Research Paper Physicochemical

Research Paper

Physicochemical, Phytochemical and Nutritional values determination of Suaeda fruticosa (Chenopodiaceae)

Accepted 20th May, 2016

ABSTRACT

A medicinal plant is any plant which, in one or more of its organs, contains substances that can be used for therapeutic purposes or which are precursors for synthesis of useful drugs. Examples of important drugs obtained from plants are digoxin from dried leaves of Digitalis lanata (Family: Scrophulariaceae), quinine and quinidine from the bark of Cinchona species (Family: Rubiaceae). Physicochemical tests on powder and phytochemical tests for determination of secondary metabolites on Suaeda fruticosa dichloromethane extract (SFD) and methanolic extract (SFM) were done. High performance liquid chromatography (HPLC) was used for the quantitative determination of flavonoids using quercetin, myricetin and kaempferol as standards. Different elements were determined by atomic absorption spectrophotometer (AAS) to evaluate the nutritive value of S. fruticosa. Physicochemical tests showed that the values of moisture contents, total ash contents, water insoluble ash, acid insoluble ash, sulphated ash, water soluble extractives and alcohol soluble extractives in S. fruticosa were 2, 74, 34, 9, 20, 46 and 16% respectively. From phytochemical studies, it is estimated that tannins and flavonoids were present in both methanolic and dichloromethane extract of S. fruticosa while saponins and terpenoids were present in methanolic extract and alkaloids and coumarins present in dichloromethane extract. Quantitative determination of flavonoids showed that quercetin, myricetin and Kaempferol present in dichloromethane extract were 19.93, 0.15 and 0.15 mg/ml respectively and in methanolic extract, quercetin and kaempferol was present in quantity of 12.40 and 0.016 mg/ml Seema Abbas, Hammad Saleem, M. Shoaib Ali respectively. Metals like lead (Pb), sodium (Na), potassium (K), nickel (Ni), Gill, Anam Mubashar Bajwa, Ammar Sarwar and copper (Cu), zinc (Zn), magnesium (Mg) and chromium were present in both M. Ovais Omer extracts. Sodium and potassium were present in higher concentrations and their Institute of Pharmaceutical Sciences, University values in dichloromethane and methanolic extract were 50.6, 68.5, 65.5 and 79.1 of Veterinary And Animal Sciences, Lahore, ppm respectively. Pakistan.

*Corresponding author. E-mail: Key words: Suaeda fruticosa, S. fruticosa methanolic extract, S. fruticosa [email protected] dichloromethane extract, high performance liquid chromatography.

INTRODUCTION

Plants and animal products were the main sources of any plant which, in one or more of its organs, contains drugs for thousands of years and the use of natural substances that can be used for therapeutic purposes or products in the treatment and prevention of diseases is as which are precursors for synthesis of useful drugs”. old as human civilization (De Pasquale, 1984). According For centuries, traditionally medicinal plants were used to World Health Organization (WHO) “A medicinal plant is because of their remedial and defensive abilities; some Academia Journal of Medicinal Plants; Abbas et al. 002

widely used plants were selected for their investigation of shade drying the plant was crushed into powder form. The chemical constituents and biological activities such as anti- powder was then stored in amber color glass bottles inflammatory, anti-bacterial and anti-cancer in an attempt to establish a scientific basis for their ethno-medical uses (Ojewole, 2005). Extraction Examples of important drugs obtained from plants are digoxin from dried leaves of Digitalis lanata (Family: Powdered plant material was used for extraction. The Scrophulariaceae), quinine and quinidine from the bark of dichloromethane and methanolic extracts were prepared. Cinchona species (Family: Rubiaceae), Vincristine and First, the powder was soaked in dichloromethane for 3 vinblastine from the dried whole plant Catharanthus days with shaking periodically. After maceration it was roseus (Family: Apocynaceae), atropine from dried leaves filtered through muslin cloth and filter paper and then and flowering or fruiting tops of Atropa belladonna concentrated under reduced pressure using a rotary (Family: Solanaceae) and morphine and codeine from the evaporator at 37°C. By using same method the methanolic air dried milky exudates obtained by incising the unripe extract was also prepared. The prepared extracts were capsules of Papaver somniferum (Family: Papaveraceae) weighed and stored in labeled airtight vials in a (Sahu and Shaw, 2009). Chenopodiaceae family comprises refrigerator and named as SFD (S. fruiticosa of 110 genera and 1700 species. These are distributed in dichloromethane extract) and SFM (S. fruiticosa arid to semi-arid saline and agricultural habitats of methanolic extract). temperate and sub-tropical regions. Few genera including Chenopodium, Halosarcia, and Suaeda are distributed in the tropics. Most of the Physicochemical analysis of powdered material Chenopodiaceae species are annuals or sub-shrubs (Kadereit et al., 2003). The English common name for Physicochemical properties that include moisture Suaeda fruticosa is ‘Alkali seepweed’ and Urdu name is contents, extractive values, total ash, water and acid Laani. It is a member of the Chenopodiaceae, which insoluble ash and sulphated ash were analyzed using the belongs to the order: Caryophyllales and Sub-class: United States Pharmacopoeia-National Formulary (2003) Caryophyllidae. It is a halophytic annual herbaceous wild methods. plant, growing in saline soil. All parts of S. fruticosa and its essential oils were reported for ethno-medicinal applications (Benwahhoud et al., 2001; El Shaer, 2010). Moisture content Halophytes are known for their ability to withstand and quench toxic reactive oxygen species (Korabecny et al., China-dish was weighed and then tare 2 g of powdered 2010) since they are equipped with a powerful anti- plant material was weighed in tared china dish. The china- oxidant system that includes enzymatic and non- dish was then put in an oven at 105°C for 30 min for enzymatic components (Ksouri et al., 2009). A substantial drying of plant material. After 30 min it was removed from number of biological and chemical studies were performed the oven and put on a china dish containing dried plant on S. fruticosa (Ullah et al., 2012). material in a desiccator. Digital balance was used to weigh china-dish. Weight of the plant material which was used to measure moisture contents was calculated by subtracting MATERIALS AND METHODS the empty china-dish weight from weight of the china-dish containing dried plant material. Moisture contents were Chemicals calculated using the formula:

Dilute Hcl, Sulphuric acid, 95% Ethanol, Potassium Moisture contents = weight of pre-dried plant material - ferricyanide, Ferric chloride, 0.1 N Hcl, olive oil, dilute weight of dried plant material. Ammonia solution, Acetic anhydride, Wagner’s reagent, 10% NaOH, Chloroform, Glacial acetic acid, Methanol, % age of moisture contents was determined at the end by DMSO, Dichloromethane, Nitric acid,quercetin, myricetin multiplying the result of the moisture content with 100. and kaempferol. All the chemicals used were of analytical grade. Total ash

Plant material Weight of the empty china dish was noted and in a tared china-dish two gram powder plant material was weighed. The whole plant was collected from kot Addu district Then, this china dish was put in furnace at temperature Muzaffargarh. Plant was spread over newspaper on 675 ± 25°C until carbon free ash was obtained. China-dish shelves for the purpose of shade drying for 30 days. After containing desired form of ash was placed in desiccator to Academia Journal of Medicinal Plants; Abbas et al. 003

cool the contents. The ash contents were weighed. The continuous stirring. The contents were filtered. 25 ml of percentage of total ash was calculated with reference to the filtrate was evaporated to dryness in china dish and sample weight. the residues dried in an oven at 105°C. Then, this china dish was weighed. The alcohol soluble extractives percentage was calculated with reference to weight of Acid insoluble ash sample.

The total ash contents obtained from two grams of powdered plant material were boiled in 25 ml dilute HCl Water soluble extractives for 5 min. The boiled material was filtered using ash less filter paper. The soluble plant material was collected as In tared flask, five gram powdered plant material was filtrate. The insoluble material was present on filter paper. weighed and macerated using 100 ml of 95% ethanol. Then, this insoluble plant material was washed with hot Maceration of the sample was done for 24 h in a closed distilled water to ensure that all soluble material was flask with continuous stirring. The contents were filtered removed. The filter paper was dried and ignited in tared and 25 ml of the filtrate evaporated to dryness in china china-dish until carbon free ash was obtained. The china dish and the residue was dried at 105°C in oven and dish was put in the desiccator for cooling. The ash contents weighed. The percentage of water soluble extractives was were weighed and the percentage of acid insoluble ash was calculated with reference to weight of sample. calculated with reference to weight of total ash used in test. Phytochemical analysis of plant extracts

Water insoluble ash Chemical tests were done on dichloromethane and methanolic extracts of S. fruticosa using standard The total ash contents that were obtained from two grams procedures to determine the phytochemicals (Njoku and of powdered plant material were then boiled in 25 ml Obi, 2009; Odebiyi and Sofowora, 1977). distilled water for 5 min. The boiled material was filtered through ash less filter paper. The soluble matter was collected as filtrate and insoluble material remained as Test for tannins residue on filter paper and washed with hot distilled water to ensure that all soluble material had been removed. This In a test tube 1 ml of the sample was taken; 1 ml of 0.008 filter paper was then dried and ignited in tared china-dish M Potassium ferricyanide was added into it. Then, 1 ml of for the time period until ash got free from carbon. It was 0.02 M Ferric chloride which contains 0.1 N HCl was then cooled in desiccator. The ash contents were weighed added. Blue-black coloration was observed for the and the percentage of water insoluble ash calculated with presence of tannins. Absence of tannins shows no colour reference to weight of total ash used in test. change.

Sulphated ash Test for phlobatannins

Sulphuric acid was mixed with two grams of powdered Crude extract of the plant was boiled with 2% aqueous plant material in a tared china-dish. The weight of the HCl. Red precipitates deposition was taken as an evidence empty china dish was noted. Paste like material was for the presence of phlobatannins. formed by the addition of sulphuric acid in the plant material. This china-dish was ignited in furnace at high temperature till white fumes stopped originating. It was Test for saponins then cooled in the desiccator. The ash contents were subjected to weighing and the percentage of sulphated ash Crude extract was taken in a test tube and mixed with 5 ml calculated with reference to weight of dried powdered distilled water. Some drops of olive oil were added to it plant material. and shaken vigorously. Formation of stable foam was an indication for the presence of saponins.

Alcohol soluble extractives Test for flavonoids Five grams powdered plant material was put in tared flak. 100 ml of 95% ethanol was poured on it for maceration. 5 ml of dilute ammonia solution was added to a portion of The maceration was done in closed flask for 24 h with the crude extract followed by addition of concentrated Academia Journal of Medicinal Plants; Abbas et al. 004

Figure 1. HPLC chromatogram for standard flavonoids (Quercetin, Myricetin and Kaempferol).

H2SO4. Observation of yellow coloration was the indication At the inter face appearance of reddish brown coloration for the presence of flavonoids. Upon standing the yellow showed positive results. colour disappeared.

Test for cardiac glycosides (Keller-Kiliani test) Test for steroids 1 ml of concentrated sulphuric acid was added to 5 ml of To 0.5 ml crude extract 2 ml of acetic anhydride was added extract and then 2 ml of glacial acetic acid containing one with 2 ml H2S04. In the samples colour change from violet drop of ferric chloride solution added. A brown ring at the to blue or green was indication for the presence of interface showed the presence of cardenolides. steroids.

Quantitative determination of flavonoids Test for alkaloids Flavonoids were quantified in the both prepared extracts 2 ml Wagner’s reagent was mixed with crude extract. of S. fruticosa. This quantification was done using HPLC Presence of alkaloids was shown by the reddish brown and standards used were quercetin, myricetin and precipitates. kaempferol. Mobile phase used was prepared mixing acetonitrile, methanol, trifloro acetic acid and water. Sample and standard solutions were prepared and run in Test for quinones minute concentration that is, 100 ug. The chromatograms were generated for both standards and samples (Figures 1, Diluted sodium hydroxide was added to the 1 ml of the 2, 3). By using the areas and retention time of the crude extract. The presence of quinones was indicated by standards peaks, the concentrations of the quercetin, blue green or red coloration. myricetin and kaempferol present in the sample was calculated (Table 1) (Oueslati et al., 2012; Saleem et al., 2014). Test for Coumarin’s

In the extract 10% sodium hydroxide and chloroform were Elemental analysis added. Presence of Coumarin’s was shown by observing yellow color. Determination of different elements in the S. fruticosa methanolic and dichloromethane extracts was done using atomic absorption spectrophotometer in ppm. For analysis Test for terpenoids (Salkowski test) of heavy metals, the S. fruticosa plant extracts were first digested. Drying of the sample was done for 48 h at 65°C 2 ml of chloroform and 3 ml of concentrated sulphuric acid before digestion. Method used for digestion of samples were added to the 5 ml of extract carefully to form a layer. was given by Hseu (2004). According to the method, two Academia Journal of Medicinal Plants; Abbas et al. 005

Figure 2. HPLC chromatogram for SFD (Suaeda fruticosa dichloromethane extract).

Figure 3. HPLC chromatogram for SFM (Suaeda fruticosa methanolic extract).

gram (2 g) of the sample was taken in crucible. This determination of different metals concentration in ppm crucible was put in the muffle furnace preheated for 30 using atomic absorption spectrophotometer (Isaac and min at 200 t0 250°C initially and for 4 h at 480°C. Ash Kerber, 1971). formed was removed from the furnace and cooled to room temperature. 2 ml of the 5 M nitric acid was added to ash and dried using hot plate. In cooled furnace sample was RESULTS placed and then heated for 15 min at 400°C. Few drops of distilled water were added for the purpose of cooling. 2 ml Physicochemical analysis of Suaeda fruticosa of concentrated hydrochloric acid was added to the sample. Sample was heated to dryness until all acid S. fruticosa powder was used as sample for all evaporated from the sample. After dryness, 5 ml of 2 M physicochemical tests. In these tests, Table 2 shows the hydrochloric acid was added to the sample and mixed. The values of moisture contents in powder stored in air tight sample was then filtered through whatman filter paper glass bottle of amber colour, total ash content, ash content and syringe filter. The filtered sample was transferred to value which was not soluble in water and acid and ash 25 ml volumetric flask and distilled water used for volume value obtained when sample was moistened with make -up. The prepared digested samples of methanolic sulphuric acid, water and alcohol soluble extractive values and dichloromethane extracts were then analyzed for calculated in percentage (%). Academia Journal of Medicinal Plants; Abbas et al. 006

Table 1. Values for retention time and area of standards and samples.

Standard Sample SFD Sample SFM Flavonoids Retention time Area Retention time Area Retention time Area Quercetin 4.372 7466 4.296 7701881 4.296 4621044 Myricetin 6.234 11531 6.023 86349 - - Kaempferol 9.493 12931 12.828 96225 12.949 10411

Table 2. Results of physicochemical analysis (%age) of Suaeda fruticosa powder.

Sample Powder of whole plant (%) Moisture contents 2 Total ash contents 74 Water insoluble ash 34 Acid insoluble ash 9 Sulphated ash 20 Water soluble extractives 46 Alcohol soluble extractives 16

Table 3. Results of phytochemical screening of dichloromethane and methanol extracts of Suaeda fruticosa.

Suaeda fruticosa plant extracts Tests performed SFD SFM Tannins + + Phlobatannins – – Saponins – + Flavonoids + + Steroids – – Alkaloids + – Quinones – – Coumarin + – Terpenoids – + Cardiac glycosides – –

+ = presence and - = absence.

Phytochemical evaluation of dichloromethane and flavonoids present in extracts were obtained and using methanolic extract these curves area the concentration of these flavonoids in plant extracts was calculated (Table 4). Different chemical tests were performed on the extracts of S. fruticosa. The extracts were named as SFD (S. fruticosa dichloromethane extract) and SFM (methanolic extract of Metals determination in Suaeda fruticosa extracts S. fruticosa) on which chemical tests were performed for the analysis of tannins, phlobatannins, saponins, Determination of different metallic compounds for flavonoids, steroids, alkaloids, quinones, coumarins, example Ni, Cu, Zn, K and Na etc in the extracts of S. terpenoids and cardiac glycosides. Table 3 presents the fruticosa which were prepared in methanol and results for the presence or absence of these dichloromethane was done with the help of atomic phytochemicals. absorption spectrophotometer (AAS). The samples used in spectrophotometer for detection of elements were digested and in diluted forms. Table 5 shows the presence Estimation of flavonoids in dichloromethane and of different metal concentrations in the extracts in ppm. methanolic extracts

Concentration of different flavonoids was estimated in DISCUSSION both prepared extracts of S. fruticosa namely; SFD and SFM. Different flavonoids concentration in plant extracts The present research work emphasizes on the was determined using HPLC. Standard flavonoids that is, physicochemical, phytochemical and biological evaluation Quercetin, Myricetin and Kaempferol and samples of both of medicinal plant namely; S. fruticosa belonging to the extracts were run. Curves for standard flavonoids and family Chenopodiaceae. The medicinal plant was taken Academia Journal of Medicinal Plants; Abbas et al. 007

Table 4. Flavonoids concentration (mg/ml) in Suaeda fruticosa extracts.

SFD SFM Flavonoids (mg/ml) Mean SD Mean SD Quercetin 19.93 ±1.59 12.40 ±1.16 Myricetin 0.15 ±0.01 - - Kaempferol 0.15 ±0.01 0.016 ±0.001

Table 5. Results of metals determination in Suaeda fruticosa methanol and dichloromethane extracts by Atomic absorption spectrophotometer.

Concentration (ppm) of elements in plant extracts Elements SFM SFD Lead 0.10 0.14 Sodium 65.5 50.6 Potassium 79.1 68.5 Nickel 0.19 0.29 Copper 0.15 0.27 Zinc 3.07 2.30 Magnesium 4.52 2.83 Chromium 0.20 0.26

Figure 4. Elemental analysis in SFD and SFM.

from kot Addu, District Muzaffargarh, Punjab, Pakistan and Physicochemical and phytochemical analysis objective research was done at the University of Veterinary and was the standardization of the natural traditionally used Animal Sciences, Lahore. Methanolic and dichloromethane medicinal plant. In physicochemical testing, different extracts of S. fruticosa was prepared. Both extracts were values were obtained for powdered material of S. fruticosa subjected for physicochemical and phytochemical following the standard testing procedures given by Janaki screening. Quantitative determination of flavonoids in and Sashidhar (1998) and Saleem et al. (2014). Moisture both extracts was done. Elemental analysis was also content was calculated to be 2%. Hot air oven was used to performed for both of these prepared extracts (Figure 4). dry the sample and weight of the pre-dried and after dried Academia Journal of Medicinal Plants; Abbas et al. 008

sample was taken and after calculation moisture content stable soap formation on shaking and are glycosides of value was obtained. Estimation of moisture content was both triterpenes and sterols. Years ago, saponins were done because it is an important factor for the preservation used as natural detergents and fish poison. Plants saponins of the medicinal plant. If the moister content value is less were also reported for biological activities such as then, the powdered herbal medicine has less chance of spermicidal, molluscicidal, antimicrobial, anti- chemical decomposition and microbial contamination. inflammatory and cytotoxic (Mahato et al., 1988; Marston Thus, it can be stored and used for a longer period of time and Hostettmann, 1985; Setty et al., 1976). effectively. For the determination of the presence of flavonoids in Different ash values were determined to check the the S. fruticosa extracts chemical test was performed and quality and purity of the powdered medicinal crude drug. appearance of yellow colour which disappeared on Acid insoluble and water insoluble ash value was obtained standing was the evidence for presence of flavonoids in by boiling small quantity of ash powder in an acid and both extracts. Flavonoids exhibit anti-oxidant property. water separately. After filtration, the filter paper on which Anti-oxidant property of flavonoids against different water insoluble and acid insoluble ash was present was oxidants was described by Larson (1988). Steroids present ignited in furnace and by this way ash values were in extracts were analyzed by colour change of extract after calculated. Carbonates, silicates and phosphates of sodium, chemical treatment from violet to blue or green. Wagner’s calcium, potassium and magnesium were present in ash reagent was used for alkaloids determination and its which was obtained after the ignition of crude herbal drug. presence was judged by a reddish brown precipitate. If the silica or calcium oxalate is present in the herbal Experiments showed that nitrogen containing compounds crude drug then acid insoluble ash test was performed. like alkaloids have potent inhibitory effect on different The values calculated for total ash, acid insoluble and oxidative procedures (Larson, 1988). water insoluble ash for powdered material of S. fruiticosa Phytochemical test for the presence of quinones is were 74, 9 and 34% respectively. These values showed indicated by a blue green or red coloration. Quinones are that silica, calcium oxalate, carbonates, silicates and toxicological intermediates which can create a variety of phosphates of sodium, calcium, potassium and magnesium hazardous effects. Coumarins identification test was were present in ash of S. fruiticosa. Sulphated ash value performed on both extracts and its presence was observed was 20%. The S. fruticosa powdered material has by a yellow colour. Coumarins known as benzopyrones extractives of 46% water soluble and 16% alcohol soluble. and mostly present in different plants have enzyme Phytochemical tests were carried out for both the inhibition properties. Biological activities reported for methanolic and dichloromethane extracts of S. fruticosa by coumarins include anti-coagulant, anti-inflammatory, anti- adopting standard procedures as described by Obadoni tumoural and anti-HIV (Borges et al.,, 2005). The presence and Ochuko (2002); Odebiyi and Sofowora (1977); Parekh of terpenoids was evaluated by a reddish brown ring and Chanda (2007) and Saleem et al. (2014). formation and was present in methanolic extract of S. Phytochemical tests determined the presence or absence fruticosa. Terpenoids present in plants was extensively of secondary metabolites in which alkaloids, steroids, used because of their aromatic qualities and have a role in flavonoids, saponins, cardiac glycosides, tannins, traditional herbal remedies. Cardiac glycosides were phlobatannins, quinones and terpenoids were present. absent in the both S. fruticosa extracts when chemical test Table 4 presents the results for the presence of secondary were performed. metabolites in the extracts of S. fruticosa. When The elements detected in the extracts were Lead (Pb), phytochemical tests were performed for tannins, blue Sodium (Na), Potassium (K), Nickel (Ni), Copper (Cu), Zinc colour appeared which indicated the presence of tannins (Zn), Magnesium (Mg) and Chromium (Cr). Table 5, shows in S. fruticosa methanol and dichloromethane extracts. the results of different concentration (in ppm) of elements Tannins are polyphenols different from other natural in the extracts. Various biologically important elements phenols and soluble in water. Tannins have the ability to involved in metabolism were present in varying precipitate proteins such as gelatin and this property is concentrations in both extracts. called astringency. Tannins are from the important Lead is known to cause neurological disorders, anemia, components involved in developing defense strategies by kidney damage, miscarriage, lower sperm count and plants. Proanthocyanidins (condensed tannins) are hepatotoxicity in higher concentration (Mudipalli, 2007). reported for their anti-bacterial, anthelmintic and anti- Sodium and potassium are important elements reported herpetic activities and provides defense (Dixon et al., for maintaining the acid–base equilibrium and osmotic 2005). Phlobatannins were absent in both extracts because pressure of body fluids. Increase in blood pressure can be when chemical test was performed no red colour prevented if potassium is taken simultaneously with precipitate deposition was observed. Chemical test sodium (Kolata, 1982). performed for saponins on extracts of S. fruticosa revealed Magnesium is an important electrolyte, responsible for that only methanolic extract show positive results which proper functioning of nerve and muscles. Magnesium were indicated by stable soap formation. works as a co-factor in more than 300 metabolic reactions Plant saponins are soluble in water, have the property of (Wildman and Medeiros, 1999). In the soft tissues, cells Academia Journal of Medicinal Plants; Abbas et al. 009

magnesium is active as a part of the protein-making Kadereit G, Borsch T, Weising K, Freitag H (2003). Phylogeny of machinery and necessary for the release of energy (Devi et Amaranthaceae and Chenopodiaceae and the evolution of C4 photosynthesis. Int. J. Plant Sci. 164(6): 959-986. al., 2008). Kolata G (1982). Value of low-sodium diets questioned. Science (USA). Another important element is zinc. Many enzymatic Korabecny J, Musilek K, Holas O, Binder J, Zemek F, Marek J, Kuca K processes require zinc and it is involved in working of (2010). Synthesis and in vitro evaluation of N-alkyl-7-methoxytacrine genetic materials, immune reactions, proteins, wound hydrochlorides as potential cholinesterase inhibitors in Alzheimer disease. Bioorg. Med Chem. Lett. 20(20): 6093-6095. healing, sperm production and development of the foetus. Ksouri R, Falleh H, Megdiche W, Trabelsi N, Mhamdi B, Chaieb K, Abdelly It is observed that zinc used in normal levels can prevent C (2009). 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