-phenylchromans (the flavonoids, including flavanones, flavones, flavonols, flavan-3-ols, and anthocyanidins) and the 3-phenylchromans

Introduction

(the isoflavonoids, including isoflavones, isoflavans, and pterocarpans. Flavonoids have been ascribed positive effects on human and animal health and are central to the current interest in

“botanicals” for disease therapy and chemoprevention (Dixon and Pasinetti, 2010).

Bacher et al., (2010), Buckingham et al., (2015) reported presence of two new flavones Atalantoflavone, 4ˊ-O-methyl atalantoflavone in the methanolic extract of the leaves of S. buxifolia (Poir.) Tenore as minor components.

Qi and Yu, (1986) isolated 4′, 5-dihydroxy, 3′-OCH3, 2″, 2″- dimethyl pyrano (5″, 6″, 7, 8) flavone from the leaves of Atalantia buxifolia (Poir) Oliv. By spectral analyses (UV, IR, NMR, MS) and chemical transformation the structures of this new compounds was elucidated and identified.

2- Coumarins in S. buxifolia The name coumarin originates from Tonka tree (Coumarouna odorata) from which coumarin itself was isolated. Coumarins have been characterized in 178 rutaceous species from 50 genera. (YangTao, 2012).

Bacher et al., 2010 founded coumarins (seselin, isomeranzin, suberosin epoxide, bergaptene, imperatorine, suberosine,

Introduction bergamottin) as the major compounds in CHCl3 fraction of the ethanolic leaf extract of S.buxifolia (Poir.) Tenore. While the remaining coumarins and the flavones have not been described for S. buxifolia yet.

Chen et al., 2001 also isolated umbelliferone, 8-Geranyl-7- hydroxycoumarin, Auraptene and 7-geranyl-6-methoxycoumarin from the methylene chloride fraction of the methanolic extract of the leaves and the root bark of Atalantia buxifolia (Poir.) Tenore and these compounds are identified by different spectral analysis.

WU et al., 1998 studied presence of umbelliferone, 8-Geranyl-7- hydroxycoumarin in the ethanolic extract of the leaves of S.buxifolia (Poir.) Tenore.

Qi and Yu, 1986 isolated eleven chemical Constituents including two new compounds were isolated from the leaves of Atalantia buxifolia (Poir) Tenore. By spectral analyses (UV, IR, NMR, MS) and chemical transformation the structures of two new compounds were elucidated as 4′, 5-dihydroxy, 3′- OCH3, 2″, 2″- dimethylpyrano (5″, 6″, 7, 8) flavone and 4-(3′-methyl-but-2′-ene) oxy, N-benzoyl phenethylamine. Seven known compounds were identified as friedelin, lupeol, α-amyrin, γ-sitosterol, β-sitosterol, 7-(6′, 7′-dihydroxy-3′, 7′-dimethyl-2-octenyl) oxycoumarin and umbelliferone.

Introduction

Dreyer, 1967 described seselin as a major component of the leaves alcoholic extract and small amounts of imperatorin, isopimpinelin and bergapten in the fruit ethanolic extracts of S.buxifolia (Poir.) Tenore.

3- Alkaloids in S. buxifolia Root and stem bark of S. buxifolia (Poir.)Tenore are rich in alkaloids.

Yang et al., 2013 isolated two new acridone alkaloids, 3- methoxy-1, 4, 5-trihydroxy-10-methyl acridone and 2, 3- dimethoxy-1, 4, 5-trihydroxy-10-methylacridone from the ethanolic extract of S. buxifolia.

Tao et al., 2012 isolated seven alkaloidss from the ethanolic extract of roots of Atalantia buxifolia (Poir.) Oliv. by means of chromatographic technique. On the basis of spectral data, their structures were identified as buxifoliadine A, 1, 3-dihydroxy-2, 4- diprenylacridone, 5-hydroxy-N-methylseverifoline, buxifoliadine B, N-methylatalaphylline, atalaphylline and atalafoline.

Acridones having unique molecular structure of two benzene rings fused together having nitrogen atom at C10 position and a keto group at C9 position. Acridone have a wide range of biological activities such as antiviral, anticancer, anti-HIV, anti- fungal, anti-microbial, antiherpes, and antimalarial and anti-

Introduction leishmanial. The journey of acridones as pharmaceuticals was started by Paul Ehrlich in late nineteenth century (Giridhar et al., 2010).

WU and Chen, 2000 isolated eight new acridone alkaloids, buxifoliadines A-H together with nine known acridone compounds from the root bark of S. buxifolia which was collected in Hainan province of China. Their structures were determined by spectroscopic methods. They studied the relationship between acridone alkaloids with the collecting area. The 13C-NMR spectra of the prenyl substituents at C-2 and/or C-4 of N-unsubstituted acridone alkaloids were also studied.

WU et al., 1998 reported a new quinolin-2, 4-dione alkaloid, severibuxine, together with 23 known compounds were isolated from the root bark of Severinia buxifolia. The structure of these compounds was determined by spectral and chemical methods. Most of them showed cytotoxic activities against P-388.

Ming, 1987 isolated two acridone alkaloids named Atalafoline and Atalafoline-B from methylene chloride fraction of the roots of Atalantia buxifolia. Atalafoline-B is a new alkaloid and its structure was proposed as N-methyl-1, 4, 5- trihydroxy-3, 6- dimethoxyacridine-9-one based on UV, IR, H-NMR and MS spectra data.

Introduction

4- Triterpenes in S. buxifolia The triterpenes are one of the most numerous and diverse groups of plant natural products. They are complex molecules that are, for the most part, beyond the reach of chemical synthesis. Simple triterpenes are components of surface waxes and specialized membranes and may potentially act as signaling molecules. Simple and conjugated triterpenes have a wide range of applications in the food, health, and industrial biotechnology sectors (Thimmappa et al., 2014).

Here, we review triterpenes reported in S.buxifolia (Poir.)Ten.

Guoa et al., (2015) isolated a new triterpenoid with apotirucallane skeleton from the ethanol extract of the roots of Atalantia buxifolia (Poir.) Oliv. The complete structural assignment of the new compound was elucidated by a combination of 1D, 2D NMR (HMQC, HMBC, COSY, and NOESY), and HR-ESI-MS analysis. Moreover, this new compound was evaluated in vitro for its cytotoxic, antimicrobial and enzymes inhibitory activities.

Yang et al., (2012) studied a new tetranortriterpenoid, named 6- deacetyl-severinolide, together with six known tetranortriterpenoids, severinolide, acetyl-isoepiatalantin, 7- isovaleroylcycloepiatalantin, cycloepiatalantin,

Introduction

7-isovaleroylcycloseverinolide, and atalantin, which isolated from the ethanol extract of the roots of Atalantia buxifolia collected in Hainan. Their structures were elucidated by spectroscopic methods including 1D and 2D NMR (HMQC, 1H–1H COSY, HMBC, and NOESY).

Wu et al., 2001 isolated two new tetranorterpenoids, namely7- isovaleroylcycloseverinolide and 7-isovaleroylcycloepiatalantin from the root bark of S. buxifolia (Poir.) and chemical structure of these compounds were elucidated using x-ray crystallography and NMR.

Chen et al., 2001 isolated also eight tetranoterpenoids as Severinolide,

7-isovaleroylcycloepiatalantin, Cycloepiatalantin, 7isovaleroylcycloseverinolide, Atalantin , Cycloseverinolide, Atalantolide& Dehydroatalantin from the methanolic extract of the root bark of Atalantia buxifolia.

Qin and Yu, 1998 isolated a new limonoid from the ethanol extract of dried roots and stems of Atalantia buxifolia (Poir) Tenore. Based on chemical properties and spectral analysis, including H1 and C13 NMR, DEPT, MS, IR and UV spectra, its structure have been identified as: 6- deoxy- 6-α acetoxyatalantin

Introduction acetate. This compound was isolated from this plant for the first time.

Wu et al., 1997 studied new tetranortriterpenoids, severinolide and cycloseverinolide, together with known atalantin & cycloepiatalantin isolated and characterized from the ethanolic extract of the root bark of S. buxifolia.

Qi and Yu., 1986 isolated eleven chemical Constituents were isolated from the leaves of Atalantia buxifolia (Poir) Oliv. By spectral analyses (UV, IR, NMR, MS) and chemical transformation the structures. Known compounds were identified as friedelin, lupeol, and α-amyrin.

Introduction

5- Sesquiterpene in S. buxifolia Table (1): Sesquiterpenes isolated from the root bark of S. buxifolia (Poir.) Tenore

Compound Ref. . Dihydro-α-santalen-12-one WU et al., 1984 . 12,13- epoxy-α-santalene Chen et al., 2001 . α-Photosantalol A . Δ13, 14-iso-α-santalol . (E)-5-(2,3-dimethyl-3- nortricyclyl)pent-3-en-2-one . (+)-α-Santalan-12-one WU et al., 1984 . α- Santalone

. Santalane-11,12,13-triol Chen et al., 2001

. α-Santalene WU et al., 1998 . α-Santalen-11-one WU et al., 1984 Chen et al., 2001

Introduction

6- Sterol in S. buxifolia (Poir.) Ten.

Qi and Yu., 1986 isolated Constituents from ethanolic extract of the leaves of Atalantia buxifolia (Poir) Tenore. By spectral analyses (UV, IR, NMR, MS) and chemical transformation the structures compounds were identified as γ-sitosterol, β-sitosterol.

7- Volatile oil constituents in the leaves

Dongxu et al., (2011) analyzed the essential oil composition in Atalantia buxifolia by (GC-MS) using area normalization method for detection of essential oil components and twenty-eight volatile compounds of Atalantia buxifolia were identified. The main components were isocyclocitral (41.598 %), guaiacol (15.234 %), β-eudesmol (10.790 %), thujopsene (2.581 %), 1, 7, 7-trimethyl-bicyclohept-2-ene (5.841%), san-talol (3.702%), ferruginol (2.657 %).

Essential oil of the leaves of S. buxifolia (Poir.) Tenore were analyzed by GC and GC / MS (Scora and Ahmed, 1994) and 59 of the 73 seperated components were identified by their MS spectra and retention indices. The MS data are given for the remaining 14 unknowns. The major constituents were the sesquiterpens α -santalene (24%), Trans β -santalol (21%),

Introduction

germacrene-B (10%), and β- caryophyllene (7%) and the monoterpene limonene (19%).

8- Miscellaneous compounds in S.buxifolia Table9- ( 2): Structures of miscellaneous compounds of S. buxifolia (Poir.) Tenore

Compound Ref. Ferulic acid methyl ester WU et al., 2001 Chen et al., 2001 Coniferyl aldehyde Tao et al., 2012 Syringaresinol YangTao, 2012 N-benzoyl phenethylamine Qi and Yu., 1986 Asparagine Chen et al., 2001

Introduction

Biological activities for S. buxifolia (Poir.)Tenore

Antibacterial activity:

Yang et al.2013 studied the antibacterial activity of the two new acridone alkaloids, 3-methoxy-1,4,5-trihydroxy-10- methylacridone and 2,3-dimethoxy-1,4,5-trihydroxy-10- methylacridone, against Staphylococcus aureus. The compounds were isolated from the ethanolic extract of the branch of Atalantia buxifolia. The studied compounds showed significant antibacterial activity (Yang et al.,2013)

Anti cough activity:

Yonggin et al., (2013) studied that the effect of ethanol extract and different extracts of Atalantia buxifolia on relieving cough and phlegm.

Methods: Atalantia buxifolia was extracted by 95% ethanol and the extract has fractionated by petroleum ether, chloroform, ethyl acetate, n-butanol and water. The murine models of concentrated

Introduction ammonia spraying and sulfur dioxide- induced coughing were established. Tracheal phenol red method was used to study the pharmacodynamics of cough and phlegum. The concentration of phenol red was increased in the high dose group of ethanol, and chloroform and n-butanol extract compared with the control group.

Conclution: Ethanol extract, chloroform and n-butanol fractions showed an expectorant effect on relieving cough and phlegm (Yonggin et al., 2013)

Cytotoxic activity:

Tao et al., 2012. reported that buxifoliadine A and aurapten isolated from the ethanolic extract of roots of S. buxifolia (Poir.) Tenore showed cytotoxic activites against human hepatoma (SMMC-7721), and 5-hydroxy-N-methyl severfoline showed significant inhibitory activity towards chronic myelogenous Leukemia (K562).

Introduction

Insect Antifeedant activity:

Wu et al., (1997) studied two new tetranorterpenoids, severonolide and cycloseveronolide together with four known compounds (atalantin, dehydro atalantin, atalantolide, cycloepiatalantin), these were isolated and characterized from the root bark of S.buxifolia (Poir.) Ten. These compounds showed signifigant antifeedant activities against Plutella xylostella.

Antimalarial activity:

Fujioka et al., (1989) tested antimalarial activity of acridone alkaloids isolated from Severinia buxifolia (Poir.) Ten. in vitro and in vivo. At concentration of 10 µg ̸ ml in vitro, seven of these alkaloids suppressed 90% or more of Plasmodium yoelii, which causes malaria in rodents. Atalaphillinine, when injected intraperitoneally in a daily dose of 50 mg ̸ kg for 3 days into mice infected with 107 Erythrocytes parasitized with plasmodium berghei or plasmodium vinckei, completely suppressed the development of malaria parasites, with no obvious acute toxic effects from the tested dose.

Introduction

Severinia buxifolia (Atalantia buxifolia) which is commonly known as the thorny, evergreen shrub. Macromorphological and micromorphological study include rootlet, stem, bark and leaf. Genetic polymorphism of S. buxifolia was revealed and indicated by RAPD-PCR analysis using eleven primers. Steam volatile substances, Carbohydrate, and ̸ or glycosides, flavonoids, either free and ̸ or combined, Saponins, Sterols and ̸ or Triterpenes, Coumarins as well as tannins of both and hydrolysable types are identified. Compounds isolated from n- hexane fraction were (H1, H2, H3) as Seselin, β-sitosterol and Friedeline . Compound isolated from methylene chloride fraction was (M1) as + (-) Ulopterol. Compound isolated from ethyl acetate fraction was (E1) as Salycilic acid. The volatile constituents isolated from the leaves, comprised 59 identified components constituents 95.9%, 89.8%, 90.8%, 87.2% of the total oil at autumn, winter, spring and summer respectively. GC ̸ Mass analysis of USM revealed presence of 35 components in leaves. α-santalene is the major component in USM , while GC ̸ Mass analysis of FAME revealed presence of 13 components in leaves and major fatty acid detected in the leaves was Pentadecanoic acid 26.07%. The total phenolic content was evaluated by spectrophotometric method using Folin- Ciocalteu reagent and the result 52.89±0.001 µg ∕ mg (µg GAE/mg) in the total alcoholic extract of leaves, while total flavonoids content was determined

Introduction by the Aluminium chloride spectrophotometric method. This study revealed 27.40 ± 0.04 µg QE/mg in the total alcoholic extract of leaves.

HPLC analysis of the butanol, ethyl acetate and methylene chloride fractions from the leaves revealed that methylene chloride fraction gave the best separation for peaks and comprises major isolated compounds. Biological evaluation revealed that essential oil, n-hexane, methylene chloride and ethyl acetate fracions of S.buxifolia are powerful anti-microbial agents and can be used as natural anti-bacterial and anti-fungal as well. None of the isolated compounds showed relevant inhibitory potential against all tested protozoa except for that Seselin. All compounds were non-cytotoxic, it can be concluded that none of the isolated constituents showed relevant antiprotozoal activity, although the crude fractions were found to have some bioactivity. The total alcoholic extract of leaves has powerful antioxidant and hepatoprotective activity.