INTRODUCTION:

Nature has a vast array of plants and is capable of providing every cure for human illness. The foundation of the healthcare system has been science since the dawn of humanity. It is impossible to locate a plant that has no medicinal properties.^1,2

Traditional medicine has remained as the most affordable and easily accessible source of treatment in the primary health care system of resource poor communities. The local people have a long history of traditional plant usage for medicinal purposes. The medicinal use of plants is very old.³ It is cleared that the natural products play the major role in the drug development system. Although uses of synthetic medicine is growing due to the cost, quick action, easy to evaluation but there was a big question mark about their safety profile.^4,5 According to the WHO, between 65% and 80% of the populations of developing countries currently use medicinal plants as remedies.⁶ The development of new products from natural sources is also encouraged because it is estimated that of the 300,000 plant species that exist in the world, only 15% have been evaluated to determine their pharmacological potential.⁷ To guarantee the quality of herbal medicines, certain steps established in the Pharmacopoeias must be followed, including correct identification of the plant species, analysis of the purity and confirmation of the presence and minimum concentration of the active ingredients (chemical marker (s)).⁸ In this regard, one of the main challenges encountered in the herbal medicine industry is ensuring unequivocal species identification of the raw material that will be used to manufacture the herbal medicine. There are several plant identification techniques, but in many cases, the identification is based mainly on botanical analysis, that can be problematic due to the high phenotypic variation among taxa, the commercialization of processed raw plant material and/or unidentifiable plant parts and the lack of highly trained professionals in plant taxonomy.^9-11

Trapa natans Linn., is the very significant medicinal plant belong to the family Trapace or Lythraceae. Trapa natans Linn., is also known as Water Caltrop or Water Chest Nut. Trapa natans Linn. is the free-floating plant, grow in ponds and lake in slightly acidic condition and in sunny position.¹² Water chestnut is an aquatic plant with a rosette of floating leaves at the tip of the submerged stem that is generally rooted in the mud. There is only one genus in the Water Chestnut Family, however depending on certain characteristics, up to thirty species have been discovered in our climate. Water-chestnut is an annual aquatic plant having both vegetative reproduction and seed production take place. It reports that seeds of water chestnut are having ample Trapa starch.¹³From fruit, cycloeucalenol and ursolic acid were extracted as chemicals. High concentrations of minerals like calcium, phosphate, iron, magnesium, and manganese can be found in the fruits of Trapa natans Linn. vitamins A, B, C, riboflavin, and nicotinic acid are also present in the fruits' inside. Moreover, this plant's high antioxidant content includes flavonoids, phenol, and flavoin.^14,15Trapa natans Linn. is a key plant in the Ayurvedic medicine. It is used to treat conditions like stomach issues, genitourinary system ailments, liver disorders, kidney damage, and more. According to multiple literature reviews, this herb is also used to treat leprosy, inflammation, diarrhoea, and five other ailments. Several pharmacological studies have demonstrated that Trapa natans Linn. has analgesic, anti-diabetic, immunomodulatory, and nortrophic properties.¹⁶

As per literature survey, it is clear that no systematic pharmacognostic, physicochemical and preliminary phytochemical research has been conducted on the Tarpa natans L. leaf. As a result, the current study examines the macroscopical, microscopical, physicochemical investigation, fluorescence study, extraction in different solvents (petroleum ether (60-80°C), chloroform, acetone, methanol, and water), phytochemical analysis of leaf in order to serve as a reference standard for differentiating the leaf from products containing adulterants.

MATERIALS AND METHODS:

Pharmacognostic Investigation:

Plant material collection and authentication:

Fresh Trapa natans Linn. plants were collected from the Umred Nagpur, Maharashtra, India. The plant was identified at the Botanical Survey of India in Rajasthan, India, under the reference number BSI/AZRC/I.12012/Tech./2019-20 (Pl. Id). The Botanical Survey of India, Arid Zone Regional Centre Jodhpur-342008 Rajasthan has deposited a voucher specimen.

Macroscopic evaluations:

The leaves were appropriately picked, cleansed with water, dried in the shade, and stored for further examination. The plant leaves size, colour, odour, taste, apex, margin, venation, presence or absence of petioles, base surface, lamina, texture, and other characteristics were observed during the macroscopical examination.¹⁷

Microscopic evaluation:

Cross sections of the leaves (the surfacing leaves – submersed leaves) were performed using the manual technique. The sections were stained with phloroglucinol and concentrated hydrochloric acid (1:1). The samples were embedded in glycerine. Histological observations and micrographs were performed with a image analyzer (OLYMPUS-BX51TF, Japan). The microphotographs were obtained from the video camera through a computer. Surface preparation was done for detailed study of nature of stomata and trichromes.^17,18

Determination of physicochemical parameter:

Foreign Matter

Weigh 100g of the original sample and spread it out in a thin layer. Inspect the sample with the unaided eye or with the use of a 6x lens and separate the foreign organic matter manually as completely as possible. Weigh and determine the percentage of foreign organic matter from the weight of the drug taken.¹⁹

Determination of Moisture Content (Loss on Drying):

Dry the evaporating dish for 30min under the same conditions to be used in the determination. Accurately weigh approximately 5g of powder into a tared evaporating dish. For a non-powdered drug, prepare about 10g of the sample by cutting and crushing it so that the pieces are about 3mm thick. Avoid using high speed grinders when preparing the samples and take care that no appreciable amount of moisture is lost during the preparation and that the portion removed is representative of the official sample. Distribute the test sample as evenly as possible to a depth of approximately 5mm in general and no more than 10mm in the case of bulk materials by gentle sideways shaking. Place the loaded bottle in the drying chamber. Dry the specimen for 3hours at 105°C and weigh it. Continue drying and weighing at half-hour intervals until the difference between two consecutive weighings is no more than 0.25 percent.²⁰

Ash values:

The ash levels give some indication of the care that was taken in the collection and manufacture of medicines for the market and the foreign matter content of natural medicines. The aim of the ash value determination is to remove all traces of organic substances that interfere with an analysis of inorganic elements. The residue left after incineration is the ash portion of the drug that is attached to it or intentionally added as a form of adulteration. Various mineral substances such as sand, earth, calcium oxalate, chalk powder or other drugs with different inorganic proportions are often added to the raw drug.^21-23

Determination of Total Ash:

In a tared platinum or silica dish, burn approximately 2g, accurately weighed, of the ground drug at a temperature not exceeding 600°C until carbon free, cool in a desiccator for 30minutes, and weigh immediately. If no carbon-free ash can be obtained in this way, suck off the charred mass with hot water, collect the residue on an ashless filter paper, burn the residue and filter paper, add the filtrate, evaporate to dryness and ignite at a temperature not above 600°C. Calculate the proportion of ash related to the air-dried drug.^21-23

Determination of Acid-insoluble Ash:

25ml of diluted hydrochloric acid are added dropwise to the crucible with the total ash. Collect the insolubles on an ashless filter paper (Whatman 41) and wash with hot water until the filtrate is neutral. Transfer the filter paper with the insoluble components into the original crucible, dry on a hot plate and glow to constant weight. Allow the residue to cool in a suitable desiccator for 30 minutes and weigh immediately. Calculate the acid-insoluble ash content of the air-dried drug.^21-23

Determination of Water-Soluble Ash:

Boil the resulting ash in total ash for 5minutes with 25 ml of water; Collect insoluble in a Gooch crucible or on an ashless filter paper (Whatman 41), wash with hot water and ignite at a temperature not exceeding 450°C for 15minutes. Subtract the weight of the insoluble from the weight of the ash; the weight difference represents the water soluble ash. Calculate the percentage of water-soluble ash based on the air-dried drug.^21-23

Extractive Values:

The extractive values were obtained by exhausting crude drugs are indicative of an approximate measure of their chemical constituents. The solvent used for extraction is in a position to dissolve appreciable quantities of substances desirable. It is employed for material to which yet to suitable chemical or biological assays exist. Percentage of dry extract was calculated in term of air-dried powder drug part.^21-23

Determination of Alcohol-soluble Extractive:

Macerate 5g of the air-dried drug, coarsely powdered, with 100ml of alcohol of specified strength in a closed flask for 24hours, shaking frequently during 6hours and allowing to stand for 18hours. Filter rapidly, taking precautions against loss of solvent, evaporate 25ml of the filtrate to dryness in a tared flat bottomed shallow dish and dry at 105°C, to constant weight and weigh. Calculate the percentage of alcohol-soluble extractive with reference to the air-dried drug.^21-23

Calculation:

If 5gm of air-dried drugs are giving ‘a’ gm of water-soluble residue. Then 100gm of air-dried drug gives (100a)/5gm of water-soluble residue. Water soluble extractive value of the sample is (100 × a)/5%.

Determination of Water-soluble Extractive:

Proceed as directed for the determination of alcohol-soluble extractive, using chloroform water (2.5ml chloroform in purified water to produce 1000ml) instead of ethanol.^21-23

Calculation:

If 5gm of air-dried drugs are gives ‘a’ gm of alcohol-soluble residue. Then 100gm of air-dried drug gives (100 × a) /5gm of alcohol-soluble residue. Alcohol-soluble extractive value of the samples is (100 × a)/ 5%.²¹

Swelling Index:

The swelling index was calculated to know that how much leaf material can swell after putting 1gm drug in water and also to know that material contains some mucilaginous content. After adding water in the leaf material two reading was taken, initial reading and the final reading after 3hours.

Take 1gm of the leaf powder in a 25ml stoppered cylinder. Adding water upto 25ml marking. Shake and keep a side for 24hours. measure the volume occupied by the drug.^22,23

Foaming Index:

Saponins gives persistent foam when shaking with water. Hence crude drugs containing saponins are evaluated by measuring the foaming ability.

Take 1gm crude drugs powder in 500ml conical flask then add 100ml boiling water and boil for 30 min. Cool, filter and collect the filtrate in the 100ml volumetric flask and adjust the volume to 100ml by adding sufficient water. Pour the decoction in to 10 stoppered test tube at 1ml, 2ml, 3ml, etc… up to 10ml. Then adjust the volume of liquid in each test tube to 10ml by adding sufficient quantity of water and stopper the tubes. Shake test tubes in lengthwise motion for 15 seconds. Allow test tubes to stand for 15min and measure the height of the foam.^22,23

Detection of inorganic constituents:

Prepare ash of drug material. Add 50% v/v HCl or 50% v/v HNO₃ to ash. Keep for 1hour or longer. Filter. With filtrate perform the following tests:

Test for calcium:

a) To 10ml filtrate, add 1 drop dil. NH₄OH and saturated ammonium oxalate solution. White precipitate of calcium oxalate forms precipitate is soluble in HCl but insoluble in acetic acid.

b) With a solution of ammonium carbonate gives white precipitate. Which is insoluble in ammonium chloride solution.

Test for magnesium:

a) Filter and separate white calcium oxalate precipitate. Obtained above. Heat and cool the filtrate which with a solution of sodium phosphate in dilute ammonia solution gives a white crystalline precipitate.

b) Gives white precipitate with ammonium carbonate solution but not with ammonium chloride solution.

Test for sodium:

a) To 2ml test solutions, add little uranyl magnesium acetate reagent, shake well and keep for a few minutes. Yellow crystalline precipitate of sodium magnesium uranyl acetate observed.

b) Flame test: Prepare thick paste of ash of drug with conc. HCl. Take on a platinum wire loop. Introduce in bunsen flame. Golden yellow flame is observed.

c) 10ml ash extract + 2ml of potassium pyroanthllollate gives white precipitate

Test for potassium:

a) To 2-3ml test solutions, add few drops sodium cobalt nitrite solution. Yellow precipitate. of potassium cobalt nitrite is observed.

b) Flame test: give violet colour to the flame.

Tests for iron:

a) To 5ml test solutions add a few drops 2% potassium ferrocyanide. Dark blue colouration is observed.

b) To 5ml test solutions, add few drops 5% ammonium thiocyanate (or 5% thiocyanate solution). Solution turns blood red.

Test for sulphate:

a) To 5ml filtrate, add few drops 5% BaCl₂ solution. White crystalline BaSO₄, precipitate. appears, insoluble in HCl.

b) With lead acetate, reagent gives white precipitate. soluble in NaOH.

Test for phosphate:

To 5ml test solutions prepared in HNO₃, add few drops ammonium molybdate solution. Heating for 10min. Cool. Yellow crystalline precipitate. of ammonium phosphomolybdate is observed.

Tests for chloride:

a) To 3ml test solutions prepared in HNO₃, add few drops 10% AgNO₃ solution. White precipitate. of AgCl₂ is observed. Precipitate. is soluble in dil. Ammonia solution.

b) To about 5 to 7ml filtrate, add 3 to 5ml lead acetate solution. White precipitate soluble in hot water is observed. Heating with MnO₂ and H₂SO₄, chlorine is liberated which gives a blue colour with starch solution.

Tests for carbonate:

a) To diluting acid liberates carbon dioxide.

b) Mercuric chloride solution produces a brownish-red precipitate.

c) With solution of magnesium sulphate, white precipitate. Is formed.

Tests for nitrates:

a) Liberates red fumes when warmed with sulphuric acid and copper.

b) With a solution of ferrous sulphate yield no brown colour but if sulphuric acid is added (slowly from the side of the test tube), a brown colour is produced at the junction of two liquids.²⁴

Fluorescence analysis:

Fluorescence analysis of powdered drug were done by placing the dry powder leaf on a slide and treated with various chemical reagents and then observed by the UV and Visible light. The developed colour were noted within 1-2 minutes to avoid drying.^25,26

Extraction with different solvents:

The leaves were collected, dried and powdered using a mechanical grinder. Powdered leaves of Trapa natans Linn. were stored in an air tight container. Coarsely powdered leaves were subjected to successive solvent extraction using Soxhlet's extractor. The powder of leaves was extracted sequentially in petroleum ether (60-80°Ϲ), chloroform, acetone, methanol, and water before being filtered. A rotary evaporator was used to concentrate the extract under vacuum, yielding semi-solid material. This was then dried in a vacuum oven dried to produce a solid residue, which was then stored in the air tight container for future studies.²⁷

Preliminary phytochemical Investigation:

Leaves material is subjected to preliminary phytochemical screening for detection of various chemical constituents.

Test for alkaloids:

To all extracts of leaves dilute hydrochloric acid will be added and filtered. The filtrate will be treated with various alkaloid reagents.

a) Mayer’s test:

The filtrate will be treated with Mayer’s reagent: appearance of cream colour indicates the presence of alkaloids.

b) Dragendroff’s test:

The filtrate will be treated with Dragendroffs reagent: appearance of reddish-brown precipitate indicates the presence of alkaloids.

c) Hager’s test:

i. The filtrate when treated with Hager’s reagent, appearance of yellow colour precipitate indicates the presence of alkaloids.

ii. The test solution was mixed with wagner’s reagent and examined for the formation of reddish-brown precipitate.

Test for carbohydrates and reducing sugar:

The small quantities of the extract will be dissolved in 4ml of distilled water and filtered. The filtrate will be subjected to.

a) Molisch’s test:

A small portion of the filtrate will be treated with Molisch’s reagent and sulphuric acid. Formation of a violet ring indicates the presence of carbohydrates.

b) Fehling’s test:

The all extracts will be treated with Fehling’s reagent A and B. The appearance of reddish brown colour precipitate indicates the presence of reducing sugar.

c) Barfoed’s reagent:

Barfoed’s reagent cupric acetate in acetic acid, is slightly acidic and is balanced so that is can only be reduced by monosaccharides but not less powerful reducing sugar. Disaccharides may also react with this reagent, but the reaction is much slower when compared to monosaccharides. Perform this test with glucose, maltose and sucrose.

Test for steroids:

Liebermann burchard’s test:

The all extracts will be treated with 3ml of acetic anhydride. Few drops of glacial acetic acid followed by a drop of concentrated sulphuric acid. Appearance of bluish green colour indicates the presence of steroids.

Test for proteins:

a) Biuret test:

The all extracts will be treated with copper sulphate solution, followed by addition of sodium hydroxide solution; appearance of violet colour indicates the presence of proteins.

b) Millon’s test:

The all extracts will be treated with Millon’s reagent; appearance of pink colour indicates the presence of proteins.

Test for tannins:

The all extracts will be treated with 10% lead acetate solution; appearance of white precipitate indicates the presence of tannin.

Test for phenolic compounds:

The all extracts will be treated with neutral ferric chloride solution; appearance of violet colour indicates the presence of phenolic compounds.

The all extracts will be treated with 10% sodium chloride solution; appearance of cream colour indicates the presence of phenolic compounds.

Test for flavonoids:

5ml of all extracts will be hydrolyzed with 10% sulphuric acid and cooled. Then, it will be extracting with diethyl ether and divided in to three portions in three separate test tubes. 1ml of diluted sodium carbonate, 1ml of 0.1N sodium hydroxide and 1ml of strong ammonia solution will be added to the first, second and third test tubes respectively. In each test tube. Development of yellow colour demonstrated the presence of flavonoids.

Shinoda’s test:

All extracts will be dissolved in alcohol, to which few magnesium turnings will beaded followed by concentrated HCL drop wise and heated and appearance of magenta colour shows the presence of flavonoids.

Test for gums and mucilage:

All extracts was treated with 25ml of absolute alcohol and filtered. The filtrate will examine for its swelling properties.

Test for glycosides:

When a pinch all extracts were treated with glacial acetic acid and few drops of ferric chloride solution, followed by the addition of conc. Sulphuric acid, formation of a ring at the junction of two liquids indicates the presence of glycosides.

Test for saponins:

Foam test:

About 1ml of the all extracts was diluted to 20ml of with distilled water and shaken well in a test tube. The formation of foam in the upper part of the test tube indicates the presence of saponins.

Test for Triterpenoids:

All extracts was warmed with tin and thionyl chloride. Pink colour indicates the presence of triterpenoids. ^28,29

RESULT AND DISCUSSION:

Pharmacognostic investigation:

Macroscopic characters:

Trapa natans Linn. is an floating annual aquatic plant with two types of leaves: finely divided feather-like submerged leaves that grow along the length of the stem and undivided floating leaves that grow in a rosette at the water's surface. The floating leaves are rhomboid, fan-shaped, and serrated on the edges, measuring 2-6.5cm in diameter, broader than long, denticulate, denate, serrate, or incised with whole base, sharp apex, and thickly pubescent or villous beneath. The following characters were observed on macroscopic observation of Leaves a) Color: Dark Green b) Odor: Characteristic c) Taste: Bitter d) Shape: rhomboid, fan-shaped finely divided feather-like submerged leaves e) Size: 2-6.5cm diameter, broader than long.

Figure 1: Trapa natans Linn. plant and dried leaves

Microscopic characters:

Cross section of the surfacing leaf lamina reveals the following structures: epidermis, mesophyll and vascular system (midrib). The lower epidermis is differentiated into an upper and a lower epidermis, each consisting of a single layer of compactly arranged thin-walled barrel-shaped cells. Externally, both types of epidermis are covered by a thin cuticle. Numerous stomata occur among the upper epidermal cells in accordance with its free-floating nature. Remarkable is the presence of many-celled hairs among the lower epidermal cells.

Figure 2: Transverse section of Trapa natans Linn. Leaves

A - The midrib vein (10X),; B - Portion of vascular bundle (10X),; C - Portion of Lamina(10X),; D – Portion between cutical and vascular bundles (45X); E – Upper epidermis and cutical portion (10X); F - Portion of lower epidermis with trichome (10X): AC– air chamber; VB – vascular bundle; Cu – cuticle; LE – lower epidermis; Ph – phloem; Xy- xylem; PC – parenchyma cell; UE – upper epidermis, T – trichome.

The spongy spongy region is represented by small and large air cavities. The air cavities are separated by a uniseriate partition. The vascular system is poorly represented by small vascular bundles. Each bundle is composed of phloem and poorly developed xylem represented by a large lacuna. The aerenchyma cells contain starch grains and numerous calcium oxalate crystals (druses), mostly facing the cavities. Shown in the Figure 2 and 3.

Figure 3: Surface preparation of Trapa natans Linn. Leaf

Powder was dark green color with characteristic odor. From the microscopy observation of powder drug it was found that Trapa natans Linn., leaves contain anomocytic stomata, multicellular trichomes. Prismatic calcium oxalate crystal, lignified vessel, starch parenchyma cell, cork and air chamber present in powder drug. Shown in the Figure 4.

Figure 4: Vessel, Calcium oxalate crystal, Starch, Parenchyma cell, Multicellular trichomes, Cork and Aerenchyma present in Trapa natans Linn. leaves powder

Physicochemical investigation:

The leaves powder of Trapa natans Linn. were evaluated for physicochemical parameters. Results are presented in Table 1. Ash values and extractive value used to detect adulteration and identification of leaf. Ash values mostly give idea about the presence of inorganic earthy matter/ impurities present along with drugs. While extractive values are useful for the determination of exhausted or adulterated drugs. Extractive value also helps in indication of the chemical nature of phytochemicals which is present in leaves and also help to check solubility of phytochemical (which solvent is used for extraction purpose). Moisture content indicates the presence of moisture in drugs which are helping for storage of crude drugs.

Table 1: Result of Physicochemical parameter

Sr. No.	Physicochemical parameter	% w/w
1	Foreign Matter	0.2
2	Loss on drying	6.5
3	Total ash	8.5
4	Acid insoluble ash	2.5
5	Water soluble ash	4.5
6	Water soluble extractive value	5.6
7	Alcohol soluble extractive value	3.2
8	Swelling Index	Nil
9	Foaming Index	111.11

Qualitative tests for determination of inorganic elements:

Inorganic components such as calcium, magnesium, potassium, sulphate, phosphate and carbonate were found in leaves as shown in Table 2.

Table 2: Result of Qualitative Test for Inorganic components

Sr. No.	Elements	Results
1	Calcium	+
2	Magnesium	+
3	Sodium	-
4	Potassium	+
5	Iron	-
6	Sulphate	+
7	Phosphate	+
8	Chloride	-
9	Carbonate	+
10	Nitrates	-

Note: ‘+’ Indicate Present and ‘-’ Indicate Absent

Fluorescence analysis:

Florescence analysis of the powder drug were determine by treating the powder drug with deferent chemical reagents and observed under UV and visible light. Fluorescence analysis result are given in Table 3

Table 3: Fluorescence Characteristic of Powder Leaves

Sr. No.	Materials and treatments	In fluorescence light	In visible light
	Leaves powder
1	Powder as such (Scraped on filter paper with powder)	Green colour fluorescence	Brown colour
2	Treated with 50% nitric acid	Green colour fluorescence	Light Brown colour
3	Treated with 5% potassium hydroxide in water	Dark Brown Fluorescence	Dark brown
4	Treated with 1N hydrochloric acid	Pale green fluorescence	Pale brown
5	Treated with dilute 80% sulphuric acid	Dark green fluorescence	Brown
6	Treated with antimony trichloride	Light brown fluorescence	Brown
7	Treated with acetone	Light green fluorescence	Black
8	Treated with methanol	Black	Brown colour
9	Treated with ethanol	Black	Brown colour

Extraction:

Extraction was carried out on leaves powder by using different solvent and distill water. The extractive values obtained are shown in Table 4. Leaves give highest extractive value in distilled water (25.18%) followed by methanol (15.52%), chloroform (19.12%), acetone (9.20%) and the lowest in petroleum ether (5.25%).

Table 4: Result of percentage extractive values of leaf of Trapa natans Linn. with different solvent

Extract	% yield (w/w)
Petroleum ether (60-80ºC)	0.80
Chloroform	8.64
Acetone	14.00
Methanol	5.60
Water	22.72

Preliminary phytochemical investigation:

Preliminary phytochemical analysis results are shown in Table 5. Trapa natans Linn. leaf contains phytochemicals such as carbohydrate, alkaloids, protein, amino acid, steroid, terpenoids, flavonoids, tannin, and phenolic compounds. As a result of the below findings, we conclude that methanol and water extracts contain maximum phytochemicals when compared to other extracts like petroleum ether, chloroform and acetone. Hence methanol and water extracts of leaves can be taken for further research.

CONCLUSION:

Trapa natans Linn. belong to the family Trapaceae, which is an annual aquatic floating plant with two types of leaves: finely divided feather-like submerged leaves that grow along the length of the stem and undivided floating leaves that grow in a rosette at the water's surface. In the current study leaves were taken for systematic pharmacognostic, physicochemical and preliminary phytochemical research. The results of this study concluded that the presences of various phytoconstituents in Trapa natans Linn. leaves which may be medicinally useful. Hence, further publishing monograph on leaves and Trapa natans Linn. leaves can be taken up.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

Authors wish to thank Nagpur College of Pharmacy. Maharashtra (India) for providing facilities to conduct this research work.

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Received on 13.07.2023 Modified on 17.08.2023

Research J. Pharm. and Tech 2023; 16(9):4341-4349.

DOI: 10.52711/0974-360X.2023.00711

Sr. No.	Phytochemical	Leaves Extract
Sr. No.	Phytochemical	Petroleum ether (60-80ºϹ)	Chloroform	Acetone	Methanol	Water
1	Alkaloids
	Dragendroff’s test	-	+	+	+	+
	Wagners test	-	+	+	+	+
	Mayers test	-	+	+	+	+
	Hagers test	-	+	+	+	+
2	Protein	-	-	-	-	-
3	Amino acids	-	-	-	-	-
4	Glycosides
	Legal’s test	+	+	+	+	+
	Keller-killani test	+	+	+	+	+
	Brontrager’s test	+	+	+	+	+
5	Flavonoids
5	Shinoda test	-	-	-	+	+
6	Carbohydrate
	Molish test	+	+	+	+	+
	Fehling test	+	+	+	+	+
	Benedicts test	+	+	+	+	+
	Bradford’s test	+	+	+	+	+
7	Steroid and triterpenoids
	Libermann-buchard test	+	+	+	+	+
	Salkowaski test	+	+	+	+	+
8	Tannins and Phenolic	-	+	+	+	+
9	Gums	-	-	-	-	-
10	Fixed oils	-	-	-	-	-
11	Saponins
11	Foam test	-	-	-	-	+