Pharmacognostical Standardisation of Ailanthus Excelsa Leaves and Randia Dumetorum Fruit Along with Antioxidant Activity and Free Radical Scavenging Capacity of Its Fractions

The world is observing an unprecedented development in the usage of herbal product at national as well as international levels. This requires the improvement of current and aimed standards for estimating the quality, safety and ef icacy of these drugs. The leaves of Ailanthus excelsa and the fruits of Randia Dumetorum are medicinal plants that are used for many diseases around the world. We then collected the lavonoids and saponin fraction extracted from the leaves of Ailanthus excelsa and the fruits of Randia dumetorum. To determine the reliability, quality and purity of these particles, we provide a crucial pharmacological pro ile along with the antioxidant activity. Pharmacological studies, such as morphological, physicochemical, TLC, and phytochemical analysis of all fractions containing total phenol and lavonoids, were performed according to speci ic methods. DPPH tests estimated the antioxidant action of all fractions, Hydrogen peroxide scavenging assay, and reducing power assay method. Previous phytochemical studies discovered the occurrence of saponins, lavonoids, tannins, and especially phenolic chemicals. All fractions have antioxidant effects, depending on the existence of a phenolic compound. The above parameters are vital to establishing pharmacological rules for the authentication of Ailanthus excelsa leaves and Randia Dumetorum fruits.


INTRODUCTION
Presently there is an enormous deal of awareness in herbal medicine today. This interest is mainly because medicinal plants do not have adverse effects. A quarter of the world's population or 1.42 trillion people are not expected to rely on traditional medicines to treat various diseases (Kadam et al., 2012). Traditional medical systems such as Ayurveda play an essential role in today's health ield, especially in the treatment of malignant diseases (Shivatare et al., 2014). However, the most signi icant barriers to the use of alternative drugs in industrialised countries are the lack of documentation and strict quality control. Research on traditional medicines needs to be documented. In this context, it is vital to try to standardise the plant substance that will be utilised as a remedy (Modi et al., 2010;Shruthi et al., 2010). Normalisation can be done through pharmacological and phyto-chemical studies. These studies help to de ine and standardise plant substance. Appropriate detection with a quality guarantee of preliminary materials is essential requirements for the quality of phytotherapy reproduction, which contributes to improving its protection and usefulness (Sharma et al., 2015;Kadam et al., 2011).
Ailanthus excelsa (family-Simaroubaceae) is called "Mahanimba" as it resembles a neem plant (Azadirachta indica). The statement Ailanthus is resulting as of Ailanthus, and it means procession plant (Lavhale and Mishra, 2007). The name of one of the species of Moluccas, in Latin, means high excelsa. Recent research has shown that it contains six lavonoids secluded from the leaves of A. excelsa (Loizzo et al., 2007). The bark of the leaves and the alcohol extracted from the stem show excellent anti-implantation activity and early abortion (Dhanasekaran et al., 1993).
A review of the literature shows that the results are bitter and sweet; warming, aphrodisiac, emetic, vaginal, carminative, antipyretic; It treats abscesses, ulcers, in lammation, wounds, tumours, skin diseases and has an antibacterial effect (Satpute et al., 2012). Many experts believe that fruit ibres have anthelmintic properties and are also used in abortion as a folk medicine (Satpute et al., 2009). Recently, the antioxidant properties of their components, usually polyphenol compounds, have been attributed to the function of medicinal plants in the manage or prevention of the disease. The intake of natural antioxidants while minimising the risk of cancer, diabetes, cardiovascular problems, and other age-related diseases (Subba and Mandal, 2015).
No information has been provided on pharmacological studies on the fruiting bodies of the leaves of Ailanthus excelsa and Randia Dumetorum. No data are provided on antioxidant activity. Therefore, this research focused on the standardisation of two plant fractions, measuring several aspects: morphology, physicochemistry, and TLC analysis. The content of qualitative and quantitative phytochemicals was also combined with antioxidant activities to investigate the presence of phytoactive ingredients.

Reagent and Chemicals
The entire reagents and chemicals required for the evaluation of pharmacognostic, phytochemical screening, TLC and antioxidant activity were analytical grade obtained from J.T. Baker, Merck and Rankem.

Procurement, authentication and extraction of sample
The leaves of plant Ailanthus excelsa and Fruit of Randia Dumetorum were collected from ields of Bhopal (Madhya Pradesh) and validated by Sa ia College of Science, Bhopal. (Madhya Pradesh) were speci ied the specimen number 157/Bot/Sa ia/2010 (Ailanthus excelsa) and 158/Bot/Sa ia/2010 (Randia Dumetorum).
The leaves of plant Ailanthus excelsa and Fruit of Randia Dumetorum were clean and dehydrated below the shade. The desiccated samples were then ground into powdered form. All parts of the plant were crushed and extracted with water, using the method of decoration. This aqueous extract was then iltered, and alcohol (ethanol) was slowly added to this extracted liquid water to make polysaccharides. The solution was then iltered, and the iltered evaporated 1/4 of the total volume.
The same amount of ethyl acetate was then added using a separatory funnel to obtain a separate fraction of constituents of the roots in ethyl acetate. The ethyl acetate extract was acidi ied with 0.1 N HCl to increase the amount of the extract. The ethyl acetate fraction was evaporated to give the residue; it was then dissolved in methanol and evaporated to give a crystalline powder. Finally, the obtained powder was also analysed to determine the presence of phytoactive ingredients. In this study, it was seen that Shinoda responded positively to the lavonoid test. Besides, TLC con irmed the positivity of lavonoids in the appropriate solvent system (EAFW).
Same plant materials were used for getting saponins rich fraction. Pulverised plant materials were treated with ethanol: water (70:30) for maceration till seven days after defatting with petroleum ether (40:60). The blend was agitated at a normal interval in this stage. Obtained extract after iltration through muslin cloth followed by ilter paper was concentrated using rotary vacuum evaporator (40°C), taking precaution that extract does not get powdered.
The concentrated extract was further treated with n-butanol to get an n-butanol soluble fraction. Nbutanol soluble fraction was further treated with chilled diethyl ether. After treating with chilled diethyl ether, the precipitate was formed. This mixture with precipitate was kept at -20°C for 24 hrs. Precipitates were further separated by centrifugation. This precipitate was further dissolved in methanol and methanol was evaporated slowly, to get crystalline powder.

Macroscopical characters
External features, dimensions and organoleptic properties of leaves and fruits were studied.

Physicochemical Evaluation
The physicochemical properties of the component were evaluated to estimate the superiority and purity of the powder form. On physical evaluation, they were ash value viz., total ash value with acid insoluble ash value and water-soluble ash value were evaluated. The value of the residue indicates that the drug contains inorganic salts. Values for the extraction of water and soluble alcohol were established. The information obtained from these tests is useful for standardising and maintaining quality standards. These chemical-physical constants are determined according to the procedures mentioned under WHO guidelines (Ibrahim et al., 2012;Baravalia et al., 2011).

Phytochemical Investigations
The leaves of the fruits of Ailanthus excelsa and Randia Dumetorum were subjected to phytochemical studies before extraction. A preliminary phytochemical test was performed to con irm that the occurrence of different pharmacologically active phytochemicals in the crude powered drug. The tests were performed by a regular procedure (Kokate, 1994;Khandelwal, 2005).

TLC characterisation
TLC is generally utilised for the quick investigation of drugs and drug preparations. Fractions were put forwarded for the characterisation through thin layer chromatography (TLC; silica gel 60 F254, Merck). Chromatograms are estimated beneath U.V. light at 254 and 365 nm to identify the existence of lavonoids plus saponin, respectively. To verify the existence of lavonoids, TLC is furthermore sprayed by an Ammonia vapour. Saponin was identi ied by Anisaldehyde-sulphuric acid reagent along with Vanillin-phosphoric acid reagent.

Total phenolics Content
The Folin-Ciocalteu technique was applied to ind out the entire phenolic content of the plant extract. Gallic acid is utilised as the standard to compare with fractions, and whole phenolic acid was articulated as mg / g gallic acid equivalent (GAE). 10, 20, 30, 40 and 50 (µg / ml) gallic acid concentrations be made in methanol. Sample of 1 mg / mL of plant extract in methanol was prepared, and 0.5 ml of every above-prepared sample was added for analysis and diluted with 2.5ml of a Ciocalteu-Folin reagent (10 times dilute) and mixed with 2 ml of 7.5 per cent sodium carbonate. The tubes are covered with paraf in and set aside at room temperature for 30 mins previous to the absorbance was read at 760 nm spectrometrically. All determination is inished three times. Folin-Ciocalteu reagent is responsive to reducing agents, as well as polyphenols. After the reaction, it turns blue. This blue colour was measured spectrophotometrically (Chang et al., 2002).

Total lavonoid content
The colourimetric technique of aluminium chloride evaluated the total lavonoids contained inside the plant extract. Brie ly, part of the sample was diluted (1 mg / ml) or standard solution (10, 20, 30, 40 and 50 µg / ml) to 75 µl. A solution of NaNO2 was added, and 0.15 ml of A1Cl3 was mixed 6 min earlier. Following 5 min, 1/2 ml of NaOH be added. The last amount was changed to 2.5 ml with puri ied water and mixed well.
The absorption of the combination is set at 510 nm compared to the similar combination, without the addition of the sample, as a blank. The entire lavonoid content was articulated as mg / g dry weight (mg / g D.W.) using the normal calibration curve. Every sample is scrutinised three times (Villaño et al., 2007).

Studies of Antioxidant Activities
In vitro antioxidant activity of the different fractions at different concentration (Table 1) was studied by three procedures: with free radical scavenging by DPPH, Hydrogen peroxide assay and reducing power assay procedures.

DPPH free radical scavenging activity
To measure the DPPH radical scavenging activity, Barros et al. (2007) was carried out to the method. The drop of DPPH radicals was measured by determining the absorption at 517 nm. The radical scavanging activity was measured as a percentage of DPPH strains by the following equation, Where A 0 is the absorbance of the DPPH solution and A 1 is the absorbance of the sample (Barros et al., 2007). Ruch et al. (1989) were used the method to determine the H 2 O 2 scavenging ability of extracts. The H 2 O 2 scavenging capacities of the extracts were calculated using formula, (Ruch et al., 1989).

Reducing power assay
The extracts were prepared in different concentrations. Phosphate buffer (2.5 ml, 2 M, pH 6.6) and potassium ferricyanide were mixed with 1 ml of each in distilled water. The blend was incubated at 50 o C for 20 min. A portion of trichloroacetic acid (TCA) was added to the combination which was then centrifuged at 2000 RPM for 20 min. The superior layer of the solution was mixed with water and FeCl 3 (0.5ml), and the absorbance was calculated at 700 nm. Increased reducing power was indicated by increased in absorbance of the reaction mixture (Yıldırım et al., 2001).

Physical Evaluation
The correct identity of the leaves of Ailanthus excelsa and the fruits of Randia Dumetorum is determined by pharmacological research. Morphological research of drugs includes evaluation of drugs by colour, odour, taste, size, shape and unique features, like touch, texture etc. It evaluates special features, among others. It is a qualitative assessment technique based on an analysis of the morphological and sensory pro iles of each drug (Table 2).
Graph 1: Effect of Fractions in DPPH free radical scavenging assay

Physicochemical Evaluation
When raw materials are burned, ash residues are formed consisting of inorganic materials (metal Graph 2: Effect of Fractions in Hydrogen peroxide scavenging assay Graph 3: Effect of Fractions in Hydrogen peroxide scavenging assay salts and silica). This value is within a reasonably wide range and is, therefore, an essential parameter for evaluating raw materials. The value of the ash identi ies more direct dirt, such as sand or dirt (Table 3).

Phytochemical Investigations
Phytochemical screening of crude extracts of leaves of A.excelsa and fruit of R. Dumetorum by using chemical method revealed the presence of bioactive constituents such as Alkaloids, Carbohydrates, Flavonoids, Saponins, Tannins and Phenolic compounds, Glycosides (Table 4 and Table 5).

TLC characterisation
TLC is primary; easy to use an analytical method, and the solvent used are not hazardous and do not require advanced tools. Depending on the solvent fraction compounds, the solvent system was chosen. TLC examined all fragments of A. excelsa and R. Dumetorum, and the results of this study are, lavonoids and saponins are present infractions (Table 6).
Water-insoluble ash 6.10 4.65 different fractions, the maximum yield was obtained for the Flavonoid fraction of R. Dumetorum (4%) followed by A.excelsa (3%). For total lavonoid content (TFC) estimation standard curve of Rutin was used, and content was estimated as Rutin equivalent (RE). For total phenol content estimation (TPC) standard curve of Gallic acid was used and estimated as Gallic acid equivalent (GAE). TFC in Ailanthus excelsa and Randia Dumetorum were found to be 26 and 19 µg/mg RE respectively. Total Phenol Content in Ailanthus excelsa and Randia Dumetorum were found to be 65.14 and 62.44 µg/mg GAE, respectively.

Studies of Antioxidant Activities
Due to the complexity of phytochemicals, the antioxidant activity of plant extracts cannot be evaluated using a single method. Therefore, it is vital to use generally accepted tests to evaluate the antioxidant activity of plant extracts. Many methods of antioxidants have been developed to evaluate antioxidant activity and explain how antioxidants work.

DPPH free radical scavenging activity
The DPPH oxidation test used in this document to measure the capacity of radical-scavenging capacity (RSC) is used worldwide. The ability of radical bio-logical agents to remove DPPH can be expressed as a unit capable of producing antioxidants. The DPPH alcohol solution has a bright purple colour, with an absorption peak at 517 nm when one of the radical scavengers disappears in the reaction system and only one electron of nitrogen is attached to the DPPH. The reaction rate and potential of the radical promoter depend on the rate and maximum value of the DPPH event (Sharma and Gupta, 2008;Gupta and Sharma, 2010 (Table 8 and Graph 1).

Hydrogen peroxide scavenging
Hydrogen peroxide itself is not very reactive, but it sometimes becomes toxic to cells because it can carry hydroxyl radicals into the cells. Therefore, the elimination of H 2 O 2 is essential to protect antioxidants in cell or food systems. It was noted that all of the samples tested in this study had the effect of removing hydrogen peroxide (Pietta, 2000). IC50 values are 88.47, 115.75, 108.4, and 181.13 for AEFF, AESF, RDFF, and RDSF respectively (Table 9 and Graph 2)

Reducing power assay
All test samples were also found to be having betterreducing power with good goodness of it (R 2 > 0.9) for all test samples. Substances with reduced potency react with potassium burkianide (Fe3 +) to form potassium burkianide (Fe2 +) and then react with iron chloride with the absorption of up to 700 nm to form a ferric, ferrous complex iron (Table 10 and Graph 3). With an increase in concentration, absorbance increased for all test samples (Wenli et al., 2004).

CONCLUSION
Pharmacological standardisation of the two plants includes physicochemical assessment, identi ication, veri ication, and detection of adulteration and quality control of the crude drug. Hot aqueous extract from the leaves of Ailanthus excelsa and the fruits of Randia Dumetorum, which have the most signi icant value for extraction. Therefore, we come to the breakdown of the aqueous extract. Analysis of the ash value shows that inorganic substances are present in very normal quantities. If physical and chemical methods are not suf icient, a phytochemical test can be used identi ied from their impurities. Phytochemical tests are helpful to detect various phyto components. Over the past two decades, the TLC method has been an essential tool for qualitative and quantitative phytochemical analysis of herbal products and preparations.
The results of this study show that the leaves of Ailanthus excelsa and the fruits of Randia Dumetorum showed free radical scavenging, Hydrogen peroxide scavenging and reducing power activity. The antioxidant activity of each fraction of the two plants can be attributed to the polyphenol content and phytochemical components. The results of this study suggest that fractions of both plants could be a natural source of antioxidants as a therapeutic agent for a biological system that is sensitive to free-reaction intermediates.