INTRODUCTION:

Medicinal plants are beneficial for healing along with curing of human illness due to secondary metabolites present in the plants. Phytoconstituents are of two types on is primary and another is secondary metabolites.¹ Primary metabolites are essential components which utilised for growth and development of plant. It includes sugars, carbohydrates, proteins, nucleic acids, lipids, hormones, etc.² Secondary metabolites are the derivatives of primary metabolic routes via various pathways. They are not essential for plants growth and development. They are not produced continuously. Frequently the secondary metabolites are produced while non-growing phase of plants. Secondary metabolites are the out-turn of primary metabolites. Secondary metabolites are such as alkaloids, glycosides, phenolics, terpenoids, flavonoids, resins, tannins, volatile oils, gums, steroids, etc. Secondary metabolites are also known as phytoconstituents or phytochemicals, etc. These constituents protect the plants from number of diseases and has tis defense mechanism against them. Every secondary metabolite is responsible for numerous pharmacological activities. The Zizyphus jujuba belongs to Rhamnaceae family and it has the vernacular names Ber, Bor, Badri, Chinese apple, Indian plum, Chinese date, etc. ^3,4

MATERIAL AND METHODS:

Collection of plant material:

Bark of the Zizyphus jujuba were collected from Loni, Ahmednagar, Maharashtra, India. It has been authenticated at the herbarium of Department of Botany and Research Centre, PVP college of Arts, Science and Commerce, Pravaranagar, Loni, Ahmednagar, Maharashtra, India 413713 with the reference number PVPC/Bot/2020-21/HD-27.

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, Sodium hydroxide, Folin Ciocalteu reagent, Tannic acid, Lead acetate solution, FeCl₃ solution, HNO_3,Chloroform, Folin-denis reagent, Gallic acid, Sodium carbonate, Sodium nitrite, 2,2-diphenyl-1-picryyhydrazl, Hydrogen peroxide, Aluminium chloride, Ascorbic acid, Quercetin, Sodium salicylate, Potassium ferric cyanide, Sodium phosphate.

Preparation of Bark extract:

The bark of Zizyphus jujuba was procured and authenticated from Department of Botany and Research Centre, PVP college of Arts, Science and Commerce, Pravaranagar, Loni, Ahmednagar, Maharashtra, India 41371. Authenticated bark was dried in shade and powdered coarsely using grinder. Using analytical grade solvents extraction has been done by following standard procedure. The coarse powder of bark was Soxhlet extracted with Methanol at 40 ^oC for 3-5 hours. The extract obtained and concentrated to yield methanolic extract under reduced pressure. 12 gm of methanolic extract was obtained from 200 gm of bark powder. ^[5]

Fig. 1: Hot continuous Extraction process

Pharmacognostic Study:

Study of macroscopic and microscopic features of crude drug were carried out using various parameters:

Macroscopic features of stem bark:

The following macroscopical characters for the fresh and dried barks were notes: size and shape, surfaces, fracture, texture, colour, odour and taste were done by observing visually or magnifying lens. ^[6]

Microscopic features:

Transverse section of the bark:

Fresh bark pieces were boiled in water for 1 to 2 hrs until the bark is ready to be get sliced into sections. Thin sections were collected. Then the sections after washing with distilled water were then transferred using No. 1 paint brush into Clearing agent Chloral hydrate 30% and kept for 10-15 minutes. Then the sections were transferred to staining agent Phloroglucinol – Conc HCl (1:1) which stains the tissues in pinkish red color. The sections were stained for 3-5 minutes and mounted on glass slide using glycerine. Then the sections have been observed using Motic Electronic Microscope. Photo micrographs of various magnifications such as 10X and 40X were taken for study of anatomical features of plant. ^[6]

Powder microscopy:

The shade dried powdered bark has been screened through sieve number 40 was used for the powdered analysis and to observe the microscopical powder characteristics. The specimens were separately treated with phloroglucinol- hydrochloric acid, ruthenium red and iodine solution for observing the presence of lignin, mucilage and starch grains in the powder. After staining of powder, the samples were observed under Motic Electronic Microscope with different magnifications. ^6,7

Physico-chemical standards:

Loss on drying:

The percentage of Loss on Drying was determined using gravimetric method. 2gm of dried bark powder was taken which was passed through sieve no. 40 in dried glass stoppered, weighed bottle. Then it is weighed and shaked for even distribution of powdered drug. Then bottle is placed in drying chamber, by removing the stopper. Then sample is dried at temperature 100^oC to 105^oC in hot air oven. then the stopper is placed over the bottle and cooled in desiccator and weighed again. ^6,7

weight of sample – weight of dried sample

Percentage of Loss on Drying = --------------------------------------------------------------- X 100

weight of sample

Determination of Foreign Content:

Approximately 100gm of dried bark powder of Zizyphus jujuba was taken and spread into a thin layer. The foreign matter was being detected by inspection with the unaided eye separated and weighed. The percent aged of foreign content was determined using the formula. ^6,7

Weight of sample – weight foreign matter

Percentage of foreign content = ---------------------------------------------------------- X 100

Weight of sample

Determination of Total Ash:

Ash depicts the inorganic matter content in the sample which is determined. Approximately 2gm dried sample in weighed tared porcelain dish. And that porcelain dish was charred over the flame until all the carbon is burnt off. Then the dish is cooled in the desiccator. Then the ash remaining in the crucible is weighed and percentage of total ash was calculated with reference to the air-dried powder. ^6,7

weight of ash

Percentage of Total Ash = ------------------------------- X 100

weight of sample taken

Determination of Acid-Insoluble Ash:

The total ash obtained above was used for determining acid-insoluble ash and boiled for 5 minutes with 25ml of dilute Hydrochloric acid. The content in the crucible was filtered by using ash filter paper (Whatman Filter Paper No:42). Residue was washed twice with hot water. Then the crucible is ignited, cooled and weighed. Then the filter paper with residue was transferred to crucible and incinerate the crucible until the vapours cease to be evolved and then more strongly until all the carbon has been removed. Cool the crucible in the desiccator. Weigh the residue and percentage of acid-insoluble ash with reference to the air-dried powder. ^6,7

100 x weight of residue after incineration

Percentage of Acid Insoluble Ash = --------------------------------------------------------------

Weight of sample taken

Determination of Water-soluble Ash:

The total ash obtained above was used for determining acid-insoluble ash and boiled for 5 minutes with 25ml of distilled water. The content in the crucible was filtered by using ash filter paper (Whatman Filter Paper No:42). Residue was washed twice with hot water. Then the crucible is ignited, cooled and weighed. After that the filter paper full of residue is being transferred to the crucible and which is heated gently until the vapours halts to be evolved and then heat it strongly until all of the carbon is being evolved. Cool the crucible in the desiccator. Weigh the residue and percentage of water-soluble ash with reference to the air-dried powder. ^6,7

100 x weight of residue after incineration

Percentage of Water-Soluble Ash = ----------------------------------------------------

Weight of sample taken

Extractive Values:

Extractive values such as alcohol soluble extractive and water-soluble extractive were determined using standard methods.

a) Determination of Alcohol-soluble Extractive Value:

5 gm of air-dried powdered drug was subjected to macerate for 24 hours in a closed flask using 100ml of 90% Ethanol with frequent shaking during first 6 hours and then allowed to stand for 18 hrs. after 24hrs, the filtrate was collected using Whatman Filter Paper no. 42. 25 ml of filtrate was evaporated to dryness on water bath, complete drying in an oven at 100 ^oC, cooled and weighed. Percentage of soluble extractive was calculated with reference to the air-dried powder. ^[6,7]

b) Determination of Water-soluble Extractive Value:

5 gm of air-dried powdered drug was subjected to macerate for 24 hours in a closed flask using 100ml of Chloroform water with frequent shaking during first 6 hours and then allowed to stand for 18 hrs. after 24hrs, the filtrate was collected using Whatman Filter Paper no. 42. 25 ml of filtrate was evaporated to dryness on water bath, complete drying in an oven at 100 ^oC, cooled and weighed. Percentage of soluble extractive was calculated with reference to the air-dried powder. ^6,7

Preliminary Qualitative Phytochemical Screening

Qualitative chemical tests were conducted for methanolic and aqueous bark extracts of Zizyphus jujuba to identify the presence of various phytoconstituents. The various tests conducted are given below and observations are recorded and tabulated. ^[6,7]

Test for carbohydrates:

Molisch’s test (General test):

To 2-3 ml extract, few drops of α- naphthol solution in alcohol was added, shaken 2 ml concentrated H₂SO₄was added from the sides of the test tube. It was observed for violet ring at the junction of two layers.

For Reducing Sugars

1. Fehling’s test:

1 ml Fehling’s A and 1 ml Fehling’s B solutions were mixed and boiled for one min. Equal volume of test solution (T.S) was added and heated in boiling water batch for 5 – 10 min and observed for a yellow and then formation of brick red precipitate.

2. Benedict’s test:

Equal volumes of Benedict’s reagent and test solution (T.S.) were mixed in a test tube and heated in boiling water for 5 min. solution may appear green, yellow or red depending upon amount of reducing sugar present in test solution.

Test for Alkaloids:

Evaporate all extracts separately. To residue, add dilute HCL. Shake well and filter. Use filtered solution for test.

1. Dragendroff’s test:

To 2-3 ml T.S, add few drops Dragendroff’s reagent and orange brown precipitate is observed.

2. Mayer’s test:

To 2-3 ml T.S, added few drops of Mayer’s reagent and observed for creamy white precipitate.

3. Hager’s test:

To 2-3 ml T.S, added Hager’s reagent and observed for yellow precipitate.

4. Wagner’s test:

To 2-3 ml T.S, added few drops of Wagner’s reagent and observed for reddish brown precipitate.

Test for Glycosides:

1. General test for glycosides:

Part A: To 2-3 ml of extract dil. H₂SO₄ was added and heated on a water bath for 1-2 min. Neutralize with 10% NaOH, check with litmus paper and to resulting solution add Fehling’s A and B. Intense red precipitate in this case indicates glycosides are present.

Part A: To the two -three mL of extract, add water and heat it. Accordingly add NaOH is being added for Neutralization of the solution simultaneously water is also added in same quantity. Then addition of Fehling’s A and B is done. Increases red precipitate in this case showed glycosides are absent.

Test for Cardiac Glycoside:

1. Baljet’s test:

Test solution was observed as yellow to orange colour with sodium picrate.

2. Sulphuric acid test:

To 2mL of extract, addition of GAA (glacial acetic acid), 1 drop of 5% FeCl₃and Conc. H₂SO₄is added side of the test tube and reddish-brown colour is being observed at the junction of two liquids and with bluish green colour upper layer is observed.

Test for Flavonoids:

1. Shinoda test:

To dried powder or extract, add 5ml 95% ethanol, few drops concentrated HCl and 0.5 gm magnesium turnings. Pink colour was observed.

2. Sulphuric acid test:

Addition of increasing amount of sodium hydroxide to the residue was observed as to whether it showed yellow coloration, which was decolorized after addition of acid.

3. Lead acetate test:

To small quantity of residue, add lead acetate solution and observed for yellow colored precipitate.

Test for Triterpenoids:

1. Liebermann – Burchard Reaction:

Mix 2ml T.S with chloroform. Add 1-2 ml acetic anhydride and 2 drops concentrated H₂SO₄along the side of walls of test tube. Formation of red/violet color.

Test for Saponins:

1. Foam test:

To dried powder or extract was shaken vigorously with water and observed for persistent foam.

Test for Tannins:

To 2-3 ml of test solution, add few drops of following solutions and observe the colour or precipitate formation:

1. 5% FeCl₃ solution: Deep blue-black colour.

2. Lead acetate solution: White ppt.

3. Gelatin solution: White ppt.

4. Acetic acid solution: Red colour solution.

5. Potassium dichromate: Red ppt.

6. Dilute Iodine solution: Transient red colour.

Test for Steroids:

1. Liebermann – Burchard Reaction:

Mix 2ml T.S with chloroform. Add 1-2 ml acetic anhydride and 2 drops concentrated H₂SO₄were added. Shake well, whether chloroform layer appear red and acid layer shows greenish yellow fluorescence.

Quantitative Estimation:

Quantitative Evaluation were estimated for methanolic bark extracts of Zizyphus jujuba to determine the total content in the drug. Following are the different contents of various metabolites using method:

Total Alkaloids Content:

5 Grams powdered bark sample was taken and transferred into 250ml beaker. Then addition of 200ml 10% Acetic acid in ethanol was done. Reaction mixture was allowed to stand for 4 hrs and then it was filtered. Filtrate was then evaporated on water bath up till it comes to ¼ of the original volume. After that, concentrated ammonium hydroxide is being added into the reaction mixture drop by drop and precipitate was obtained. Then that solution with precipitate was allowed to stand for some time. Then precipitate is washed using dilute ammonium hydroxide and filtered. Then the filtrate was dried and weighed. ⁸

Total Phenolic Content:

Folin-Ciocalteu method has been used to determine total phenolic content of the extract. Crude extract about 200 μl (1mg/mL) was diluted up to 3ml using distilled water and mixed completely with 0.5 ml of Folin-Ciocalteu reagent for about 3 min. addition of 2ml of sodium carbonate (20% w/v) was done. Mixture was allowed to stand for next 60 min away from the light in dark, then the absorbance is measured at about 650 nm. Hence the total phenolic content was determined by calibration curve and results were expressed using gallic acid as equivalent per gm dry weight. ⁹

Total Flavonoid Content:

Aluminium chloride colorimetric method has been used to determine total flavonoid content of the extract. 50 μl (1 mg/mL ethanol) of crude extract was diluted up to 1ml using methanol, also mixed with 4 ml of D.W. and then addition of 0.3 ml of sodium nitrite solution (5%); 0.3 ml of aluminium chloride solution (10%) done after incubation for 5 minutes. Then the mixture is allowed to stand for 6 minutes. Then the addition of 1 mol/L sodium hydroxide solution about 2 ml was done. Then the volume was made up to 10ml using double-distilled water. Then that mixture is allowed to stand for about 15 min. Absorbance was measured at 510 nm. Then the total flavonoid content was calculated using calibration curve and results were expressed using quercetin as equivalent per gm dry weight. ⁹

Total Terpenoid Content:

100mg (wi) dried plant extract was taken and immense in 9mL of ethanol for about 24 hours. Then filtration was done and extract was extracted again with 10 mL of Petroleum ether using separating funnel. The ether extract was isolated in pre-weighed glass vials and waited for its complete drying (wf). Ether was evaporated and yield (%) of total terpenoids contents was measured by the formula ¹⁰

Total Terpenoid Content = [wi – wf / wi X 100]

RESULTS AND DISCUSSION:

Pharmacognostical Study:

Macroscopy:

Table No 02: Macroscopical Characters

Sr. No	Color	Dark Brown
1	Odor	Characteristics
2	Taste	Astringent
3	Size	30-40 cm long and 0.8-1 cm thick
4	Shape	Flat and Thick
5	Fracture and Fissure	Long Fissures, Furrowed, Scaly and Laminated
6	Inner surface	Corrugated

Figure 05: Bark of Ziziphus jujuba

Microscopy:

· Cork cells are observed polygonal in shape. Phellodermal cells are generally parenchymatous in the nature. Lenticels are observed in the periderm, mostly in the old trees which has similar function to the stomata. These are open pores with absence of guard cells. The cork cells are impregnated with a layer of suberin. ⁶

· Sclerenchyma is a hardened, dead, tissue with thickened and lignified. Cells are generally long, narrow and pointed at both ends likes Fibre’s and in TS it appears polygonal with thickening and without intercellular. Function of sclerenchyma is mechanical strength. Found in all hard wood parts of the plant, whenever mechanical strength is needed. Sclerenchyma appears reddish pink after treatment with phloroglucinol and hydrochloric acid (1:1).⁶

· Phloem is the living tissue which conducts food from leaves o the plant parts. Phloem comprises of sieve elements, companion cells, phloem parenchyma and phloem fibres.⁶

· Xylem is a dead tissue conducts water from roots to leaves. Xylem comprises of tracheid’s, vessels, parenchyma and xylem sclerenchyma or wood fibres. ⁶

· Sclereids or stone cells are isodiametric or irregular, lignified, pitted or stratified cells, occurring simply or in bands, with varying lumen (the middle space left over after the lignification). Typically, U shaped thickening.⁶

· Sclerenchymatous Fibres are narrow, often elongated Fibre’s with pointed ends, lignified cells, named according to tissue they represent i.e. phloem fibre’s, xylem fibre’s, pericyclic fibres. ⁶

· Medullary Rays: When parenchymatous cells run diagonally or radially and extend from the pith to the cortex through the secondary xylem and secondary phloem, it is termed as a medullary ray. Width of medullary rays may vary from uniseriate to multiseriate. Medullary rays perform this function of conduction of food and water in lateral section. ⁶

· Calcium Oxalate Crystals: Single prismatic crystals occurred in small groups have large size and well developed. crystal sheath or crystal fibre, parenchymatous cells surrounding the Fibre’s contain calcium oxalate crystals. Crystals in the parenchymatous cells form crystal sheath. In each cell one crystal is present and a large number of such cells are arranged in row. ⁶

Figure 06: Photomicrograph of Ziziphus jujuba stem bark (Transverse Section)

Figure 07: Photomicrograph of Ziziphus jujuba stem bark (Longitudinal Section)

Microscopic characters (TS) of the bark:

The outer hard and woody exfoliating old bark consists almost entirely of the dead elements such as phellogen, phelloderm, phloem elements, secondary tissues. The outer layer is brown colored cork tissue. It gets stained pinkish to red by phloroglucinol and hydrochloric acid stain. Below cork, phellogen and phelloderm layers are present. Lenticels are present in the periderm which plays same role as stomata plays in leaves. They are open with no guard cells. Cork cells are impregnated with a layer of suberin. Cortex layer is present where phloem elements are found. Secondary phloem tissues are arranged and within them the stone cells and Sclereids layers is also observed. Also, medullary rays are observed. Thin-walled polygonal shape cells of cork tissues are observed. Lignified pitted ‘U’ shaped thickening and with narrow lumen Sclereids were observed. Polygonal Lignified stone cells were also being observed. Prismatic calcium oxalate crystals are also being observed in good amount. Sheath of the crystals is also being observed around sclerenchyma. Narrow elongated fibres with pointed ends also lignified were observed. Lignified Xylem pitted vessels were observed. ^6,7

Powder Characteristics:

Figure 08: Photomicrograph of Ziziphus jujuba stem bark Powder Characters

Powder Microscopy:

The bark powder is dark brown in color with astringent taste. Powder consists of thin-walled polygonal lignified cork cells, lignified phloem fibres, lignified xylem pitted vessels, lignified stone cells, U shaped stratified lignified Sclereids and prismatic calcium oxalate crystals.

Microchemical Test:

Table No 03: Microchemical Test of Powder

Sr. No.	Reagent	Observation	Characteristics
1	Phloroglucinol + Conc. HCl (1:1)	Pinkish	Lignified cells: fibre’s, stone cells, Sclereids, cork cells
2	Iodine	Blue	Starch
3	Ruthenium red	No Pink Colour	No Mucilage cells
4	Acetic acid	Insoluble	Calcium oxalate crystals
4	Dil. Hydrochloric acid	Soluble	Calcium oxalate crystals
5	Alcoholic Picric acid	No Yellow Colour	No Aleurone grains found
6	Sudan red III	No Red colour	Oil globules absent

Physico-chemical standards:

Table No 04: Physico-chemical standards

Parameter	Result	IP Limits
Loss on Drying	2.3 % w/w	-
Total Ash	Not more than 9.3 % w/w	Not more than 10% w/w
Water soluble ash	Not more than 4.2 % w/w	Not more than 3% w/w
Acid Insoluble ash	Not more than 3.36 % w/w	Not more than 6%
Water soluble extractive	Not less than 13.33 % w/w	Not less than 10%
Alcohol soluble extractive	Not less than 16 % w/w	Not less than 20%

Quantitative Estimation:

Phytochemical Screening:

Table No 05: Preliminary Phytochemical Screening

Sr. No	Type of Phytoconstituent	Methanolic extract	Aqueous extract
1	Carbohydrates	+++	+++
2	Reducing sugars	+++	+++
3	Alkaloids	+++	+++
4	Glycosides	+++	+++
5	Cardiac glycosides	+++	+++
6	Flavonoids	+++	+++
7	Triterpenoids	+++	+++
8	Saponins	+++	---
9	Tannins	+++	+++
10	Steroids	---	---

Quantitative Estimation

Table No 06: Quantitative Estimation of Extract

Sr. No	Phytochemical Content	Content Amount	Method	Calculated using
1	Total Alkaloid Content	22 mg/gm	Standard Method using Ammonium Hydroxide	Formula
2	Total Phenolic Content	123.7 mg/gm	Folin-Ciocalteu Method	Calibration Curve
3	Total Flavonoid Content	83.29 mg/gm	Aluminium Chloride Colorimetric Method	Calibration Curve
4	Total Terpenoid Content	19.21 mg/gm	Standard Method Using PET ether	Formula

Figure No 09: Quantitative Estimation of Extract

CONCLUSION:

The proposal of existing project was generated from the literature review of the plant; which suggested that the various parts of the plant have being used as traditional medicine and alternative system of medicine by medicinal practitioners such as leaves, flowers, fruits, root, bark, stem, seeds, etc. Botanical Validation of the plant relied entirely on the macroscopical characters and microscopical characters. Macroscopical characteristics such as Loss on drying, Ash Values, Extractive values, Size, Shapes, etc. Macroscopical characters are determined for identifying the purity of the drug. Microscopical characters were observed such as cork cells, phloem and xylem tissues, sclereids, etc. These characters are evaluated for identification of the drug.

Extraction of the bark of the Zizyphus jujuba has been done using Hot Soxhlet extraction method. Distilled water and HPLC grade methanol have been used as solvent for extraction. Phytochemical constituents were also observed using various phytochemical dentification test. Phytochemicals identified such as alkaloids, glycosides, tannins, carbohydrates, terpenoids, flavonoids, phenolic acids, saponins, etc. The qualitative estimation of the phytochemical was also being observed. Total phenolic, flavonoid, alkaloids and tannin contents were evaluated.

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Received on 12.10.2021 Modified on 06.12.2021

Research J. Science and Tech. 2022; 14(1):10-20.

DOI: 10.52711/2349-2988.2022.00002