Phytochemical and Biological Study of Pelargonium peltatum (L.) L’Her. Family Geraniaceae Cultivated in Egypt

Thesis submitted by

Fatma Hassan Mohammed Nossier

For the Degree of Master in Pharmaceutical Science (Pharmacognosy)

Under supervision of

Prof. Dr. Aly Mohamed El Sayed Professor of pharmacognosy Faculty of pharmacy Cairo University

Prof. Dr. Azza Ramy Abd El Moneam Professor of Pharmacognosy Faculty of Pharmacy Cairo University

Dr. Rehab Mohammed Seif El Din Ashour Lecturer of Pharmacognosy Faculty of Pharmacy Cairo University

2019

Abstract

are noted with considerable economic (عتر :Several herbs belong to genus Pelargonium (Arabic importance in ornamental plants market. Pelargonium originates from South Africa, where more than 200 species are cultivated. In Egypt, Pelargonium species are cultivated in Upper Egypt, mainly Beni Suef and Faiyum. Some Pelargonium species contain volatile oil called geranium oil which has economic value in perfume industry and cosmetics. Also, geranium oil has biological importance as it was reported to possess antibacterial, antifungal, anti-oxidant, insect repellent and other activities. Pelargonium peltatum (L.) L‟Her. cv. Balcon lilla is characterized by green, succulent and palmately lobed leaves. The color of the flowers is pale pink. The preliminary phytochemical screening followed by TLC of the 70% ethanolic extract and its fractions revealed that n-hexane and methylene chloride fractions were rich in sterols and triterpenes, while ethyl acetate and n-butanol fractions were rich in flavonoids and tannins. This is the first study of the volatile components of Pelargonium peltatum (L.) L‟Her. and the first time to use head space GC/MS for analyzing the oil of Pelargonium species. Five compounds were identified, namely; β- myrcene (44.86%), (-)-β-pinene (35.89), (-)-α-pinene (10.4%), p-mentha-1(7),8-diene (5.14%) and camphene (3.71%). Four compounds were isolated and identified from the ethyl acetate fraction; 1- O-monogalloyl-β-glucose, gallic acid, ethyl gallate and 1,2,3,4,6-pentagalloyl-ß-D-glucopyranose (PGG). The total phenolic and flavonoids contents were 19.3992 mg GAE/g and 2.7203 mg QE/g of dry weight, respectively. HPLC/MS profiling revealed the presence of thirty one compounds, twelve flavonoids and nineteen phenolic acid derivatives. Seventeen compounds were identified for the first time in the Pelargonium species. It is the first HPLC profiling of Pelargonium peltatum (L.) L‟Her. cv. Balcon lilla. In vivo and in vitro screening of biological activities of the 70% ethanolic extract of Pelargonium peltatum (L.) L‟Her. leaves and its fractions revealed that n- hexane fraction showed the most potent analgesic activity. The 70% ethanolic extract showed analgesic, anti-inflammatory and anti-hyperglycemic activities. Methylene chloride and ethyl acetate fractions showed high anti-inflammatory and anti-oxidant activities. The most effective cytotoxic fraction on the three tested cell lines was methylene chloride fraction.

Introduction

For thousands of years, natural compounds, mostly from plants, have been the mainstay of traditional medicine. They also have been the source of lead compounds for modern medicine (Ginsburg and Deharo 2011). In fact more than 25% of modern pharmaceuticals are plant derived are noted with considerable (عتر :Duke 1993). Several herbs belong to genus Pelargonium (Arabic) economic importance as ornamental plants in the market, gardens and forest free area. Pelargonium comes from Greek word “pelargos” (stork) because of the beak like fruits. Pelargonium commonly called geranium and originates from South Africa where more than 200 species are cultivated (Adams et al., 2006). In Egypt, Pelargonium species are cultivated in Upper Egypt, mainly Beni Suef and Fayoum. Geranium oil is secreted from glandular hairs located on both surfaces of the scented leaves of several species and cultivars. The volatile oil constitutes valuable commercial value in perfumery, cosmetics and aromatherapy. The main sites of production of geranium oil are Egypt, China and Reunion (Wells and Lis-Balchin 2002). European countries such as Spain, France and Italy produce smaller scale. The best quality of geranium oil comes from Reunion and Egypt where climate, soil and other vital factors contribute to quality of the oil (Demarne 2002). Geranium oil is among the top twenty of plant volatile oils; so many researches were conducted on volatile oils of different species to explore their chemical composition and biological activities. The oil was reported to contain monoterpenoid (Cavar and Maksimovec 2012) and sesquiterpenoid constituents (Kayser et al., 1998). Best oil composition is obtained with citronellol/geraniol ratio of 33/26% in January planted crops (Williams and Harborne 2002). Geranium oil is applicable in aromatherapy for massage as relaxant agent. For skin disorders, it heals fungal infections, athlete's foot, eczema, hemorrhoids (Saraswathi et al., 2011), bruises and burns. It has antiseptic and haemostatic activities, and relieves tiredness, general fatigue and convalescence (Lis-Balchin 2002). Geranium oil is also a wonderful insect repellent (Alipour et al., 2015). Pelargonium species were reported as rich source of tannins, flavonoid derivatives (El-Sherei et al., 2004a), coumarins (Hauer et al., 2010) and phenolic acids (Bauer and Treutter 1990). Pelargonium peltatum (L.) L‟Her. (family Geraniaceae), known as ivy-leaved geranium, hanging is one of the species cultivated in Egypt and used for ,(جارونيا مدادة أو جارونيا لير :geranium (Arabic ornamental purpose. It has faint odor and attractive flowers which last from May to July. It is used in folk medicine for treatment of oral ulcer and toothache (Scott et al., 2004; Walt 1977).

Aim of work

The current literature revealed the importance of Pelargonium species concerning their commercial value as oil producing, chemical constituents and biological activities. Although, available literature about this genus was numerous, little was reported on Pelargonium peltatum (L.) L‟Her. No published data was found concerning the chemical composition and biological activities of the Egyptian Pelargonium peltatum (L.) L‟Her. cultivar Balcon lilla. This encouraged us to investigate this ornamental species chemically and biologically and explore its medicinal value. The current present study includes reviewing the literature of different Pelargonium species, determination of the macro-morphological characters of Pelargonium peltatum (L.) L‟Her. cultivar Balcon lilla. plant, phytochemical screening of the 70% ethanolic extract of the dried leaves and its fractions (n-hexane, methylene chloride, ethyl acetate and n-butanol) to determine the different constituents, investigation of the volatile constituents of the leaves using GC/MS, isolation and identification of major phenolic compounds by chromatographic and spectroscopic techniques, determination of total phenolic and flavonoids, profiling the polar secondary metabolites of the 70% ethanolic extract by HPLC/MS/MS, finally; the biological activity of the 70% ethanolic extract and its different fractions (n-hexane, methylene chloride, ethyl acetate and n-butanol) were evaluated for their analgesic, anti-inflammatory, anti-hyperglycemic, cytotoxicity and antioxidant properties.

Review

Geuns Pelargonium was rich in compounds from different classes such as essential oil flavonoids tannins and coumarins. Also this genus showed diversity of biological activities such as antimicrobial, anti-oxidant, anti-cancer, anti-inflammatory and other biological activities.

Essential oil Some Pelargonuium species have distinct fragrance and contain essential oil. The essential oil distilled from Pelargonium species is called geranium oil. The oil was reported to contain monoterpenoid and sesquiterpenoid constituents. Pelargonium graveolens L'Herit. constitutes the main source of geranium oil. The major components of geranium oil are citronellol, geraniol, linalool and 10-epi-γ-eudesmol Geranium oil is important in aromatherapy for massage and for skin disorders. Verma et al., (2010) studied the changes in the essential oil composition of rose scented geranium Palergonium graveolens L‟Herit. cv. „CIM-Pawan‟ due to the date of transplanting in India (from December to April). The oil was analyzed by GC. The main components of the oil were citronellol (21.3-28.7%), geraniol (23.1-38.4%), linalool (4.2-6.5%), iso-menthone (6.5-7.9%), citronellyl formate (6.3-8.3%), 10-epi-γ-eudesmol (4.7-5.6%) and geranyl formate (3.3-4.3%).

Boukhris et al., (2013a) analyzed the essential oil of the aerial parts of Pelargonium graveolens L'Herit. by GC/MS. The main constituents of the oil were β-citronellol (21.9%), citronellyl formate (13.2%), geraniol (11.1%), 10-epi-γ-eudesmol (7.9%), geranyl formate (6.2%) and linalool (5.6%).

Koheil et al., (2012) analyzed the composition of the essential oil of leaves and flowers of Pelargonium zonale L. cultivated in Egypt by GC/MS. Hydrodistillation of the leaves and flowers yielded 0.12% and 0.06% v/w of the fresh plant, respectively. The essential oil of the leaves was typified by the presence of β-caryophyllene (24.6%), α-humulene (23.8%), caryophyllene oxide (8.5%) and α-cedrol (8.3%); while that of the flowers was characterized by α-humulene (36.73%). Meanwhile, the major monoterpene hydrocarbon was β-myrecene (7% & 1.6%, respectively) in both oil samples.

Andrade et al., (2011) identified the chemical composition of the essential oil from the leaves of Pelargonium odoratissimum (L.) L‟Her. in Brazil by GC/MS. The essential oil from the leaves of Pelargonium odoratissimum (L.) L‟Her. furnished methyleugenol (96.80%), methylisoeugenol (1.7%), bicyclogermacrene (0.9%) and germacrene B (0.3%).

Phenolic Compounds Several Pelargonium species were reported to be rich in phenolic compounds such as flavonoids, tannins, phenolic acids and coumarins. Flavonoids such as myricetin, quercetin, kaempferol and their glucosides were identified in addition to methylated flavonoids and anthocyanins. Both hydrolysable and non-hydrolysable tannins were reported to represent major constituents in many species of Pelargonium. Phenolic acids and coumarins were also reported in Pelargonium species. Compound name Species name [part used] Ref. P.denticulatum [aerial parts] El-Sherei et al., (2004a) Quercetin P. quercifolium [leaves] Williaims et al., (1997) P. radula [aerial parts] Pepeljnjak et al., (2005) Isoquercitrin P. graveolens [leaves and flowers] Boukhris et al., (2013a) P. purpureum Proestos et al., (2008) Rutin P. hispidum and P. radens [leaves] Iancu et al., (2016) 6-Methyl quercetin P. crispum [leaves] 8-Methyl quercetin Williaims et al., (1997) Quercetin-3-methyl ether P. quercifolium [leaves] Quercetin 3,7-dimethyl ether P. fulgidum[leaves] P. reoseum [leaves and flowers] Gumbaridze et al., (1980) Kaempferol P. asperum [aerial parts] Kokkalou and Souleles (1988) P. denticulatum [aerial parts] El-Sherei et al., (2004a) Astragalin P. graveolens [leaves and flowers] Boukhris et al., (2013a) P. graveolens [leaves] Hamed et al., (2015) Gallic acid p. purpureum Proestos et al., (2008) P. sidoides and P. reniforme Kayser and Kolodziej (1997) Methyl gallate [aerial parts and roots] P. reniforme [roots] Kim et al., (2009) 1,2,3,4,6-Pentagalloyl-ß-D- P. inquinans [leaves] Ji et al., (2005) glucopyranose 4 1,2,6-Tri-O-galloyl-β-D C1- glucopyranose P. graveolens [leaves] Hamed et al., (2015) 4 1,2,3,6-Tetra-O-galloyl-β-D C1- glucopyranose Protocatechuic acid P. x hortorum [leaves] Contour and Louguet (1985) Gentisic acid P. x hortorum [leaves] Contour and Louguet (1985) Vanillic Pelargonium purpureum Proestos et al., (2008) Fraxinol P. sidoides 8-Hydroxy-5,7- Latte et al., (2000) P. reniforme dimethoxycoumarin-6-sulfate Umckalin P. denticulatum El-Sherei et al., (2004b)

Other constitiuents

Pelargonium species was reported to have saturated and unsaturated fatty acids as Palmitic, oleic, and caprylic acids.

Siedel and Taylor (2004) reported the presence of palmitic, oleic and linoleic acid in the n-hexane fractions of roots of Pelargonium reniforme (Andr.) Curt. and Pelargonium sidoides DC. by (GC/MS).

Meselhy (2004) analyzed the light petroleum extract of Pelargonium denticulatum Jacq. by GC. n- Eicosane and n-decosane were the major hydrocarbons while β-sistosterol, stigmasterol and cholesterol were the major sterol in the unsaponifiable matter (USM). The major saturated fatty acids were palmitic and caprylic acids while tridecanoleic and caprioleic acids were the major unsaturated fatty acids. α-Amyrin, friedelin (3-oxo-friedelane) and β-sistosterol were isolated and identified by spectral data.

Saad El-Deen (2010) analyzed the light petroleum extract of aerial parts of Pelargonium zonale L. by GC. n-Decosane followed by n-octadecane were the major hydrocarbons while stigmasterol was the major sterol followed by β-sistosterol. The major saturated fatty acid was palmitic acid followed by lauric acids while oleic acid was the major unsaturated fatty acid followed by palmitoleic acids.

Biological activities

Pelargonium species are rich in phenolic compounds and some species contain essential oils which contribute to their different biological activities. Several Pelargonium species were reported to possess antimicrobial) and anti-oxidant activities. Other Pelargonium species could be used as natural insect repellent due to the presence of essential oil. Many other activities of Pelargonium such as anti-inflammatory, anti-coagulant and anti-cancer were also reported.

1- Antimicrobial activity Mativandlela et al., (2006) reported that the ethanol and acetone extracts of the roots of Pelargonium sidoides DC. inhibited the growth of Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumonia. Both acetone and ethanol extracts of Pelargonium reniforme (Andr.) Curt and only the ethanol extract of Pelargonium sidoides DC. inhibited the growth of Aspergillus niger and Fusarium oxysporum. Growth of Rhizopus stolonifer was suppressed by the ethanol

extracts of Pelargonium reniforme (Andr.) Curt. and Pelargonium sidoides DC. Acetone, chloroform and ethanol extracts of Pelargonium reniforme (Andr) Curt. showed activity against Mycobacterium tuberculosis.

Lalli et al., (2008) observed that crude extracts of Pelargonium glutinosum (Jacq.) L'Herit., Pelargonium pseudoglutinosum Knuth., Pelargonium scabrum (Burm. f.) L'Herit. and Pelargonium sublignosum Knuth. exhibited considerable antimicrobial activity against all the tested Gram positive bacteria with Pelargonium pseudoglutinosum Knuth. demonstrating the highest inhibitory activity.

Chraibi et al., (2016) indicated that essential oil of Pelargonium asperum Ehrh. exhibited significant antimicrobial activity against all the tested microorganisms with the strongest inhibitory effect against yeasts.

2- Anti-oxidant activity Sun et al., (2005) reported the antioxidant effect of essential oil and its monomers as well as residue and waste water after distillation from buds, stems and leaves of Pelargonium graveolens L'Herit. by DPPH method. The extract of the leaves collected at noon had the strongest antioxidant effect.

Piao et al., (2008) stated that geranium (Pelargonium inquinans Ait.) has an excellent antioxidative potential and that 1,2,3,4,6-penta-O-galloyl-ß-D-glucose (PGG) was considered to be the active component.

3- Insecticidal activity Bouzenna and Krichen (2013) showed that the essential oil of Pelargonium graveolens L'Herit. has insecticidal effects towards the insect Rhysopertha dominica.

Tabari et al., (2017) reported that Pelargonium roseum Eckl. & Zeyh. essential oil and two of its constituents, citronellol and geraniol could be helpful in developing novel and safe mosquito control tools and also offer an environmentally safe and cheap tools for reducing Culex pipiens mosquito populations.

4- Anthelmintic activity Meselhy (2013) stated that umbelliferone, umckalin and 4-methyl-5,8-dimethoxy-coumarin-7-O- glucoside in ethyl acetate fraction of acetone extract of Pelargonium fragrans Willd. roots recorded high activity as antiwormal against Allolobophora caliginosa.

Kozan et al., (2016) revealed that methanolic extract of Pelargonium endlicherianum Fenzl. demonstrated anthelmintic effect against the eggs, the first stage larvae and the adult stage of Haemonchus contortus.

5- Respiratory tract infesction Lizogub et al., (2007) concluded that EPs 7630 represents an effective treatment for the common cold. It significantly reduces the severity of symptoms and shortens the duration of the common cold compared with placebo.

Bao et al., (2015) stated that EPs 7630 showed significant and dose-dependent reduction of cough frequency.

6- Anti-sickling activity Fakim et al., (1992) observed that in experiment with haemoglobin AA erythrocytes suspension of the aqueous-methanol extract of the aerial parts of Pelargonium graveolens L'Herit. reversed blood crenation in acidic citrate-dextrose saline; in separate experiments with haemoglobin AS erythrocytes, sickling of the red blood cells to normal shape was also reversed.

El-Sherei et al., (2004a) reported that the methanolic extract of the aerial parts of Pelargonium denticulatum Jacq. exhibited antisickling activity.

7- Spasmolytic activity Lis-Balchin and Roth (2000) stated that the essential oils of the leaves of Pelargonium exstipulatum (Cav.) L‟Herit., Pelargonium odoratissimum (L.) L‟Herit. and a hybrid Pelargonium fragrans Willd. had a post-synaptic spasmolytic effect on smooth muscle in vitro.

Hart and Lis-Balchin (2002) demonstrated that the majority of Pelargonium oils and their components produced a relaxation of smooth muscle on isolated guinea pig ileum through a mechanism that involved the enzyme adenylate cyclase and a rise in cAMP.

8-Hypoglycemic activity Boukhris et al., (2012) reported a hypoglycemic effect of the leaves essential oil of Pelargonium graveolens L'Herit. together with glibenclamide in alloxan induced diabetic rats in Tunisia.

Afifi et al., (2014) concluded that Pelargonium graveolens L'Herit. aqueous extract and volatile oil as well as their purified phyto-constituents (menthone, linalool, geraniol and caryophyllene) inhibited α-amylase, α-glucosidase and pancreatic triacylglycerol lipase in vitro.

9- Anti-cancer activity Hamed (2007) stated that the leaves essential oil of Pelargonium zonale L. exhibited anti-tumor activity against HepG2 cell (Liver carcinoma cell) with IC50 7.49 µg.

4 Hamed et al., (2015) stated that 1,2,3,6-tetra-O-galloyl-β-D C1-glucopyranose and 1,2,3,4,6-penta- 4 O-galloyl-β-D C1-glucopyranose isolated from methanolic extract of dried leaves of Pelargonium zonale L. induced significantly high cytotoxic activity against HepG2 tumor cell line compared to 4 6-O-monogalloyl-(α/β)-D C1-glucopyranose.

10- Other activities Lis-Balchin and Hart (1994) investigated the pharmacological activity of Pelargonium grossularoides (L.) L'Herit. leaves extracts on in vitro preparations of smooth, skeletal and cardiac muscle. Most extracts contracted or increased the tone of guinea pig ileum, rat uterus and rat diaphragm with organic extracts (hexane and methanol) having a greater activity than aqueous extracts. A negative inotropic effect was observed on the rabbit heart. The spasmogenic action induced by the extracts on isolated uterus of the rat was consistent with the reported abortifacient effect of the plant.

Kayser et al., (2001) studied the effects of extracts and isolated constituents (coumarins and phenols) of Pelargonium sidoides DC. on non-specific immune functions. All Pelargonium extracts, gallic acid and its methyl ester significantly reduced the intracelluar survival of leishmania parasites. Gallic acid and its methyl were identified as the prominent immunomodulatory principle for this herbal medicine.

El-Sherei et al., (2004a) reported that both ethanolic and aqueous extracts of the aerial parts of Pelargonium denticulatum Jacq. exhibited a significant anticonvulsant activity.

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Summary

Introduction

Several herbs belong to genus Pelargonium are noted with considerable economic importance in ornamental plants market, gardens and forest free area. Pelargonium originates from South Africa, where more than 200 species are cultivated. In Egypt, Pelargonium species are cultivated in Upper Egypt, mainly Beni Suef and Fayoum. Some Pelargonium species contain volatile oil called geranium oil which has economic value in perfume industry and cosmetics. Also, geranium oil has biological importance as it was reported to possess antibacterial, antifungal, antioxidant, insect repellent and other activities. Pelargonium peltatum (L.) L‟Her. cultivated in Egypt has faint odor and attractive flowers and used for ornamental purpose. Although numerous researches were carried out on different species of this genus little no reports were available on the Egyptian Balcon lilla cultivar of Pelargonium peltatum (L.) L‟Her. which encouraged us to investigate it chemically and biologically and explore its medicinal value.

Part I: Macro-morphological characters

Pelargonium peltatum (L.) L‟Her. cv. Balcon lilla is characterized by being herbaceous perennial plant. The leaves are green, succulent and palmately lobed in shape. The stem is soft, not erect when getting long but curved down. The color of the flowers is pale pink.

Part II: Phytochemical study

A- Phytochemical screening

The preliminary phytochemical screening followed by TLC of the 70% ethanolic extract and its fractions revealed that n-hexane and methylene chloride fractions were rich in sterols and triterpenes, while ethyl acetate and n-butanol fractions were rich in flavonoids and tannins. The % yield of these fractions was 1.5, 0.5, 5.5 and 1.2%, respectively.

B. Study of the volatile components of Pelargonium peltatum (L.) L’Her. This is the first study of the volatile components of Pelargonium peltatum (L.) L‟Her. and the first time to use headspace GC/MS for analyzing the oil of Pelargonium species. The chromatogram of the plant collected in November showed five compounds, β-myrcene (44.86%) and (-)-β-pinene (35.89) were found to be the major components, alongside with (-)-α-pinene (10.4%), p-mentha- 1(7),8-diene (5.14%) and camphene (3.71%). The five identified compounds were classified as monoterpene hydrocarbons, whereas no oxygenated compounds were detected.

C. Isolation and identification of major constituents of ethyl acetate fraction of Pelargonium peltatum (L.) L’Her.

The 70% ethanolic extract of the dried leaves was fractionated using different organic solvents to obtain a dry fraction of ethyl acetate. The ethyl acetate fraction was fractionated on VLC column. The obtained fractions were separately subjected to rechromatography on silica gel 60 for further purification which affords four compounds. Their structures were identified based on their physicochemical and spectral analysis as 1-O-monogalloyl-β-glucose, gallic acid, ethyl gallate and 1,2,3,4,6-pentagalloyl-ß-D-glucopyranose (PGG). Ethyl gallate was isolated for the first time here from Pelargonium species. While 1-O- monogalloyl-β-glucose, gallic acid and 1,2,3,4,6-penta-O-galloyl-ß-D-glucose were isolated before from other Pelargonium species.

D. Quantitative estimation of total phenolics

1. Quantitative estimation of total phenolic content

The phenolic content of the leaves was spectrophotometrically determined by folin-ciocalteu colorimetric method. The total phenolic content recorded 19.3992 mg Gallic Acid Equivalent (GAE)/g of dry weight.

2. Quantitative estimation of total flavonoids content

The total flavonoids content of the leaves was determined for the first time here using aluminum chloride colorimetric method. It was found to be 2.7203 mg/g of dry weight expressed as quercetin equivalent.

E. HPLC-PDA-ESI-MS/MS profiling of polar secondary metabolites present in 70% ethanolic extract of the leaves of Pelargonium peltatum (L.) L’Her.

HPLC for Pelargonium peltatum (L.) L‟Her. cv. Balcon lilla is reported here for the first time. Thirty one compounds were identified including twelve flavonoids and nineteen phenolic acid derivatives. Seventeen compounds were identified for the first time in the genus Pelargonium including quercetin-3-O-pentosyl (1-2) neohesperidoside, quercetin-3-O-rhamnosyl (1-2) neohesperidoside, quercetin-7-O-glucuronide, quercetin-7-O-galloyl-neohesperidoside, quercetrin- 3-O-glucuronide butyl ester, eriodictyol-7-O-glucoside, chebulic acid, protocatecheuic acid hexoside, two isomers of digallate rhamnoside, ethyl gallate, tetra-O-galloyl hexoside, protocatecheuic acid di-hexoside, protocatecheuic acid rhamnosyl hexoside, in addition to two digalloyl glycerol and tri-galloyl glycerol derivatives. HPLC/MS profiling confirmed the presence of the previously isolated compounds.

Part III: Biological activity

1. Determination of analgesic activity

The 70% ethanolic extract and the n-hexane fraction showed the most potent analgesic activity compared to aspirin as standard at the three tested concentration levels. While, methylene chloride and ethyl acetate fractions showed less potent analgesic activity.

2. Determination of anti-inflammatory activity

At the first hour of treatment, the 70% ethanolic extract showed the highest % of inhibition of edema (62.4%) induced by carrageenan followed by ethyl acetate fraction (52.3%) and then methylene chloride fraction (51.0%) compared to indomethacin as standard. At the second hour of treatment, ethyl acetate fraction showed the highest % of inhibition followed by the 70% ethanolic extract and then methylene chloride fraction. Meanwhile, after three hours of treatment n-hexane fraction was the most potent followed by the 70% ethanolic extract and then ethyl acetate fraction.

3. Determination of anti-hyperglycemic activity The 70% ethanolic extract at low dose was the most effective in decreasing the cholesterol and triglycerides levels while at the high dose (250 mg/kg) was the most potent as anti-hyperglycemic compared to Gliclazide.

4. Determination of cytotoxic activity

The 70% ethanolic extract and its fractions were evaluated for their cytotoxicity against three carcinoma cell lines (breast, colon and lung carcinoma cell lines). The results showed that methylene chloride fraction recorded the highest cytotoxic activity against all the tested cell lines compared to doxorubicin as standard.

5. Determination of antioxidant activity

The obtained data revealed that methylene chloride fraction showed the highest antioxidant activity with IC50 (14.38 µg) followed by ethyl acetate fraction with IC50 (16.38 µg) as compared to vitamin C (16.77 µg).

Conclusions

 The phytochemical screening of Pelargonium peltatum (L.) L‟Her. revealed the presence of sterols, triterpenes, flavonoids and tannins.

 The volatile components of the leaves of Pelargonium peltatum (L.) L‟Her. were analyzed using head space GC/MS technique. Five compounds were identified, all were monoterpene hydrocarbons.

 Chemical investigation of the ethyl acetate fraction resulted in isolation of four compounds, identified as 1-O-monogalloyl-β-glucose, gallic acid, ethyl gallate and 1,2,3,4,6-penta-O- galloyl-ß-D-glucose (PGG).

 Total phenolic content in the dried leaves was found to be 19.3992 mg GAE/g dry weight. While the total flavonoids content was found to be 2.7203 mg/g dry weight.

 HPLC-PDA-ESI-MS/MS profiling of secondary metabolites in the 70% ethanolic extract of the leaves resulted in identification of thirty one compounds, being twelve flavonoids and nineteen phenolic compounds. Seventeen compounds were identified for the first time in Pelargonium species. This is the first HPLC profiling for Pelargonium species.

 Pelargonium peltatum (L.) L‟Her. cv. Balcon lilla revealed significant analgesic, anti- inflammatory, anti-hyperglycemic, cytotoxic and antioxidant activities.

 Pelargonium peltatum (L.) L‟Her. should be widely cultivated not only for ornamental purposes but also for their discovered biological importance.