INTRODUCTION:

Herbal plants have been source of wide variety of biologically active constituents for many of centuries and used mainly as crude material or as pure compounds for treating various disease and disorder conditions¹. By using of herbal medicines becoming popular due to toxicity and side-effects or adverse effect of allopathic medicines. For preparation of different kinds of Ayurvedic, formulation herbal medicines are important base, which ultimately plays an important role in the development for drug discovery of effective therapeutic agents^2,3. Casuarina equisetifolia is a handsome tree with drooping branches, having 10 to 50 meter height and it is found in dry hill sides and open forests of India, Srilanka and Australia⁴. This species is an important raw material for many major paper mills in India⁵. It has been widely cultivated in Florida for coastal landscaping, as an ornamental shade tree and agricultural promotion - whether as fuel wood, timber or planted as windbreaks bordering groves⁶. Casuarina equisetifolia traditionally used for various types of disease and disorder such as diabetic, hyperlipidimic, Astringent, diahorreha, muscle weakness, gastric problems, microbial disease and nervous disorder, in the parts are medicinal used such as aerial parts, root, and seed^7,8. The stem-inner bark is brownish pink in colour and is a tonic and astringent. It is useful in treating diarrhoea and dysentery⁹. Pharmacognostic account of stem-inner bark as well as physiochemical, phytochemical evaluation helps to show which secondary metabolites are present in stem-inner bark and due to presence of various secondary metabolites such as alkaloids, flavonoids, phenols, tannins, terpenoids, and carbohydrates, stem-inner bark of Casuarina equisetifolia is helpful in showing various Pharmacological activity.

MATERIAL AND METHODS:

Collection of plant material:

The stem-inner bark of Casuarina equisetifolia plant (Family: Casuarinaceae) was collected from Kolhar (BK) Ahmednagar, Maharashtra, India. The plant sample was terminologically identified and authenticated at the herbarium of Department of Botany and Research Centre, Padmashri Vikhe Patil College of Arts, Science and Commerce, Loni, Pravaranagar-413 713.With the Ref No: PVPC/Bot/2020-21/HD-19.

Chemicals and Reagents:

To carry out whole research work various chemicals and reagents were used. Methanol, Ethanol, Phloroglucinol, Hydrochloric acid, Picric Acid, Iodine, alpha-naphthol, H₂SO₄, Benedict’s reagent, Fehling’s A and Fehling’s B Solutions, Barfoed’s reagent, Selwinoff’s reagent, Dragendroff’s reagent, Mayer’s reagent, Hager’s reagent, Wagner’s reagent, tannic acid, sodium hydroxide, lead acetate solution, FeCl₃ solution, HNO_3,Chloroform, Folin ciocalteu reagent, folin-denis reagent, sodium carbonate, gallic acid, sodium nitrite, aluminium chloride, quercetin, 2,2-diphenyl-1-picryyhydrazl, hydrogen peroxide, ascorbic acid, potassium ferric cyanide, sodium salicylate, sodium phosphate

Preparation of Stem-inner Bark extract:

The drug of Casuarina equisetifolia was collected. Then the dried stem-inner bark material is pulverized in grinder. The coarse powder was used for extraction.

Method: Hot continuous Extraction, Cold Percolation Method and Maceration.

Solvents: Using solvent Aqueous and Organic solvents like Methanol and Ethanol.

Powdered bark samples were extracted using water, organic solvents like methanol and Ethanol for 48 hrs, 6 hrs and 36 hrs by Maceration, Hot continuous Extraction and Cold Percolation Method Respectively (Fig. 1 and Fig. 2). After completion of extraction, the extract itself in round bottom flask (RBF) was evaporated to the half of total extract and then by taking small-small amount of extract from RBF into the porcelain dish, concentrated the extract on water bath and dried and stored at 4^oC until further use.

Figure 1: Hot continuous Extraction process

Figure 2. Cold Percolation Extraction process

PHARMACOGNOSTIC STUDY:

The plant materials were studied for different parameters as given below; macroscopic features and microscopic examination.

Macroscopic features of stem-inner bark:

Various organoleptic and morphological characters of C. equisetifolia stem-inner bark like colour, shape, size, taste, odour, fracture and configuration etc. were studied¹⁰.

Microscopic features:

In this study, transverse sections of stem-inner bark were studied under photomicrograph. Phloroglucinol-HCl, Picric Acid, Iodine are the Staining reagents which was used for microscopic evaluation as per standard procedures^11,12.With or without staining reagents the various identifying features of the drug were studied and finally recorded. The test tube containing sufficient water was boiled for few minutes with inner-stem bark. The softened stem-inner bark was transversally cute into fine sections, which were initially kept for staining using 0.1% w/v phloroglucinol reagent followed by concentrated HCL. The stained sections were observed under microscope^13,14. Different layers of cells and identifying characters were observed and thereafter, photomicrography was done.

Powder Microscopy:

The dried stem-inner bark of C. equisetifolia was powdered and observed under microscope. Different staining reagents such as iodine and phloroglucinol for detection of starch grains and for detection of lignified components respectively were used. To a little quantity of stem bark powder taken over a microscopic slide, 1 to 2 drops of 0.1% w/v phloroglucinol solution and a drop of concentrated hydrochloric acid were added and covered with a cover slip. Glycerol is used for mounting slide and then examined under microscope¹⁵.The characteristic structures and cell components were observed and their photographs were taken using photomicrography.

PHYSICO-CHEMICAL STANDARDS:

Physico-chemical standards of stem–inner bark sample were carried out using Aqueous and organic solvents like Methanol and Ethanol solvent. Various Physico-chemical standard was carried out that include Determination of moisture content (Loss on drying), Determination of Total Ash in that Water-soluble Ash and Acid- Insoluble Ash, and Extractive Values in that Alcohol-soluble Extractive Value and Water-soluble Extractive Value¹⁵.

PRELIMINARY QUALITATIVE PHYTOCHEMICAL SCREENING FOR VARIOUS EXTRACTS:

Preliminary Phytochemical screening of stem–inner bark sample were carried out using Aqueous and organic solvents like Methanol and Ethanol solvent. Various phytochemical tests was carried out that include Molisch’s test for carbohydrate, Benedicts test and Fehling’s test for reducing sugars, Barfoed’s test for monosaccharides, Selwinoff’s test for hexose sugar, Dragendroff’s test; Mayer’s test; Hager’s test, Tannic acid test and Wagner’s test for alkaloids, Shinoda test; Sulphuric acid test for Flavonoids, Ferric chloride test; Lead acetate test; Bromine water test, Nitric acid test; Potassium dichromate test for Tannins, Salkowski test, Liebermann- Burchard reaction for Steroid using standard procedure¹⁵.

QUANTITATIVE ANALYSIS:

Estimation of total phenol content:

Folin-Ciocalteu Reagent (FCR) method is used to determine the Total phenolic content in aqueous and organic solvent like methanol, Ethanol extract of C. equisetifolia stem-inner bark¹⁶. About 1ml of Folin- Ciocalteu reagent was added to 1ml of the all extracts, and then for 5 minutes mixture was incubated. About 10ml of 7% Na₂CO₃ solution was added to this resultant solution and again incubated at 23^OC in dark for 90 min. At 765nm using UV- visible spectrophotometer such as UV 1650 Pc, Schimadzu, the absorbance of the mixture was read against the blank and Standard, which was used, is Gallic acid. The total phenolic content in the extracts was expected in Gallic acid equivalent i.e. GAE.

Estimation of total flavonoid content:

Aluminium chloride colourimetric method is used to determine the total flavonoid content of all three extract i.e. aqueous, methanol and ethanol Extract of C. equisetifoila stem-inner bark¹⁷.About 0.5ml of all stem-inner bark extracts were mixed individually with 2ml of solvent and 0.30ml of 5% sodium nitrite solution. To this solution mixture, 2ml of 1M NaOH, 0.3ml of 10% Aluminium chloride and 2.8ml of DW was added and incubated for 30 min at room temperature. The absorbance was taken at 510nm using UV-visible spectrophotometer means UV 1650 Pc, Schimadzu. The standard, which was used in this flavonoid determination, is Quercetin and Quercetin equivalent (mg of QAE/g of extract) is considered for final determination of Total flavonoid concentration.

Estimation of tannin:

Folin Denis method was used to determine the total tannin content in aqueous and organic solvent like ethanol, methanol extracts of C. equisetifoila stem-inner bark,¹⁸with slight modifications in the actual procedure. To 1ml of all three stem-inner bark extracts, 3ml of distilled water, 0.5ml of Folin-Denis reagent and 1.0ml of 1N Na₂CO₃ solution were added. This mixture was further diluted with H₂O. The absorbance was taken at 515nm, by mixing thoroughly. The standard which was used for this method is Tannic acid. Total tannin content was expressed as mg of tannic acid equivalents per gm of dried sample i.e. mg of TAE/g of extract.

Estimation of total terpenoids:

Total terpenoid content was estimated¹⁹for all three extract i.e. ethanol, methanol and aqueous extract of C. equisetifolia stem-inner bark. For about 24 hrs, the crude extract was soaked in 95% ethanol (20ml). The petroleum ether was used to extract filtrate (60°C - 80°C). Then the residue of the extract is dried which was obtained from the petroleum ether and finally weighed in order estimate the total terpenoids content of C. equisetifoila stem-inner bark.

Weight of terpenoid in gm

Terpenoid content % =------------------------------------------- × 100

Weight of the sample in gm

RESULTS AND DISCUSSION:

PHARMACOGNOSTICAL STUDY:

Macroscopic study of stem-inner Bark:

Morphological examination of the stem bark (Fig. 3) shows that the bark consists of flat and thick, usually 0.3-0.9 cm thick, Outer surface of the stem-inner bark is Brownish Pink and internal surface light pink to dirty brown coloured with short fracture. Taste is astringent and smell is odourless.

Figure 3. Casuarina Equisetifolia Stem-Inner Bark

Microscopic study of stem-inner Bark:

Transverse section study of the stem bark (Fig. 4) depicts that,

Phellogen (cork cambium) is 3-5 layered thick having polygonal and tangentially elongated thin-walled parenchymatous cells.

Secondary cortex (Phelloderm) consists of 12-18 layers having oval to polygonal, tangentially elongated thin-walled parenchyamatous cells. Stone cells are arranged in a tangential manner in a large group having oval to elongated shape, present below secondary cortex, which forms continuous as well as discontinuous band.

Medullary Rays are thick walled parenchymatous cells containing a few cubical, rhomboidal and hexagonal crystal of calcium oxalate.

Secondary phloem is composed of phloem parenchyma, phloem fibers and stone cells alternating with lignified stone cells.

Lignified fibers are small thin-walled polygonal cells present in cork.

In secondary phloem, a few stone cells are found which are scattered in secondary cortex.

Figure 4. T.S. of C. equisetifolia Stem-inner bark. Pg- Phellogen (Cork cambium), Pd- Phelloderm (secondary cortex), Pf- Phloem fibre, Pp- Phloem parenchyma, Sc- Stone cells.

Powder Study:

Inner-Stem bark powder appears brownish showing thick walled hexagonal phelloderm cells; rectangular thin walled cortex cells; thick walled elongated phloem fibres; lignified stone cells and Prismatic crystals of calcium oxalate, lignified phloem fibres, Sclerides, Tracheid’s. Powder characteristics of the Stem-Inner bark have been shown in. (Fig. 5)

Figure 5. Powder Characteristic of Casuarina equisetifolia Stem-inner bark

PHYSICO-CHEMICAL STANDARDS:

In this study, various physicochemical parameters like loss on drying, total ash, acid insoluble ash, water-soluble ash and extractive values were determined in triplicate (Average of three reading) as mentioned in Table 1.

Table 1. Physico-Chemical Analysis of C. equisetifolia Stem-Inner Bark

Parameter	Value obtained on dry weight basis (% w/w)
Loss on Drying	2.7
Total Ash	11.43
Water soluble ash	0.85
Acid Insoluble ash	10.2
Water soluble extractive	5.6
Alcohol soluble extractive	0.82

Phytochemical Screening:

Qualitative Analysis:

Preliminary qualitative phytochemical screening of aqueous and organic solvent extracts of Casuarina equisetifolia stem-inner bark showed the presence of varied phytochemicals (table 2). All the extracts showed the presence of proteins and phenols. Aqueous, ethanol and methanol extracts showed the presence of alkaloids, glycosides, carbohydrates, flavonoids and tannins. Ethanolic and methanolic extract of stem-inner bark results as a there is presence of all the phytochemicals such as alkaloids, flavonoids, tannins, sugar, saponins. From the phytochemical study, it is concluded that the polarity of the compounds being extracted by each solvent such as methanol, ethanol and aqueous.

Table 2. Preliminary Phytochemical Screening

Type of Phytoconstituents	Aqueous	Methanol	Ethanol
Alkaloids	+	+	+
Carbohydrates	+	+	+
Tannins	+	+	+
Flavonoids	+	+	+
Protein	+	+	+
Phenols	+	+	+
Glycosides	+	+	+
Steroids	+	+	+
Saponins	+	+	+

Quantitative Analysis:

The total phenolic content in aqueous, ethanol and methanolic extracts of Casuarina equisetifolia stem-inner bark was determined by Folin ciocalteu method and was expressed in Gallic acid equivalent for that Standard Curve equation is y = 0.175x-0.118, R² = 0.995. Among the three extracts, methanol extract contained maximum phenol content i.e. 69.2±0.14 mgGAE/g of extract, ethanol extract contained 65.4±0.12mg GAE/g of extract and aqueous extract contained 39.5±0.22mg GAE/g of extract. (fig. 6a) The total flavonoid content in aqueous, ethanol and methanolic extracts of Casuarina equisetifolia stem-inner bark was determined by the aluminium chloride colourimetric method and was expressed in Quercetin equivalent and for that Standard Curve equation is y = 0.013x-0.161, R² = 0.990. Among the three extracts, methanol extract contained maximum flavonoids content and is 36.14±0.12 mg of QE/g of extract, followed by ethanol extract contained 30.4±0.11mg of QE/g of extract and aqueous extract contained 25.66±0.13mg of QE/g of extract. (fig. 1a). The total tannin content was calculated using standard curve of Tannic acid i.e. y = 0.003x-0.007 R² = 0.994 and expressed in Tannic acid equivalents/g of extract. Among the various solvents, used methanolic extract contained the highest content of tannin 76.42±0.31mg TAE/g of extract followed by ethanol and aqueous extracts with 65.25±0.36 and 23.29±0.15mg TAE/g of extract respectively (fig. 6a).

The methanol stem-inner bark extracts showed maximum terpenoid content (8%) followed by ethanol extract (4.5%) and aqueous extract (3.2%) (fig. 6b).

Figure 6(a). Quantitative analysis (total phenol, flavonoid and Tannin) data presented are the means of three replicates

Figure 6(b). Quantitative analysis–Total Terpenoid Values are expressed as mean of three replicates

CONCLUSION:

Casuarina equisetifolia is one of the evergreen plant responsible for different pharmacological activity and ultimately useful as herbal drug. Different parts of this plant C. equisetifoila shows various Pharmacological activity such analgesic, anti-inflammatory, wound healing, anti-histaminic activity. Stem-inner bark of Casuarina equisetifolia shows different secondary metabolites such Phenol, alkaloids, tannins, flavonoids, terpenoids, Carbohydrate, Glycosides. As stem- inner bark of C. equisetifolia phytochemically evaluated using three different extract, conclusion is that Methanol extract shows maximum Phenol, Tannin, flavonoid and Terpenoid content followed by Ethanol extract and aqueous extract.

ACKNOWLEDGEMENT:

The authors are thankful to our Friends and Faculty of Pravara Rural College of Pharmacy, Pravaranagar, Loni, Maharashtra, India for their valuable support and encouragement throughout the entire period of research.

CONFLICT OF INTEREST:

Authors have declared that no conflict of interests exists.

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Received on 17.05.2021 Modified on 19.06.2021

Research J. Science and Tech. 2021; 13(3):193-199.

DOI: 10.52711/2349-2988.2021.00029