INTRODUCTION:

The vegetables are the chief and they are extremely useful for human health and in the presence of illness and diseases. The use of vegetables for medicinal purposes is still important for primary healthcare even at a time when modern medicine is widely accepted.¹ In the present studies the indian tree of C. auriculatum shown in figure 1, we have focus our investigations on one of the frequently available plant in Maharashtra i.e. Cyphostemma auriculata (Roxb.) having family Vitaceae. C. auriculatum (Roxb.) is medicinally important plant which is used by tribals Maharashtra. Cyphostemma species is reported for the treatment of vomiting, typhoid, nausea, flu and fatigue, headache or as beauty products and also to cure of liver damage. Some people use it for polishing precious metals and jewellery by rubbing the fresh leaves over the surface.^3,4,5

The results of every study done on traditional medicines need to be documented. Due to this weakness, it is important to ensure the standardisation of plants and plant parts for use in medications. To accomplish our goal in a staged manner using the standardization method, we can employ a variety of methodologies such as pharmacognostic, phytochemical and in vitro antioxidant activity was study on petroleum ether and ethanolic extract. These procedures and actions aid in the standardization and identification of plant material. An important element to ensuring the reproducible quality of herbal medicine, which will help us to establish its safety and efficacy, is the detailed categorizing and quality assurance of preliminary material.^5,6,7

Classification of this plant is as follows-

Figure 1. Cyphostemma auriculatum (Roxb.) Plant

MATERIALS AND METHODS:

Chemicals:

All analytical grade chemicals used in this study were purchased from modern science Nashik, Maharashtra, conc. Hydrochloric acid, chloral hydrate, Phloroglucinol, H₂SO₄, NaOH, FeCl₃, Distilled water, glycerine water, potassium hydroxide etc.

Procurement of plant: The fresh plant of C. auriculatum Roxb. will be collected from area of Trimbakeshwar, District Nashik. The plant material collected during the flowering and fruiting season for proper botanical identification. The plant material was identified by Botanical survey of india, western regional centre, koregaon road, pune with certification no. BSI/WRC/Iden,Cer./2021/1111210001787

Processing of the plant material: The whole plant was washed and 3/4th part was shade dried and then pulverized while 1/4th part was subjected to separation of leaves.

Pharmacognostic study:

Macroscopy, Microscopy and powder characteristics are performed according to Khandelwal and Mukherjee. (Khandelwal, 2008) (Mukherjee, 2019)

Macroscopic and organoleptic Characterization:

Macroscopic parameters of leaves were noted for colour, Odour, taste, shape and texture. Leaves evaluation include absence or presence of petioles and different characters of lamina which as shown in table 1.

Microscopic evaluation:

Transverse sections of the plant's leaves were cut out, stained properly, and mounted in accordance with standard operating procedure after photomicrographs were taken. Every assessment was carried out using a compound microscope.

Powdered microscopy:

Shade-dried leaves taken and make fine powdered and examined under a microscope. On each slide, a small amount of leaf powder was placed individually. Then, 2-3 drops of chloral hydrate were added, and the slides were stained as well. Each slide had a cover slip applied before being inspected under a microscope. Various cell components observed such as epidermis, stomata, covering trichomes and calcium oxalate crystal were photographed taken using a digital camera.

Evaluation of Analytical Parameter:

The evaluation of physicochemical properties was carried out in accordance with the methodologies recommended by the Indian Pharmacopoeia (2010) and the World Health Organization's guidelines for quality control of medicinal plant materials.

Ash value:

Determination of total ash:

About 2 g of dried crude drug was weighed accurately in a tared silica crucible and incinerated at a temperature not exceeding 450^oC until free from carbon. It was then cooled in desiccator and weighed. The percentage of ash was calculated with reference to the air dried drug.

Determination of water-soluble ash:

The total ash was boiled for five minutes with 25 mL of water. The insoluble matter was collected in an ash less filter paper, washed with hot water and ignited for 15 minutes at a temperature not exceeding 450^oC. The weight of the insoluble matter was subtracted from the weight of the ash and the difference in the weight of the ash represents the water-soluble ash. The percentage of water-soluble ash was calculated with reference to the dried drug.

Extractive values:

Determination of alcohol soluble extractive:

About 5 g of the powder was macerated with 100 mL of alcohol of the specified strength in a closed flask for 24 h, shaking frequently for 6 h and allowed to stand for 18 hr. It was filtered rapidly, and 25 mL of the filtrate was evaporated to dryness at 105^oC and weighed. The percentage of alcohol soluble extractive was calculated with reference to the dried drug.

Determination of water-soluble extractive:

About 5 g of the powder was macerated with 100 mL of distilled water in a closed flask for 24 hr, shaking frequently for 6 hr and allowed to stand for 18 hr. It was filtered rapidly and 25 mL of the filtrate was evaporated to dryness at 105^oC and weighed. The percentage of water-soluble extractive was calculated with reference to the dried drug.

Loss on drying:

Loss on drying is the loss of mass expressed as percent w/w and was determined by the following procedure:

About 2 g of drug was weighed and transferred to a dry stoppered weighing bottle. The weight of the bottle and the drug was taken accurately. After removing the stopper, the bottle containing drug was placed in an oven for 1 h at 120^oC. After 1 hr, the bottle was removed and cooled in a desiccator and weighed by replacing the stopper which was continued until difference between two successive weighing was not more than 0.25 % of constant weight.

Preliminary phytochemical analysis:

The leaves of the plant were shade dried, coarsely powdered, and extracted with petroleum Ether and Ethanol were subjected to the following chemical tests for identification of phytochemical constituents.^8,9,10

Tests for alkaloids:

(a) Dragendorff’s test, An orange-red precipitate was produced by adding 1 mL of Dragendorff's reagent to 2 mL of extract, confirming the presence of alkaloids.

(b) Mayer’s test, To 1 mL of extract, a few drops of Mayer's reagent were added. Alkaloids have been identified because a yellowish or white precipitate formed.

(c) Hager’s test, Extract were treated with few drops of Hager’s reagent. A yellow precipitate was formed, indicating the presence of alkaloids.

Tests for flavonoids:

(a) Alkaline reagent test, To 2 mL of the extract, two to three drops of sodium hydroxide were added.

After adding a few drops of diluted HCL, the initial strongly yellow colour gradually becomes colourless, indicating the presence of flavonoids.

(b) Shinod’s test, 1 mL of the extract was mixed with ten drops of diluted HCl and a piece of magnesium; the resultant deep pink colour indicated the presence of flavonoids.

Test for phenolic compounds and tannins:

(a) Ferric chloride test, 1 mL of the extract was mixed with 2 mL of a 5% neutral ferric chloride solution, and the dark blue colouring indicated the presence of tannins and phenolic chemicals.

(b) Lead tetra acetic acid test, The presence of phenolic compounds and tannins was detected when one mL of lead tetra acetate solution was treated with 0.5 mL of the extract.

Tests for proteins:

(a) Biuret test, In 1 mL of extract, two drops of 3% copper sulphate and a few drops of 10% sodium hydroxide were added; an appearance of violet or red colour indicated the presence of proteins. (b) Ninhydrin test. To 1 mL of extract, two drops of a freshly prepared 0.2% ninhydrin solution were added. Purple colour indicates the presence of proteins.

Test for carbohydrates:

(a) Molish test, To 2 mL of extract, a few drops of an alcoholic a-naphthol solution were added. A few drops of concentrated H₂SO₄ were subsequent to added to the test tube walls. A violet coloured ring developed where two liquids junction, indicating the presence of carbs.

(b) Benedict’s test. The presence of carbohydrates was determined by adding 8–10 drops of extract to 5 mL of Benedict's reagent, heating it for 5 minutes, and then observing the dark red precipitate that formed.

(c) Fehling’s test, The presence of carbohydrates was determined by adding an equivalent volume of Fehling's (A and B) solution to 2 mL of extract and heating it for 5 minutes. The resulting crimson/dark red precipitate was obtained.

Tests for glycosides:

Keller Killiani test,The extract was combined with 0.5 mL of a solution that contained glacial acetic acid and a few drops of ferric chloride. 1 mL of concentrated H₂SO₄ was then added along the test tube's walls. The presence of cardiac glycosides was revealed by the emergence of a deep blue colour at the intersection of two liquids.

Tests for saponins:

The 5 mL of extract in a test tube was mixed with one drop of Na₂CO₃ solution. It was shaken vigorously and then allowed to rest for five minutes. Saponins were detected by the formation of foam.

Test for triterpenoids:

Horizon test, 1 mL of the extract was mixed with 2 mL of trichloroacetic acid. The development of a red precipitate provided indication that terpenoids were present.

Test for steroids:

Salkowski test, when concentrated H₂SO₄ was added along the test tube walls and the test extract was shaken with chloroform, a red colour developed, indicating the presence of steroids.

Estimation of Total Phenolic Content:

(Folin-Ciocalteu method):

The total phenolic content was estimated using the Folin-Ciocalteu method. A stock solution of the pet ether and ethanolic extract was prepared by dissolving 10 mg of the each extract in 10 mL of distilled water, resulting in a concentration of 1000 μg/mL respectively. This stock was further diluted to obtain concentrations of 500 μg/mL and 750 μg/mL for pet ether and ethanolic extract.

A standard stock solution of gallic acid (100 μg/mL) was prepared by dissolving 1 mg of gallic acid in 10 mL of distilled water. From this, serial dilutions were made to prepare standard solutions at concentrations of 10, 20, 40, 60, 80, and 100 μg/mL.

For both standard and each of extract samples, 0.5 mL of the solution was mixed with 2 mL of 10% Folin-Ciocalteu reagent after 5 min followed by 2 mL of 7.5% sodium bicarbonate solution was added. The mixture was incubated in the dark at room temperature for 30 minutes. Absorbance was then measured at 765 nm using a UV-Vis spectrophotometer, against a blank.

All measurements were performed in triplicate. A calibration curve was constructed using the absorbance values of the standard gallic acid concentrations. The total phenolic content in the extract was calculated from the linear regression equation and expressed in terms of mg gallic acid equivalents (GAE) per gram of extract.¹²

To calculate Total Phenolic Content (expressed as mg Gallic Acid Equivalents (GAE) per g of extract), use the following formula: ¹³

C = C1 × V/m

Where,

C= Concentration of phenolic from the calibration curve (µg/mL or mg/mL)

C1 =concentration of Gallic acid established from the calibration curve in mg/ml,

V= Volume of extract used in the reaction (mL)

m = weight of the plant extract in g.

In vitro Antioxidant Study:

After preliminary phytochemical screening of Petroleum ether and ethanolic extract, several concentrations ranging from 20-100 μg/mL of C. auriculatum were tested for their antioxidant activity through DPPH free radical scavenging activity.

DPPH free radical scavenging activity:

The free radical scavenging activity was measured in terms of hydrogen donating or radical scavenging ability using the stable radical DPPH. For preparing DPPH solution about 0.1 mM solution of DPPH in methanol is taking. 1 mL of DPPH solution was added to 3 mL of the different concentration PECA and ECA extract (20-100 μg/mL) separately in different test tubes. 1 mL of DPPH solution was added to standard Ascorbic acid solution of varying concentrations ranging from 20-100 μg/mL separately in different test tubes. This control solution (a solution without the test compound, but with an equivalent amount of methanol) used was also allowed to react with 1 mL of the DPPH solution separately in different test tubes. These mixtures were shaken and allowed to stand at room temperature for 30 min and the absorbance was measured at 517 nm using a spectrophotometer. The IC50 value (50 % of inhibitory concentration in μg/mL) of the crude extract and its fractions were compared with that of vitamin E, which was used as the standard. When the DPPH radical is scavenged, the colour of the reaction mixture changes from purple to yellow with decreasing of absorbance at wavelength 517 nm. Decrease in absorbance of the reaction mixture indicates higher free radical scavenging activity.^14,15,16

The percentage inhibition of DPPH radical was calculated using the formula:

Absorbance of control -Absorbance of test

Percentage inhibition (%) = -------------------------- x 100

Absorbance of control

RESULT AND DISCUSSION:

Table 1: Result of Macroscopy

Part of Plant	Observations
Leaves	Leaflets are ovate with wedge shape base.
	Margins are toothed.
	Lamina is elliptical, ovate or broadly ovate.
	Terminal leaflets obovate to rhomboidal or elliptical.
	Arrangement of leaves is five foliolate.

Microscopical Investigation:

The upper epidermis is composed of polygonal cells and typically lacks both stomata and covering trichomes, except in the region of the midrib, where trichomes may be occasionally present. The lower epidermis, however, contains anomocytic stomata and is densely covered with multicellular trichomes, typically ranging from 2 to 10 cells. A cross-sectional view of the leaf shows a cutinized epidermis, beneath which lies a single layer of palisade parenchyma cells directly under the upper epidermis. The mesophyll is distinctly differentiated into one row of chloroplast-rich palisade cells and 7–8 layers of spongy parenchyma cells, which are generally isodiametric or irregular in shape.

Some spongy parenchyma cells contain needle-like (acicular) calcium oxalate crystals arranged in clusters. Additionally, both the mesophyll and midrib regions exhibit a considerable presence of rosette-type calcium oxalate crystals.

In the midrib region, a well-developed vascular bundle is evident, surrounded by parenchymatous cells embedded with rosette crystals. Both upper and lower epidermal layers are present in this area, under which lies a band of collenchyma tissue. These structural details are depicted in Figures 2 and 3.

Figure 2: Transverse section (T.S) of part of leaf passing through midrib

Figure 3: T.S. of part of lamina showing upper epidermis, palisade cells, mesophyll, lower epidermis and covering trichomes

Powder Characterization:

Powdered leaf material was treated with glycerine and stained using a combination of phloroglucinol and hydrochloric acid for microscopic analysis. The examination of powder characteristics revealed the presence of leaflet epidermal cells, anomocytic stomata, pitted, lignified vessels, and covering trichomes are showed in figure 4.

Figure 4: (1a- Upper epidermis in surface view and underlying palisade cells, 2a- Lower epidermis in surface view showing stomata, 2b- Lower epidermis in surface view showing stomata and trichomes, 3a- A part of a leaf showing covering trichomes and rows of cluster crystals of calcium oxalate, 3b- Covering trichomes, 4a- Rows of cluster crystals of calcium oxalate, 4b- Cluster crystal of calcium oxalate, 4c- Bunch of acicular crystals of calcium oxalate)

EVALUATION OF ANALYTICAL PARAMETERS:

Ash Value:

Ash values are helpful in determining the quality and purity of a crude drug, especially in the powdered form. The ash content of a crude drug is generally taken to be the residue remaining after incineration. It usually represents the inorganic salts naturally occurring in the drug and adhering to it, but it may also include inorganic matter fielded for the purpose of adulteration.²⁰The total ash, the water-soluble ash and Acid-insoluble ash determined for the powdered drug of C. auriculatum (Roxb.) is listed in Table 2.

Extractive value:

Extractive value of crude drugs is useful for their evaluation, especially when the constituents of a drug cannot be readily estimated by any other means. These values are indicative of approximate measures of their chemical constituents and the nature of the constants present in crude drugs.²¹ The water-soluble and alcohol soluble extractive values determined for the powdered drug of C. auriculatum (Roxb.) are listed in Table 2. The extractive values of the dried crude drug of C. auriculatum (Roxb.) were found to be less than 19 % approximately.

Table 2: Estimation analytical parameters

Sr. No.	Parameters	Values (% W/W)
1.	Total Ash	7.96
2.	Water-soluble ash	0.90
3.	Acid-insoluble ash	1.84
4.	Water-soluble extractive value	10.02
5.	Alcohol-soluble extractive value	19.35
6.	Loss on drying	2.16

Loss on drying:

From the loss on drying study listed in Table II, it is evident that the powdered drug of C. auriculatum (Roxb.) can hold moisture of ~2 % w/w.

Preliminary phytochemical screening of Petroleum ether and ethanolic extracts:

The petroleum ether and ethanolic extract of C. auiculatum (Roxb.) were tested for various phytochemicals and the results are presented in Table 3

Table 3: Preliminary phytochemical screening

Sr. No.	Phytochemical	Petroleum ether extract (PECA)	Ethanol (ECA)
1.	Alkaloid	-	+
2.	Glycoside	+	-
3.	Flavonoid	-	++
4.	Tannin	+	+
5.	Carbohydrates	+	+
6.	Proteins	+	++
7.	Steroids	+	++
8.	Terpenoids	+	++
9	Saponins	-	+

+ Presence and – Absence

Total Phenolic Content (Folin-Ciocalteu method):

Table 4 displays the absorbance for different concentrations of standard gallic acid (10-100 µg/mL) while Figure 5 illustrates the corresponding calibration curve generated from these data and Total Phenolic Content in Table 5.

Table 4: Calibration of Gallic Acid (Standard)

Sr. No.	Standard	Concentration (μg/ml)	Absorbance
1.	Gallic acid	10	0.265± 0.024
2.		20	0.499± 0.056
3.		40	0.745± 0.028
4.		60	0.985± 0.068
5.		80	1.164± 0.064
6.		100	1.325± 0.042

Values are given as Mean±SD. The observations are repeated in triplicate.

Figure 5: Calibration curve of standard Gallic acid

Table 5 Total phenolic content of the C. auriculatum Roxb.

Sample solution	Phenolic Content(μg/ml)
Pet ether Extract	82.56
Ethanolic Extract	46.69

DPPH free radical scavenging activity

Figure 6: Graphical representation of DPPH free radical scavenging activity results of C. auriculatum Roxb.

Control, STD- Standard (Ascorbic acid), PECA- Petroleum extract of C. auriculatum, ECA- Ethanolic extract of C. auriculatum (Values are shown as mean ±SEM, n=3, (*p < 0.05, ***P < 0.001)as compared against standard ANNOVA followed by Dunnet’s ‘t’ test)

The ethanolic extract of C. auriculatum Roxb. exhibited strong DPPH free radical scavenging activity in a concentration-dependent manner. At 100 µg/mL, it showed 87.25 % inhibition, which was comparable to the standard antioxidant, ascorbic acid, which exhibited 93.47% inhibition at the same concentration. The calculated IC₅₀ value for the Pet ether extract 91.32 µg/mL and ethanolic extract was approximately 52.24 µg/mL, while that of ascorbic acid was 33.56 µg/mL, indicating that the ethanolic extract possesses significant antioxidant potential, likely due to its high content of phenolic and flavonoid compounds. The antioxidant activity was depending on dose as increases activity also increases. Fig. 5 gives the data for graphical representation of radical scavenging activity. And the results of DPPH free radical scavenging activity listed in Table 5.

Table 5: DPPH free radical scavenging activity results

Sr. no.	Concentration μg/mL	% Inhibition
Sr. no.	Concentration μg/mL	STD	PECA	ECA
1.	20	42.56± 1.25	12.57± 1.56	31.25± 1.36
2.	40	61.58± 1.28	21.25± 1.45	49.58± 1.25
3.	60	76.85± 1.27	32.58± 1.25	65.47± 1.65
4.	80	88.56± 1.25	43.89± 1.65	78.45± 1.42
5.	100	93.47± 1.56	52.68± 1.58	87.25± 1.25
IC50		33.56	91.32	52.24

DISCUSSION:

The goal of the study was to investigate the in-vitro antioxidant activity of C. auriculatum Roxb and standardise the plant according to physicochemical and phytochemical estimations. The leaf's transverse section indicates that the upper epidermis has polygonal cells. In the lower epidermis, it additionally shows the presence of anomocytic stomata with covering trichomes. It also consists of calcium oxalate crystals and parenchymatous cells. Powder microscopy revealed the presence of trichomes, stomata, calcium oxalate crystals.

The analytical parameters such as ash value, extractive value, loss on drying were studies. The phytochemical investigation of extracts indicates a presence of alkaloids, glycosides, flavonoids, tannins, carbohydrates, proteins, steroids, terpenoids and saponins etc.

The total phenolic content was estimated using the Folin-Ciocalteu method. The Pet ether and Ethanolic extracts of 46.69 and 82.56 μg/ml.

The in-vitro antioxidant DPPH free radical scavenging activity was determined by the decrease in its absorbance as concentration increases at 517nm which induced antioxidants. ethanolic extract shows the most significant activity when it compare to standard.

CONCLUSION:

As one of the standardizing parameters, this study provides a detailed description of the morphology, microscopy, and physicochemical characteristics of the powdered drug of Cyphostemma auriculatum Roxb. Additionally, analytical parameters and phytochemical investigations were carried out. The in vitro antioxidant activity study demonstrated that the ethanolic extract exhibited the most significant free radical scavenging activity when compared to the standard. Furthermore, the total phenolic content was found to be highest in the ethanolic extract, which correlates with its strong antioxidant potential. These findings suggest that the phenolic compounds may contribute significantly to the observed activity. Further research involving the isolation and characterization of individual phytoconstituents.

ACKNOWLEDGEMENT:

The Author is thankful to the Principal and Faculty of MVP’s College of Pharmacy for providing facility and support to carry out the study.

CONFLICT OF INTEREST:

None.

AUTHORS CONTRIBUTION:

Ashlesha Wakchaure has done research under the guidance of Dr. Manoj Kumbhare.

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Received on 05.02.2025 Revised on 09.05.2025

Accepted on 12.08.2025 Published on 05.09.2025

Available online from September 08, 2025

Research J. Pharmacy and Technology. 2025;18(9):4047-4054.

DOI: 10.52711/0974-360X.2025.00581

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Pharmacognostic Standardization and Correlation of Total Phenolic Content with Antioxidant Activity in Cyphostemma auriculatum Roxb.