INTRODUCTION:

Nature has been a resource of therapeutic mediators for numerous years. Modern drugs that are used even today have been separated from natural resources. The isolation of the bioactive molecules from organic sources were achievable as the herbs were used in conventional treatement¹. According to World Health Organization (WHO), about three-quarter of the world population depends upon conventional remedies as preventive medicine². The plants and their extracts are popular elements in Indian systems of medicine. Major drug companies are presently performing extensive research on plant materials for their potential medical value. The plant-based, traditional medicine systems proceed to play an important role in health care³. The global market for natural medicines currently at more than $60 billion annually⁴. The latest global resurgence in herbal medicines has led to a rise in the requirement for them.

The commercialisation of these medicines to satisfy this enhancing need has caused in a decline in their quality, primarily due to a shortage of adequate regulations relating to this industry of medicine. The essential of the hour is to progress a systematic approach as well as to create well-designed approaches for the standardisation of herbal resources and their formulations. Various methods of phytochemical standardisation, such as preliminary phytochemical screening, fingerprint profiling and quantification of the marker compound with an endorsement to plant essences are utilised⁵. Regulation is necessary to ensure the accessibility of a consistent product in all component of the world. It guarantees a continually stronger product with guaranteed constituents⁶.

The Mimusops elengi Linn is very famous for its shade, fragrant flowers. M. elengi is effectively reported for several therapeutic properties like antinociceptive, diuretic effects, gastroprotective, antibacterial, antifungal, anticarcinogenic, free radical scavenging, antihyperglycemic etc.^7,8,. M. elengi consists of a number of active phytoconstituents and thus have various type of biological and pharmacological activities.. It possesses activities like antibacterial⁹, anti hemorrhoidal, antifungal, anticarcinogenic, free radical scavenging¹⁰, antihyperglycemic¹¹, antineoplastic, gastroprotective^12,13,, antinociceptive and diuretic effects, antiviral¹⁴, cognitive enhancing activity¹⁵ and cytotoxic activities¹⁶. The objective of the current research study was to find the physicochemical, phytochemical and HPTLC fingerprinting examine of EMEL.

MATERIALS AND METHODS:

Plant collection and authentication:

The plants were gathered from in and around Tiruchirappalli district, Tamil Nadu, India. They were botanically authenticated by Rapinet Herbarium, St. Joseph’s College, Tiruchirappalli, Tamil Nadu. The specimen was banked in the Herbarium for future reference (voucher number: SY001). The plant samples were completely cleaned under running tap water to eliminate sticking dust and shade dried for about fourteen days, ground into coarse powder and kept in an airtight container until further investigations.

Preparation of extracts:

The pulverized plant samples of leaves and flowers (100 g) were used for successive solvent extraction (500 ml) with low polarity to high polarity nature of solvents like hexane, chloroform, ethyl acetate, ethanol and water. The extraction was carried out for 48 h. The extract was concentrated by a Rotary flask evaporator. Every time before extracting with the following solvent the excess was desiccated completely to confiscate the solvent utilised. The extracted samples were collected and used for phytochemical screening.

Physicochemical properties:

Fluorescence Evaluation:

Fluorescence of the powder was observed under normal light and UV light (254nm) treating with acids and alkaline solutions of the drug¹⁷. Fluorescence study was also carried out for the powder and also with extracts with different solvents (Hexane, Chloroform, Ethyl acetate, Ethanol and Water).

Physiochemical Evaluation:

Physiochemical studies are evaluated to determine the quality and purity of the leaf powder of M. elengi. The percentage of moisture content (Loss of dry), total ash, water soluble and acid insoluble ash were calculated as per Indian Pharmacopoeia^18,19,. The extract of the pulverized leaves were made with the various solvents for the study of extractive value.

Preliminary Phytochemical screening:

The leaf powder of M. elengi and extraction from different solvents (Hexane, Chloroform, Ethyl acetate, Ethanol and Water) were studied for the presence and absence of secondary metabolites like, alkaloids, glycosides, saponins, phytosterols, phenolics, terpenoids, flavonoids, coumarins, steroids, sugars, quinines. lignin, starch, protein and tannins by qualitative chemical tests^20-26.

HPTLC finger printing analysis of EMEL

Test solution preparation

10 mg of the plant samples was weighed and dissolved in 250ml of respective solvent and centrifuged at 3000rpm for 5 min. This solution was utilized as a test solution for HPTLC analysis²⁷.

RESULTS AND DISCUSSION:

As a part of standardization procedure, the leaf sample of M. elengi was tested for relevant physiochemical, phytochemical parameters and HPTLC fingerprinting analysis.

Fluorescence Evaluation:

The fluorescence of pulverized drug and extracts of (Table 1 and 2) different solvents helps in the determination of quality and purity of the drug. The powder and extracts exhibited fluorescence, in the visible range If the substances themselves are not fluorescent, they may often be converted into fluorescent by-products by reagents, thus it is an vital parameter of pharmacognostical evaluation.

Table 1: Fluorescence analysis of leaves powder of M. elengi

Drug + Chemicals	After 24 hours		After 48 hours
Drug + Chemicals	Normal light	UV light	Normal light	UV light
Powder	Green	Green	Green	Green
Powder + 50% H₂SO₄	Black	Black	Black	Black
Powder + Aq.1N NaOH	Dark brown	Dark brown	Brown	Red
Powder + CHCl₃	Green	Dark green	Green	Yellow green
Powder + Ethylacetate	Green	Green	Dark green	Dark green
Powder + Hexane	Brown	Brown	Dark brown	Dark brown
Powder + Acetone	Green	Green	Dark green	Dark green
Powder +Benzene	Green	Green	Dark green	Dark green
Powder + 1N HCl	Yellow	Light Yellow	Yellow	Light Yellow
Powder + Alc. 1N NaOH	Green	Dark green	Light green	Dark green
Powder + Alcohol	Green	Dark green	Dark green	Dark green
Powder + Water	Green	Dark green	Dark green	Dark green

Table 2: Fluorescence Analysis of different extracts of M. elengi leaves

Extracts of Different solvents	After 24 hours		After 48 hours
Extracts of Different solvents	Normal light	UV light	Normal light	UV light
Hexane	Brown	Brown	Dark brown	Dark brown
Chloroform	Green	Dark green	Green	Dark green
Ethyl acetate	Green	Green	Dark green	Dark green
Ethanol	Dark green	Dark green	Dark green	Dark green
Water	Dark green	Dark green	Dark green	Dark green

Physiochemical Evaluation:

The determination of physiochemical parameter is important to determination of adulterants and improper handling of drugs. Table 3 showed total ash content was 3.49±0.27% (w/w). The amount of acid insoluble ash and water soluble ash were found to be 0.25±0.03 and 3.42±0.30 % (w/w) respectively. Ash values are useful in governing the quality and purity of powdered form of crude drugs. The total ash frequently contains of inorganic radicals like silicates, phosphates, carbonates and silica of potassium, sodium, magnesium and calcium. Occasionally, inorganic variables like calcium oxalate, silica, carbonate content of crude drug impacts "total ash" values, such variables are then removed by treating with acid (as they are soluble in HCl) and then acid insoluble ash value is determined. The values vary within fairly broad limits and are therefore an important parameter for the purpose of evaluation of crude drugs. Ash insoluble in HCl is the excess acquired after extracting the total ash with HCl. This acid insoluble ash value mostely specifies adulteration with silicious materials like earth or sand. Here, Low acid insoluble ash (0.25±0.03%) indicates less silicious materials like earth or sand. Water soluble ash is that portion of the total ash subsance which is soluble in water. It is a good indicator of either extraction of water soluble salts in the drug or incorrect preparation. While determining the total ash, very high temperature (> 650° C) may result in the conversion of carbonates to oxides. Ash values of a drug give a suggestion of the earthy matter or the inorganic composition and other impurities current together with the drug²⁸. The moisture content of M. elengi leaves was 5.32±1.07% w/w (Table 3). Loss on drying determines both water and volatile matter. Excessive of water in medicinal plant materials will stimulate microbial growth and deterioration following hydrolysis. This is especially significant for components that absorb moisture easily or degrade quickly in the existence of water. The less value of moisture content could protect against bacterial, fungal or yeast growth.

Table 3: Determination of Ash Values of leaves powder of M. elengi

Ash Value % (W/W)
Total Ash	3.49±0.27
Acid Insoluble Ash	0.25±0.03
Water Soluble Ash	3.42±0.3
Moisture content	5.32±1.07

Table 4: Determination of Extractive Value of leaves powder of M. elengi

Extractive Value % (W/W)
Hexane	5.1±0.36
Chloroform	7.91±0.23
Ethyl Acetate	10.09±0.50
Ethanol	11.68±0.46
Water	15.12±0.32

The the crude drug’s extractive value decides the quality as well as purity of the drug²⁹. In present study the extractive values of the leaves of Mimusops elengi Linn in Hexane, Chloroform, Ethyl acetate, Ethanol and Water of were determined. Extractive values of M. elengi leaves showed very high quantity of polar constituents than non-polar constituents (Table 4). Extractive values identify the amount of active constituents extracted with solvents from a given amount of medicinal plant product and are useful for evaluation of crude drugs and provide a suggestion about the nature of chemical components existing in them. The quantity of extractive drug yield of a given solvent is often an approximate measure of a certain component or group of related constituents the drug contains. Sometimes the quantity of drug soluble in a presented solvent is an index of its purity. The solvent used for extraction should be in a position to dissolve significant quantities of substances preferred. Taking into consideration the diversity in chemical nature and properties of contents of drugs, various solvents are utilized for determination of extractive values These experiments were repeated thrice in order to arrive at standard values.The results showed greater extractive values in water, ethanol, ethyl acetate, chloroform and hexane extractions indicating the effect of chemical compounds present in the plant.

Table 5: Preliminary phytochemical investigation of different extracts of leaves powder of M. elengi

S. No	Phyto chemicals	Extraction of different solvents
S. No	Phyto chemicals	Hexane	Chloroform	Ethyl acetate	Ethanol	Water
1	Terpenoids	-	-	-	-	-
2	Flavones	+	-	-	+	+
3	Steroids	+	+	+	+	+
4	Anthroquinones	-	-	-	-	-
5	Glycosides	+	+	-	-	-
6	Alkaloids	+	+	-	+	+
7	Quinone	-	-	+	-	-
8	Phenol	-	+	+	-	+
9	Tannin	-	-	-	-	-
10	Saponin	-	-	-	-	+
11	Coumarin	-	-	-	-	-
12	Starch	-	-	-	-	-

+ - Presence; - - Absence

Development of HPTLC methods for extract:

HPTLC is a powerful analytical technique. This method is visual, rapid and economical as it utilises smaller volumes of solvents with minimum sample clean up. Above all, in a short duration, a large number of samples are analysed simultaneously³⁰.

Figure 1: High performance thin layer chromatography study of Mimusops elengi Linn.

(a) Represent as EMEL at 336 nm (b and c) Densitogram of EMEL and flavonoid standard respectively.

HPTLC of the ethanolic extract of Mimusops elengi Linn (Figure 1) show six peak areas, thereby showing six Rf values. Thus it illustrates the presence of six different constituents in EMEL. Figure 1a and 1b shows the chromatogram of EMEL and the standard compound Quercetin, respectively. Figure 1c exhibited the densitogram for a flavonoid profile of M. elengi L. leaves, and 1d exhibited the densitogram of Quercetin used for checking the quality of extracts. The HPTLC observations of Mimusops elengi Linn shows 6 peaks, (Figure 1c), comparing the height and area of the peaks with the standard the presence of flavanoids is confirmed (Figure 1d). Blue and yellowish blue coloured fluorescent zone at the UV 366nm mode after derivatization confirms the presence of flavonoids in the sample (Figure 1a). Flavonoids are the very important and possess a broad spectrum of chemical and biological activities including free radical scavenging properties³¹. The flavonoids and phenolic compounds in plants have been proved is possess various biological effects including antioxidant, free radical scavenging abilities, anti inflammatory, anti carcinogenic etc³².

Typical HPTLC fingerprinting of specific plant types will not only assist in the identification and quality control of a particular types, but also provide basic information helpful for the isolation, purification, characterization, and identification of marker chemical compounds of the species and also employed for taxonomic categorization. Thus, the present study provides adequate data in the identification, standardization, and quality control of medicinal plant

CONCLUSION:

In this study, phytoconstituents were identified on the basis of their physicochemical, phytochemical and HPTLC analysis. Identified phytoconstituents were confirmed by the visualization at 366nm wavelength of densitometric analysis of the plate. A conclusion is drawn from this study that the EMEL are more effective than the other extracts. The reported data based on the HPTLC fingerprint approach can also be suggested as a quick and dependable analytic model for the pharmacognostic research study of plant raw materials used in industrial products.

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Received on 22.08.2019 Modified on 05.10.2019

Research J. Pharm. and Tech 2020; 13(5):2091-2095.

DOI: 10.5958/0974-360X.2020.00376.5