Chemical and Biological Assessment of aegyptiaca Pers. Growing in Egypt

A thesis submitted by

Alaa Mohamed Abd-El-Moniem Mohamed Shaheen

In partial fulfillment of the requirements for the degree of Master in Pharmaceutical Sciences (Pharmacognosy) Under the Supervision of

Prof. Dr. Essam Abdel-Sattar Chairman of Pharmacognosy Department Faculty of Pharmacy Cairo University Prof. Dr. El-Sayeda Ahmed El-Kashoury Professor of Pharmacognosy Faculty of Pharmacy

Cairo University

Assoc. Prof. Dr. Mohamed El-Amir Fathey Hegazy Associate Professor of Medicinal Chemistry National Research Center

Pharmacognosy Department Faculty of Pharmacy Cairo University A.R.E. 2017 تقييم كيميائي وبيولوجي لنبات ستاكيس ايجيبتياكا (القرطم( الذي ينمو في مصر رسالة مقدمة من أالء محمد عبد المنعم محمد شاهين

إلستيفاء متطلبات الحصول على درجة الماجستير في العلوم الصيدلية )عقاقير( تحت إشراف أ.د/ عصام عبد الستار أستاذ ورئيس قسم العقاقير كلية الصيدلة-جامعة القاهرة

أ.د/ السيدة أحمد الكاشورى أستاذ العقاقير كلية الصيدلة- ج امعة القاهرة

أ.م.د/ محمد األمير فتحي حجازي أستاذ مساعد بقسم كيمياء النباتات الطبية المركز القومي للبحوث

قسم العقاقير كلية الصيدلة- جامعة القاهرة جمهورية مصر العربية 2017

Abstract

Genus Stachys is one of the largest genera of family consisting of about 300 species. It is centered in the Mediterranean region and south western Asia. Stachys aegyptiaca Pers. is a perennial aromatic plant growing wild in Wadi Jilbal, Sainte Catherine, Sinai. Preliminary phytochemical screening and estimation of the total phenolic and flavonoid contents were performed. Essential oil of Stachys aegyptiaca was obtained by two different techniques, conventional hydrodistillation (HD) and microwave assisted extraction (MAE). Gas chromatography-mass spectrometry analysis of the essential oil obtained by both techniques was carried out. Phytochemical investigation of the methylene chloride/methanol (1:1) extract of the air-dried aerial parts of the plant resulted in isolation and characterization of three new diterpenes (Stachyaegyptin A, B and C) together with four known neo-clerodane diterpenes (Stachysolone, 13- Mono-acetyl-stachysolone, Diacetyl-stachysolone and Rosestachenone). Seven methoxylated flavonoid aglycones were isolated. Three methoxy flavones were isolated for the first time from genus Stachys identified as 3, 7, 3', 4'- Tetramethyl-quercetin, Eupatilin-7-methyl ether and 5, 8-Dihydroxy-3, 6, 7, 4'- tetramethoxy flavone. 5-Hydroxy-3, 6, 7, 4'-tetramethoxy flavone was isolated for the first time from S. aegyptiaca while Calycopterin, 5-Hydroxy auranetin and Xanthomicrol were previously isolated from S. aegyptiaca. The extract and isolated compounds were evaluated for their anti-inflammatory, cytotoxic and antioxidant activities.

Keywords: Stachys aegyptiaca, Lamiaceae, essential oil, diterpenes, methoxylated flavones, biological activities. Introduction

People have always suffered from diseases by bacteria, fungi, viruses, parasites, cold, digestive problems, inflammation and pain. Modern medicines, depending on synthetic drugs and antiinfectives, became only available during the last 150 years. Previously, people had to rely on drugs from nature especially from , fungi and animals. Medicinal systems, developed thousands of years ago, heavily relied on herbal medicine. The treatment of infections and health disorders with herbal medicines is due to their content of active natural products (Wink, 2015). Family Lamiaceae (Labiatae or mint family) is one of the most diverse and widespread plant families. It contains about 236 genera and 6,900 to 7,200 species of flowering plants. Its medicinal value is mainly based on high essential oil content. Many species belonging to the family are highly aromatic due to the presence of external glandular structures that produce essential oil. Members of family Lamiaceae are widely cultivated due to their aromatic nature and their ease of cultivation as they can propagate by stem cuttings (Raja, 2012). Genus Stachys is one of the largest genera of family Lamiaceae consisting of 300 species and centered in the Mediterranean region and south western Asia (Jassbi et al., 2014). Many species of this genus have been widely used in traditional medicine, as long ago as the times of early Egyptian civilization. Aerial parts of several species have been used as infusions and decoctions to treat skin disorders, stomachache, ulcer, asthma, rheumatic pains and cancer (Gören, 2014). Many species of the Stachys genus are used to prepare a wild tea, also known as ‘mountain tea,’ that is believed to be sedative, antispasmodic, diuretic and emmenagogue (Gören, 2014). In addition, several Stachys species are also used as a food, as their edible are an important source of carbohydrates such as Stachys affinis Bunge. They were also used in traditional medicine in treatment of diarrhea, sore throat, internal bleeding and weakness of heart and liver (Venditti et al., 2013a). Pharmacological studies on extracts or pure compounds of several Stachys species demonstrated cytotoxic, antimicrobial, antioxidant, anti- inflammatory and anxiolytic effects. Phytochemical reports on the genus revealed the presence of flavonoids, essential oils, phenylethanoid glycosides, diterpenes and irridoids as secondary metabolites (Tundis et al., 2014). Stachys aegyptiaca is a growing wild in Sinai deserts.

Aim of work

Several flavonoid compounds were isolated from the 70% ethanolic extract (El-Ansari et al., 1991 and 1995) or the methanolic extract (El- Desoky et al., 2007) of S. aegyptiaca while few reports were found on diterpenoid content of S. aegyptiaca (Melek et al., 1992; Mohamed and Mohamed, 2014) despite of the numerous types of diterpene skeletons identified in the genus Stachys (Piozzi and Bruno, 2011). In this study, the aerial parts of the plant were extracted with the less polar solvent mixture methylene chloride/methanol (1:1) with the aim of:  Isolation of new natural products with an important biological profile.  Chemical characterization of the isolated compounds.  Comprehensive bioassay testing of lead compounds, including cytotoxicity, antioxidant and anti-inflammatory activities.

This study includes the following

1. Review of literature regarding the phytoconstituents and bioactivity of the members of the genus Stachys. 2. Preliminary phytochemical screening of the aerial parts of Stachys aegyptiaca. 3. Estimation of the total phenolic and flavonoid contents of the aerial parts of the plant. 4. Comparative investigation of the essential oils of the aerial parts prepared by conventional hydrodistillation and microwave assisted extraction. 5. Isolation, purification and characterization of the chemical constituents of the aerial parts using different chromatographic and spectral techniques. 6. In-vivo evaluation of the anti-inflammatory activity of the total extract as well as the in-vitro evaluation of the total extract and the isolated compounds for their cytotoxic, anti-inflammatory and antioxidant activities.

Review of literature Stachys is one of the largest genera of family Lamiaceae, containing about 300 species. It is a subcosmopolitan genus distributed in warm temperate regions of Mediterranean, Europe and in almost all types of habitats and at all altitudes (Munoz et al., 2001). Phytochemical investigations of the genus Stachys revealed the presence of essential oils (Halim et al., 1991; Grujic-Jovanovic et al., 2011; Karami and Dehghan-Mashtani, 2015), terpenoids (Fazio et al., 1994a and 1994b; Paternostro et al., 2000; Mohamed and Mohamed, 2014), flavonoids (El-Ansari et al., 1991 and 1995; Vundać et al., 2005), phenylethanoid glycosides (Ikeda et al., 1994; Nishimura et al., 1991; Delazar et al., 2011), iridoids (Munoz et al., 2001; Murata et al., 2008) and triterpenoid saponins (Yamamato et al., 1994; Cho et al., 2014a and 2014b). The different species of the genus were also investigated for their antimicrobial (Farjam et al., 2011; Yildirim et al., 2013; Jassbi et al., 2014;), antioxidant (Morteza-Semnani et al., 2006; Bilušić Vundać et al., 2007; Ebrahimabadi et al., 2010), anxiolytic (Rabbani et al., 2003 and 2005; Kumar et al., 2013) and anti-inflammatory activities (Benmebarek et al., 2013a; Sadeghi et al., 2014; Barreto et al., 2016).

Major active constituents of the genus Stachys I. Essential oil constituents The essential oil yield of Stachys species is low compared to other members of family Lamiaceae (e.g. Origanum, Satureja, Sideritis, Salvia and Thymus) however, the consumption pattern of the Stachys species is quite similar to the other members of the family. Most species of the genus Stachys have pleasant smell except few species have unpleasant smell due to their essential oil (Gören, 2014). They are consumed as herbal tea in the major parts of the world due to their moderate antibacterial, antifungal, anti-inflammatory and anti-oxidant capacity. They are widely used as herbal remedy in alternative medicine (Gören, 2014). The composition of the essential oil of the genus Stachys has been studied extensively. Investigation of the essential oil of S. aegyptiaca obtained from the leaves by hydrodistillation revealed the presence of fourteen monoterpenes hydrocarbons, four oxygenated monoterpenes and seven sesquiterpene hydrocarbons (Halim et al., 1991). Published data concerning the chemical composition of the essential oils of different species of the genus are summarized in Table (1) and the structures of selected oil constituents are represented in Figure (1).

II. Diterpenes Different diterpenes of ent-clerodane, kaurane, labdane and rosane types were isolated. The published data regarding these constituents are summarized in Table (2) and their structures are shown in Figure (2).

III. Triterpenoids and Sterols Triterpenoid compounds of oleanane and ursane types as well as sterols have been reported from the genus Stachys. The published data concerning these triterpenes are listed in Table (3) and their structures are displayed in Figure (3).

IV. Iridoids The published data regarding these iridoids are summarized in Table (4) and the structures of the isolated compounds are shown in Figure (4).

V. Phenylethanoid and phenylpropanoid glycosides There is much confusion regarding names and structures of phenylethanoid glycosides in the literature (Jimenez and Riguera, 1994). For example, Compound number (126) in Table (5) was isolated in 1988 from Stachys lavandulifolia by Basaran et al., 1988 and named Lavandulifolioside. later in 1990, it was isolated from the tubers of Stachys sieboldii and named Stachysoside B (Miyase et al., 1990), and again in 1991 from the leaves of the same plant, but this time named Stachysoside A (Nishimura et al., 1991). This explains the presence of several names for the same compound as shown in Table (5) while some other phenylethanoid constituents were isolated but given no common name (Ikeda et al., 1994). Very few reports are found on phenylpropanoid glycosides. The published data regarding these constituents are summarized in Table (5) and the structures of the isolated compounds are represented in Figure (5).

VI. Flavonoids Flavonoid aglycones and glycosides of different classes are widely distributed in the genus Stachys. Published data of these constituents are displayed in Tables (6&7) and the structures of the isolated compounds are shown in Figures (6&7).

VII. Phenolic acids The published data on phenolic acids reported in different Stachys species are summarized in Table (8) and their structures are represented in Figure (8).

VIII. Miscellaneous constituents Other constituents including simple phenolic glucosides, lignans, organic acids, fatty acids, sugars and sugar derivatives were also detected in different species of the genus. Published data regarding these constituents are displayed in Table (9) and Figure (9).

Biological Activities Different Stachys species have been used in folk medicine for healing wounds, disinfectant, treating abdominal pains and asthma. It is also used in treating rheumatic pains, inflammation, spasm and fever. The essential oils of some Stachys species were also used as anti-cancer. Other biological activities reported for different Stachys species including antibacterial, antioxidant, antinephritic and antianxiety activities were also investigated (Khanavi et al., 2012). The available scientifically based reports on biological activities of the genus Stachys are summarized in Table (10).

General Summary

Family Lamiaceae is one of the largest and most diverse plant families consisting of 236 genera and about 6900-7200 species. The members of the family are widely cultivated due to the ease of cultivation and high essential oil content.

Genus Stachys is one of the largest genera of family Lamiaceae. It consists of about 300 species distributed in Mediterranean region and south western Asia. The plants of the genus are used in treatment of diarrhea, sore throat, heart and liver weakness and internal bleeding. Many species of this genus have been widely used in traditional medicine, as long ago as the times of early Egyptian civilization. Aerial parts of several species have been used in infusions and decoctions to treat such problems as skin disorders, stomachache, ulcer, asthma, rheumatic pains and cancer. Many species of the Stachys genus were named as mountain tea as they were used for preparation of wild tea. It was believed to be sedative, antispasmodic, diuretic and emmenagogue. In addition, several Stachys species are eaten due to high carbohydrate content such as Stachys affinis Bunge. Stachys aegyptiaca Pers. is a perennial plant growing wild in Sinai deserts. It is a bushy plant about 1 meter height with pink corolla and lanceolate to elliptic entire leaves.

Reviewing the current literature, several studies were done on Stachys aegyptiaca. The essential oil of leaves was obtained by hydrodistillation and its major constituents were identified. Several flavonoid compounds have been isolated from the ethanolic (70%) and the methanolic extracts of S.

aegyptiaca. However, few reports were traced on the diterpene content of S. aegyptiaca. This study includes the following. 7. Review of literature regarding the phytoconstituents and bioactivity of the genus Stachys. 8. Preliminary phytochemical screening of the aerial parts of S. aegyptiaca. 9. Comparative investigation of the essential oils of the aerial parts prepared by conventional hydrodistillation and microwave assisted extraction. 10. Estimation of the total phenolic and flavonoid contents of the aerial parts of the plant. 11. Isolation, purification and characterization of the chemical constituents of the aerial parts using different chromatographic and spectral techniques. 12. In-vitro and in-vivo evaluation of the anti-inflammatory activity of the total extract as well as the in-vitro evaluation of the total extract and the isolated compounds for their cytotoxic and antioxidant activities. Part Ⅰ Phytochemical investigation of Stachys aegyptiaca Pers.

Chapter Ⅰ: Preliminary phytochemical screening.

Preliminary phytochemical screening of the air dried powdered aerial parts of S. aegyptiaca revealed the presence of essential oils, carbohydrates and/or glycosides, sterols and/or triterpenes, flavonoids, tannins and saponins and absence of alkaloids, anthraquinones and cardiac glycosides. Chapter Ⅱ: Investigation of the essential oil

Conventional hydro-distillation (HD) and microwave-assisted extraction (MAE) were used for the extraction of S. aegyptiaca essential oil. Gas chromatography-mass spectrometry analysis of the essential oils obtained

revealed variation in the percentage yield and chemical composition of the oils. The analysis showed the presence of 30 and 48 components constituting 99.82% and 99.31% of the total composition of the oils obtained by HD and MAE, respectively. The main components found in the oil were α-pinene (24.65 and 41.14%), trans-caryophyllene (14.65 and 8.63%), 6-epi-shyobunol (14.61 and 11.15 %), α-cadinol (11.08 and 5.55%), δ-cadinene (5.75 and 2.39%) and (-) spathulenol (5.41 and 4.32%) and germacrene D (5.1 and 4.19 %) extracted by HD and MAE, respectively. Oxygenated sesquiterpenes and monoterpene hydrocarbons are the main classes of compounds of the extracted S. aegyptiaca essential oils representing 37.91 and 49.56% of the oil composition using HD and MAE, respectively. MAE offered reduction in the extraction time (60 min vs 3 hr) with better oil yield (1.4 % w/v) when compared to HD (0.9%) w/v. Eighteen constituents were identified in the oil obtained by MAE but were absent in the hydrodistilled oil.

Chapter Ⅲ: Estimation of the total phenolic and total flavonoid contents

The total phenolic content was estimated by Folin-Cicalteu assay method. Gallic acid was used as a standard. The total phenolic (TP) content of the 70% methanol extract of the aerial parts of S. aegyptiaca expressed as gallic acid equivalent (GAE)/g extract was calculated from the standard calibration curve. The results revealed that the total phenolic content of the aerial parts of S. aegyptiaca was 63.04 mg GAE/g extract.

The total flavonoid content of the 70% methanol extract of the aerial parts was estimated by AlCl3 method. Rutin was used as a standard. The total flavnoid (TF) content of the aerial parts expressed as rutin equivalent (RE)/g extract was 25.40 mg RE/g extract.

Chapter Ⅳ: Isolation and characterization of the main active constituents

The air-dried powdered aerial parts of S. aegyptiaca were extracted with

CH2Cl2/ MeOH (1:1). The extract (110 g) was then fractionated on silica gel flash column eluted with n-hexane (3L) and the polarity is gradually increased with CH2Cl2 and methanol (25% increments) till 100% methanol. Fractions (500 ml each) were collected and monitored by TLC to give 9 main fractions. Fractions 5, 6 and 7 showed better chromatographic profiles. Therefore they were selected for further fractionation by reversed phase chromatography on octadecylsilyl (ODS) column, eluted with 80% methanol and decreasing the polarity by 10% increments of methanol till 100% to give 3 subfractions for each fraction.

Subfractions 5-1 (6.5 g) was subjected to semi-preparative separation on HPLC, to afford four compounds:  Stachyegyptin A (isolated for the first time from nature).  Stachyegyptin B (isolated for the first time from nature).  Di-acetyl stachysolone (isolated before from S. aegyptiaca).  Calycopterin (isolated before from S. aegyptiaca). Subfractions 5-2 (6.3 g) was subjected to semi-preparative separation on HPLC to afford five compounds. These compounds are:  Stachyegyptin C (isolated for the first time from nature).  5, 8-Dihydroxy-3, 6, 7, 4'-tetramethoxy flavone (first report on its isolation from Stachys genus).  3, 7, 3', 4'-Tetramethyl-quercetin (first report on its isolation from Stachys genus).  5-Hydroxy-3, 6, 7, 4'-tetramethoxy flavone (first report on its isolation from S. aegyptiaca).

 5-Hydroxy auranetin (isolated before from S. aegyptiaca). Subfractions 6-1 (4.8 g) was subjected to semi-preparative separation on HPLC to afford two compounds:  13-Mono acetyl stachysolone (isolated before from S. aegyptiaca)  Rosestachenone (isolated for the first time from S. aegyptiaca) Subfractions 7-1 (3.6 g) was subjected to semi-preparative separation on HPLC to afford three compounds  Stachysolone (isolated before from S. aegyptiaca).  Xanthomicrol (isolated before from S. aegyptiaca).  Eupatilin-7-methylether (first report on its isolation from Stachys genus). The isolated diterpenoid compounds were identified by 1D (1H-NMR and 13C- NMR), 2D NMR (COSY, DEPT, HMQC, HMBC and NOESY) techniques and comparison with the published data. While the flavonoidal compounds were identified by 1D NMR, mass spectroscopy, UV shift reagents as well as comparison of their data with those published in the literature. Part Ⅱ Biological evaluation of Stachys aegyptiaca A. Evaluation of antioxidant activity (DPPH assay).

The antioxidant activity of the total extract CH2Cl2/ MeOH (1:1) and the isolated compounds were estimated by DPPH assay using quercetin dihydrate as a standard antioxidant drug. Tested samples showed a weak radical scavenging activity. The total extract produced the strongest DPPH scavenging activity among other samples with IC50 of 83.0 µg/ ml. The antioxidant activity exhibited by the total extract may be due to other

constituents of the plant like iridoids, phenylethanoids, flavonoid glycosides and phenolic acids.

B. In-vitro anti-inflammatory activity.

The anti-inflammatory activity of the total extract CH2Cl2/ MeOH (1:1) and the isolated compounds was estimated by inhibition of LPS-induced nitric oxide release in RAW 264.7 macrophage using sulphoraphane as a standard anti-inflammatory drug. The anti-inflammatory activity performed with nitric oxide inhibition assay revealed that the tested samples have a weak to moderate anti-inflammatory activity. The highest activity was obtained by compound 4 (Calycopterin) which caused about 50% inhibition of NO (IC50 =62.3 µM).

C. Antiproliferative activity against HepG2.

The anti-proliferative activity of the total extract CH2Cl2/MeOH (1:1) and the isolated compounds were estimated by MTT assay against HEPG2 cells. Results revealed that none of the tested samples have a promising cytotoxic effect. The highest cytotoxic activity was established by compound 1 (Stachyaegyptin A) which caused about 50% loss of HepG2 cell viability.

D. In-vivo anti-inflammatory activity

The anti-inflammatory activity of the total extract CH2Cl2/MeOH (1:1) was estimated by carageenan induced paw edema model using indomethacin as standard anti-inflammatory drug. The total extract was orally administered in doses of 50, 100, 200 mg/kg. Results revealed that the total extract of S. aegyptiaca in doses of 50, 100, 200 mg/kg have 37.89, 43.37 and 47.02 % of indomethacin potency, respectively.

General Conclusion and Recommendations

 The air-dried powdered aerial parts of S. aegyptiaca were subjected to preliminary phytochemical screening for the presence of different types of secondary metabolites. Results revealed the presence of essential oils, carbohydrates and/or glycosides, sterols and/or triterpenes, flavonoids, tannins and saponins while alkaloids, anthraquinones and cardiac glycosides were absent.  Estimation of the total phenolic and flavonoid content of the 70% methanolic extract of the aerial parts of S. aegyptiaca was performed. The investigation revealed that the total phenolic content was 63.04 mg (GAE)/g extract, while the total flavonoid content was 25.40 mg (RE)/g extract.  Essential oil of S. aegyptiaca was obtained by two different techniques, conventional hydrodistillation (HD) and microwave assisted extraction (MAE). MAE offered reduction in the extraction time of the essential oil from 3 hrs in HD to 1 hr in MAE, with better oil yield (1.4 %w/v) when compared with HD (0.9% w/v). Forty eight components were identified in microwave extracted oil representing (99.31%) of the oil composition, while 30 components were identified in hydro-distilled oil representing 99.82% of the oil composition. The main components found in the oil were α-pinene, trans-caryophyllene, 6-epi-shyobunol, α-cadinol, δ-cadinene, (-) spathulenol and germacrene D. Oxygenated sesquiterpenes and monoterpene hydrocarbons are the main classes of compounds representing 37.91 and 49.56% of the oil composition using HD and MAE, respectively. These results recommended the use of MAE as an alternative tool for isolation of essential oils from their natural sources.

 Fourteen compounds were isolated from the CH2Cl2/MeOH (1:1) extract including

. Seven diterpenes  Stachyegyptins A, B and C (isolated for the first time from nature).  Stachysolone, 13-Mono-acetyl-stachysolone and Di-acetyl-stachysolone (isolated before from S. aegyptiaca).  Roseostachenone (isolated for the first time from S. aegyptiaca). . Seven methoxylated flavonoids

 3, 7, 3', 4'-Tetramethyl-quercetin, eupatilin-7-methylether and 5, 8- Dihydroxy-3, 6, 7, 4'-tetramethoxy flavone (isolated for the first time from the genus Stachys).  5-Hydroxy-3, 6, 7, 4'-tetramethoxy flavone (first report for its isolation from S. aegyptiaca).  Calycopterin, 5-Hydroxy auranetin and Xanthomicrol (previously isolated from S. aegyptiaca).  The total extract showed a moderate in-vivo anti-inflammatory and good antioxidant activities.  A moderate anti-inflammatory in vitro was elicited by compound 4 (Calycopterin) in the RAW 264.7 model.  All isolated compounds were not antiproliferative against HepG2 and have no antioxidant activity.  The activity of the total extract may be due to other constituents such as iridoids, phenylethanoid glycosides, flavonoid glycosides and phenolic acids.  Other biological activities reported for the genus Stachys should be performed to support its tradition uses.