INTRODUCTION:

Traditional herbal medicines are naturally occurring; plant-derived substances with minimum or no industrial processing that have been used to treat illness within local or regional healing processes. Traditional herbal medicines are getting importance in global health debates¹. Herbal medicines involve the use of various plant parts such as seeds, berries, roots, leaves, bark, or flowers for medicinal purposes². Traditional herbal medicinal practice has been known for centuries in different parts of the world. Herbal medicines are gaining interest because they are cost effective and eco-friendly nature³. Traditional medicine, including herbal medicine, provide their knowledge and practice of herbal healing for the prevention, diagnosis and elimination of physical, mental or social imbalance⁴. The pharmacological studies explain that plant have anticancer, antiulcer, antidiabetic, anthelmintic, antibacterial, immunological, hypolipidemic and antioxidant, haematinic and hepatoprotective effects.

Plant consists of fat, alkaloids, carbohydrates, glycosides, flavonoids⁵. The complex mixture of an extract from an herbal product may contain organic compounds such as fatty acids, sterols, alkaloids, flavonoids, saponins, glycosides, tannins, lignans, terpenes, peptides and carbohydrates and determination of the bioactive component(s) is very difficult⁶.

Due to the demand for fibrous material in whole world, global shortage of trees in many areas and environmental awareness, non-woods have become one of the important alternative sources of fibrous material for the 21st century⁷. Hibiscuss cannabinus is one of the most potential sources of fiber in the cottage industry in world. Recently kenaf production has been increased throughout the world for its elevated fiber content⁸. This plant was traditionally prescribed in traditional folk medicine in Africa and India; reported to contain several active phytoconstituents such as tannins, saponins, polyphenolics, alkaloids, lignin’s, essential oils and steroids⁹. Hibiscus cannabinus L. (Malvaceae) is a woody to herbaceous annual plant. It produced large cream coloured flowers characterized by a reddish purple or scarlet throat, which are popular in the western world as “Kenaf” and widely grown as a fibre crop¹⁰.

PLANT PROFILE:

Taxonomic classification:

Kingdom : Plantae

Subkingdom : Viridiplantae

Infrakingdom : Streptophyta

Super division : Embryophyta

Division : Tracheophyta

Subdivision : Spermatophytina

Class : Magnoliopsida

Superorder : Rosanae

Family : Malvaceae

Genus : Hibiscus

Species : Hibiscus cannabinus¹¹.

Chemical Constituents:

Kenaf contains several active phytoconstituents namely polyphenols, tannins, steroids, alkaloids, saponins, lignans such as boehmenan K, boehmenan H, threo-carolignan K and threocarolignan H, essential oils such as (E)-phytol (28.16%), (Z)-phytol (8.02%), N-nonanal (5.70%), benzene acetaldehyde (4.39%), (E)-2-hexanal (3.10%) and 5-methyl furfural (3.00%), ethyl alcohol, isobutyl alcohol, limonene, phellandrene¹²^,¹³.

MATERIAL AND METHODS:

1. Collection and drying:

Leaves of Hibiscus cannabinus were collected form Loni. Cleaned and dried at room temperature in shade and away from direct sunlight. The dried leaves were powdered coarsely in grinder. Large difference in particle size of crude drug result in long extraction time as the course particle increase the extraction time and fine powdered material was sieved through 60-120 mesh to remove fines and large particles and the powder was subjected for further study.

2. Authentication:

The plant was authentication by Department of Botany and Research Centre, Padmashri Vikhe Patil College of Arts, Science and Commerce, Pravaranagar on 4/12/2020 herbarium has been deposited in the department.

PHARMACOGNOSTIC STUDY:

The plant materials were studied for different parameter as given below, macroscopic feature and microscopic examination.

Macroscopic Study¹⁴:

The macroscopic study is morphological description of the plant parts. Which were seen by naked eyes or magnifying lens.

Powder Microscopic feature¹⁵:

In the study of the powder microscopy of leaves of Hibiscus cannabinus the Upper epidermis was covered by cuticle and contains elongated tubular cells. Lower epidermis was also covered by smooth cuticle and it also contains elongated tubular cells. Unicellular trichomes were seen. Stomata were seen which is Anisocytic type. Vascular bundle is present and various identifying characters were observed under Motic electronic microscope. Calcium oxalate crystals present.

EXTRACTION:

Ethanolic Extract¹⁵:

The leaves of Hibiscus cannabinus were dried under shade at room temperature for seven days and powdered it by the use of grinder and were sieved through sieve no.40 to get the coarse powder (100gm) and were extracted with ethanol as solvent by Soxhlet apparatus and filtered then obtained extract was concentrated and stored in vacuum desiccator. The obtained yield was calculated. Then extract of Hibiscus cannabinus was subjected for qualitative and phytochemical analysis.

Aqueous Extract¹⁶:

The leaves were air-dried in shade and ground to a powder form. The ground material (100g) was then transferred in to boiling water (1.5litres) for 15 min while stirring. After cooling, the resulting suspension was get filtered and the residue obtained was further extracted two times as before. The filtrate was concentrated using an evaporator. The concentrate was further evaporated upto its dryness in an oven at 40°C to obtain a powder. The aqueous extract was stored in a refrigerator for use.

DETERMINATION OF PHYSICAL CONTENT¹⁷^,¹⁸:

1. Loss on Drying¹⁷:

Weight of powder about 1.5 gm into a weighted flat and thin porcelain dish. Dry in oven at 105°C until the weighing is constant. Cool in desiccator and take its final weight. The loss in weight on drying is noted as moisture.

2. Ash value:

1. Total ash:

Determination of ash value is used to determine quality and purity of a crude drug and it is the identity of it.

Procedure:

Place about 2gm of ground air dried powdered drug accurately weighed in a crucible heat on of 2cm high flame heat till vapours almost cease. Allow the ash to cool in suitable desiccators for 30min, and then weigh without delay. Calculate the percentage content of total ash in mg/g of the dried material.

A sample of 2gm was weighted and air dried in a tarred silica dish. It was incinerated at a temperature not exceeding 450°C until it was free from carbon, cooled and the ash was weighted and percentage of ash was calculated.

2. Acid insoluble ash:

Method:

The ash was obtained as per the method described above for total ash. The ash obtained was boiled with 25ml 2M hydrochloric acid for 5minutes.Filtered and the insoluble matter was collected in Gooch crucible, washed with hot water, ignited, cooled, in a desiccator and weighed. The percentage of acid insoluble ash was calculated with reference to the air-dried drug.

3. Water soluble ash:

Method:

The ash was obtained as per the method described above for total ash. The ash obtained was boiled for 5 minutes with 25ml of water. Filtered and the insoluble matter was collected in a Grouch crucible, washed with hot water and ignited for 15 minutes at a temperature not exceeding 450°C. The weight of the collected insoluble matter was subtracted from the weight of the ash. The difference in that weight represented the water- soluble ash. Percentage of water – soluble ash was calculated with the air-dried drug.

4. Extractive value determination

This method determines the number of active constituents in given amount of medicinal plant material when it extract with solvents. The extraction of any crude drug with a specific solvent yields a solution which contains different phytoconstituents. The composition of these Phyto-constituents in that particular solvent yields a solution congaing different phytoconstituents. The composition of these Phyto-constituents in that particular solvent depends upon the nature of the nature of the drug and solvent used.

1. Alcohol soluble extractive value:

Accurately weight 5gm of air-dried crude drug was taken in closed flask and macerated with 100ml of 95% ethanol for 24hrs. It was shaken frequently during the first 6hrs and filter rapidly taking precaution against loss of alcohol. 25ml of the filtrate was taken and evaporate to dryness on a water bath and complete drying at 105°C. Then cool in desiccator and weighed. The percentage of alcohol soluble extractive value was calculated with reference to air dried drug.

2. Water soluble extractive value¹⁸:

5 gm of the air-dried drug, coarsely powdered have to be macerated with 100mi lf water closed flask for 24 hours, shaking frequently during the first 6 hours and allowing to stand for 18 hours. Thereafter, filter rapidly taking precautions against loss of water, evaporate 25ml of the filtrate to dryness in a tarred flat-bottomed shallow dish, dry at 105°C and weigh. The percentage of water-soluble extractive value with reference to the air-dried drug has to be calculated.

PHYTOCHEMICAL STUDY¹⁷:

Characterization of Extracts by chemical Methods:

· Test for alkaloids:

A) Dragendorff’s Test: About 2ml of filtrate sample was treated with Four to five drops of Dragendorff’s reagent and formation of Orange-brown precipitate was observed.

B) Mayer’s test: 2ml of filtrate mixed with few drops of Mayer’s reagent then ppt was observed.

· Test for Carbohydrates:

A) Fehling’s test: 1ml of Fehling’s A and 1ml of Fehling’s B solutions mix then boiled for 1 min, Added equal volume of test solution. Then heated in boiling water bath for 5-10 min. Brick red ppt was observed.

B) Benedict’s test: Same volume of Benedict’s reagent and test solution was mixed in test tube. Heated in boiling water bath for 5 min. Solution appeared in green colour.

· Test for steroids:

A) Salkowski reaction: In 2ml of extract, added 2ml of chloroform and 2ml of conc. H₂SO₄_.Shake well Chloroform layer appears red and acid layer shows greenish yellow fluorescence.’

· Test for Flavonoids:

A) Sulphuric acid test: 2ml of extracts was treated with few drops of sulphuric acid solution. Formation of deep yellow colour indicates the presence of flavonoids.

· Test for Tannin and phenolic compounds:

A) Lead acetate solution : Add 2ml of extract in lead acetate solution then white ppt was observed for presence of tannins and phenols.

B) Dilute HNO₃Solution: Mix 2-3ml of extract in dilute HNO₃then reddish to yellow colour taken as evidence for the presence of tannins and phenolic compounds.

· Test for Protein:

A) Biuret test: 3ml of test solution mix with 4% NaOH and few drops of 1% CuSO₄solution. Pink colour was observed for the presence of proteins.

· Test for Saponins:

A) Foam test: To 2ml of plant extract was treated with 8ml of water in a test tube. The mixture was shaken vigorously and observed for the formation of stable foam for 5 mins that confirms the presence of saponins.

QUANTITATIVE ANALYSIS OF PHYTOCONSTITUENTS:

· Total Phenolic Content¹⁹:

Phenolic content was measured fusing the Folin-Ciocalteu colourimetric with some modification. 1mL of kenaf leaf plant extracts was taken into a test tube and poured with 2.5mL of FolinCiocalteu phenol reagent (10%) and 2mL of Na2CO3 solution (2%). The test tube was shaken and the solution was remained in the dark for 2 hours at room temperature. The blue-green colour appearance formed in the incubated test tube indicated the presence of phenolics which was recorded at 765 nm. Thus, gallic acid (0.1mg/mL) was used as standard and serial dilutions were made with the range from 10 to 50 µg/mL. Result was presented as gallic acid equivalents (GAE) (mg/g of the extracted compound).

· Total Tannin Content¹⁸:

Procedure by WHO:

First taken powdered drug of Hibiscus cannabinus leaves, add 150ml of water then heated for 30min then cooled it. Transferred in 250ml of volumetric flask. Then diluted by using water. Filtered it. Discarded first 50ml of filtrate.

Then evaporate 50ml of plant material extract to dryness. Kept it for 4 hours at 105°C and weighted.

After that taken 80ml of plant material extract. Added 6 gm of hide powder and shake well for 60 min. Filtered and evaporated 50ml filtrate upto dryness at 105°C.

Taken 6gm of hide powder R than add 80ml of water and shake well for 60 min. Then filtered and evaporate 50ml of clear filtrate to dryness at 105°C and finally weighted.

[T₁-(T₂-T₀)]

Total tannin content= ------------------------- X 500

W=weight of plant material in grams

PHARMACOLOGICAL ACTIVITY²⁰:

Animal Grouping:

Application for the permission for the animal experiments was submitted to send either to CPCSEA (address in form A above) or institutional animal ethics committee (IAEC) with (Protocol No.: 1942/PO/Re/S/17/CPCSEA/2020/02/14).

The animals were allowed to acclimatize for a period of two weeks before treatments commenced. The animals were divided into groups [A, B, C, D, E, F, G, H] of six animals each. Group A served as control, while B served as standard. Group C, D, E. received 50 mg/kg, 100 mg/kg, 150mg/kg of ethanolic extract respectively and Group F, G, H received 50mg/kg, 100mg/kg, 150mg/kg of aqueous extract respectively.

Drug administration:

Dosages were calculated based on body weight of each animal in mg/kg body weight of the animal. The animals were weighed and the average weights of the animals were used in the calculation of the dosage. The extracts were administered by oral intubation through orogastric tube. The administration lasted for 10 days.

No of Groups	No of Rats
Group-Control	6
Group-Standard	6
Group-Test (03 Ethanolic Extract)	18
Group-Test (03 Ethanolic Extract)	18
Total	48

Anxiolytic activity:

Anxiolytic activity study on Elevated plus maze:

The elevated plus-maze study was carried-out using the Wistar albino mice weighing 150-250g was used. They was maintained in standard laboratory conditions and fed standard diet and water ad libitum. The elevated plus maze model consist of two open arms (15x5cm) and two closed arms (15x5x12cm), with open pair perpendicular to the closed one. Five groups (6 animals in each, n=6) was prepared for Control, standard (Diazepam 1mg/kg) and Three Test of ethanolic extract and Three test for aqueous extract (Hibiscus cannabinus) and administered with extract and standard before 30min of experimentation once a day for 10 days. The behaviors that were typically recorded when rodents were in the elevated plus maze are the time spent and entries made on the open and closed arms. After treatment of the Wistar albino mice with the drugs, the mice were placed in the center of the elevated plus-maze, facing one of the open arms. During a 5 min test period the following parameters were taken: the number of entries and time spent in the open and closed arms. Entry into an arm was recorded when the rats crossed the demarcation of respective arm with its four paws, and was considered to be on the central platform whenever two paws were on it. After each test, the maze was carefully cleaned up with a wet tissue paper (normal saline) to eliminate the interference of the olfactory cues on the next mice. The time in seconds, spent by the animals in the open arms and closed arms was noted. Anxiolytic compounds was reducing the animal’s natural aversion to the open arms and promoting exploration.

RESULT AND DISCUSSION:

The Pharmacognostic study of leaves of Hibiscus cannabinus macroscopic, microscopic and physiochemical parameter were studied.

Sr. No	Parameter	Morphological character
1.	Color	Green
2.	Odor	Characteristics
3.	Taste	Sour
4.	Size	7-15cm long
5.	Shape	3-5 lobed

Powder microscopy:

Micro Chemical Test-

Sr.No	Test	Observation	Inference
1	Powder+Phloroglucinol+ con.HCL (1:1)	Pink color	Elongated cells present.
2	Powder+ Acetic acid	Insoluble	Calcium oxalate crystals present.

Color, consistency, percentage yield of different extracts of leaves of Hibiscus cannabinus from successive solvent extraction

Extract	Color of extract	Extract of twig
Ethanol	Dark Green	8.5%
Water	Dark Green	6.85%

Physicochemical Parameter in Hibiscus cannabinus leaves

Sr. No.	Parameter	Value(%w/w)
1	Loss on drying	5.5
2	Total ash	32
3	Acid insoluble ash	24
4	Water soluble ash	8
5	Alcohol Soluble extractive value	1
6	Water Soluble extractive value	3

Preliminary phytochemical screening of extracts:

Phytochemical	Test	Ethanol	Aqueous
Alkaloids	Dragendorff’s test	+++	+++
Alkaloids	Mayer’s test	+++	+++
Carbohydrate	Fehling’s test	+++	+++
Carbohydrate	Benedict’s test	+++	+++
Steroids	Salkowski test	+++	---
Flavonoids	Sulphuric acid test	+++	+++
Tannin	Lead acetate test	+++	+++
Phenols	Dilute HNO₃solution	+++	+++
Proteins	Biuret test	+++	---
Saponins	Foam test	+++	+++

QUANTITATIVE ANALYSIS OF PHYTOCONSTITUENTS

Sr.No.	Phytoconstituents	Value
1.	Total Phenolic Content	4.6mgGAE/mg
2.	Total Tannin Content	20%

PHARMACOLOGICAL STUDY:

Group	Open Arm		Closed Arm
Group	No. of Entries	Time	No. of Entries	Time
Control	1.3 ±0.12	28.16±0.85	3±0.25	271.83±0.12
Standard	4.1±0.25 **	269.1±0.45**	2.33±0.11**	30.83±0.45**
EE 50mg/kg	2.5±0.32	178.16±0.12	3.5±0.33	121.83±0.48
EE 100mg/kg	2.83±0.25*	218.0±0.13*	3.1± 0.11*	82±0.12*
EE 150mg/kg	3.16±0.33*	233.3±0.21*	2.16±0.85*	66.16±0.96*
AE 50mg/kg	1.66± 0.22	166.1±0.36	2.83±0.23	133.83±0.45
AE 100mg/kg	2.16±0.41*	185.5±0.23*	3.22±0.36*	114.5±0.21*
AE 150mg/kg	2.5±0.63**	226.6±0.45**	2.16±0.11**	73.3±0.21*

All values are mean ± SEM, (n =6), one way ANOVA, followed by Dunnet’s test. * P< 0.05, **P< 0.01, ns-not significant when compared to vehicle treated group.

Graph 1.-Showing number of entries in open arm and closed arm

Graph 2-Showing Time spent in seconds

DISCUSSION:

In the present study, anxiolytic effects of Hibiscus cannabinus have been studied in experimental model of anxiety as Elevated plus maze model. The anxiolytic makers commonly associated with anxiolytic agents in the elevated plus maze model are increase in time spent in the open arms as well as increase in open arm entries²¹. These makers are important parameters that validate test agents with anxiolytic property. The use of Hibiscus cannabinus as an anti-anxiety agent in traditional medicine have not been investigated scientifically, so this study was carried out to investigate the anxiolytic effect of ethanolic and aqueous leaf extract of Hibiscus cannabinus leaves. The extract treated animals showed increase in open arm entries and time spent in open arm that was compared with the standard drug (diazepam). This is an anxiolytic-like effect. The phytochemical screening of the extract revealed the presence of flavonoid, alkaloid, phenol, tannin. Flavonoids (flavanones) have shown anti-anxiety activity in various studies. Further, the anxiolytic effect of flavonoids has been attributed to its effect on central nervous system and BZD receptors, as it was found that flavanones bind with high affinity BZD site of GABA A receptors²²^,²³. From the above study it showed that the ethanolic extract of the Hibiscus cannabinus leaves showed anxiolytic effext than aqueous extract.

CONCLUSION:

· The above study concludes that the Hibiscus cannabinus leaves shows the significant anxiolytic activity on elevated plus maze model.

· Traditionally plant was used as antioxidant and Immunomodulatory activity. The ethanolic extract shows the more anxiolytic activity as compare to aqueous extract

· Which need of feature from the Preliminary Phytochemical screening Alkaloid, Carbohydrates, Steroid, Proteins, Phenol, Tannins, Saponins in both extracts.

ACKNOWLWDGEMENT:

Authors are grateful to Dr. Sanjay Bhawar Principal of Prvara Rural College of Pharmacy and other Staff members for providing necessary facilities to carry out this work.

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Received on 12.07.2022 Modified on 22.08.2022

Res. J. Pharmacognosy and Phytochem. 2022; 14(4):265-271.

DOI: 10.52711/0975-4385.2022.00046