Obsahové Látky a Ich Biologická Aktivita Vybraných Druhov Euphorbiaceae- Euphorbia Trigona

FARMACEUTICKÁ FAKULTA

Obsahové látky a ich biologická aktivita vybraných druhov Euphorbiaceae- Euphorbia trigona

Diplomová práce

REBEKA DEMJANOVIČ MAGYAROVÁ

Vedoucí práce: prof. PharmDr. Karel Šmejkal, Ph.D.

Ústav přírodních léčiv Program Farmacie

Brno 2021

OBSAHOVÉ LÁTKY A ICH BIOLOGICKÁ AKTIVITA VYBRANÝCH DRUHOV EUPHORBIACEAE- EUPHORBIA TRIGONA

Bibliographic record

Author: Rebeka Demjanovič Magyarová Faculty of Pharmacy Masaryk University Department of Natural Drugs

Title of Thesis: Contain compounds and the biological activity of selected species of Euphorbiaceae - Euphorbia trigona

Degree Programme: Pharmacy

Supervisor: prof. PharmDr. Karel Šmejkal, Ph.D.

Year: 2021

Number of Pages: 93

Keywords: active substances, biological activity, Euphorbiaceae, Euphorbia trigona

2 OBSAHOVÉ LÁTKY A ICH BIOLOGICKÁ AKTIVITA VYBRANÝCH DRUHOV EUPHORBIACEAE- EUPHORBIA TRIGONA

Bibliografický záznam

Autor: Rebeka Demjanovič Magyarová Farmaceutická fakulta Masarykova univerzita

Ústav přírodních léčiv

Název práce: Obsahové látky a ich biologická aktivita vybraných druhov Euphorbiaceae- Euphorbia trigona

Studijní program: Farmacie

Vedoucí práce: prof. PharmDr. Karel Šmejkal, Ph.D.

Rok: 2021

Počet stran: 93

Klíčová slova: obsahové látky, biologická aktivita, Euphorbiaceae, Euphorbia trigona

3 OBSAHOVÉ LÁTKY A ICH BIOLOGICKÁ AKTIVITA VYBRANÝCH DRUHOV EUPHORBIACEAE- EUPHORBIA TRIGONA

Abstract

The diploma thesis is focused on finding and collecting verified information from trusted sources about the use of plants of the genus Euphorbia - specifically Euphorbia trigona, their content substances, and their biological activity This plant genus and the selected specie represent a huge potential for the treatment of many difficult-to-treat diseases and at the same time can pose a great risk if used unjusti- fiably. The theoretical part discusses the basic information about genus Euphorbia and specifically E. trigona, focusing mainly of the content substances, their biological activity and experiments performed using this succulent. The experimental part is focused on obtaining the extract from E. trigona and its fractions, and subsequently preparing a suitable mobile phase for further investigation of the substances and their potential use contained in the obtained fractions.

4 OBSAHOVÉ LÁTKY A ICH BIOLOGICKÁ AKTIVITA VYBRANÝCH DRUHOV EUPHORBIACEAE- EUPHORBIA TRIGONA

Abstract

Diplomová práca je zameraná na vyhľadanie a zozbieranie overených informácii z dôveryhodných zdrojov o obsahových latkách, ich biologickej aktivite a použití rastlín z rodu Euphorbia a následne konkrétne Euphorbia trigona. Tento rastlinný rod aj vybraný druh predstavujú obrovský potenciál pre liečbu mnoho ťažko liečiteľných chorôb a zároveň môžu predstavovať veľké riziko pri ich neopodstatnenom použití. V teoretickej časti sú rozobraté základné informácie o rode Euphorbia a E. trigona, zamerané hlavne na obsahové látky, ich biologickú aktivitu a experimenty prevedené s použitím tohto sukulentu. Experimentálna časť je zameraná na získanie extraktu a jeho frakcií z tejto rastliny a následne prípravu vhodnej mobilnej fázy pre ďalší prieskum látok obsiahnutých v získaných frakciách a ich potencionálne využitie v praxi.

OBSAHOVÉ LÁTKY A ICH BIOLOGICKÁ AKTIVITA VYBRANÝCH DRUHOV EUPHORBIACEAE- EUPHORBIA TRIGONA

Čestné prohlášení

Prohlašuji, že jsem diplomovou práci na téma Obsahové látky a ich biologická aktivita vybraných druhov Euphorbiaceae- Euphorbia trigona zpracovala sama. Veškeré prameny a zdroje informací, které jsem použila k sepsání této práce, byly citovány v textu a jsou uvedeny v seznamu použitých pramenů a literatury.

V Brně 13. března 2019 ...... Rebeka Demjanovič Magyarová

OBSAHOVÉ LÁTKY A ICH BIOLOGICKÁ AKTIVITA VYBRANÝCH DRUHOV EUPHORBIACEAE- EUPHORBIA TRIGONA

Poďakovanie

V prvom rade by som rada poďakovať profesorovi Šmejkalovi za jeho ochotu viesť ma pri písaní tejto diplomovej práce, za jeho odborné rady, kontrolu a trpezlivosť. Ďalej by som rada poďakovať pracovníkom ústavu prírodných liečiv, ktorí mi boli vždy k dispozícii, keď som potrebovala poradiť alebo niečo nájsť. Veľká vďaka patrí môjmu manželovi, ktorý mi bol oporou a podporoval ma v dopísaní tejto práce. Ďakujem aj moji rodičom, za ich podporu, rady a povzbudenia, mojej sestre, ktorá mi neváhala pomôcť pri formátovej stránke diplomovej práce, Carlovi Pflederovi za gramatickú korektúru a môjmu pár týždňovému dieťaťu, ktoré ma občas nechalo niečo napísať. A najväčšia vďaka patrí Pánu Bohu, bez ktorého by táto diplomová práca nebola.

CONTENTS

1 INTRODUCTION 15

2 THEORETICAL PART 16 2.1 Taxonomic classification of Euphorbia trigona ...... 16 2.2 Synonyms of E. trigona ...... 16 2.3 Taxonomy of genus Euphorbia ...... 17 2.4 Morphology of genus Euphorbia ...... 18 2.5 History of the genus Euphorbia ...... 19 2.6 Occurrence and use of Euphorbia plants ...... 19 2.7 Biochemical substances included in latex of Euphorbia species ...... 22 2.8 Biological activity of substances from Euphorbia plants ...... 28 2.9 Euphorbia trigona ...... 39 2.10 Occurrence and use of Euphorbia trigona ...... 42 2.11 Controlled human dermatological experiments and case reports with E. trigona ...... 44 2.12 Biochemical substances included in latex of E. Trigona ...... 46 2.13 Biological activity of E. trigona ...... 58 2.14 Potential biological activity of E. trigona ...... 60

3 EXPERIMENTAL PART 62 3.1 Chemicals used ...... 62 3.2 Material used for TLC ...... 62 3.3 Devices ...... 62 3.4 Herbal material used and its processing ...... 62 3.5 Methods ...... 65

4 DISCUSSION 71

5 CONCLUSION 73

6 LITERATURE 74

List of figures 87

List of tables 89 13 CONTENTS

List of Acronyms 90

14 INTRODUCTION

1 INTRODUCTION

Family Euphorbiaceae includes many different species with a wide variety of content substances. These substances also vary in biological activity. Some of them are used in traditional medicine, but most are still not thoroughly examined by science, and their potential for medical usage is largely unexplored. One of these less-examined species is Euphorbia trigona Mill., a spurge from the genus Euphorbia. This succulent plant is often seen as a houseplant, but in some regions of Asia it is also used as medical plant for treating various diseases. (1)(2) The purpose of this thesis was to find any medicinal properties of Euphorbia trigona Mill., as well as identifying any possible dangers involved with improper usage. These conclusions will be based on the content substances of Euphorbia trigona Mill., their biological activity and the provided experiments. However, because of the epidemiologic situation the experiment could not be completed, so it was decided instead to research the entire genus Euphorbia to identify possible active substances, their biological activity and use of E. trigona.

15 THEORETICAL PART

2 THEORETICAL PART

2.1 Taxonomic classification of Euphorbia trigona

Kingdom: Plantae – plantes, Planta, Vegetal, plants Subkingdom: Viridiplantae – green plants Infrakingdom: Streptophyta – land plants Superdivision: Embryophyta Division: Tracheophyta – vascular plants, tracheophytes Subdivision: Spermatophytina – spermatophytes, seed plants, phanéro- games Class: Magnoliopsida Superorder: Rosanae Order: Malpighiales Family: Euphorbiaceae – spurge, euphorbes Genus: Euphorbia L. – spurge Subgenus: Euphorbia Species: Euphorbia trigona Mill. (3)

2.2 Synonyms of E. trigona

Euphorbia hermentiana Lem. Euphorbia trigona Roxb. Euphorbia trigona Haw. African Milk Tree Friendship cactus African Milk-bush Saiunkaku (Japan) High Chaparall Cathedral caktus Abyssinian Euphorbia

16 THEORETICAL PART

Hermentiana triangularis (4)

2.3 Taxonomy of genus Euphorbia

The Euphorbiaceae family includes about 2000 species and is named after Euphor- bus, the physician to King Juba II of Mauritania in AD 18, who discovered the therapeutic properties of this family. (5) This family is the largest in the Spurge sub-category and contains trees, succulents and herbaceous plants, which usually contain milk latex. There is a suggestion that the Euphorbiaceae is closely related to Malvales, in particular the Sterculiaceae, because of the presence of monoadelphous stamen, rudiments of stames and the na- ture of the embryo. It is also believed that Euphorbiaceae arose from Malvales. (6)(7)(8) The family Euphorbiaceae contains many genera and because of the huge number of species, strong adaptive system, extreme morphological diversity and almost cosmopolitan distribution, it is difficult to complete infrageneric classification of genus Euphorbia. However, there is one characteristic morphological sign which unites the whole genus - the cyanthium. (8) The treatment by Boisseier in de Candolle’s Prodromus is the most recent complete monograph of Euphorbia and it is more than 150 years old. 740 species and 27 sections are recognized in this monograph. Following Boisseier, systematic treatments of the genus have been produced by Bethan, Pax and Hoffmann, Wheeler and Prokhanov. (8) Prokhanov divided the genus Euphorbia in three subgenera: Chmaesyce, Cysti- dospermum and Paralis. After that a great effort has been made to separate subgenera from the genus Euphorbia. However, the last molecular studies demonstrated that even though most of these genera are monophyletic, after separation they would end as paraphyletic Euphorbia. The last separation ended with the presence of four major subgenera: Esula, Rhizanthium, Chamaesyce and Euphorbia. (8) Euphorbia trigona is a plant classified in the genus Euphorbia, subgenus Euphor- bia. This genus is also known by the common name of Spurge. Subgenus Euphorbia

17 THEORETICAL PART

is the most diverse subgenus of the four subgenera and contains about 650 species. There are 21 sections within this subgenus divided into clades. The largest clade is Euphorbia sect. Euphorbia – the “spine-shield” Euphorbias. There are approximately 340 species, and their defining characteristic is the spine-shield. E. trigona has a paired spine. (8)(9)

2.4 Morphology of genus Euphorbia

Plants of genus Euphorbia are mostly herbs, rarely shrubs and trees, often filled with milky latex. (8) Leaves are opposite or alternate, sometimes replaced by hairs, thorns and glands. Flowers of the spurge are unisexual (male or female) and small-sized. Male flower contains single anther, filament, and pedicel. Female flower consists of a pedicel and three-parted ovary. They are often aggregated into an inflorescence or clus- ter of flowers known as cyathium. This feature is unique to spurge. Cyathium is a pseudanthial inflorescence which looks like a typical dicot flower. It can form beau- tiful variations in different groups within the genus. Each group of flowers is sur- rounded by floral envelope or involucre. There are glands attached to the involucre, which differ in size and shape. Sometimes specialized leaves known as cythophylls or cyathial leaves appear surrounding the cyathium (7)(10)(11) However, a new study by Prenner and Rudall suggest that the cyathium is nei- ther flower nor an inflorescence, but rather a “hybrid” whose genes maintain regulation that normally controls features of single flowers and have overlapped into the inflorescence itself. (11) Spurge fruit form capsules that typically split open immediately when ripe. There are usually three seeds in each capsule, which vary in size, shape and surface features. Seeds of some spurges have a fleshy appendage known as the caruncle above the point of attachment to the central column of the fruit. (10) One of the most salient features of spurge are variations in habits or physical forms. Some species are small, living just a few weeks. Conversely, there exists

18 THEORETICAL PART

species which reach 20 meters in height. Some of the succulents of genus Euphorbia have developed in underground tubes. (10) Spurge contains the milky sap-latex, which is suggested to have a protective and defensive role in helping heal wounds and in deterring potential plant-eaters. Latex is composed of many different chemical compounds, and some of them are toxic and potentially carcinogenic. Other can cause irritation by direct contact with the skin, eyes or mucous membrane. (10)

2.5 History of the genus Euphorbia

As previously mentioned, the family Euphorbiaceae is named after Euphorbus, and it contains many various species of Euphorbia. Euphorbus was the physician to King Juba II of Mauritiania in AD 18. He was examining spurge growing in the Atlas Moun- tains and from his works comes the first mention of plant Euphorbia. John Goodyear later wrote in his 1655 translation of AD Greek herbal by Dioscorides, that the latex of spurge removes hanging warts and is good for pterygia and carbuncles, gangrenes, fistulas. Years later it was said in England that the sap from spurge can remove all blemishes of the skin. (12)

2.6 Occurrence and use of Euphorbia plants

The Euphorbia plants grow all over the world, either wild or cultivated, and in the house or garden. Many members of this genus originate from Africa or Madagascar. Ninety-one Euphorbia species are found in Turkey. (13) Plants of genus Euphorbia have been used as traditional medicine for many years. Some of them are used as spices or food plants. The parts of the Euphorbia species used include roots, seeds, latex, leaves, flowers, stems or whole plants (14)(15) In India and Africa is the latex of spurges used for treatment against warts and in China against skin diseases. (12)

19 THEORETICAL PART

Some species of this genus have been used for the treatment of migraines, gonorrhea, skin diseases and against intestinal parasites. In particular, E. neriifolia Linn. has been used as cure against bronchitis, tumors, leukoderma, inflammation, abdominal troubles or enlargement of spleen. (15) E. hirta Linn. is also popular in traditional medicine, because of its ability to fight infections caused by susceptible bacteria. It has been used as oral drug for treating asthma, chronic flu and other respiratory diseases such as bronchitis. In parts of Af- rica and Asia it is used against gastrointestinal disorders, ocular diseases and intestinal parasitosis. Extract of E. hirta has shown anti-inflammatory and anti-asthmatic activities. (15)(16)(17) The flowers of E. virgata Waldst. & Kit. are used to treat eczema, and the flowers from E. helioscopia Linn., E. hirta and E. characias Linn. are used for their antifungal and antimicrobial activity. (13) The Euphorbia plants E. macroclada Linn. and E. coniosperma Boiss. are used for healing wounds and bites of snakes and scorpions. (13) E. esula contains a major antileukemic substance, the diterpenoid diester ingenol-13,20-dibenzoate. The milky latex of this plant has shown significant antineoplastic activity against the sarcoma 180 in rodents, specifically Walker 256 carcino- sarcoma, Lewis lung carcinoma and P-388 lymphocytic leukemia cell lines. (18) Another Euphorbia, E. neriifolia has been used as cure against bronchitis, tumors, leukoderma, inflammation, abdominal troubles or enlargement of spleen. (15) Traditional medicine has used E. nivulia as a treatment for many diseases such as syphilis, dropsy, general anasarca, leprosy, dyspepsia, jaundice, rheumatism, colic, bronchitis, whooping cough and as remedy for enlargement of liver and spleen. (17) There have been cases reported where latex of Euphorbia plants causes immediate burning, hyperemia and irritation in eyes or skin. Eyes are especially sensitive to the substances contained in the latex of Euphorbia plants- this contact results in burning pain, lacrimation, uveitis, photophobia, conjunctivitis, cornea ulceration, keratitis, cornea oedema, sloughing of the central corneal epithelium or even blind- ness. (5) One specific example is a report of a 44-year-old man who has a corneal epithelial defect in the eye caused by E. tirucalli sap. He and the other patients hurt

20 THEORETICAL PART

by Euphorbia sap reported that the symptoms began immediately after exposure and worsened after several hours. The contact between the sap of spurge and eyes caused corneal punctate epitheliopathy, which continued to increase corneal epithelial defect. This could have been caused by the antineoplastic effects of spurge sap, which can prevent the replication of corneal epithelia resulting in slowing of the healing process. Fortunately, after nine or more days of immediate treatment the visual outcome was good. (12) Another study maintains that ocular toxicity can by caused by other three species of Euphorbia genus. These are E. trigona, E. neriifolia and E. milli. (19) In contrast, there is a case in which Euphorbia latex was used as drug to treat itching in eyes. (20) Because of all these different facts, further examination of the chemical substances obtained in Euphorbia plants and their biological activity is obviously required to gather information for their appropriate potential usage.

21 THEORETICAL PART

2.7 Biochemical substances included in latex of Euphorbia species

As previously mentioned, Euphorbia species contains a milky liquid called latex. This colloidal suspension contains many different substances, mainly secondary metabolites and proteins. They are contained in specialized cells known as laticifers. Laticif- ers are internal secretory structures which may contain a variety of cellular components, among them nuclei, mitochondria, ribosome-like particles and lutoids as well as nucleic acids. In latex we can find various enzymes, alkaloids, terpenes, vitamins, carbohydrates, lipids and free amino acids. The function of latex in many plants is not proven, but some say that it serves as a protection against microbial attacks and herbivore consumption. Many studies concluded that members of Euphorbia are rich sources of lectins, which are able to inhibit protein synthesis in tumoral cells. (4)(21)(22)

2.7.1 Monoterpenes

Monoterpenes are terpenes composed of two isoprene units with ten carbon atoms. These arise from geranyldiphospate. Monoterpenes are the major components of essential oils and are often used in human health care products. Both the natural monoterpenes and their synthetic derivatives show various pharmacological properties such as antibacterial, antifungal, antioxidant, anticancer, anti- inflammatory, antihis- taminic, local anesthetic and anti-spasmodic. (23)(24) Plants of genus Euphorbia contain various monoterpenes, for example α-terpineol and linalool. (13)

2.7.2 Sesquiterpenes

The basic skeleton of sesquiterpenes contains 15 carbon atoms. Sesquiterpenes often form one of the main constituents of essential oils. From the carbon skeletons, we can distinguish several types of sesquiterpenes, namely acyclic, mono-, bi- and tricyclic. The substituents can be divided into alcohols, ketones and sesquiterpene

22 THEORETICAL PART

lactones. From a therapeutic point of view the most important are probably azuleno- genic compounds. (23) Sesquiterpenoids: β-caryophyllene, germacrene-D, α-humulene, clovandiol, eu- phanginol, euphorbioside A and B were found in the extract of Euphorbia plants. (13)(25)(26)

2.7.3 Diterpenes

Diterpenes are natural compounds consisting of four isoprene units and having twenty carbon atoms. Their precursor can be considered geranylgeraniol (and its di- phosphate). (23) The diversity of their chemical structure and distribution in the organisms can be used in chemotaxonomic classification. Diterpenes show also different biological and pharmacological activities which could be used in various treatments. By contrast, many diterpene esters isolated from the latex of Euphorbia plants are able to poison human and animals, cause cell proliferation, tumor promotion and skin dermatitis. These esters are usually esters of phorbol, and because of their ability to activate protein kinase C, are able to initiate uncontrolled cancerous growth. (25) Protein kinase C (PKC) is the name of the family of serine/threonine enzymes. This family has three subclasses of enzymes- conventional, novel and atypical PKC. This family of enzymes is involved in different cell signaling events that induce cell proliferation, growth, apoptosis, metastasis and regulation of gene expression. These enzymes, specifically atypical PKCs, participate in treatment of many types of cancer, such as breast cancer, colon cancer, ovarian cancer and melanoma. According to one study, inhibitors of atypical PKC in combination with rapamycin could be potential treatment against bladder cancer by activating tumor suppressors in bladder cancer cells. (27) Diterpenoids have been found in the latex of Euphorbia species with the basic skeletons of abietane, jatrophane, lathyrane, myrsinane, daphnane, ingenane, tigliane, paraliane, pepluane and segetane. Some of these diterpenoids are unusual and could contain potential pharmacological activities. (13)(23)(26)

23 THEORETICAL PART

One of these specific groups of diterpenoids are abietanes. These diterpenoids have usually an α, β-unsaturated γ-lactone ring, which stands between C-12 and C- 13. The importance of these substances is in their inhibitory activity on growth of different tumour cells, for example Jurkat cells, ANA-1, B16, K562 cells and LNCaP cells. Some of the Euphorbia species have abietane diterpenoids with an epoxy ring system usually at C-8 a C-14 or C-11 and C-12. This system is responsible for ob- served cytotoxicity. (26) Another type of diterpenoid is ingenane. These substances have a specific 5/7/7/3- tetracyclic ring system and a ketone bridge between C-8 and C-10. It is thought that these diterpenoids show antinematode and termiticidal activity. For antinematode activity the presence of one free hydroxy group at C-3 or C-5 is crucial. (26) Another study has reported tumor promoting and proinflammatory activity, but also irritant effects. This effect is probably caused by the presence of a hydroxyl on C-20, because when it has been replaced with acetyl group the irritancy was lowered. Furthermore, the length of the aliphatic chain in ester group of the ingenol-3-esters influences the irritant properties and the skin tumor promoting activities. (26) One of these diterpenoids is ingenol-3-angelate. This active compound found in E. antiquorum and E. peplus represents a potential future treatment of cancer. Extracts of these plants have been already used in traditional medicine in Thailand and Aus- tralia, and a specific study showed that ingenol-3-angelate is a PKC ligand. This ingenane ester was able to cause down-regulation of classical and novel PKC isoforms in three tested cancer cell lines. (28) The third specific group of diterpenoids are tiglianes. These substances have 5/7/6/3-tetracyclic ring system and almost all of them have hydroxy groups located on C-4, C-9, C-13 and C-20. These hydroxy groups could be easily esterified, resulting in higher tumor promotion, irritant and proinflammatory activity. (26) All these three groups of diterpenoids - abietane, ingenane and tigliane - are the main substances with anticancer properties from genus Euphorbia. (29) A final mention of note is the diterpenoid with a myrsinol-type skeleton, 3,7,15- tri-O-acetyl-5-O-nicotinoyl-13,14-dihydroxymyrsinol. This substance was isolated from E. decipiens and it has inhibitory activity against jack bean urease and also

24 THEORETICAL PART

against Bacillus pasteurii urease. Excessive levels of urease in soils reduces soil fer- tility in an agricultural context, and excess in the body can promote development of kidney stones, pyelonephritis and peptic ulcers. Due to these facts, it is desirable to develop new inhibitors of urease levels. Extract of E. decipies, with great efficacy against urease, presents a potential and safe solutions for these problems. (30)

2.7.4 Triterpenes

Triterpenoids are phytochemicals found in plants, biosynthesized by the cyclization of squalene. They are metabolites of isopentenyl pyrophosphate oligomers and can be subclassified into different groups. After extensive research, it is plausible to maintain that triterpenoids have a large biological, and they are already used as medicine for their anti-inflammatory, analgesic, antipyretic, hepatoprotective, sedative or tonic activities. Additionally, some of them show antioxidant, antimicrobial, antiviral, antiangiogenic, anticancerogenic or spasmolytic activity. (28) Extracts of Euphorbia plants contain triterpenes such as lupeol, betulin, tarax- erone, taraxerol, lupeol, lupeol acetate and β-amyrin. (13)(31)(32)(33)(34)(35)

2.7.5 Steroids

Steroids are natural substances derived from triterpene squalene. Their structure contains tetracyclic hydrocarbon sterane – cyclopentanoperhydrophenanthrene. (23) In Euphorbia plants the phytosterols: β-sitosterol, cycloartenol 24-methylenecycloartanol, daucosterol, 24-hydroperoxycycloart-25-en-3β-ol, 25-hydroperoxycycloart-23-en-3β-ol and (23E)-cycloart-23-en-3β,25-diol were found. (13)(31)(36)(37)(38)

2.7.6 Stilbenes

Stilbenes are secondary metabolites with the structure of 1,2-diphenylethylene backbone with hydroxyl groups substituted on the aromatic rings. They exist in

25 THEORETICAL PART

monomer or oligomer form. Some of them, for example trans-resveratrol, have a con- trolling influence on cell proliferation and apoptosis. (39) Piceatannol and resveratrol were found in this group of compounds in Euphor- bia. (40)(43)

2.7.7 Flavonoids

Flavonoids are aromatic substances with two aromatic rings and structure C6–C3– C6. Located in their skeleton is a chromane ring connected to second aromatic ring in position 2,3 or 4. (41) Flavonoids are subdivided into flavones, flavonols, flavanones, flavanols, anthocyanins and isoflavones. Some of them are often found in human diet and could be useful in prevention of cancers, cardiovascular diseases and neurodegeneration. (41) Kaempferol, myricetin, avicularin, tricetin, myricitrin, rutin, quercitrin, di- methoxy quercetrin, quercetin, isoquercetin, ampelopsin, baicalein, wogonin and their derivatives, such as kaempferol 3-O-glycopyranoside and others were found in the flavonoids of Euphorbia plants. (31)(42)(43)

2.7.8 Tannins

Tannins are organic substances found in plants. They are polyphenols with the phenolic group bound to the amino groups of the polypeptide chains of the protein molecules. (23) In Euphorbia plants tannins such as euphorbin E, euphorbin F, tirucallin A and tirucallin B were detected. (13)(44)

2.7.9 Phenolic acids

Phenolic acids are non-flavonoid phenolic compounds derived from benzoic and cin- namic acids and further divided into two groups, derivatives of hydroxybenzoic acid and derivatives of hydroxycinnamic acid. Phenolic acids are found in plants and ce- reals as products of secondary metabolism in plants. Some of them have antioxidant properties. Their molecules contain a benzene ring substituted with one or more car- boxylic groups, side chain with carboxyl and hydroxyl or methoxyl groups. The

26 THEORETICAL PART

hydroxycinnamic group of acid shows stronger antioxidant effect, because they have a double bond in their structure, improving scavenging of radicals. (45) Phenolic acids were also found in the extracts of various Euphorbia plants. These acids are vanillic acid, p-hydroxycinnamic acid, ellagic acid, 3,3'-di-O-methylellagic acid, 3, 3’, 4'-tri-O-methylellagic acid, gallic acid, 4-O-methylgallic acid, 3,4-di-O-gal- loylquinic acid, caffeic acid, chlorogenic acid, chrysophanic acid, protocatechuic acid, dihydroxybenzoic acid, p-coumaric acid, ferulic acid, caffeic acid and octadecanoic acid. (46)(47)(48)

2.7.10 Coumarins

Coumarins are natural products found in plants with vanilla-like odor. They are ben- zopyrones, lactones of O-hydroxycinnamic acid. Coumarins are subdivided into four subclasses: simple coumarins, dimer coumarins, furanocoumarins and pyranocou- marins. They show various biological activities, such as antimicrobial, antiviral, anti- inflammatory, antidiabetic, antioxidant and enzyme inhibitory. (49)(50) The coumarins scopoletin, isoscopoletin, 6,7-dihydroxycoumarin, umbellifer- one, 6,7,8-trimethoxyl-coumarin, scoparone, esculetin, daphnoretin, aesculetin-6-O- (6’-O-galloyl)-β-D-galactopyranoside, fraxetin-8-O-(6’-O-galloyl)-β-D-galactopyranoside and isocoumarin ethylbrevifolin carboxylate were found in Euphorbia plants. (51)(52)

27 THEORETICAL PART

2.8 Biological activity of substances from Euphorbia plants

The substances detected in Euphorbia plants have various pharmacological properties. Some of these properties and activities have great potential for use as treatment against various diseases. However, some useless and even dangerous effects were discovered within the latex compounds of some Euphorbia plants. These dangerous effects included high skin and eye irritation, prostaglandin production activation, blood platelets activation and tumor promotion. (53) Because this work is primarily oriented to the relatively unstudied species E. trigona, it was necessary to collect information on the entire Euphorbia genus to ac- curately estimate the pharmacological significance of E. trigona. Many Euphorbia species are used in traditional medicine for their analgesic, anti-inflammatory, antifungal, cytotoxic, diuretic, laxative and wound healing activity. However, some of these effects remain scientifically unproven, so further investigation on Euphorbia genus and its biological activity is required. (13)

2.8.1 Antibacterial and antimicrobial activity

The Euphorbia plants whose extracts showed antibacterial and antifungal activity are E. fusiformis, E. hyssopifolia, E. helioscopia, E. peplus, E. hirta, E. neriifolia and E. characias. Several studies confirmed that flavonoids and their derivatives are responsible for antimicrobial activity. For example, the extract of E. characias contains flavonoids quercetin-3-O-glucoside, quercetin-3-O-rhamnoside, quercetin-3- O-arabinoside and quercetin-3-O-xyloside with antimicrobial and antioxidant activities. (13) Another study determined that the extract of E. hyssopifolia is effective against gram-positive Staphylococcus aureus, gram-negative Salmonella typhi and Esche- richia coli. A different spurge, E. hirta, was found to be effective against gram-negative S. typhi and E. coli and could be used as treatment against typhoid fever and urinary tract infections. Two flavonoids, quercitrin and myricitrin, were detected in this

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spurge (E. hirta). These are the most responsible compounds for antibacterial activity of the extract from E. hirta. (54)(55) The methanol extract from the stem of E. neriifolia showed significant antimicrobial activity. This extract was used against S. aureus, S. aeruginosa, E. coli, Pseudo- monas aeruginosa, S. typhi, P. vulgarius, Aspergillus niger and Candida albicans, and compared to the effects of the standard drugs streptomycin and ampicillin. The results showed that the antimicrobial activity of this extract is comparable to these drugs. (56)

2.8.2 Analgesic and antipyretic activity

Plants of genus Euphorbia were found to have a large potential for isolation and development of natural analgesic and antipyretic drugs – with less side effects than chemical compounds used for the same purpose. The spurges E. hirta, E. antiquorum, E. bicolor and E. neriifolia all showed analgesic activity. (55)(56)(57)(58)(59) E. hirta has antipyretic activity at the sedative dose of 100 and 400 mg/kg, when used against yeast-induced hyperthermia in a rat model. This spurge also has analgesic, dose-dependent effects in doses of 20 and 25 mg/kg. Additionally, E. hirta was effective against acute pain in a carrageenan-induced edema model. Antipyretic and analgesic activity of E. hirta is similarly effective as diclofenac and acetylsalicylic acid - nonsteroidal anti-inflammatory drugs (NSAIDs). Further investigation into the mechanism of action of this extract is required before safe human medical usage can be approved. (55)(57) A study of ethanolic extract of E. antiquorum, a spurge used in traditional medicine for various diseases, was found to have dose-dependent analgesic activity. The dose used in this experiment was 250 and 500 mg kg−1 body weight of mice. Tannins, flavonoids, terpenoids and alkaloids detected in extract of E. antiquorum were responsible for this analgesic activity. (58) Extract of E. bicolor was able to induce long-lasting analgesia by non-opioid peripheral mechanism. This experiment was provided on the rat model of inflammatory pain. (59)

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Finally, a hydro-alcoholic extract isolated from leaves of E. neriifolia showed strong analgesic activity similar to diclofenac sodium. This activity was evaluated on an Eddy’s hot plate by tail-flick method in albino rats, using thermal, mechanical and chemical stimulus. The inhibition of pain by this extract in concentration 400 mg/kg was 432% after 60 min. (56)

2.8.3 Anti-inflammatory activity

Anti-inflammatory activity has been reported for extracts from E. australis, E. drum- mondii, E. hirta, E. neriifolia, E. macroclada, E. antiquorum, E. characias subsp. wulfenii, E. heyneana, E. royleana and E. kansui. Flavonoids and quercetin glycosides are the primary responsible compounds for this activity. The methanol extracts of E. macroclada and E. characias subsp. wulfenii had the best results in a study on the anti-inflammatory activity in Euphorbias. The flavonoids found in the extract from E. macroclada are rutin, hyperosid, quercetin, apigenin, kaempferol, myricetin, naringenin and hesperetin. (13)(60) In another animal study the ethanol extract of E. prostrata was able to inhibit first phase of the acute inflammatory reaction. (61) As previously mentioned, anti-inflammatory activity was also detected in E. hirta. The mechanism of action of this activity is probably mast cell membrane stabi- lization and inhibition of the release of inflammatory mediators. The extract of this spurge is also able to inhibit enzyme 5-lipoxygenase. Two flavonoids, quercetin 3-O- rhamnoside and myricetin 3-O-rhamnoside, isolated from E. hirta, were tested for inhibition effect. Despite the strong inhibition effect of this plant’s extract, all the isolated compounds exhibited a lower inhibition effect. (62)(63) Another study found that β-amyrin, a triterpenoid from E. hirta, was able to inhibit nitric oxide production. It can be concluded that this plant and β-amyrin have potential use as a treatment against arthritis inflammation. Additionally, this active compound is not showing cytotoxicity within the effective concentration range. (55) Two other triterpenoids from E. hirta, 24-methylencycloartenol and β-sitosterol, showed anti-inflammatory activity in a TPA-induced ear model. (54)

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Several studies confirmed several terpenoids were responsible for anti-inflammatory activity, in addition to the previous flavonoids. The responsible terpenoids were β-amyrin, 24-methylencycloartenol, and β-sitosterol from E. hirta and several diterpenoids from E. neriifolia. (60)(64) In a study on the analgesic and anti-inflammatory activity of E. antiquorum it was discovered that the ethanolic extract of this plant has dose-dependent anti-inflammatory activity. The biochemicals detected in the extract of E. antiquorum that were responsible for this activity are tannins, flavanoids, terpenoids and alkaloids. (58) The petroleum ether fraction of latex from E. neriifolia showed anti-inflammatory activity at the doses 750 and 500 mg/ml in albino rat model. The steroids euphol, cycloartenol and nerifoliol are most likely responsible for this activity. (56)

2.8.4 Pro-Inflammatory Activity

Interestingly, despite the anti-inflammatory effects of some of the substances contained in Euphorbia latex, other substances were conversely found to have pro-inflammatory effects. There are toxic substances located in the milky latex of Euphor- bia plants called phorboids. These substances are a type of specific diterpenes, such as the tigliane, ingenane and daphnane diterpene derivatives. These phorboids may cause pain and inflammation in eyes, nose, mouth or skin, mainly because of the activation of protein kinase C enzyme. Various Euphorbia plants contain different amount of phorboids. These Euphorbias are E. trigona, E. kansui, E. petiolate, E. iberica, E. broteri, E. esula, E. cyparissias, E. paralias, E. cauducifolia, E. kamerunica and E. acrurensis. (24)

2.8.5 Wound-healing activity

Another property of the extracts of Euphorbia plants is wound healing activity. Fla- vonoids and quercetin glycosides are the substances which exhibit this activity. The n-hexane, ethyl acetate and methanol extracts from the aerial parts of Euphorbia virgata, E. hirta, E. caducifolia Haines, E. nivulia, E. nerifolia, E. characias subsp. Wulfenii, E. helioscopia, E. sequieriana subs. sequieriana and E. macroclada display wound-

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healing activity. The most effective wound-healing activity is displayed by the methanol extract of E. characias subsp. wulfenii, which contains hydroxyproline and flavonoids. (13) Another study found that the latex of E. nivulia can reduce bleeding and can reduce the amount of time for blood clotting. In these latex alkaloids, cyanogenic glycosides, phenolics, saponins and tannins were found. (17) Extract of E. virgata containing flavonoids and their derivatives such as quercetin-3-glucoside, kaempferol, kaempferol-3-O-glucoside, kaempferol-3-rutinoside and rutin displayed wound healing properties. (13) The ethanol extract of E. hirta also showed significant burn wound healing activity. Extracts of this spurge showed antimicrobial activity against the microbes P. aeruginosa and S. aureus, which commonly cause burn and wound infections. (55)

2.8.6 Antioxidant activity

Some of the Euphorbia plants, especially E. helioscopia, E. characias, E. heyneana, E. macroclada, E. hirta, E. neriifolia and E. rigida showed antioxidant activity. Because free radicals can cause lipid peroxidation that results in cell damage, antioxidant activity is connected to healing process. (13)(55)(56)(65) Flavonoids such as quercetin-3-O-glucoside, quercetin-3-O-rhamnoside, quercetin-3-O-arabinoside and quercetin-3-O-xyloside, which show antioxidant activity, were found in E. characias. (13) The extract from E. hirta also showed strong dose dependent antioxidant activity. Phenolic substances, flavonoids, tannins, alkaloids, steroids, terpenoids and reducing sugars were found in this extract. Phytochemicals with antioxidant activity have an advantage over synthetic antioxidants because synthetic antioxidants have side effects in vivo. (55) Saponins isolated from the leaves of E. neriifolia showed significant antioxidant activity compared to that of ascorbic acid at 75.6%, butylated hydroxyanisole at 60.8% and butylated hydroxytoluene at 75.6%. Euphol, a steroid isolated from leaves of this spurge, showed antioxidant activity comparable to toa-tocopherol. (56)(65)

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2.8.7 Anti-allergic and anti-asthmatic activity

Some species of Euphorbia were also found to display anti-allergic and anti-asthmatic activity. These are E. hirta, E. helioscopia and E. kansui. All three are used in traditional medicine for treating various diseases. (54)(55)(66)(67) In one experiment, E. hirta showed significant antiallergic activity and was able to prevent early and late phase allergic reaction. Specifically, the extract of this spurge inhibited eosinophil accumulation and eosinophil peroxidase activity and also reduced the protein content in bronchoalveolar lavage fluid (BALF). The effec- tivity of this extract is similar to ketotifen and cetirizine, making this spurge a potential anti-allergic drug. Additional clinical studies are needed before implementation. (54) The anti-asthmatic activity of E. hirta is probably caused by the synergistic effect of anti-inflammatory and antioxidant activities of the flavonoids, sterols and triterpenoids which were detected in the extract of this spurge. The main active component is probably the flavonoid quercitrin. Effectiveness of the extract from E. hirta is similar to corticosteroid in the asthmatic mouse model. Because of other effects caused by ethanol extract from E. hirta, such as an inhibitory effect on interleukin-4 release and compound 48/80 (N-methyl-p-methoxy-phenethylamine) release inhibition, this plant could be a potent natural medicine used as anti-asthmatic drug. (55) E. helioscopia, another unstudied spurge, also displayed anti-asthmatic activity. The polyphenolic compound helioscopinin-A, which has an inhibitory effect on leukotriene D4-induced tracheal contraction, was responsible for this effect. This compound was also able to inhibit antigen-induced bronchial constriction and capillary permeability in passive cutaneous anaphylaxis responses. Both reactions were tested on rats and guinea pigs. While the main mechanism action of helioscopinin-A is in inhibition of leukotriene D4-induced responses, the partial inhibitory effect on release of allergic mediators is likely also involved. (66) It was concluded that ingenane-, tigliane- and daphane- type diterpenoids are most likely responsible for the anti-allergic activity of E. kansui. (67)

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2.8.8 Anticarcinogenic activity

Euphorbia species with anticarcinogenic activity are E. lagascae, E. kansui, E. tirucalli, E. esula E. fischeriana, E. poisonii, E. pulcherrima, E. splendens, E. macrostegia, E. neriifolia, E. grandides and E. hirta. (18)(29)(53)(55)(67)(68)(69)(70)(71)(72) Extracts of these spurges contain terpenes, steroids and anthocyanins as active substances, but it is primarily diterpenes and triterpenes which are responsible for anticarcinogenic activity. From diterpenes, those with abietane, tigliane, and ingenane skeletons were particularly effective, and from triterpenes the effective substances were the cycloartane derivatives. (24)(27) The seeds from E. lagascae contain picatannol, a hydroxy derivative of resveratrol, which has antiproliferative and antiinvasive effects against hepatoma AH109A cells. This stilbene is also strong inducer of HO-1 expression, via effect on protein kinase C and tyrosine pathways. (68)(69) Another Euphorbia, E. fischeriana, has potential as an anticancerogenic drug. Bi- oactive substances with strong antitumor effects were detected in its latex. The most potent of the substances from this spurge were the low-polarity fractions with triterpenoids, abietane- and tigliane-type diterpenoids. E. fischeriana has been used in traditional medicine as treatment against liver and lung cancers. It was determined that additional clinical trials are necessary before using these bioactive substances in treatment of cancer. (55) 25 terpenoids were isolated from the extract of E. kansui, including ingenane and jatrophane-type derivatives. More than half of the derivatives, mostly the ingenane-type, showed strong anti-proliferative activity in MDA-MB-435 and Colo205 cells. While more clinical studies are necessary, the extract of this spurge and ingenane-type diterpenoids are potent anti-cancer compounds. (67) E. kansui has also a toxic effect on leukemic, non-small cell lung cancer, colon cancer, melanoma, and renal cancer. It also showed antileukemic activity against the P-388 lymphocytic leukemia in mice. (53) Similarly, extract of E. esula has antileukemic activity against the P-388 lymphocytic leukemia in mice. (53) Diterpenoids, especially diterpenoid ingenol-3,20-dibenzoate and phorbol-12-tiglate-13-decanoate are responsible for this activity. (18)

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E. stracheyi, a spurge used in traditional Chinese medicine, contains substances with anticarcinogenic activity in its extract. These diterpenoids were found to have anti-proliferative activity and cytotoxicity against Hela cell line and Hep G2 cell line with varying effective rates. (29) Cycloartane triterpenoids with cytotoxic activity against two human breast cancer lines were found in the extract of E. macrostegia. These lines are MDA-MB48 and MCG-7 line. (70) Latex of E. tirucalli showed antineoplastic activity in an in vitro study. The latex of this spurge was used in a dilution of 9 drops of latex per 1 liter of saline solution. This concentration proved the latex to be effective against B16/F10 melanoma cells in a time-dependent dose. This concentration was not found to be toxic. Based on this study, it can be proposed that E. tirucalli could be potentially used as anticancerogenic drug, but further clinical studies are needed. (71) E. poisonii, E. pulcherrima and E. splendens also showed antitumor activity. (53) A small amount of cytotoxic activity against human epidermoid carcinoma KB 3-1 cells was detected in E. hirta. Three isolated flavonoids, afzelin, myricitrin and quercitrin were responsible for this activity. (72) E. neriifolia is very popular in traditional medicine for supposed healing effects on many diseases. Anticancerogenic activity of the extract from this spurge was proven by studying its saponin and flavonoid fractions against CCl-4 induced hepatotoxicity and by diethyl nitrosamine induced renal carcinogenesis. Saponins contained in this extract were able to restore the depleted hepatic super-oxide dismutase and by improving the antioxidant status of liver, reduce glutathione levels. The flavonoids isolated from leaves of this spurge were able to neutralize the oxidative stress by reducing the formation of free radicals. Therefore, the extract of E. neriifolia has hepatoprotective and renal protective effects. While establishment of the molecular mechanism of this extract and further clinical studies are needed, E. neriifolia could potentially be used in cancer treatment. (56)

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2.8.9 Anti–malarial activity

Even though modern medicine is very progressive and well- developed, there are still diseases which cause death of many patients worldwide. One of the worst of these diseases is malaria. It is the world’s deadliest infectious illness, and the cures available are still not sufficient. Africa is the continent which suffers the most from this illness. Because of the lack of access to medicine, the Ethiopian people use plants as treatment for various diseases, including malaria. E. hirta and E. prostrata are plants from the genus Euphorbia and are used for these malaria treatments. In the study with mice the anti-malarial activity of these plants was confirmed and speci- fied- they both had strong anti-plasmodial activities against Plasmodium falciparum in vitro. Flavonol glycosides afzelin, quercitrin and myricitrin are responsible for antimalarial activity in E. hirta. (72) Another Euphorbia, which is unused even in traditional medicine, is E. abys- sinica. From this Euphorbia the alkaloids, glycosides, phenols, indoles, tannins, saponins and steroids were isolated, and under study their singe or synergic anti- ma- larial activity against Plasmodium berghei infection in mice was proven. Another very important result obtained in this study is that the extract of this Euphorbia caused no death or toxicity at limit dose of 2000mg/kg and mice treated with 600 mg/kg of this extract live much longer than mice treated by only distilled water. However more experiments are needed to estimate the full antimalarial activity and safety of this plant.(35)

2.8.10 Immunomodulatory and immunosuppressive activity

Some of the Euphorbia species have immunomodulatory properties. These properties are connected to diterpenes found in the latex of Euphorbias. Euphorbias with this activity are E. ebracteolate, E. pubescens, E. neriifolia, E. aellenii and E. trigona. (2)(60) Specifically, the alcoholic extract of E. neriifolia has good phagocytic potential and increased the number of lymphocytes. It also improved haemopoietic activity and increased the number of surviving rats. (56)

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However, some Euphorbia plants have immunosuppressive activity. One of them is E. royleana, from which an ethyl acetate fraction which displayed immunosuppressive activity was isolated. (2) Immunomodulatory or immunosuppressive activity sometimes depends on the concentration of extracts from Euphoria plants, specifically, E. hirta. The ethanol extracts of this spurge have immunostimulatory activity at concentration of 80 mg/ ml and 160 mg/ml. However, at concentration of 40 mg/ml this extract has immunosuppressive activity. (63)

2.8.11 Anti-HIV activity

E. neriifolia is a poisonous but ethnomedical plant from genus Euphorbia. In the study examining its contained substances it was discovered that this plant contains many different diterpenoids, some with anti-HIV or cytotoxic activity. Obvious anti-HIV-1 effect were shown by two compounds: ent-16α,17-dihydroxyatisan-3-one and euri- foloid R. However, more experiments and clinical studies are needed to confirm the usage of these compounds as treatment against HIV. (60)

2.8.12 Antidiabetic activity

Diabetes mellitus is accompanied by hyperglycaemia, resulting in dysfunction and long-term damage of organs, especially the blood vessels, kidneys, nerves, heart and eyes. The ethanol and petroleum ether extracts from flowers of E. hirta used daily for three weeks were able to reduce alloxan-induced hyperglycaemia, triglycerides and cholesterol. These extracts also showed significant antioxidant activity at dose dependent effect. This antioxidative side effect of E. hirta presents a benefit in reducing oxidative-induced complications in patients with diabetes mellitus. (55) The methanolic extract from E. helioscopia showed antidiabetic and antioxidant activities. (73)

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2.8.13 Proteolytic activity

Some of the Euphorbia plants contain proteolytic enzymes in their latex, which are responsible for proteolytic activity. This activity can be used as chemotaxonomic marker for genus Euphorbia. One of these enzymes is serine protease, which can be found in E. milli. var milli, E. cyparissias and E. tirucalli. There are also other types of endopeptidases involved, such as Euphorbia protease B, which belongs to cucumisin- like serine proteases. One of the methods for determining the proteolytic activity is to measure the change in fluorescence compared to trypsin. Trypsin is serine endo- peptidase. Many species of Euphorbia genus have proteolytic activity, but Euphor- bias with proteolytic activity higher than an activity compared to 2.5 mg/ml trypsin are E. pilosa, E. ornithopus, E. ledienii, E. heterochroma, E. hamata, E. esula, E. evansii and E. dulcis. (74)

2.8.14 Other biological activities

Euphorbia species vary greatly in the structure of their chemical compounds and many of them are still not thoroughly researched. Outside the previously mentioned biological activities, Euphorbia species also have anxiolytic, antidiarrheal, antiproliferative, anti-JEV and cytotoxic activities. (13) Specifically, extracts from E. hirta showed anxiolytic, antidiarrheal and anti-JEV activities. Flavonoid myricetin isolated from this spurge was responsible for anti-JEV activity. (31) E. neriifolia, the plant, which was already mentioned to have anti-HIV effect, also shows the cytotoxic activity for HepG2 and HepG2/Adr cells which is caused by diterpenoids. This cytotoxic activity had IC50 of 0.01 μM. (11)(60) In traditional medicine the plants of genus Euphorbia are used for their potential diuretic, laxative and antifungal effects. However, there is no credible evidence which could prove these effects. (11)

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2.9 Euphorbia trigona

Euphorbia trigona is one of the succulent plants which grows in Africa. It is commonly known as the African milk tree because it contains a high amount of milky latex. (75) E. trigona belongs to family Euphorbiaceae in the genus Euphorbia. It has a thick, straight stem with three ribs as seen in Figure 2. The stem has dark green color and the leaves growing out from rips are oblong- lanceolate and they have similar, a slightly lighter color. (4)(76)

Figure 1 (76)

39 THEORETICAL PART

Figure 2 (76)

Figure 3 Euphorbia trigona (own picture)

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2.9.1 Latex

A latex is a colloidal suspension containing secondary metabolites and proteins. Even people who do not know all functions and dangers hidden in this specific milk use latex of E. trigona for different purposes. In southeast Asia, this latex has been used as a poison for catching fish, or an ingredient of arrow poison. (75)(77) The latex of E. trigona is composed of 25.28 mg/ml of proteins, 1.3 mg/ml of carbohydrates and 44 mg/ml of total solids with pH 4.9. This latex was proven to have enzymatic activity. The enzymes found were (units/mg protein): Protease (azo- casein) 0.01, protease (BSA) 0.05, acid phosphatase 0.31, alkaline phosphatase 0.02, N-acetyl-�-glucosaminidase 0.2, leucine-aminopeptidase 0.01, α-galactosidase 0.03, �-galactosidase 0.01. (78)

Figure 4 E. trigona and its latex (own picture)

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2.10 Occurrence and use of Euphorbia trigona

2.10.1 Medical plant

E. trigona has a history of folk use as a medicinal plant in southern Asia in the treatment of earache and boils. In the traditional Indian medicinal system of Ayurveda, E. trigona is used to treat respiratory infections, gonorrhea and urinary tract infections. It is also used for treating tumors, warts, intestinal parasites, hepatitis, rheu- matoid arthritis and inflammation. (2)

2.10.2 House plant

In the United States it is sold as a houseplant and is commonly found as a houseplant in Europe. (1)

2.10.3 In milk production

Two specific uses of the spurge are in milk production and as meat tenderizer. (15) The latex of E. trigona contains many enzymes. One of them is protease – proteases are hydrolytic enzymes, which hydrolyze the peptic bonds and transform proteins into shorter peptides. This can be used in many industrial processes, especially beer fermentation, cheese processing, meat tenderization, bread-baking, flavor en- hancement, as well as in the textile and pharmaceutical industries. The activity of crude protease from E. trigona was the highest among the three tested Euphorbias, 812.50 U/ml. The optimum pH was 7.0 and temperature 60°C, but protease obtained from E. trigona retained its activity over a wide range of pH (5.0-9.0) and temperature up to 65°C with casein as a substrate. This protease proved milk clotting and caseinolytic activity ranged from 0.58 U/ml to 1.01 U/ml. In conclusion, there is a high potential for the protease from E. trigona, but further study on enzyme purification and characterization is required before its utiliza- tion. (15)

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2.10.4 Meat tenderizer

The most important factor for consumer satisfaction and perception of taste of meat is tenderness. Tenderizing meat can be done either physically or chemically. One of the popular methods of meat tenderization is using proteolytic enzymes such as papain, bromelain and ficin. Papain can be replaced by ginger and Cucumis. Even though there is lack of evidence and experiments of utilizing Euphorbia species proteases for meat tenderizing, they have great potential to be used in this application or in cheese production. (15)

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2.11 Controlled human dermatological experiments and case reports with E. trigona

2.11.1 Controlled human dermatological experiments

One controlled human dermatological experiment on the latex of E. trigona was performed in the United States. A small amount of the latex of E. trigona was applied on the flexor forearms of a volunteer. The result of open patch tests was the generation of irritant follicular dermatitis with residual hyperpigmentation remaining for over a week after application. The results of the closed testing to the upper layer of the skin on forearms were bullae and severe vesiculation. These results proved that E. trigona is a potential health hazard if held and suggest that manual manipulation of this plant should be performed with care. (1) Another experiment was performed in Chicago in 1980. There were five Cauca- sian volunteers participating in this experiment, all of them healthy and without any history of atopic dermatitis. The testing was divided in two parts: open patch and closed patch. In open patch testing fresh latex from E. hermentiana (synonymous with E. trigona), was applied to the right flexor forearm of each volunteer. In closed patch testing strips with latex were applied to flexor surfaces of both arms of volunteers and were left there for 24 hours. After 30 minutes the open test showed no reactions, but after 24 hours all volunteers had erythematous, macules or papules. In closed patch testing all volunteers received bullae on at least one site and vesiculation on the another, followed by hyperpigmentation. This experiment concluded that latex from E. hermentiana causes irritant follicular dermatitis. Due to wide local availability of this plant E. hermentiana should be considered as a potential human health hazard. (20)

2.11.2 Case reports

In September 2008, a 52-year-old man was hospitalized for intense eight eye pain after contact with the latex of E. trigona, which had been accidentally sprayed into his eye. The persistent pain remained even though the patient had his eye cleaned

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with fresh water. At the hospital his eye was treated with normal saline under anes- thesia, followed by chloramphenicol eye ointment and an eye patch. After that the patient was transferred to the ophthalmology clinic, where fluoromethanole (0.1%) eye drops, levofloxacin (0.5%) eye drop and Solcoseryl (20%) eye gel were applied. The patient had intense pain for two days, but after one week his eye was full recov- ered. Because of this report, people working with E. trigona should take precautions, such as wearing eye protection and avoiding direct skin and eye contact with the latex of E. trigona. (5)

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2.12 Biochemical substances included in latex of E. Trigona

E. trigona contains many different substances, most of which are still unidentified. There are substances considered to be irritating or cancer-causing. However, some of them can be used in medicine for treating different health problems. Because there is a close relationship between the structures and biological activity, it would be useful to know the content substances of E. trigona. This knowledge is important for discovering any new biological activity and the potential for wider usage of E. trigona extracts. (26) By phytochemical analysis, the extract of E. trigona was found to contain sterols, alkaloids, tannins, flavonoids and saponins. Not all of the bioactive compounds were present in all extracts- the table below lists the extracts with detected compounds. (2)

Table 1 Phytochemical analysis of the extracts from E. trigona (Adapted from: (2))

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2.12.1 Diterpene esters

As previously mentioned, diterpene esters are often found in the latex of Euphorbia species. E. trigona contains latex in its diterpenes as well. These are the 17-hydroxyingenol esters (1-3), the ingenol ester (4), the 20-deoxy-17-hydroxyingenol ester (5), the 8-methoxyingol esters (6-8), the 19-hydroxyingolester (9), and the ingol esters (10-12). (79)(80) The 17-hydroxyingenol and ingenol derivatives (1-4) are considered as skin- irritating. The 20-deoxy-17-hydroxyingenol derivative (5) is not expected to have this activity. It has been hypothesized that diterpenes of the phorbol ester and bio- genetically related types (daphane, ingenane, lathyrane) are irritant defense substances of plants. This hypothesis was confirmed by the experiment on different in- sects. The most potent substance tested was 12-0-tetradecanoyl-phorbol-13-acetate (TPA), which causes a mortality rate of 100 % house mosquito at a dose of 0.6 ppm. This substance is also most potent tumor- promoting substance of croton oil. (79)

1-5 Compound R1 R2 R3 R4 Deca-2,4,6- 1 H O-Angelate OH trienoate 2 Angelate Ac OAc OAc 3 Angelate H OAc OAc 4 Angelate H H OAc 5 Angelate H OAc H Adapted from: (79)

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6-12 Compound R1 R2 R3 6 Benzoate Me H 7 Tiglate Me H 8 Angelate Me H 9 Ac Benzoate OH 10 Ac Benzoate H 11 Ac tiglate H 12 tiglate H H Adapted from: (79) In another experiment two new ingol esters, 3,12-O-diacetyl-7-O-benzoyl-8- methoxyingol (13) and 3,12-O-diacetyl-7-O-tigloyl-8-methoxyingol were isolated (14). These esters were obtained from an acetone-soluble fraction of the latex of E. trigona. Respectively, a third methoxylated ingol ester and a fourth new compound were identified as 3,12-O-diacetyl-7-O-angeloyl-8-methoxyingol (15) and 3,7,12-O- triacetyl-8-O-benzoyl-18-hydroxyingol (16). (79)(80) Three other ingol ester compounds were in the latex of E. trigona, whose structures match the latex of another two Euphorbias. The first of them is 3,7,12-O-triacetyl-8-0-benzoylingol (17), next was 3,7,12-O-triacetyl-8-0-tigloylingol (18) and finally 3,12-O-diacetyl-7-O-tigloylingol (19). These substances all were easily sepa- rable by a newly developed isolation process using droplet counter-current chromatography. (79)(80)

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13-19 Compound R1 R2 R3 R4

13 COMe COC6H5 Me H

14 COMe Me H

15 COMe Me H

16 COMe COMe COC6H5 OH

17 COMe COMe COC6H5 H

18 COMe COMe H

19 COMe H H

Adapted from: (80)

2.12.2 Triterpenoids

Triterpenoids, active substances with various biological activities, were also isolated from the stem of E. trigona. The first of them was taraxeryl acetate (20). This compound showed anti-diabetic activity in the study with Artemisia roxburghiana. (82)(83)(84) Another triterpenoid isolated from E. trigona is friedelin (21). Friedelin is bioactive substance with antioxidant, free-radical scavenging, gastro and liver protective, anti-diabetic and anti-diarrhea activities. It also showed a significant protective effect on lungs affected by lipopolysaccharides. Friedelin (21) has potential to be

49 THEORETICAL PART

useful in the treatment of diabetes type 2 because of its insulin-mimicking action. (82)(85)(86)(87)(88)(89)(90) The third triterpene compound from E. trigona is friedelan-3-ol (22) as α and β isomer. Both were suspected to have anticancerogenic activity, but the biological study did not prove this activity. (82)(91)(92) The next triterpenoid found in E. trigona is taraxerol (23). Taraxerol (23) is a compound with anti-cancer activity and potent anti-inflammatory effects. It inter- feres with the activation of TAK1 and Akt and deregulates the expression of proinflammatory mediators. (82)(93)(94)

20 21

22 23

α-Amyrin (24) is another triterpenoid found in E. trigona. This active substance shows anti-inflammatory, antineoplastic and anti-hyperlipidemic activity.

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It is a trypsin and a chymotrypsin inhibitor and also induces proliferation of human keratinocytes. Because of its hepatomodulatory potential, it could become a liver curative drug. (82)(95)(96)(97) The isomer of α-amyrin is β-amyrin (25). This compound has antiviral, hepatoprotective, antinociceptive and anti-inflammatory activity. It shows sedative, anxiolytic and antidepressant effects. The antidepressant effect is probably caused by nor- adrenergic mechanism, although the sedative and anxiolytic effect is more likely caused by an action on benzodiazepine-type receptors. This compound can also en- hance the total sleeping behavior in pentobarbital-induced sleeping by mechanism through activation of GABAergic neurotransmitter system. (82)(98)(99)(100)(101) Another point of interest is the mixture of 7 and 8, which has gastro-protective activity because of the activation of capsaicin-sensitive primary afferent neurons. (98)(100)

24 25

The following triterpene compound is lupeol (26). It has potential as an anti- inflammatory, anti-microbial, anti-protozoal, anti-proliferative, antiangiogenic, antiinvasive and cholesterol lowering substance. An important fact about lupeol is that it can selectively target diseased human cells while sparing the healthy cells. It also regulates the expression and activity of some molecules such as cytokines IL-2, IL-4, IL-5, IL-β, NFκB, proteases, α-glucosidase, cFLIP and Bcl-2. (102)(103) Lupeol has selective anticancerogenic potential on different human cancer cells. It inhibits cell

51 THEORETICAL PART

proliferation of hepatocellular carcinoma HCCLM3 cells by inhibiting BDNF secretion and phosphorylation of GSK-3β(Ser-9). (104) Another ability of lupeol is the reduction of oxidative stress and inhibition of critical control points of apoptosis, resulting in prevention of AAP-induced hepatotoxicity. It could significantly prevent hepatic damage and reduce serum trans-ami- nases. (105) Lupeol also shows antimalarial activity, and scientists suspect that after structure diversification more potent and stronger derivatives for treating malaria could be found. (106) Another study has proven lupeol’s antidiabetic activity by suppressing the progression of diabetes after 21 days. The treatment with lupeol decreased glycated hemoglobin, serum glucose and nitric oxide while increasing serum insulin level. Antioxidant level is also important regarding Diabetes and is usually decreased. But the treatment with lupeol successfully increased antioxidant level and decreased the level of thiobarbituric acid-reactive oxygen species. (107) Lupeol could also be very useful for hypercholesterolemic conditions. Usually in this condition the lipemic-oxidative and inflammatory aberrations results in athero- genesis. But the treatment with lupeol is able to normalize nitrosamine stress and improve inflammatory aberrations. (108)

The following triterpenoid is known as betulin (27). This triterpene has proven anti- HIV activity and it is able to inhibit pro-inflammatory responses produced by the gram-negative stimuli LPS and E. coli. Specifically, betulin suppressed LPS- induced tumor necrosis factor α (TNF-α) and levels of interleukin (IL) 6 and increased

52 THEORETICAL PART

the level of IL-10. This resulted in lowering pro-inflammatory cytokines, myelop- eroxidase activity and bacterial loads in long tissue during gram-negative pneumonia. Therefore, it could be inferred that treatment with betulin could be used on lung inflammation. (33) Another study found that betulin can inhibit the maturation of SREBP (sterol regulatory element-binding protein), which leads to reduction of biosynthesis of cholesterol and fatty acid. Simply put, the SREBP are main transcription factors able to activate the expression of genes involved in biosynthesis of cholesterol, fatty acid and triglyceride. Because of this ability betulin can improve diet-induced obesity, increase insulin sensitivity and lower the lipid content in tissues and serum. It is also able to reduce the size of atherosclerotic plaques. These findings should lead to the development of drug for hyperlipidemia or could lead to a new treatment of metabolic diseases such as diabetes type II and atherosclerosis by using betulin as a treatment. (109) Another interesting fact about betulin is that it is involved in the activation of the caspase cascade and leads to cell death. It inhibits cell proliferation in a concentration-dependent function, and it could be used in induction of apoptosis in a human melanoma cell line. A modified derivative, 28-O- propynoylbetulin, is twice as effective as unmodified betulin and has greater proapoptotic and antiproliferative effect and could therefore be used as potent an- timelanoma agent. (110) Another source mentions that betulin (lup-20(29)-ene-3- β,28-diol) is easily isolable, but as a raw material is not a very usable compound. Its derivatives and biological active compounds such as betulinic acid are much more interesting. Betulin can be easily converted to this acid, which has rich spectrum of biological and pharmacological activities. Betulinic acid has anti-HIV activity, and it is specifically cytotoxic to some tumor cell lines. It induces apoptosis in tumor cells, but it is non-toxic up to 500mg/kg body weight in mice. This makes it potentially safe and useful as treatment of cancer and HIV infection. Additionally, there was heavy interest in developing a new more potent anti- HIV compounds based on betulinic acid, resulting in the discovery of highly active derivatives with higher potency and better therapeutical effect than current clinical anti-HIV compounds. (34)Chyba! Nenašiel sa žiaden zdroj odkazov. This acid shows also antimalarial and anti-inflammatory activities. (34)

53 THEORETICAL PART

2.12.3 Steroids

As previously mentioned, various natural steroids were found in the genus Euphor- bia. Extract of E. trigona also contains some steroids- phytosterols, too. One of them is sitosterol (28). Sitosterol, or β-sitosterol, is derived from the sitostane. This phy- tosterol can inhibit proliferation and induce apoptosis in solid human tumors – especially in colon and breast cancers. The mechanism of this action is managed by the selective activation of caspase-3 and induction of Bax/Bcl-2 ratio. (36)(112) Sitos- terol has also a potent angiogenic activity, which could be used as management of chronic wounds. (112) Interestingly, sitosterol shows anti-inflammatory and antipyretic activities. Its antipyretic activity was similar to acetylsalicylic acid. Sitosterol has a high potential to be used as a safe therapeutical drug. (113). Sitosterol is also an antioxidant, and it regulates activity of some enzymes. Its inhibition of enzyme 5- α-reductase, which transfers testosterone to dehydrotestosteron, is the most important feature. Because of this action, sitosterol showed good results in treatment of benign prostatic hypertrophy and prostatic cancer. (112) Sitosterol could be used as antihyperlipidemic agent, because it competes with cholesterol for absorption. The reason why it can compete with cholesterol is that they both have very similar structure. (114) β-sitosterol and other phytosterols present a potential immunity modulator, which could be used as prevention against COVID-19. Also, β-sitosterol is known to have an antioxidant and immune boosting role especially in viral infection

54 THEORETICAL PART

including pneumonia. A recent study found that β-sitosterol can boost immunity against SARS-CoV-2 and restrict the viral invasion into the host cell. The mechanism of this action works by inhibiting the SARS-CoV-2 spike glycoprotein. (115)

Cycloartenol (29) is a precursor of some functional sterols and it is a plant me- tabolite. It has inhibitory effects on TPA- induced Epstein-Barr virus early antigen (EBV-EA) activation in Raji cells. Cycloartenol also has therapeutical effect against skin neoplasms and shows significant antidiabetic activity. (116)(117) The next steroid is a 24-methylenecycloartanol (30). This compound is often found in plant extracts together with cycloartenol. It also showed significant antidiabetic activity and inhibitory effect on TPA- induced Epstein-Barr virus early antigen activation in Raji cells with activity value 0.0088 µM. (118)(119)

55 THEORETICAL PART

One new special molecule isolated from n-hexane extract of E. trigona was identified as cycloart-25-ene-3ß,24-dioldiacetate (31). (82)

56 THEORETICAL PART

2.12.4 Glycoproteins

Glycoproteins involve many different groups of structures and substances, including the special group known as lectins. These lectins bind specifically and reversibly to carbohydrates, leading to cell agglutination and precipitation. Because of this activity, lectins are part of many different cellular mechanisms. The lectins of E. trigona especially showed strong hemagglutination of erythrocytes. There were three different lectins isolated from latex of E. trigona, all are heat stable up to 60°C. These lectins are believed to belong to the type II RIP family. This special group of proteins “II RIP family” contain substances which can inhibit protein synthesis in a cell-free system. All three lectins from E. trigona were proven to have this activity and placed among the most toxic type RIPs. But the most important fact is that these lectins might play a role in antitumor defense, because certain tumoral cells are more sensitive to type II RIPs that normal cells. (75)

57 THEORETICAL PART

2.13 Biological activity of E. trigona

E. trigona is still a mostly unresearched plant. Because of some independent experiments some of E. trigona’s biological activities are already known. There is also information available about biological activity of genus Euphorbia, so we can expect that some of this activity will be also identical for E. trigona. However, more experimentation on the content substances of E. trigona is required to collect more information. Information about confirmed biological activity of E. trigona is contained in the following sections.

2.13.1 Antimicrobial Activity

In one study it was confirmed that major latex lectin from E. trigona has antimicrobial activity. This lectin is strongly concentrated and similar to known plant lectins- ricin such as ricin from Ricinus communis and agglutinin from Viscum album colora- tum. These lectins are bound to D-galactose and N-acetyl-D-glucosamine, which block many fungal cell walls. Antimicrobial activity was tested on three phytopatho- genic filamentous ascomycetes- Aspergillus niger, Fusarium graminearum and Botry- tis cinerea. Even though the effect of lectin from E. trigona on germination of the melanized conidiospores of Botrytis cinerea was not very significant, the concentration 0.1 mg.cm-3 of this lectin was able to inhibit germination and the hyphal growth of A. niger and Fusarium graminearum. (79)(80) Another study showed that extract of E. trigona is able to inhibit hemolysin production managed by Proteus mirabilis. Hemolysins are cytotoxic for human blood leukocytes and they are also able to cause release of inflammatory mediators from mast cells and granulocytes. Also, P. mirabilis can cause urolithiasis and blockage of catheters because it causes urease-mediated urea hydrolysis. In this study the extracts of E. trigona were able to significantly inhibit the activity of urease. There is evidence that these extracts are helping to control biofilm formation caused by P. mirabilis, which is very important for prevention against infection. These abilities could be considered antiquorum-sensing properties. (120)

58 THEORETICAL PART

2.13.2 Antioxidant activity

It was proven that extract from E. trigona shows antioxidant activity. Because the antioxidant properties of plant extract cannot be measured by one single method, this study used two different methods- DPPH radical scavenging assay and Fe3+ reducing power assay. The results were compared to standard antioxidant – ascorbic acid. Both methods have shown that extracts from whole plant E. trigona have antioxidant activity. Flavonoids, saponins, quinones and phenolic compounds were responsible for this activity. (120)

2.13.3 Immunomodulatory activity

In another study it was found that E. trigona and its latex extracts can improve the innate and adaptive sides of the immune system in vitro. This study was based on studying lymphocyte proliferation and NETs formation respectively. It is important to mention that the immunomodulating activity of E. trigona could only be used when it is safe and not cytotoxic. Only the ethanol and petroleum ether extract were tested because they showed strong immunomodulatory activity. Results showed that all the extracts at the concentrations with significant immunomodulatory activity are non-toxic to normal mouse fibroblast cell line L292, PBLs and Jurka. Furthermore, none of the extracts induced formation of TNF-α, which is often part of adverse reaction. Even though further in vivo tests are needed, it is rea- sonable to assume that these extracts are safe for usage and have good potential for development of new anti-infective and immunomodulatory treatment. (2)

2.13.4 Anti-swarming activity

The swarming activity is very important for spreading of infections by pathogens such as P. mirabilis and P. aeruginosa. Extracts from E. trigona were obtained by four solvents: petroleum ether, chloroform, ethyl acetate and methanol. The concentration of these solutions was 50μg/ml. All these solutions were found to be able to inhibit swarming motility of P. mirabilis and P. aeruginosa. The most effective solution against P. aeruginosa was the chloroform solution, and the petroleum ether was the

59 THEORETICAL PART

least. Against P. mirabilis the petroleum ether and methanol solutions were the most effective while the chloroform solution was the least. Even though the antibacterial activity at the same concentration was not significant, E. trigona could clearly be used to control spreading of pathogens. This activity together with the immunomodulatory activity would be very useful in treatment of different infections. (2)

2.13.5 Agglutinating ability

K. R. Lynn and others studied the agglutinating ability of latex from different Euphor- bia species. They isolated lectins from these plants and determined the aminoacids of the seven lectins. They noted that the lectins from E. trigona, E. lactea, E. lactea cristata and Eleuphorbia drupifera are related. However, purified lectins from E. trigona were much more effective in agglutination than other lectins and showed the same effect on all types of human blood: 0, A, B, AB. (121)

2.14 Potential biological activity of E. trigona

As previously mentioned, E. trigona is very potent plant from Euphorbia genus. Re- cent studies confirmed its antimicrobial, antioxidant, immunomodulatory, anti- swarming and agglutinating activities. (2)(79)(120)(121) However, the extract of E. trigona was found to contain biochemical compounds with more biological activities, which were not tested and confirmed for this spurge. We can assume that E. trigona may have also antidiabetic activity, because in its extract compounds such as taraxeryl-acetate, friedelin, lupeol, botulin, cycloartenol and 24-methylenecycloartanol were found. These compounds showed significant antidiabetic activity. (84)(87)(107)(109)(119) One of the other potential biological activities of E. trigona is antihyperlipidemic activity. The compounds in the latex of this Euphorbia with this activity found are betulin, α-amyrin and lupeol. (96)(97)(108)(109) Finally, E. trigona could have anticancerogenic potential, because it contains lupeol, betulinic acid, taraxerol and β-sitosterol. (34)(36)(94)(104)

60 THEORETICAL PART

It is obvious that more experiments and clinical trials are needed, but we can assume that E. trigona could be used in medical practice.

61 EXPERIMENTAL PART

3 EXPERIMENTAL PART

3.1 Chemicals used

Ethyl acetate Chloroform Toluene Methanol n-Hexane Acetone Sulfuric acid

3.2 Material used for TLC

Aluminium plate Silicagel (F254 20x20 cm, thickness of layer 200 µm) (Merck)

3.3 Devices

Analytic weighing-machine OHAUS ANALYTICAL Plus (Ohaus) Lyophilisator (Christ) Ultrasound water bath (Bandelin) Vaccum rotary evaporator Rotavapor R-3 (Büchi) Nitrogene evaporator (Evaterm)

3.4 Herbal material used and its processing

The experiment used hexane extract, methanol extract, ethyl-acetate extract and water extract from stem of Euphorbia trigona, spp. First the stem of E. trigona was cut from a household plant and dried in a her- barium for two months. At the end of the two months the stem was cut into small

62 EXPERIMENTAL PART

pieces (approx. 3 cm), weighed and put into a glass bottle. The total weight of herbal material was 158 grams. The herb material was steeped in the bottle with 800 mL of methanol and left for maceration for 24 hours. After that time, it was decanted through the cottonwool and the excess methanol was evaporated by vacuum evaporator. The process of extraction was repeated 3 times.

Figure 5 Vacuum rotary evaporator Rotavapor R-3 (Büchi) with methanol extract from E. trigona (own picture)

The last obtained extract was put into the sonication bath, filtered, methanol was removed, and the extract was left in freezer. The weight of obtained pure extract was 6.67 g. 0.200 g. was taken from this amount and saved as a sample. The rest of the pure extract was dissolved in 500 mL of methanol. Following this, 100 mL of water and 500 mL of n-hexane were added. The resulting solution was left in a dividing bank for 30 minutes. This solution was left in separatory funnel for 30 minutes to obtain two separated liquid phases. The n-hexane extract was taken, evaporated

63 EXPERIMENTAL PART

using vacuum rotary evaporator and stored. The extraction with n-hexane was repeated three times, always with new hexane as solvent. The n-hexane extracts were combined, the n-hexane was removed, and the extract was transferred into vial and finally dried by using nitrogen evaporator.

Figure 6 Nitrogene evaporator EVATERM with n-hexane extract from E. trigona (own picture)

Methanol portion was also evaporated by vacuum rotavapor, 200 ml water and chloroform in ratio 1:1 was added to the obtained concentrated extract. This solution was thoroughly shaken and left to separate in separatory funnel for 24 hours. Next, obtained chloroform portion was taken and evaporated by vacuum rotavapor. The water residue was again shaken with new chloroform. The process was repeated together 3 times, the extracts were combined, evaporated, transferred into vial and finally evaporated by using nitrogen. Water fraction was mixed with ethyl acetate and left to separate for 24 hours in separatory funnel to obtain ethyl acetate and water portion. Ethyl acetate extract was

64 EXPERIMENTAL PART

taken and evaporated by vacuum rotavapor. The process of ethyl acetate extraction was repeated 3 times with new ethyl acetate. The process resulted in ethyl acetate and water extract. The water fraction was evaporated by vacuum extractor and then put into the lyophilisator.

Figure 7 Lyophilisator (Christ) with water fraction of extract from E. trigona (own picture)

Four fractions of extract were obtained from E. trigona: n-hexane, chloroform, ethyl acetate, and water. From all of them, samples were prepared at a concentration of 10 mg/ml. These samples were used for finding suitable mobile phase by using TLC for separation and UV detector for detection of content compounds.

3.5 Methods

3.5.1 Analytic thin-layer chromatography (TLC)

Stationary phase: aluminium plate Silicagel F254 20x20 cm, thickness of layer 200 µm

65 EXPERIMENTAL PART

Mobile phase: chloroform, toluene, ethyl acetate, acetone and methanol in various ratios (v/v/v/v/v) Detection: UV at the wavelength of 253 nm and 366 nm, sulfuric acid

Thin layer chromatography (TLC) is a simple, highly sensitive and inexpen- sive chromatographic technique with high speed of preparation. This technique is used to separate a mixture into components by using a thin layer of stationary phase, which is supported by an inert backing. TLC can be used to monitor the progress of a reaction and to purify small amount of a compound. The principle of this method is based on different affinities for the mobile and stationary phases, which affects the speed of compound’s migration and results in well-defined and well-separated spots. (122) Next, sulfuric acid was applied to the TLC plate and a UV detector was used to find the results.

3.5.2 Herbal material

The weight of dried stem from E. trigona was 158 grams.

3.5.3 The extracts isolated from E. trigona

The chloroform extract isolated from E. trigona weighted 1.575 grams. The amount of the obtained n-hexane extract was 1.056 grams, and the amount of obtained ethyl acetate extract was 0.498 grams. The obtained water extract weighted 0.9832 grams.

3.5.4 Mobile fraction selection for column chromatography

The mobile phase was selected by TLC analysis followed by UV detection. There were more variations tested for methanol and hexane extract separately, displayed in table 2, and again for ethyl acetate extract, displayed in table 3. For methanol and hexane extract a fraction composed of chloroform, toluene, ethyl acetate and methanol in ratio 96:3:0.5:0.5 (v/v/v/v) was selected, by which the

66 EXPERIMENTAL PART

substances were separated the most likely. For ethyl acetate extract a fraction composed of chloroform and methanol in ratio 90 : 10 was selected.

Table 2 Mobile phase selection for (column chromatography) HPLC for chloroform and n- hexane extract from E. trigona

Mobile phase Ratio Result

chloroform : methanol (v/v) 90 : 10 not suitable

chloroform : ethyl acetate (v/v) 90 : 10 not suitable

chloroform : methanol : ethyl acetate (v/v/v) 60 : 30 : 10 not suitable

chloroform : methanol : ethyl acetate (v/v/v) 90 : 5 : 5 not suitable

chloroform : ethyl acetate : methanol (v/v/v) 96 : 2 : 2 not suitable

chloroform : methanol (v/v) 96 : 4 not suitable

chloroform : ethyl acetate (v/v) 96 : 4 not suitable

chloroform (v) 100 not suitable

chloroform : acetone (v/v) 94 : 6 not suitable

chloroform : toluene (v/v) 96 : 4 not suitable

chloroform : toluene :ethyl acetate : methanol 94 : 2 : 2 : 2 not suitable (v/v/v/v)

chloroform : toluene : methanol (v/v/v) 95.5 : 4 : 0.5 not suitable

chloroform : toluene : ethyl acetate : methanol 94 : 5 : 0.5 : 0.5 suitable (v/v/v/v)

67 EXPERIMENTAL PART

Table 3 Mobile phase selection for (column chromatography) for ethyl acetate extract from E. trigona

Mobile phase Ratio Result

chloroform : toluene :ethyl acetate : methanol 95 : 2 : 1 : 2 not suitable (v/v/v/v)

chloroform : methanol (v/v) 70 : 30 not suitable

chloroform : methanol : ethyl acetate (v/v/v) 80 : 10 : 10 not suitable

chloroform : methanol (v/v) 80 : 20 not suitable

chloroform : methanol (v/v) 85 : 15 not suitable

chloroform : methanol (v/v) 90 : 10 suitable

Figure 8: TLC result of chloroform and n-hexane extracts of E. trigona under UV long wavelength (366 nm) (own picture)

68 EXPERIMENTAL PART

Figure 9: TLC result of chloroform and n-hexane extracts of E. trigona under UV short wavelength (254 nm) (own picture)

Figure 10: TLC result of chloroform and n-hexane extract of E. trigona (own picture)

69 EXPERIMENTAL PART

Figure 11: TLC result of ethyl acetate extract of E. trigona under UV short wavelength (254 nm) (own picture)

Figure 12: TLC result of ethyl acetate extract of E. trigona under UV long wavelength (366 nm) (own picture)

4 DISCUSSION

This thesis concerns Euphorbia trigona from the genus Euphorbia and the family Eu- phorbiaceae. This succulent plant occurs worldwide, often being used as a houseplant. However, information about its obtained substances and effect on human health is lacking. I have been interested in this succulent plant since my childhood and was suspicious about the danger of the milky latex found in this Euphorbia. I decided to make an experiment to gather more information about this succulent plant. In the research part of my thesis, I collected and wrote down all available information about the occurrence of E. trigona, obtained substances and their proven biological activity, and performed a few experiments with this Euphorbia. I also researched basic information about the genus Euphorbia, content substances found in plants of this genus and the biological activity of these substances. Some of the information proved to be quite interesting and described a rich potential for E. trigona. There is still a need for more experiments with this plant to evaluate all available ways to use it for treatments of various illnesses. In some countries Euphorbia is already used as medicinal plant against infections, tumors, warts, and parasites in addition to other uses described in this thesis. Contrastingly, there have been reports of skin and eye irritation caused by the latex from this Euphorbia. From these incidents we could assume that the latex from this Euphorbia must be used carefully and in correct amounts because of its strong effect. The anticancerogenic activity of the substances from this plant was also explored, but there are not enough human experiments to prove this activity and in order to use it as treatment for cancer we must discover correct dosages, as well as what dangers and benefits could be connected with this treatment. Another interesting point is the usage of the milk latex in milk production. As previously mentioned, the latex of E. trigona contains the hydrolytic enzyme protease, which could be used in industrial processes such as milk clot formation or as a meat tenderizer. Even though these possibilities have been discovered, more research is required before it could be used in food production.

I have listed all discovered substances found in the latex of E. trigona and connected them to biological activity to hypothesize their effects or potential usage in treatment against various diseases. Diterpene esters found in this latex are responsible for toxic reactions in the human body such as tumor promotion, proliferation and skin dermatitis. (51) Because of this information we could suspect that the first step in finding a cure is to get rid of these substances and isolate the other non- toxic ones. In experimental part, I planned to isolate various extracts of E. trigona and ex- amine them using HPLC method. However, because of the epidemiological situation this plan was unachievable. I managed to obtain four extracts of this Euphorbia and find a suitable mobile phase for HPLC. These extracts were methanol, hexane, ethyl acetate and water based. The most suitable mobile phase for methanol and hexane extract was composed of chloroform, toluene, ethyl acetate and methanol in a ratio 94 : 5 : 0.5 : 0.5. For ethyl acetate extract a mobile phase composed of chloroform and methanol in ratio 90 : 10 was chosen. This forms the basis on which the further experimentation was done. Based upon the findings of the combined research, I am convinced that E. trigona has potential to be developed into a medical drug after further experimentation.

5 CONCLUSION

The mission of my thesis was to explore and confirm the potential of using Euphorbia trigona as a medicine based on its content substances, their biological activity and the provided experiments. Furthermore, I was supposed to isolate specific biochemical substances from the extract of E. trigona and to prepare them for biological activity studies. I found that E. trigona and the substances contained in its extract represent a high potential for medical use. However, more experiments and clinical trials are needed for creating useful and safe medicine. I managed to isolate four extracts from E. trigona, methanol, hexane, ethyl acetate and water extract. A suitable mobile phase for HPLC for methanol and hexane extracts were determined as solutions of chloroform, toluene, ethyl acetate and methanol in ratio 94:5:0.5:0.5. For ethyl acetate extract a mobile phase composed of chloroform and methanol in ratio 90 : 10 was suitable. These findings represent a firm basis upon which further experimentation is possible.

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(18) KUPCHAN S.M. et al. Antileukemic principles isolated from euphorbiaceae plants. Science (New York, N.Y.). 1976, 191(4227), 571–572.

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LIST OF FIGURES

List of figures

Figure 1 (76) ...... 39 Figure 2 (76) ...... 40 Figure 3 Euphorbia trigona (own picture) ...... 40 Figure 4 E. trigona and its latex (own picture) ...... 41 Figure 5 Vacuum rotary evaporator Rotavapor R-3 (Büchi) with methanol extract from E. trigona (own picture) ...... 63 Figure 6 Nitrogene evaporator EVATERM with n-hexane extract from E. trigona (own picture) ...... 64 Figure 7 Lyophilisator (Christ) with water fraction of extract from E. trigona (own picture) ...... 65 Figure 8: TLC result of chloroform and n-hexane extracts of E. trigona under UV long wavelength (366 nm) (own picture) ...... 68 Figure 9: TLC result of chloroform and n-hexane extracts of E. trigona under UV short wavelength (254 nm) (own picture) ...... 69 Figure 10: TLC result of chloroform and n-hexane extract of E. trigona (own picture) ...... 69 Figure 11: TLC result of ethyl acetate extract of E. trigona under UV short wavelength (254 nm) (own picture) ...... 70 Figure 12: TLC result of ethyl acetate extract of E. trigona under UV long wavelength (366 nm) (own picture) ...... 70

LIST OF TABLES

List of tables

Table 1 Phytochemical analysis of the extracts from E. trigona (Adapted from: (2)) ...... 46 Table 2 Mobile phase selection for (column chromatography) HPLC for chloroform and n-hexane extract from E. trigona ...... 67 Table 3 Mobile phase selection for (column chromatography) for ethyl acetate extract from E. trigona ...... 68

89 LIST OF ACRONYMS

List of Acronyms

AAP – acetaminophen

AH109A – rat ascites hepatoma cell line

Akt – Protein kinase B

ANA – antinuclear antibody anti-JEV – anti-Japanese encephalitis virus

BALF – bronchoalveolar lavage fluid

Bcl-2 – B-cell lymphoma 2

BDNF – brain-derived neurotrophic factor cFLIP – cellular caspase-8 (FLICE)-like inhibitory protein

Colo205 – human Caucasian colon adenocarcinoma cell line compound 48/80 – N-methyl-p-methoxy-phenethylamine

DENA – diethylnitrosamine

DPPH – 2,2-diphenyl-1-picryl-hydrazyl-hydrate

EBV-EA – Epstein-Barr virus early antigen

GABA – gamma- aminobutyric acid

GSH – reduced glutathione

GSK-3 – glycogen synthase kinase-3

HCCLM3 – human hepatocellular carcinoma cell line

Hep G2 – hepatoma cell line

HepG2/Adr – DOX-resistant human hepatocellular carcinoma cells

HIV – human immunodeficiency virus

90 LIST OF ACRONYMS

HO-1 – heme oxygenase-1

IL – interleukin

LNCaP – androgen-sensitive human prostate adenocarcinoma cells

LPS – lipopolysaccharides

MCF-7 – breast cancer cell line

MDA-MB-435 – breast cancer cell line

MDA-MB48 – breast cancer cell line

NETs – neutrophil extracellular traps

NFκB – nuclear factor κB

NSAIDs – nonsteroidal anti-inflammatory drugs

PKC – protein kinase C

SOD – super-oxide dismutase

SREBP – sterol regulatory element-binding protein

TAK1 – mitogen-activated protein kinase 7

TLC – thin layer chromatography

TNF-α – tumor necrosis factor α

TPA – 12-0-tetradecanoyl-phorbol-13-acetate