INTRODUCTION:

Herbal crops have been used in cure of disease from the prehistoric time. It is assessed by the World Health Organization that approximately 75-80% of the world's population uses plant drugs either partly or completely as medicine. ¹Lately there has been a shift in worldwide trend from synthetic to herbal medicine, which we can say ‘Return to Nature’.

Medicinal plants have been known for eras and are highly esteemed all over the world as a rich source of healing agents for the hindrance of diseases and ailments. Nature has bestowed our country with a huge wealth of medicinal plants, hence India has often been mentioned to as the Remedial Garden of the world. ² Prosopis cineraria (L.) Druce (family: Fabaceae) frequently identified as “Khejari” in Rajasthan. It is the State tree of Rajasthan, India because it can be found in desert places where it can survive. It is a well-known hosted class in parts of Southeast Asia, as well as Indonesia.³P. cineraria is a short tree, reaching in height from 3–5 m (9.8–16.4 ft). Plant leaves shape are bipinnate, contain seven to fourteen leaflets on every of one to three pinnae. Subdivisions are thorned along the internodes. Flowers are minor and creamy-yellow, and enclosed by seeds in pods. The tree is originate in extremely arid conditions, with rainfall as low as 15 cm (5.9 in) per annum; but is revealing of the presence of a deep water table. As with some other Prosopis spp., P. cineraria has revealed a tolerance of exceedingly alkaline and saline environments. ⁴

The magnitude of the beneficial value of this tree has been underlined in our prehistoric nonfiction and different types of pharmacological actions such as analgesic, antipyretic, antihyperglycemic, antioxidant, antihypercholesterolemic, antitumor, nootropic, anticonvulsant etc. due to presence of numerous primary and secondary metabolites.12-26 The leaf of the tree is known as loom and they have excessive nutrient content like Carbohydrate, Protein, Fat, Minerals and Vitamins. ^[5,6] Leaf mixture of P. cineraria is theputicaly active on boils and blisters, including mouth ulcers in livestock ⁷ and smoke of the leaves is considered good for eye troubles. ⁸Different phytoconstituents like tannins, steroids, Flavone derivatives, alkaloids etc. has been isolated from the plant. ^{9, 10, 11}leaves extracts are used for open sores on the skin, prevent miscarriage, help in preventing protein calorie malnutrition iron calcium deficiency in blood, muscular tremors and wandering of the mind. ^{12,
13, 14}

MATERIAL AND METHODS:

Collection and Authentication:

The fresh leaves of Prosopis cineraria (L.) Druce (family: Fabaceae) were collected from Jaunpur, Amdendakar Nager and Lucknow during the month of March 2017. The fresh seed were used for the study of macroscopic and microscopical characters. However collected plant material were shade-dried and coarsely powdered. This coarse powder was used for the determination of ash values, extractive values, and preliminary phytochemical investigation was studied as per standard methods.dried leaves were blended to powder with a clean electronic blender then pass through sieves no.40 and obtained powder stored in airtight glass containers kept in laboratory cupboard, until required for preparation. For identification and taxonomic authentication sample were given to Integral University, Kursi Road, Lucknow, Uttar Pradesh and authentication no. IU/PHAR/HRB/20/01

Extraction of plant material:

100 gm of coarse powdered of air dried Prosopis cineraria leaves. Were filled coarse powder in muslin cloth and exposed to soxhlet extractor for continuous hot extraction with hexane, ethyl acetate, ethanol and distilled water for 12hrs separately. Then after the completion of extraction process each extracts were filtered and filtrate was vaporized to dryness. The percentage yield of hexane, ethyl acetate, ethanol and the aqueous extracts were calculated. ^{15, 16, 17}

Macroscopic and microscopic studies:

The macroscopic of the leaves was carried out according to standard methods. Representative diagrams were taken with the help of inverted microscope for photo documentation ^{18, 19.}

Physicochemical analysis:

Physicochemical analysis i.e. extractive values of hexane, ethyl acetate, ethanol and aqueous, ^{20, 21 and 22} fluorescent analysis, total ash, acid-insoluble ash, water-soluble ash, foreign matter and moisture content were carried out. ²³ Standardized digital pH meter was used to measure the pH of 1 and 10% aqueous extracts.

Preliminary phytochemical screening:

Preliminary phytochemical screening of hexane, ethyl acetate, ethanol and aqueous was carried out for the detection of various compounds by using standard procedures described ^{24, 25, 26}.

TLC analysis:

Powder of silica gel G was taken and dissolved in distilled water with the help of stirring rod a then poured over a glass plates to form a thin layer. The ready plates were air dried for setting and then retained in an oven at 100-120°C (30min) for initiation. The n-hexane and ethanolic exact were dissolved in respective solvents and spotted over an activated plate (1cm above from the bottom). The marked plates were kept in a previously saturated developing compartment containing mobile phase, and permitted to run 3/4th of the height of the ready plate. The plates were air dried and number of spots were noted and Rf value were calculated ^{27, 28, 29}. Spots were visualized by respective spraying agents. Numbers of solvents systems were tried but the maximum resolution was shown in Toluene: ethyl acetate: Formic acid and n-hexane: Acetone for the ethanolic and hexane extract respectively.

HPTLC analysis:

HPTLC fingerprinting is an appreciated tool for the estimation of herbal medicine. This study was conducted for the quantitative determination of phytochemicals present in the plant extract ^{30, 31, 32,
33 and 34}.

Quercetin Estimation by HPTLC:

Conditions:

· Stationary phase: Pre-coated silica gel 60 F₂₅₄aluminium plates

· Mobile phase: solvents used in differ combination Toluene: Ethyl acetate: Formic Acid (5:4:1).

· Chamber Saturation Time: 20 mins.

· Test Solution: 3.5 gm of drug extract dissolved in methanol and then filter the liquid extract. Make the volume up to 50 ml with methanol.

· Standard Quercetin Solution: Prepare 1 µg/ µl solution in methanol.

· Visualization and Detection: 254 nm (Short wavelength

Procedure:

Take previously washed with methanol and dried TLC plate and fix dimension at X position and mark from base with help of pencil at 10 mm and 90 mm. and also left 15 mm from both sides of plate. Apply the test sample solution 10 µl and 15 µl in the form of bands with the programming of Linomats applicator. Apply the standard quercetin solution in 0.1 to 0.6 µl concentration.Allow the solvent to be evaporated and place the plate in the saturated tank, possibly vertical and so that spots or bands are above the level of mobile phase. Close the tank and allow to stand at room temperature until mobile phase ascended to the marked line.Remove the plate and dry and visualize as in UV-Vis light at 254 nm and 366 nm.Prepare the scanning programme for completely dried plate at wavelength of 254 nm.

Plot the calibration curve by scan data of different spots of standard varied for concentration. Hence graph between AUC and concentration.Calculate the concentration of unknown test sample bands by linear plot of cal. curve.

Calculation:

Percentage of Quercetin: C₀ × purity of std.

C₁

Where, C₀= Concentration of sample found from cal. curve in µg and C₁= Concentration of sample applied in µg.

Gallic Acid Estimation by HPTLC-

Conditions:

· Stationary phase: Pre-coated silica gel 60 F₂₅₄aluminium plates

· Mobile phase: solvents used in differ combination Toluene: Ethyl acetate: Formic acid (5:5.5:0.5).

Chamber Saturation Time: 20 mins.

· Reference Standard: Dissolve 25 mg of Gallic acid in 25 ml of methanol and take 1 ml of above solution and diluted up to 10 ml of methanol.

· Test Solution: 20 mg of extract sample by dissolved in 25 ml of methanol by sonication process for 20 min.

· Visualization and Detection: 254 nm (Short wavelength)

Procedure:

Take previously washed with methanol and dried TLC plate and fix dimension at X position and mark from base with help of pencil at 10 mm and 90 mm. and also left 15 mm from both sides of plate. Apply the test sample solution 10 µl and 15 µl in the form of bands with the programming of Linomats applicator. Apply the standard Gallic acid solution in 0.1 to 0.6 µl concentration. Allow the solvent to be evaporated and place the plate in the saturated tank, possibly vertical and so that spots or bands are above the level of mobile phase. Close the tank and allow standing at room temperature until mobile phase ascended to the marked line. Remove the plate and dry and visualize as in UV-Vis light at 254 nm and 366 nm.Prepare the scanning programme for completely dried plate at wavelength of 254 nm.

Plot the calibration curve by scan data of different spots of standard varied for concentration. Hence graph between AUC and concentration. Calculate the concentration of unknown test sample bands by linear plot of cal. curve.

Calculation:

Percentage of Gallic acid: C₀ × purity of std.

C₁

Where, C₀= Concentration of sample found from cal. curve in µg and C₁= Concentration of sample applied in µg.

RESULTS AND DISCUSSION:

Macroscopical characteristics:

The leaves of Prosopis cineraria (L.) Druce are dark green in colour. They are better in taste and have specific odour and pungent. Leaves are bipinnate, by seven to fourteen leaflets on every of one to three pinnae. Branches are thorned along the internodes. Between 16 and 20 pairs of leaflets are present, margin is entire with acuminate apex. The ventral surface is smooth while the dorsal surface is rough. The leaves have reticulate venation, oblong shaped and petiole is 0.5–5 cm long.

Microscopic characters:

Microscopy of leaf:

A transverse division of P. cineraria leaf (midrib and lamina) revealed the existence of upper epidermis, lower epidermis and mesophyll (Figure 2). The upper epidermis was single layered, slightly rectangular, with a distinct cuticle and simple trachoma’s. Just below the upper epidermis, a single palisade layer with 2 to 3 layers of spongy parenchyma is present. The cells of upper epidermis and lower epidermis are similar in size. The central portion showed the presence of a bundle sheath. The leaf surface (upper and lower surface) study detected the presence of epidermal cells and paracytic stomata, which followed two subsidiary cells are parallel to that of stoma and simple trichomes on margin (Figure 3).

Fig. 2: Transverse section of P. Cineraria leaf.

Fig. 3: Lower leaf surface study of P. Cineraria leaf.

Microscopy of leaves powder:

Powder characteristic of P. cineraria leaves revealed the presence of vessels, simple trichomes, calcium oxalate crystals, epidermal cells and paracytic stomata etc.

Fig. 4: Powder Microscopy.

Physicochemical parameters:

The physico-chemical characters of powdered drug of air dried such P. cineraria leaves as hexane, ethyl acetate, and ethanol, and water soluble extractive, ash value, acid insoluble ash, water-soluble ash, loss on drying, and foreign matter are presented in Table 1. The fluorescence analysis of the powdered drug of Prosopis cineraria (L.) Druce in various solvents was performed under normal and Ultra Violet (254nm and 366nm) light and powdered drug reaction with different reagents were evaluated in Table 2 and Table 3 respectively. The pH of 1% and 10% aqueous solution of powered drug of Prosopis cineraria (L.) Druce are noted in Table 4.

Table 1: Physico chemical parameters of leaves of Prosopis cineraria.

Quantitative parameter	Values obtained (%) w/w
Quantitative parameter	Cold extractive	Hot extractive	Successive extractive
1.Hexane Extractive	4.8 ± 0.28	8.8±0.03	7.06±0.25
2.Ethyl Acetate Extractive	9.092±0.39	15.62±0.10	17.21±0.13
3.Ethanol Soluble Extractive	12.19±0.13	18.27±0.15	14.93±0.04
4.Water Soluble Extractive	24.82±0.26	34.06±0.33	26.27±0.20
5.Total Ash	7.06±0.28
6.Acid Insoluble Ash	3.14±0.16
7.Water – Soluble Ash	19.3±0.28
8.Loss on Drying	3.78±0.3
9.Foreign Matter	1.7±0.03

Table 2: Fluorescence analysis of powdered Prosopis cineraria.

Solvents Used	Observation
Solvents Used	Day Light	UV 254nm	UV 366nm
Methanol	Light green	Green	Green
Petroleum ether	Yellow	Yellow	Orange
Chloroform	Yellow	Green	Dark green
Ethyl acetate	Green	Green	Dark green
Toluene	Light green	Light green	Green
AcetoneIodine	Colourless	Light green	Dark green
Ethanol	Green	Dark green	Dark green
ConcHCl	Light green	Green	Green
Distilled water	Green	Green	Dark green
Conc. H₂SO₄	Yellow	Green	Dark green
Conc.HNO₃	Brown	Dark brown	Black
NaOH in methanol	Yellow	Dark yellow	Orange
Picric acid	Green	Green	Dark green
Hexane	Green	Green	Dark green

Table 3: Powdered drug reaction with different reagents.

Treatment Observation	Treatment Observation
Distilled water	Green
NaOH (1N)	Yellow
Conc H2SO4	Yellow
ConcHCl	Brownish
Conc HNO3	Brownish
Glacial acetic acid	Greenish
Petroleum ether	Greenish
Benzene	Greenish
Ethanol	Dark green

Table 4: Determination of pH of The Drug.

Sample pH	Sample pH
pH of 1% solution	6.4
pH of 10%solution	6.8

Preliminary phytochemical screening:

The preliminary phytochemical investigation of the hexane, ethyl acetate, ethanol and aqueous extracts of Prosopis cineraria leaves showed the presence of sterols, flavonoids, amino acids, glycosides, phenolic compounds, carbohydrates, saponins and alkaloids are present. (Table 5)

Table 5: Qualitative analysis of phytochemicals in Prosopis cineraria leaves.

Phytochemicals	Hexane	Ethyl acetate	Ethanol	Aqueous
Sterols	-	-	-	-
Tannins	-	+	+	+
Proteins and amino acids	+	+	+	+
Glycosides	-	+	+	+
Phenolic compounds	+	+	-	-
Carbohydrates	+	+	+	+
Saponins	-	-	-	-
Alkaloids	+	+	+	+
Flavonoids	+	+	+	+
Terpenoids	-	+	+	+

Table 6: TLC of Prosopis cineraria leaves.

Test extracts	Solvent system	Number of spots	Rf value
Hexane extract	Acetic acid:Chloroform	2	0.88, 0.91
Ethanolic extract	Toluene: Ethyl acetate: Formic acid	4	0.15, 0.88, 0.96, 0.98

+ = Present - = Nil

Thin layer chromatography:

Thin layer chromatography of the hexane and ethanolic extracts were carried out separately using toluene: ethyl acetate: formic acid (5:4:1) for the ethanolic extract and hexane: acetone (9:1) for the hexane extract as mobile phase respectively and the Rf values were recorded and depicted in Table 6. The visualizing method employed was UV chamber used to effect visualization of the resolved spots.

High performance thin layer chromatography fingerprinting:

HPTLC methods are commonly applied for the documentation and analyze the content uniformity of herbal raw materials and their formulations. In this study, numerous solvent systems were used for individual estimation of these poly phenolics and flavonoids to evaluate the combinatorial separation of these compounds in a single solvent system and between different components of the extract. From the solvent system studies, mobile phase consisting of toluene: ethyl acetate: formic acid in the ratio of 8: 2: 0.1 v/v/v established the finest resolution between other peaks of the extract. The procedure for the separation and determination of different compounds in ethanolic fraction of P. cineraria using HPTLC-densitometry is reported calibration curve in which Gallic acid and Quercetin were observed. The identification of Gallic acid and Quercetin were confirmed by the bands which obtained from the test sample and standards. The peak of Gallic acid and Quercetin the test sample was very near to standard.

In the part of standardization study, the macroscopical examination of the leaves of Prosopis cineraria (L.) Druce was studied. Macroscopical assessment is a performance of qualitative assessment recognized on the study of morphological and organoleptic characters of the medicines. The microscopical characters determine the histological profile of the leaves and can serve as diagnostic parameters. The plants leaves extracts extractive value, ash value, loss on drying and fluorescent analysis have been carried out by standard method. The extractive values in different solvents specify the quantity and nature of ingredients in the extracts. The extractive values are also accommodating in assessment of definite ingredients soluble in specific solvent. The fluorescence analysis of the powdered drug of the leaves of Prosopis cineraria (L.) Druce in various solvents was performed under normal and UV light to detect the fluorescent compounds. Thin layer chromatography (TLC) is principally appreciated for the preliminary separation and determination of plant constituents.HPTLC studies carried out for determination of phytochemicals present in the plant extract.

CONCLUSION:

After present investigation it can be concluded that the standardization and preliminary phytochemical investigation of Prosopis cineraria (L.) Druce yielded a set of standards that can serve as an essential basis of evidence to ascertain the identity and to determine the quality and purity of the plant material in future studies. This study is a substantial step and it further requires a long term study to evaluate therapeutic efficacy and toxicity of leaves, to establish as the drug. Further studies are required on the safety issues of synthetic compound because herbal products are preferred as a symbol of safeguard in comparison. The advance phytochemical analysis entail us the presence of variety of active molecules which may be responsible for its different activity. so as to avoid the usage of harmful synthetic chemicals and can avoid resistant development.

ACKNOWLEDGEMENTS:

The authors are thankful to Director Dr.Udaivir Singh SaraHygia Institute of Pharmaceutical Education and Research, Ghaila Road, Balrampur, Lucknow.Dr. Aleza Rizvi (Associate Professor and Head of Department) Hygia Institute of Pharmacy, Ghaila Road, Balrampur, Lucknowfor providing all the facilities for our research work.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 14.09.2020 Modified on 30.12.2020

Research J. Pharm.and Tech 2022; 15(2):621-627.

DOI: 10.52711/0974-360X.2022.00102