Pharmacognostical parameters, sphytochemical screening, TPC, TFC and evaluation of in-vitro anti-oxidant activity of leaves of Cymbidium aloifolium L (SW)

Tahid Alam*, Bapi Ray Sarkar, Pooja Sharma Luitel, Arup kumar Gorai

Department of Pharmaceutical Technology, University of North Bengal,

Raja Rammohunpur, Darjeeling, West Bengal-734013, India.

*Corresponding Author E-mail: tahidalam108@gmail.com

ABSTRACT:

Natural drugs are one of the most important needs in daily life and their history is as old as human civilization. Orchids are nature's most beautiful and widely dispersed flowering plant groupings. Cymbidium aloifolium (L) SW family (Orchidaceae) is an epiphytic herbaceous orchid. The orchidaceae family is one of the oldest and largest flowering plant families in the world, with over 800 genera and between 25000 and 35000 species. The current study aims to examine in vitro antioxidant activity from different solvent extracts of Cymbidium aloifolium (L) SW leaves. The examination was conducted by several factors, including pharmacognostical, physicochemical, and phytochemical details of the leaf extracts of Cymbidium aloifolium (L) SW, which plays a vital role in laying down standardization and pharmacopoeia parameters. The antioxidant capacity of various solvent extracts of Cymbidium aloifolium (L) SW leaf extract was investigated using ferric reducing antioxidant potential (FRAP) tests and DPPH radical scavenging activities. A microscopic investigation exposes the leaf's cellular structure. To standardize the aim, physicochemical investigations were established, including swelling index, fluorescence analysis, total ash, acid insoluble ash, water soluble ash, and loss on drying. Antioxidant potential was determined by comparing the results with ascorbic acid standard. Leaves of Cymbidium aloifolium (L) SW methanolic extract showed the best result, followed by chloroform, ethyl acetate, pet ether, and water. These findings support the identification of Cymbidium aloifolium (L) SW and quality control measures. Further investigation is required to identify the specific phytochemical and the way in which it generates antioxidant activity.

KEYWORDS: Cymbidium aloifolium, Orchidaceae, Epiphytic, Pharmacognostic parameter, Antioxidant.

INTRODUCTION:

Nature, which holds commendable importance, is a source of wellness and goodness for humankind. Natural drugs are one of the most important needs in daily life and their history is as old as human civilization. The history of herbal plants has been known since decades which were medicinally used in India, China, Greece and Egypt and until today most part of the universe still depends on indigenous system of medicine¹. Orchids are nature's most beautiful and widely dispersed flowering plant groupings on the planet.

According to Vagbhata, Sushruta and Charaka orchids were used as medicine in Ayurvedic, Siddha, and Unani systems of medicine^2,
3. Use of various metabolites from extracts of different species of orchids relates to several health benefits which includes anti-inflammatory, anti-rheumatic, diuretic, hypoglycemic, anti-microbial, relaxation, anti-carcinogenic, antivirus, anticonvulsive, and neuroprotective properties⁴. Many orchids are used as diet and herbal drugs in diverse parts of universe by several different cultures and tribes even though orchids are primarily grown as ornaments^5,6. Cymbidium aloifolium (L) SW or Aloe-leafed cymbidium, home-grown to Asia and member of Orchidaceae family is an epiphytic herbaceous species of orchid. It is mainly found in Southeast Asia and China, having originated in Burma to Sumatra, one of the world's oldest and largest flowering plant families, with over 800 genera and between 25000 to 35000 species⁷. It can be found growing between rocks or on another plant an altitude ranging from 300m to 2900m⁸. From ages the Orchidaceae family has enormous therapeutic activity and is used till date⁹ for curing skin diseases, wounds, ear ache, fever, paralysis, vertigo, joining fractured bones, boils and weakness of eyes¹⁰. Based on the prerequisite for evaluation of phytoconstituent from indigenous medicine various pharmacological, phytochemical screening, and analytical methods is required¹¹. The first step in this direction is the characterization of the herb, or a thorough pharmacognostic examination that provides the physical characteristics, exterior appearance, and microscopy of the crude medication^12,
13. The present study therefore comprises analyses of the leaves' anatomy and structure as well as evaluations of their physicochemical parameters (loss on drying, foreign matter, ash values, extractive values), fluorescence, preliminary phytochemical properties, total phenolic contents, total flavonoid contents, and in-vitro antioxidant evaluation.

MATERIALS AND METHODS:

Collection and authentication of the plant:

The plant was collected from the University of North Bengal, Siliguri, West Bengal on October, 2021 in, it was then authenticated at the Botanical Survey of India, Gangtok, Sikkim with specimen no is -09-NBU-2022

Chemicals and reagents:

Analytical grade reagents and chemicals were used for the research work.

Pharmacognostic evaluations:

Morphological evaluation:

Five samples of leaves were taken to conduct the morphological evaluation of the plant, and according to the data provided by different books, the taxonomic description was depicted^{12, 13}.

Microscopic evaluation:

To perform microscopic evaluation, fresh leaves were immersed in formalin: acetic acid: 70% alcohol (5:5:90) for 24 h and transverse section of leaves using blade and razor method was done. Stains like safranin and fast green dye were used to stain the section¹⁴.

Powder study:

Prior to the evaluation of the powdered drug, a solution of Chloral hydrate (75%) was used as a clearing reagent, and slides of powdered leaves for microscopy were set as per the prescribed procedures¹⁴.

Fluorescence analysis:

In separate test tubes, add a little amount of dried powder and a few milliliters of each reagent. Let stand for a few minutes. Observe each test tubes under ordinary light and UV light (short wavelength 254nm and long wavelength 366nm) and note down the colour emmited^15,16.

Phytochemical analysis:

Phytochemical screening was carried out using a documented approach on Cymbidium aloifolium L (Sw) leaf extracts to qualitatively detect several phyto-constituents such as alkaloids, glycosides, carbohydrates, phenolic and tannins, flavonoids, saponins, gums, and mucilage^17,18.

Physicochemical analysis:

Factors like extractive value, loss on drying, and total ash (water-soluble ash and acid-insoluble ash) were performed by taking powdered samples for Physicochemical analysis according to the official book¹⁹.

In- vitro antioxidant assay:

In-vitro antioxidant assay will be checked by using some models like as- DPPH & FRAP.

DPPH radical scavenging activity²⁰:

To assess each extract's capacity to neutralize radicals using the DPPH technique, DPPH scavenging activity was carried out. To carry out this procedure, add 1 ml of DPPH to an extract solution of 1 ml (50-700 g/ml) in methanol. Solution of (0.3mM). The extract solution must incubate for 30 minutes in the dark after adding DPPH. At 517 nm, absorbance was measured in a UV-vis spectrophotometer against a blank sample. Following percentage of scavenging activity was calculated using an equation.

% Scavenging of DPPH = [(A₀ -A₁ ) /A₀ ] × 10

Where, Asorbance of the control = A₀

Absorbance of the test extracts = A₁

FRAP reducing power activity²¹:

Reducing power activity was perform to examine the reducing power activity of each extracts using FRAP method. 1ml (100-1000 µg/ml) of extract solution in methanol were added to 2.5 ml of phosphate buffer (pH 6.6, 0.2 M) and 1% of 2.5 ml potassium ferricyanide. After that solution kept in 50⁰C in water bath for 20 mins. On this solution add 2.5ml 10% TCA and then pipette out from this mixture, add 2.5 ml distilled water and 0.5 ml ferric chloride. Measure the absorbance at 700 nm at UV-Vis spectrophotometer.

Total phenolic content²²:

Using the established Folin-ciocalteu method, total phenolic content of each extract was carried out by mixing 0.4ml of extract (100-1000µg/ml) solution with 0.4ml of 10% (w/v) Folin -ciocalteu reagent and adding 4ml of Na₂CO₃ (7%) to the above mixture and incubate it at 37⁰C for 90 min. Using a UV-vis spectrophotometer (Shimatzu, UV-1980) the absorbance was measured at 730 nm against blank. The result was expressed as mg/g of gallic acid equivalents in milligrams gallic acid equivalent per gram (mg GAE/g) of dry extract.

Total flavonoid content²³

Total flavonoid content was performed by taking 1 ml of extract solution (100-1000 µg/ml) to 0.2 ml of 10% (w/v) AlCl₃ in methanol. Add 5.6 ml of distilled water and 0.2ml (1M) potassium acetate and incubate it at room temperature for 30 minutes and measure the absorbance at 415 nm against blank. The findings were presented as milligrams per gram of Quercetin equivalents (mg QE/g) of dry extract.

RESULT AND DISCUSSION:

Macroscopic evaluation:

Table 1: Macroscopic evaluation of leaves of Cymbidium aloifolium (L) Sw

Sl No	Characters	Observation of leaf
1	Arrangement	Cluster
2	Odour	Characteristic
3	Colour	Dark Green
4	Size	L 40-60 cm, W 2-4 cm
5	Shape	Oblong
6	Taste	Light at start bitter at end
7	Appearance	Smooth
8	Margin	Entire
9	Apex	Obtuse – Retuse
10	Base	Cuneate
11	Surface	Smooth
12	Texture	Smooth
13	Venation	Parallel

Microscopic evaluation :

Transverse Section (T.S) of leaves of Cymbidium aloifolium (L) Sw (Figure-1).

Powder Microscopy of leaves of Cymbidium aloifolium (Figure-2).

Physicochemical analysis:

Different values obtained from the proximate analysis of the present drugs are recorded as follows:

Total ash, water soluble ash, acid insoluble ash, swelling index, Moisture content and Alcohol soluble Extractive Value, Water Soluble Extractive Value of leaves of C. aloifolium are shown on Table 2.

Table 2: Physicochemical parameters (%W/W) of C. aloifolium (Values are presented as mean ± standard deviation)

Physical parameter	Result
Alcohol soluble extracts (methanol : water), 9:1	12±0.423
Water soluble extracts (water : methanol ), 9:1	20.5 ±0.210
Moisture content	8.6±1.14
Total ash	6.15±0.263
Water soluble ash	3.4±0.362
Acid insoluble ash	1.8±0.092
Swelling index	0.8±0.002

Extractive values play an important role in pharmacognostical study. A crude drug's moisture content determines whether it breaks down due to chemical changes or microbial attack. The extract values provide information about the chemical constituents present in the plant and are useful for estimating which constituents are soluble in the specific solvent used for the extraction as well as determining the amount of materials that are exhausted. If a crude drug contains too much moisture at a relatively high temperature, it will activate its enzymes and create an environment that is conducive to the growth of microorganisms. The purity and quality of C. aloifolium can be ascertained with the help of the ash values found in this study. Gum or mucilage is present because of the swelling qualities.

Determination of Fluorescence analysis:

The fluorescence analysis of palnt has been given in table-3.

Phytochemical Screening:

The phytochemical Screening of palnt has been given in table-4.

In- vitro antioxidant assay:

a) DPPH radical scavenging activity:

DPPH assay aids in the detection of the reduction of DPPH to DPPH₂ due to the presence of an antioxidant compound present in the extract because of which colour changes from purple to yellow and can be easily detected in absorbance at 517 nm. Figure 3 represents the graph between the inhibitory concentrations of standard drug with different solvent extract. The IC₅₀values of Ascorbic acid were found to be 28.817 and IC₅₀ values of different extract of leaves of Cymbidium aloifolium was found as follows - Pet ether (136.229), Chloroform – (92.966), Ethyl acetate – (56.323), Methanol - (41.868), Water (212.562).

Table 3: Result of Fluorescence analysis of powder of Cymbidium aloifolim (L) Sw

SL No	Treatment Of Powder/Test	Visile Rays	UV Rays
SL No	Treatment Of Powder/Test	Visile Rays	Short Wave	Long Wave
1	Powder as such	Light brown	Black	Brown
2	Powder + 50% H₂SO₄	Greenish brown	Light brown	Light brown
3	Powder + 50% HNO₃	Light brown	Light brown	Light brown
4	Powder + 5% KOH	Light brown	Black	Black
5	Powder + Methanol	Light brown	Deep brown	Light brown
6	Powder + 1N HCL	Light brown	Black	Faded brown
7	Powder + Cold water	Light brown	Brown	Light brown
8	Powder + Hot water	Light brown	Black	Light brown
9	Powder + Picric acid	Yellow	Black	Light brown
10	Powder + Ammonia solution	Light brown	Deep brown	Light brown
11	Powder + Chloroform	Brown	Brown	Greenish brown
12	Powder + Glacial acetic acid	Light brown	Light brown	Light brown
13	Powder + 5% Iodine	Light brown	Black	Black
14	Powder + Fecl₃	Light brown	Black	Light brown
15	Powder + Pet ether	Light brown	Light brown	Black

Table 4: Preliminary Phytochemical test result of leaves extract of Cymbidium aloifolium (L) Sw

Test	Reagent	Petroleum ether extract	Chloroform	Ethyl acetate	Methanol	Water
Alkaloid	Dragendorff reagent	-	-	-	+	-
	Hagers reagent	+	+	-	+	-
	Wagners reagent	-	-	-	+	-
Glycoside	Legal test	-	-	-	+	+
Glycoside	Keller-kiliani test	+	+	-	+	-
Saponin	Foam test	-	-	-	-	-
Saponin	Lead acetate	-	-	-	-	-
Tannin	Ferric chloride
Steroid (cholesterol)	Salkwaski test	+	+	+	+	+
Flavonoid	Shinoda	+	+	+	-	-
	Ferric chloride	+	-	+	+	+
	Lead acetate	+	-	+	-	-
Phenol	Ferric chloride	-	+	+	+	+
Carbohydrate	Molish	-	+	+	+	+
	Benedict	-	-	-	+	-
	Fehling	+	-	-	+	-

** “+”indicates presence & “-” indicates absence

Figure 3: DPPH radical scavenging activity of Ascorbic acid, Pet Ether, Chloroform, Ethyl Acetate, Methanol and Water Extracts. Errors bars represent ± standard deviation.

b) FRAP reducing power activity:

Depending on the concentration of the extract/standard, colour changes from pale yellow/white to greenish blue or dark blue and the mechanism behind the colour change is due to the antioxidant group present in the plant which reduced ferricyanide (Fe₂+) to ferrocyanide (Fe₃+) addition of ferric chloride produces ferric-ferrous complex measured at 700 nm. Figure 4 displays the assay for lowering the power of both the extract and the standard, which indicates that while the assay for reducing power of plant extract is higher than that of standard (ascorbic acid), it still contains the compound that is responsible for antioxidant activity. The extract was follows as Pet ether, chloroform, Ethyl acetate, Methanol and Water and in comparison with standard ascorbic acid Methanolic extract showed good result followed by chloroform, ethyl acetate, pet ether, water.

Figure 4: Reducing power assay of Ascorbic Acid, Pet Ether, Chloroform, Ethyl Acetate, Methanol and Water Extracts. Errors bars represent ± standard deviation. (Series 1 = Concentration 100, Series 2 = Concentration 150, Series 3 = Concentration 200, Series 4 = Concentration 250, Series 5 = Concentration 300.)

Determination of Total phenolic content:

Using the Folin–Ciocalteu technique, the total phenol content of several extracts of Cymbidium aloifolium leaves was assessed. This reagent is made by combining two chemicals, phosphotungstic acid and phosphomolybdic acid. The blue color of the oxidation occurs when the acid is reduced to tungsten and molybdenum in the presence of phenol groups. Figure 6 displays the calibration curve of the standard that was established using gallic acid as the standard. Using the linear equation A = 0.0023C - 0.0056, R2 = 0.9929, the phenolic component concentration was estimated as gallic acid equivalents based on the calibration curve. C stands for gallic acid equivalents (μg), while A stands for absorbance. The total phenolic content of the extract was expressed in milligrams of gallic acid equivalent per gram of dry weight.

Figure 5: Standard curve of Gallic acid

Figure 6: Total phenolic content. Errors bars represent ± standard deviation

The level of Total Polyphenolic Content of the Gallic acid which was used as standard found was 950.40 g. and the Total Polyphenolic Content of the different extract was as follows - Pet ether (205.29), Chloroform – (449.37), Ethyl acetate – (620.99) , Methanol (783.70) , Water (43.86) of Gallic acid equivalent per gram of the extract

Determination of Total flavonoid content:

The total flavonoid content of different extract of Cymbidium aloifolium leaves was assessed by utilizing the aluminium chloride colorimetric method, where this reagent forms a complex with flavones and flavonol which a UV spectrophotometer can easily detect at 415 nm. Here, Quercetin was used as a standard whose calibration curve was established in Figure 7.

Figure 7 : Standard curve of Quercetin

Linear equation based on the calibration curve is: A=0.0018c, R² = 0.989. A is the absorbance, and C is Quercetin equivalents (μg/ml).

Figure 8: Total Flavonoid Content. Errors bars represent ± standard deviation

The level of total flavonoid content of the Quercetin which was used as standard found was 963.82 g. and the Total flavonoid content of the different extract was as follows – Pet ether (290.65), Chloroform (395.11), Ethyl acetate (720.42) , Methanol (812.77) ,Water (182.61) of Quercetin acid equivalent per gram of the extract.

CONCLUSION:

Phytochemical investigation of leaves of Cymbidium aloifolium (L) Sw confirmed the presence of flavonoid, alkaloid, carbohydrate, tannin, glycoside, and triterpenoid compounds. Furthermore, In this experiment, phenols and flavonoid groups of compounds were found in the leaves of Cymbidium aloifolium (L) Sw. The presence of these compounds is responsible for the entrapment of free radicals and acts as a natural antioxidant activity of the leaves of Cymbidium aloifolium (L) Sw. This evaluation helps in the investigation of compounds that may help combat many harmful diseases.

Methanolic extract of leaves of Cymbidium aloifolium (L) Sw showed potent antioxidant activity by inhibiting DPPH. The reducing power of the extract increased with increasing amount of sample. Total Phenolic and Flavonoid content showed the highest potency in methanolic extract followed by ethyl acetate, chloroform, pet ether, and water. Further investigation into the separation and characterization of certain chemical moieties from Cymbidium aloifolium (L) Sw leaf extracts and their biological tests may one day lead to the development of powerful, nontoxic antioxidants.

CONFLICT OF INTEREST:

There are no conflicts of interest for the authors in relation to this study. The paper's writing and content are solely the authors' responsibility.

ACKNOWLEDGEMENT:

The author would like to thank Ganesh Dey, a Research scholar of the Department of Pharmaceutical Technology, University of North Bengal, for his valuable suggestion and guidance.

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Received on 12.12.2023 Revised on 16.04.2024

Accepted on 28.06.2024 Published on 24.12.2024

Available online from December 27, 2024

Research J. Pharmacy and Technology. 2024;17(12):5743-5749.

DOI: 10.52711/0974-360X.2024.00874