Pharmacognostical Study of Some Species of Family cultivated in Egypt

A THESIS SUBMITTED BY Aya Mohamed Faisal Mohamed Teaching Assistant Faculty of Pharmacy – Ahram Canadian University

For the Degree of Master in Pharmaceutical Sciences (PHARMACOGNOSY)

Under the Supervision of

Prof. Dr. Prof. Dr. Seham Salah El-Din El-Hawary Ibrahim Ibrahim Mahmoud

Professor of Pharmacognosy Professor of Pharmacognosy Faculty of Pharmacy Dean of Faculty of Pharmacy Cairo University Ahram Canadian University

Dr. Manal Mahmoud Sabry Lecturer of Pharmacognosy Faculty of Pharmacy Cairo University

PHARMACOGNOSY DEPARTMENT FACULTY OF PHARMACY CAIRO UNIVERSITY 2018

Abstract

A preliminary phytochemical screening of the aerial partsof T. pallida (Rose) D.R.Hunt, T. zebrinaHeynh ex.Bosse and T. spathaceaSwartz.(Commelinaceae) cultivating in Egypt was carried out. Lipoidal contents was investigated in the petroleum ether extractive of T. pallida (Rose) D.R.Hunt. The phytochemical investigation of the flavonoidal and phenolic compounds using HPLC of the three species indicated that T. zebrinaHeynh ex. Bosse possessed the highest percentage of flavonoid and phenolic content where Kamp3, (2-p-comaroyl) glucoside was the most abundant compound (2740 mg/100g). Analysis of the secondary metabolites of the three Tradescantia species using HPLC-PDA-MS/MS led to the identification of 27 phenolic compounds. The phytochemical investigation of T. pallida (Rose) D.R.Hunt led to the isolation of 1 steroidal compound. β-sitosterol compound was isolated from the petroleum ether extractive. In addition to the separation and identification of seven phenolic compounds from the n-butanol fraction by using LC-ESI-MS/MS (trans-cinnamic acid, caffeic-O-pentoside, p- coumaroyl-O-pentoside, quercetin, syringic-O-hexoside, naringenin and apigenin). Using the in-vivo and in-vitro biological studies, the total ethanolic extracts of the three species were safe, exhibiting potent anti-inflammatory, antihyperglycemic, hepatoprotective and antioxidant activities. The ethanolic extracts of the threeTradescantiaspecies showed no cytotoxic activity against the tested carcinoma cell lines (HEPG-2, MCF-7 and HCT-116). The botanical features of the stems and of the three species have been studied in order to find out the diagnostic characters that help in identification of the either in the entire or the powdered form.

Introduction Commelinaceae: dayflower or spiderwort family, is a family. Members of this family are ornamentals. The stems of these are generally well-developed, and often swollen at the nodes. are often short-lived, lasting for a day or less.

The family includes about 40-50 genera and 731 species. Commelina(170 species), Tradescantia (75-80 species), Murdannia (55 species), are well-known genera of the family. The family is diverse in both the Old-World tropics and the New World tropics, with some genera present in both.

In traditional medicine, plants of genus Tradescantiawere used for the treatment of mycosal infections, venereal diseases , urinary tract infections, hemorrhoids, tuberculosis and cough (Other traditional uses viz; anti-inflammatory, anti-toxic supplement and improve blood circulation, antidiarrheal, expectorant, hypoglycemic agent and against snakebiteswere also reported.

Pharmacological activities of different extracts of various Tradescantiaspecies had been experimentally proved viz; antibacterial, anti-hyperuricemic, analgesic and anti-inflammatory effects. Moreover, the cytotoxic and hepatoprotective activitiesof T. spathaceaSw. were reported.

Aim of Work

Tracing the current literature, there was a little reported about the active constituents regarding genus Tradescantiaand their biological activity, thus the present study includes a pharmacognostical study of three species of genus Tradescantia: T. pallida (Rose) D.R.Hunt., T. zebrinaHeynh. ex Bosse and T. spathaceaSw., their main active constituents and to find the way to differentiate between them.

This work comprises four parts:

Part I: A detailed genetic profiling of the three Tradescantia species, as well as, macro and micromorphological studies of the aerial parts of the three species.

Part II: Includes Phytochemical investigation, involving Preliminary Phytochemical Screening, Qualitative and Quantitative determination of Flavonoids and Phenolic compounds using spectrophotometric and HPLC analyses.

Part III: Extraction, Fractionation, as well as, Isolation and Identification of the main constituents of Tradescantia pallida (Rose) D.R. Hunt

Part IV: Includes Biological Study, including Toxicological study (LD50 determination), in vitro studies (Cytotoxic and

Antioxidant screening) and in vivo studies (Hepatoprotective, Anti-inflammatory and Anti-hyperglycemic activities) of the alcohol extracts of the three species.

Review of Literature

Tradescantia species (syn. Spiderwort, Family: Commelinaceae) are succulent perennial plants. There are about eighty species of Tradescantia, native to the New World from southern Canada to northern Argentina including the West Indies.

The genus possesses numerous medicinal properties such as antimicrobial, hepatoprotective, anti-oxidant, antiviral, analgesic, and anti-inflammatory activities. In herbal medicine, they are used to treat sore throats, skin problems, stomach problems, expectorant, and anti-diarrheal. Bioactive constituents as phenolics, flavonoids, glycosides, sterols were reported about the genus.

General Summary

Genus Tradescantia (syn. Spiderwort, Fam: Commelinaceae) comprises succulent perennial plants. There are over seventy-five species of Tradescantia, native to the New World, but have naturalized throughout the tropics.

In traditional medicine, plants belonging to genus Tradescantiawere used for the treatment of mucosal infections, venereal diseases, urinary tract infections, hemorrhoids, tuberculosis and cough. Other traditional uses viz; anti-inflammatory, anti-toxic supplement and improve blood circulation, antidiarrheal, expectorant, hypoglycemic agent and against snakebites were also reported.

The present study has been planned with the aim to evaluate the potentialities of three Egyptian Tradescantia species: T. pallida (Rose) D.R. Hunt, T. zebrinaHenyh. ex Bosse and T. spathacea Sw. to illucidate their main active constituents and to find the way to differentiate between them. This study comprises three parts.

Part I: Genetic and Botanical Study Chapter I DNA Fingerprinting The extracted DNA from each of the three Tradescantiaspecies was amplified using ten decamer primers to detect their genetic variability. The ten primers of arbitrary sequences generated a total of 55 fragments in T. pallida (Rose) D.R. Hunt and 55 in T. spathacea Sw., while 64 fragments were generated in T. zebrinaHeynh. ex Bosse

The highest degree of similarities (92%) was recorded using primer A1 while the lowest degree of similarity (71.5%) was recorded using primer A2.

The genetic characterization of the three Tradescantiaspecies showed that primer A1 could be used as an indicator for obtaining genetic markers for genus Tradescantia. In addition, primers A7 and A10 were found to be the most effective in generating unique bands on application of the RAPD technique to both plants, and therefore can act as species-specific markers in addition to the morphological and anatomical characteristics.

Chapter II Botanical Study A.Macromorphology

I. Macromorphology of Tradescantia pallida (Rose) D.R. Hunt.

Tradescantia pallida (Rose) D.R. Hunt. is an ornamental, succulent, perennial herb, native to Mexico. It reproduces by sprouting (when a is removed from the plant and placed in a warm moist place, young plants will very soon appear in the notches around the margin of the leaf) or by cutting.

It carries lanceolate, fleshy leaves and pink, small, petaled flowers in the leaf axills.

The Stem: The stem is thick, succulent and cylindrical. The stem is smooth, glabrous, violet purple in color. The stem has no odor but has an astringent taste.

The Leaf: The leaf is simple, alternate, sessile, fleshy, green in color and turned purple when exposed to sunlight. The lamina is lanceolate, glabrous, with an acute apex and asymmetric base. It has an entire margin with parallel

venation. It measures from 5-12 cm in length, 2 cm in width. The leaf has no odor but has an astringent taste.

II. Macromorphology of TradescantiazebrinaHeynh. ex Bosse

TradescantiazebrinaHeynh. ex Bosse is an ornamental, succulent, perennial herb, native to southern Mexico. It reproduces by sprouting or by cutting.

The plant cultivated in Egypt, reaches up to 7 cm in height.

The Stem: short, easily produce at their nodes, succulent. Branching stems are glabrous and usually tinged with purplish red. It has no odor but has an astringent taste.

The Leaf: simple, opposite, sessile, exstipulate. The upper surface of the leaf is green and/or purplish in color with broad silvery stripes and the lower surface is dark purple. The lamina is ovate, glabrous, acute to acuminate apex. It has an entire margin and parallel venation. Lamina measures from 1- 3-7 cm in length, 1.5-3 cm in width. The leaf has no odor but has an astringent taste.

III. Macromorphology of Tradescantiaspathacea Swartz

Tradescantiaspathacea Sw. is an ornamental, succulent, perennial herb native to Central America, Mexico and the Caribbean. It is reproduced by division or by leaf cutting.

The plant cultivated in Egypt reaches up to 16 cm in height.

The Stem: erect, short and unbranched. It has no odor but has an astringent taste.

The Leaf: The leaf is simple, alternate, sessile and rosette-forming succulent herb. The lamina is lanceolate, glabrous with an entire margin and parallel venation. The upper surface is green in color, the lower surface rich reddish- purple; inflorescence axillary and boat-shaped. The leaf has no odor but has astringent taste.

B. Micromorphology

I. Micromorphology of Tradescantia pallida (Rose) D.R.Hunt,Tradescantia zebrine Heynh. ex Bosse and Tradescantia spathacea Sw.

The Stem

The epidermal cells are polygonal, axially elongated with straight beaded anticlinal walls, covered with thin cuticle; showing few paracytic stomata, and no trichomes.

The cortex consists of several rows of rounded, thin-walled parenchyma cells. Differentiated into two zones, one zone consisting of angular collenchyma cells and another area made up of several cells of chlorenchyma. Calcium oxalate crystals were scattered (clusters and raphides).

The vascular bundles were scattered all over the stele, the larger one was present near the center. They were poorly developed with phloem to the exterior and xylem to the interior. Schlerenchymatous elements were present ad pericycle was not distinguished.

The Leaf

A transverse section of the leaf is isobilateral. The mesophyll is formed of undifferentiated spongy tissue. The stomata are present on both epidermal

cells. The upper epidermis of the lamina is made up of hexagonal cells. The mesophyll is homogenous. The Epidermis of both surfaces are pentagonal, elongated, with straight anticlinal walls. The upper epidermal cells were larger than those of the lower one. Transverse section, revealed that the epidermal cells of both surfaces were arranged in a single layer, covered by a thin cuticle. In terms of size, upper epidermal cells of the lamina were larger than those of the lower ones. Beneath the upper epidermis was an area of typically multilayered upper hypodermis. It was followed by the palisade parenchyma, towards the hypodermis, and by a many-layered spongy tissue towards the lower epidermis.

The vascular tissue consists of small, closed vascular bundles formed of few spiral and annular xylem vessels and few phloem tissues formed of sieve tubes and companion cells.

Part II: Phytochemical Investigation Chapter I Preliminary Phytochemical Screening The preliminary phytochemical screening of the aerial parts of T. pallida (Rose) D.R. Hunt, T. zebrinaHeynh. ex Bosse and T. spathacea Sw. revealed the presence of carbohydrates and/or glycosides, flavonoids, phenolic acids, alkaloids and/or nitrogenous compounds, tannins, sterols and/or triterpenes. Cardiac glycosides, saponins, anthraquinones and were not detected.

Chapter II

Qualitative and quantitative analysis flavonoids and phenolic compounds

I. Spectrophotometric method

Total phenolic and flavonoid contents were determined in the ethanolic extracts of the leaves by Folin-Ciocalteu and aluminum chloride colorimetric methods, respectively. A Shimadzu spectrophotometer (UV-1650PC) was used.

For total phenolic content: the absorbance of the blue color was measured at 765 nm. Gallic acid was used as standard and equivalents determined (w/w) from a pre-established concentration curve (y = 0.0041x + 0.0594, R² = 0.9959). The total phenolic acids recorded for T. pallida (Rose) D.R. Hunt, T. zebrinaHeynh. ex Bosse and T. spathacea Sw., were 35.35±0.14, 62.17±1.62 and 85.11±0.61 respectively, corresponding to µg (GAE) / mg dried extract. These results obviously indicate that the total phenolic content of the leaves of T. spathacea Sw. estimated as µg/ mg dried extract GAE, exceed that present in those of the other two species.

For total flavonoid content: The absorbance of the yellow color was measured at 415 nm. Quercetin was used as standard and equivalents determined (w/w) from a pre-established concentration curve (Y = 0.0127x – 0.0306, R2 = 0.9993). the total flavonoids recorded were 11.54 ± 0.01, 29.52 ± 0.12 and 26.87 ± 0.04 for the extracts of the leaves of T. pallida (Rose) D.R. Hunt, T. zebrinaHeynh. ex Bosse and T. spathacea Sw., respectively, corresponding to µg/ QE/mg dried extract. These results indicate that the total flavonoid content, estimated as QE, of the leaves of T. zebrinaHeynh. ex Bosse leaves is higher than those present in the other two species.

II. High Performance Liquid Chromatography Analysis

HPLC fingerprinting was conducted to investigate the degree of similarities and/or differences among the phenolic and flavonoid profiles of the 3 species. The experimental results revealed that the concentrations of total identified phenolic compounds were 757.187, 1199.792 and 242.344 mg/100g, respectively, of the dried extracts of the leaves of T. pallida (Rose) D.R. Hunt, T. zebrinaHeynh. ex Bosse and T. spathacea Sw. Salicylic acid, catechin and pyrogallol were the major identified phenolic compounds with concentrations 315.7, 139.3 and 176.8 mg/100g, respectively, in the dried ethanol extracts of the investigated leaves.

While the experimental results revealed that the concentrations of total identified flavonoids were 2226.85, 4851.7 and 779 mg/100g, respectively, of the dried extracts of the leaves of T. pallida (Rose) D.R. Hunt, T. zebrinaHeynh. ex Bosse and T. spathacea Sw. Kamp3, (2-p-comaroyl) glucose and Naringin were the major identified flavonoid compounds with concentrations 2740.6 and 369.3 mg/100g, respectively, in the dried ethanol extracts of the investigated leaves.

Chapter III

Identification of different compounds in the alcoholic extract of Tradescantia species using HPLC-PDA-MS/MS

Lyophilized dried powder of the three Tradescantia species were subjected to HPLC-PDA-MS/MS in negative ionization mode.

• The study resulted in identification of twenty-seven secondary metabolites of the three species

• T. pallida (Rose) D.R.Hunt revealed 14 phenolic compounds

• T. zebrinaHeynh ex. Bosse revealed 9 phenolic compounds

• T. spathacea Sw. revealed 22 phenolic compounds

Part III: Phytochemical Investigation of the Leaves of Tradescantia pallida (rose) D.R.Hunt

Chapter I

A. Fractionation and Chromatographic Screening

Fresh samples of the aerial parts of T. pallida (Rose) D.R. Hunt were extracted with ethanol 80%. The ethanolic extract was successively fractionated with petroleum ether (60-80 oC), methylene chloride and n- butanol. The different extractives were physico-chemically examined. Chemical examination revealed the presence of sterols and /or triterpenes in the petroleum ether fraction. Flavonoids, phenolic acids and carbohydrates /or glycosides were detected in the n-butanol fraction.

B. Thin-Layer Chromatographic Screening of Extractives

TLC investigation of the lipoidal and flavonoids and/ or other phenolics in the different extractives revealed the presence of sterols and/or triterpenes in the petroleum ether fraction. Phenolic compounds were also detected in the n-butanol extractive.

Chapter II

Investigation of the Lipoidal Content

Gas Chromatography/ Mass Spectrometry (GC/MS) analysis of lipoids

• The unsaponifiable and saponifiable lipoids were prepared from one gram of the petroleum ether extracts of the leaves of T. pallida (Rose) D.R. Hunt.

• Constituents identified by GC/MS analysis of the fatty acids methyl esters in the saponifiable fractions of the petroleum ether extract represented SFA with 12.47% and USFA with 5.6% of the total composition. Constituents identified by GC/MS analysis of the unsaponfiable fraction extract represented hydrocarbons with 64.98% and sterols with 3.32% of the total composition. Unusual chloride compound “9,12-Octadecadienoyl chloride” was detected and predominated the sample (21.59%).

Chapter III

Isolation and Identification of the Main Constituents

A. Study of the Petroleum-ether Extractive

Isolation and identification of the constituents of the petroleum ether extractive were carried out on Tradescantia pallida (Rose) D.R.Hunt.

The petroleum ether-soluble fraction of the alcohol extract of Tradescantia pallida (Rose) D.R.Hunt was fractionated on a silica gel column. Gradient elution was carried out using petroleum ether, petroleum ether-methylene chloride, methylene chloride, methylene chloride-methanol. After purification by repeated fractionation on silica columns, three compounds were isolated and identified based on physico-chemical examination, spectral analysis, direct comparision with published data and/or reference samples.

Compound P: White needle crystals, soluble in CHCl3 was identified as β- sitosterol by determination of spectral data (1H-NMR and 13C-NMR) and comparing with published data.

B. Study of the n-Butanol Extractive

The n-butanol extractive of T. pallida (Rose) D.R.Hunt was fractionated on a VLC column. Elution was started with CH2Cl2 followed by increasing polarity through 5 % increments of CH3OH until 100% CH3OH was reached.

Three main fractions were obtained and fraction III demonstrated 8 spots then subjected to LC-ESI-MS. LC-ESI-MS profiling led to the identification of 7 phenolic compounds; 4 phenolic acids (trans-cinnamic acid, caffeic pentoside, p-coumaroyl pentoside and syringic hexoside) and 3 flavonoids (quercetin, naringenin and apigenin).

Metabolic assignments were tentatively identified by comparing the retention times and mass spectrum with reported data.

Part IV: Biological Study Chapter I Toxicological Study Determination of Median Lethal Dose (LD50)

Oral administration of the alcoholic extracts of the aerial parts of T. pallida (Rose) D.R. Hunt, T. zebrinaHeynh. ex Bosse and T. spathacea Sw. in doses up to 7 g/Kg b.wt. failed to kill mice within 24 hours. We conclude that the tested extracts were not toxic.

Chapter II In-Vitro Studies A. Cytotoxic Screening

The alcoholic extract of the leaves of the three Tradescantia species showed no cytotoxic activity against HEPG-2 (human liver cancer cell line), MCF-7 (human breast cancer cell line) and HCT-116 (human colon carcinoma cell line) when compared to doxorubicin.

B. Antioxidant Activity

The alcoholic extract of T. zebrinaHeynh. ex Bosse had significant scavenging effects on the DPPH radical, more than the alcoholic extract of T. pallida (Rose) D.R. Hunt and T. spathacea Sw. that possessed a moderate antioxidant activity when compared to ascorbic acid.

Chapter III

In-Vivo Studies

A. Hepatoprotective Activity

Alcoholic extracts of the aerial parts of the three Tradescantia plants were administered for 14 days, then liver damage in rats was induced by intraperitoneal injection of 5 ml/Kg b.wt. of 25 % CCl4. The alcohol extracts of T. spathacea Sw. showed significant decrease for AST, ALT, ALP, while T. pallida (Rose) D.R. Hunt and T. zebrinaHeynh. ex Bosse showed significant decrease from 10th d (after CCl4 injection) on liver enzymes. After histopathological study of the three species, the results support a potent hepatoprotective activity for T. spathacea Sw. and a moderate significant hepatoprotective activity for the other two species.

B. Anti-inflammatory Activity

The alcoholic extracts of the three plants showed a significant strong anti- inflammatory activity at a tested dose (100 mg/Kg b.wt.) when compared to the Indomethacin (20 mg/Kg b.wt. by sub plantar).

C.Antihyperglycemic Activity

The alcoholic extracts of the three plants showed significant antihyperglycemic activity after four weeks of diabetes induction by i.p. injection of alloxan at a tested dose (150 mg/Kg b.wt.) when compared to metformin (100 mg/Kg b. wt.). It was obvious that the alcoholic extracts of the three species of Tradescantia showed significant antihyperglycemic activity after 4 weeks. The most potent extract was the alcoholic extract of Tradescantiaspathacea Sw.

Conclusion

 One of the main goals achieved in this thesis was the comparative botanical and genetic characterization of T. pallida (Rose) D.R. Hunt, T. zebrinaHeynh. ex Bosse and T. spathaceaSw.

 The results obtained indicated a high degree of taxonomical and botanical similarity among the three plants. Although, distinct criteria for discrimination were observed.

 HPLC-PDA-MS/MS negative ionization mode identified 27 secondary metabolites for the three species under investigation.

 The phytochemical investigation of T. pallida (Rose) D.R. Hunt led to the isolation of 1 steroidal compound and identification of 7 phenolic compounds of Tradescantia pallida (Rose) D.R.Hunt .

 Assessment of the biological activities of the alcoholic extracts of the leaves of the three plants was also considered an important goal. This was

achieved by evaluation of toxicological study (LD50), as well as, investigation of different in-vivo studies (hepatoprotective, anti- inflammatory and antihyperglycemic activities) and in-vitro studies (cytotoxic and antioxidant activities).

 The results showed that the alcoholic extracts of the leaves of the three plants possessed potent anti-inflammatory activity. T. spathacea Sw. extract possessed potent hepatoprotective and antihyperglycemic activities. The other two plants showed moderate response to other biological

activities. T. zebrinaHeynh. ex Bosse showed significant antioxidant activity.