Phytochemical and Biological Studies of Different Crassula Species Family Crassulaceae

Phytochemical and Biological Studies of Different Crassula species Family Crassulaceae

A thesis submitted by Omneya Eid Mohamed M.Eid

B.Sc.Pharm.

2013

Submitted for partial fulfillment of requirements for M.Sc. Degree in Pharmaceutical Sciences

(Pharmacognosy)

Under supervision of

Prof. Dr. Shahira Prof. Dr. Mariam Mohammed Ezzat Hussein Gonaid

Professor of Pharmacognosy Professor of Pharmacognosy Faculty of Pharmacy Faculty of Pharmacy Cairo University Future University

Dr. Mouchira Aly

Choucry Lecturer of Pharmacognosy Faculty of Pharmacy

Cairo University

Department of Pharmacognosy

Faculty of Pharmacy Cairo University Egypt 2018

Abstract Phytochemical and Biological studies of Different Crassula species family Crassulaceae Key Words: Crassulaceae, Crassula tetragona L, Crassula ovata (Mill), DNA, GC, terpenoidal compounds, Phenolics, Flavonoids, LC/MS/MS, antioxidant, antidiabetic, antimicrobial. The Crassulaceae (stonecrop family) is a very diverse group, and plants range from tiny insignificant annual herbs to perennial shrubs and trees. Many of the species have attractive flowers. The entire family consists of 33 genera and 23 hybrid genera with a total of 1410 species and 305 intraspecific taxa. The genus Crassula includes around 200 species of succulent plants, ranging in size from less than one inch in height to 6 foot shrubs. Throughout the last period, researchers gave an attention for the compounds isolated from cactus plants (Ornamental Plants). But, there was no much work concerning the chemical composition or biological activity of genus Crassula or species tetragona nor ovata. Aim of the work: a) - to investigate DNA fingerprint of Crassula tetragona and Crassula ovata, b) -Preliminary phytochemical screening to identify the nature of the active constituents. C)- Investigation of the lipid content of the two plants. D)- Quantitative estimation of total flavonoid, phenolics and triterpenes contents. E) - Isolation, identification and structure elucidation of the major isolated constituents from the methylene chloride fractions. F)-LC/MS/MS of n-Butanol fractions of both plants. G) - Determination of the plant safety (the acute toxicity study). H) - Determination of the anti- hyperglycemic activity of the total ethanolic extracts of the aerial parts of both plants. I) - Determination of the anti-

2 hyperlipidemic activity of the total ethanolic extracts of the aerial parts of both plants. I)-A study of the antioxidant activity of the total ethanolic extracts, methylene chloride and n-butanol fractions of both plants. J)-Determination of anti-microbial activity of the ethanolic extracts, methylene chloride and n- butanol fractions of both plants. Results: A) -Isolation and identification of 6 compounds from both plants, B) -screening of lipid content of both plants and identification of 7 steroidal components, 21 fatty acid methyl ester and 32 hydrocarbon compounds. C) -triterpenoidal content of both plants was greater than phenolics and flavonoid content. D) – Identification of 21 phenolic compound using LC/MS/MS, E) – both plants had significant antidiabetic, antimicrobial and antioxidant effect Conclusion and Recommendation: A) -Further study on the total extracts and individual components for understanding the mechanism of these biological activities. B) - Clinical trials should be performed in order to support the above investigation and to facilitate their pharmaceutical formulation. Introduction

For many years, natural preparations had an important role in the cure of a widespread sort of ailments and have increase of the quality of human beings lives. Medicinal plants represent an important part of our natural wealth. They serve as important therapeutic agents, as well as, valuable raw material for manufacturing numerous traditional and modern medicines. The history of the use of medicinal plants for treatment of diseases and ailments probably dates back to the beginning of human civilization. Our forefathers were compelled to use any natural substance that they could find to ease their sufferings caused by acute and chronic illness, physical discomforts, injuries, and even terminal illnesses. Since ancient times, plants with therapeutic properties have secured an important place in the healing practices and treatment of diseases (Akanda et al. 2014).

The Crassulaceae is a large family including 1250–1500 species. It exhibits a degree of leaf succulence allocated into six subfamilies and about 35 genera (Thiede and Eggli 2007). The Crassulaceae plants are succulent and of cosmopolite distribution. This family is divided in 35 genera and the most important are Sedum, Crassula, Echeveria and Kalanchoe. These are commonly used as ornamental plants (López-Angulo et al. 2016). Crassula is one of the largest genus of family Crassulaceae that comprises about 150 species (El-Hawary et al. 2016).

Crassula tetragona L is a succulent plant native to Southern Africa. It is also a common ornamental plant, susceptible to mealy bugs and fungal diseases as with all succulents (worldofsucculents.com).

Crassula ovata (Mill). (also called jade plant ) is a native plant to South Africa. It is also a common houseplant all over the world, but it is mostly located in the Northern Hemisphere particularly in cold and/or dry areas where water is rare. Crassula ovata is a vibrant part together with a variety of euphorbias, aloes, Portulacaria afra and other succulent plants (Muiruri and Mwangi 2016). In Africa, jade leaves are boiled in milk and consumed to stop diarrhea. The Khoi and other African tribes used to eat the roots and stems of the jade plant. The plant was grated and cooked after which they were eaten with thick milk. The leaves were also boiled in milk as a remedy for diarrhea, treating epilepsy, corns and as a purgative (Muiruri and Mwangi 2016). There was no much work concerning the genus Crassula or species tetragona nor ovata so we will shed light on both plants constituents and their biological activities, choosing the antidiabetic as a main biological activity for both plants due to its great importance nowadays and according to the review of the literature, family Crassulaceae have an interesting antidiabetic activity

Aim of work

1. Collection and authentication of the plant material. 2. DNA fingerprint of the two species. 3. Determination of the lipid content in both plants. 4. Quantitative estimation of total Flavonoids, phenolics and triterpene content. 5. Successive extraction of the plant material with different solvents. 6. Methylene chloride fractions will be subjected to phytochemical investigation followed by isolation of their main constituents by using different chromatographic techniques. 7. Structure elucidation of the isolated compounds. 8. LC/MS/MS of the n-butanol fractions. 9. Investigation of the biological activity of different fractions (anti-microbial, anti-diabetic & anti-oxidant)

General Summary

The Crassulaceae plants are succulent and of cosmopolite distribution. This family is divided in 35 genera and the most important are Sedum, Crassula, Echeveria and Kalanchoe. These are commonly used as ornamental plants. Crassula is one of the largest genus of family Crassulaceae, comprises about 150 species. Crassula tetragona L is a succulent plant native to Southern Africa. It is also a common ornamental plant, susceptible to mealy bugs and fungal diseases as with all succulents. Crassula ovata (Mill). (Also called jade plant) is a native plant to South Africa. It is also a common houseplant all over the world, but it is mostly located in the Northern Hemisphere particularly in cold and/or dry areas where water is rare. Crassula ovata is a vibrant part together with a variety

5 of euphorbias, aloes, Portulacaria afra and other succulent plants. Chapter I DNA Fingerprint of Crassula species

 RAPD-PCR fingerprinting: In order to evaluate the efficiency of RAPD-PCR fingerprinting for two species, the DNA was used as a template for the 6 RAPD primers banding pattern. Total of 94 bands template for all primers, 41 of them were polymorphic (43.617%). The highest band number was produced with primer OP-A02 (22 bands) while the lowest was produced with primer OP- B02 (10 bands) Primer OP-D01 showed the highest polymorphic percent (68.421%) while primer OP-B07 showed the lowest polymorphic percent (7.143%).  ISSR-PCR fingerprinting: The results of ISSR fingerprinting of 6 ISSR primers banding pattern showed 60 bands for all primers, 28 of them were polymorphic (46.666%) The highest band number was produced with primer HB-15 (15 bands) while the lowest was produced with primer 44B (7 bands). Primer HB-14 showed the highest polymorphic percent (81.818%) while primer HB- 15 showed the lowest polymorphic percent (13.333%).

Chapter II Preliminary Phytochemical screening Phytochemical screening of the Crassula species under investigation revealed the presence of carbohydrates and/or glycosides, tannins, flavonoids and unsaturated sterols and/or terpens. Trace amounts of saponins also were found while alkaloids and/or nitrogenous bases, anthraquinone and cardiac glycosides were absent. Chapter III Investigation of lipoidal matter of both Crassula tetragona and Crassula ovata

 Investigation of the unsaponifiable matters of n-hexane extract of C. tetragona revealed the presence of 13 hydrocarbons with the presence of n-tricontane (57.65%) as the major hydrocarbon. In addition to: Four steroidal components; with the presence of campesterol (19.77%) as the major steroidal compound. On the other hand investigation of the unsaponifiable matters of n-hexane extract of C. ovata revealed the presence of 19 hydrocarbons with n-tricontane (57.44%) as the major hydrocarbon. In addition to: Three steroidal components; with the presence of β-sitosterol (13.98%) as the major steroidal compound.  Investigation of FAME revealed the presence of 12 components in C. tetragona and 9 components in C. ovata respectively. 

Chapter IV

Determination of Total Phenols, Flavonoids and total triterpene Activity

I. Colorimetric determination of total phenolic content:

Spectrophotometric determination of the total phenolic content was carried out using the Folin-Ciocalteu colorimetric method.

Total phenolic of C. tetragona = 3.667 μg/100μg ± 0.01 Total phenolic of C. ovata = 1.732 μg/100μg ± 0.01

II. Colorimetric determination of flavonoidal content:

The method adopted was based on measuring the intensity of the color developed after reaction of flavonoids with aluminium chloride reagent.

Total flavonoid of C. tetragona = 9.814 μg/100μg ± 0.01 Total flavonoid of C. ovata = 11.873 μg/100μg ± 0.01

III. Colorimetric determination of triterpene content Total triterpene of C. tetragona = 63.18889 μg/100μg ± 0.01 Total triterpene phenolic of C. ovata = 87.06667μg/100μg ± 0.01

Chapter V

Isolation, identification and structure elucidation of the major isolated constituents from the active fractions.

This is results in the isolation of terpenoidal compounds which are: The identification and structural elucidation of the compounds was through HNMR, CNMR, ESI-Mass, DEPT, HSQC, HMBC, COSY 5 compounds isolated for the first time from Crassula tetragona

1. 28 Methyl-5α-cycloart-12, 20, 24-trien-3 β, 15β-di-ol This compound is a novel compound isolated for the first time from Crassula tetragona (identified through HNMR, CNMR, ESI-Mass, DEPT, HSQC, and HMBC) 2. Campesterol This compound identified through Mass and HNMR with comparison with authentic and isolated for the first time from the plant 3. Cyclolaudenol This compound identified through Mass, HNMR, CNMR with comparison and isolated for the first time from the plant

4. Lupeol This compound identified through Mass and HNMR, with comparison and isolated for the first time from the plant

5. β-sitosterol This compound identified through Mass and HNMR, with comparison and isolated for the first time from the plant

3 compounds isolated for the first time from Crassula ovata 1. α-amyrin This compound identified through Mass, HNMR, CNMR with comparison and isolated for the first time from the plant 2. Lupeol 3. β-sitosterol Chapter VI

LC/MS/MS of butanol fractions of both plants In this study a comprehensive qualitative analysis of the phenolic constituents present in the n-Butanol fraction of Crassula species under investigation were achieved by UPLC/MS/MS. A total 45compounds, 24 known and 21 unknown tentatively identified based on mass measurements and product ions compared with revealed data before, and this study was investigated for the first time for both plants.

Chapter VII

Biological Study

1. Determination of the safety of the plant.

The acute toxicity studies were performed on mice with uniform weights. Animals were subjected to fasting condition overnight with access to drinking water. They were given different doses of the alcoholic extracts and observed for 24h for mortality. All the animals were observed closely for 24h

10 and daily for 14 days and no mortality was observed when taken a doses up to 2000 mg/Kg. Hence, 200 mg/kg was selected as a maximum safety dose.

2. Determination of the antidiabetic activity of the ethanolic extracts of the aerial parts of both plants. -hyperglycemic effect. The alcoholic extract of C. tetragona (200 mg/kg) reduced the blood glucose level of STZ induced diabetic rats after 2 weeks by 57.8% followed by C. tetragona (100 mg/kg) 56.3% compared to Glyclazide (10 mg/kg) 58.1%. From these results it can be concluded that C. tetragona has significant anti- hyperglycemic activity than C. ovata.

The alcoholic extract of C. tetragona (200 mg/kg) increase the insulin level of STZ induced diabetic rats after 2 weeks more than the other extracts and more than Glyclazide followed by C.ovata (200 mg/kg). From these results it can be concluded that C .tetragona has significant increasing in the insulin level than C. ovata.

All the tested extracts lower triglyceride and cholesterol levels. The ethanolic extract of C .tetragona and C. ovata (200 mg/kg) reduced triglycerides level by 21.6% compared to Glyclazide (10mg/kg) 18.5%, and the alcoholic extract of C. ovata (200mg/kg) reduced Cholesterol level by 28.3% followed by C. tetragona (200 mg/kg) 24.9% compared to Glyclazide 31.1%

concluded that C. ovata and C. tetragona nearly have the same anti-hyperlipidimic activity.

3. Determination of anti-microbial activity of the alcoholic extracts, methylene chloride and butanol fractions of the both Plants.

All the tested extracts have a significant non-selective anti- microbial activity against gram positive, gram negative and fungi. The potency of each tested sample was calculated relative to the inhibitory power of reference drugs (considered as 100%). Among the most noticeable results, the effect of total alcoholic extract of C. tetragona and C. ovata upon the pathogenic fungi Aspergillus fumigatus (97.8 & 89.8 % of Amphotericin B inhibitory power, respectively). The effect of total alcoholic extract of C. tetragona and C. ovata upon the pathogenic bacteria Streptococcus pneumoniae (94.9 & 89.4% of Ampicillin inhibitory power, respectively). The effect of total alcoholic extract of C. tetragona and C. ovata upon the pathogenic bacteria Escherichia coli (95.7 & 82.4% of Ciprofloxacin inhibitory power, respectively). The alcoholic extract of both plants gives the highest broad spectrum anti-microbial activity. 4. A study of the antioxidant activity of the alcoholic extracts, methylene chloride and n-butanol fractions of the both Plants.

The 50% scavenging activity of DPPH (IC50) was attained with 50 μg/ml for the methylene chloride fraction of C. tetragona followed by the alcoholic extract and n-butanol fractions of C. ovata (100μg/ml). Comparison revealed that the methylene chloride fraction of C. tetragona exhibited higher antioxidant activity (lower IC50).

Conclusion From the previous summary it can be concluded that Craaaula tetragona L. and Crassula ovata (Mill). Which are cactus plants gown in South Africa had a similarity in DNA finger print by using ISSR and RAPD technique approximately about 50%

The Phytochemical screening of the Crassula species under investigation revealed the presence of carbohydrates and/or glycosides, tannins, flavonoids and unsaturated sterols and/or terpens. Trace amounts of saponins also were found while alkaloids and/or nitrogenous bases, anthraquinone and cardiac glycosides were absent. Investigation of the unsaponifiable matters of n-hexane extract of C. tetragona revealed the presence of 13 hydrocarbons.In addition to: Four steroidal components.

On the other hand investigation of the unsaponifiable matters of n-hexane extract of C. ovata revealed the presence of 19 hydrocarbons. In addition to: Three steroidal components

Investigation of FAME revealed the presence of 12 components in C. tetragona and 9 components in C. ovata respectively.

The quantitative estimation of total phenolics, flavonoids and triterpenes showed the high mount of triterpene than flavonoids and triterpenes

The isolation of terpenoidal compounds which are:

5 compounds isolated for the first time from Crassula tetragona 1. 28 Methyl-5α-cycloart-12, 20, 24-trien-3 β, 15β-di-ol 2. Campesterol 3. Cyclolaudenol 4. Lupeol 5. β-sitosterol 3 compounds isolated for the first time from Crassula ovata 1. α-amyrin 2. Lupeol 3. β-sitosterol

All the tested extracts have anti-hyperglycemic effect. The alcoholic extract of C. tetragona (200 mg/kg) reduced the blood glucose level of STZ induced diabetic rats after 2 weeks by 57.8% followed by C. tetragona (100 mg/kg) 56.3% compared to Glyclazide (10 mg/kg) 58.1%. From these results it can be concluded that C. tetragona has significant anti- hyperglycemic activity than C. ovata.

14 pathogenic fungi Aspergillus fumigatus (97.8 & 89.8 % of Amphotericin B inhibitory power, respectively). The effect of total alcoholic extract of C. tetragona and C. ovata upon the pathogenic bacteria Streptococcus pneumoniae (94.9 & 89.4% of Ampicillin inhibitory power, respectively). The effect of total alcoholic extract of C. tetragona and C. ovata upon the pathogenic bacteria Escherichia coli (95.7 & 82.4% of Ciprofloxacin inhibitory power, respectively). The alcoholic extract of both plants gives the highest broad spectrum anti-microbial activity.