1. INTRODUCTION:

The traditional herbal medicine shows importance in global health debate¹. Many traditional herbal medicines are playing an important role in global health^1-12. Lots of Industry has shown interest in investment for medicinal herbs and novel chemical compounds^13-14. The various parts of plants such as seeds, berries, roots, leaves, bark, or flowers are used for medicinal purposes¹⁵. As per reports many herbal plants used in the treatment of individual health conditions or multiple health conditions. Multiple plant parts used for herbal medicines have been successfully studied¹⁶.

In the current scenario herbal medicine has become a popular for healthcare; even though there are some differences between herbal and conventional pharmacological treatments,

herbal medicine needs to be tested for efficacy by using conventional methods which were used for trial and specific herbal extracts has been demonstrated which were efficacious for specific conditions¹⁷. Herbal medicines are gaining interest because they were cost effective and eco-friendly nature¹⁸. Recorded uses of plants as medicine from 5,000 years to the Sumerians and the Vedas¹⁹. The written text of Rig-Veda came about 2000 years ago²⁰.

There were all types of natural medicine practically overall approach for the treatment of a specific medicinal condition in that the physical, mental as well as social well-being of an individual all are collectively considered. All ancient medicine, including herbal medicine, provides their knowledge and practice of prevention, diagnosis and elimination of physical, mental or social imbalance²¹. Herbs and different plants were various chemicals for their metabolic activities and to protect them from various disease and predators also. Initially metabolites like carbohydrates and fats are found in all plants; whereas secondary metabolites are toxins which are used to prevent predation²². Herbal medicine and other herbal preparations gives assurance about the safety and efficacy of many herbal products but lack of proper scientific evaluation resulted in the IUPAC supporting a project with the objective of preparing protocols on efficacy, safety, standardization and documentation of herbal medicine²³. Herbal medicines and oral literature knowledge are considered interlinked and therefore, some countries justify less strict regulations. In the developing countries most of the population depends on herbal medicine. So it is necessary to have regulations and proper legislation to ensure that required standards of safety and efficacy are met. A balance between herbal industry and regulation is needed to protect public health^24-25.

Plant History:

The plant extract shows presence of primary and secondary compounds such as carbohydrates, fatty acids, steroids , alkaloids, flavonoids, saponin, glycosides and tannins²⁶.

2. MATERIALS AND METHODS:

2.1 Plant Materials:

The leaves of Hibiscus Cannabinus (Malvaceae) were collected from local area and subjected for natural drying and was authentified by Mrs. Priyanka A Ingle, Scientist ‘C’ by comparing morphological features.

The plant Hibiscus cannabinus leaves were collected naturally dried leaves used for coarse powder preparation with mesh size 150.

2.2 Pharmacognostic Studies:

Macroscopic: The above macroscopic characters were performed for size, shape, color, odour and taste.

Microscopic: The plant was collected from Ahmednagar district (Maharashtra), India. Transverse section of leaf was taken, stained with different micro chemical reagents and observed at 10X.

Powder Microscopy: Powder microscopy were performed the powdered characteristics of leaves shows lignified cells, pericyclic fibers, stone cells, cuticles, calcium oxalate crystals.

2.3 Physical-chemical Properties: The different Physiochemical studies like LOD of total ash, acid insoluble, and water soluble ash were performed.

2.4 Phytochemical Studies: The various phytochemical tests were performed for presence of secondary metabolites like carbohydrates, proteins, alkaloids, flavonoids, tannins, phenols, steroids, and glycosides^27-29.

2.5 Extraction:

The 50gm dried powder of the plant Hibiscus cannabinus L., was extracted successively with various solvents viz. aqueous, hydro-alcoholic and ethanolic in Soxhlet extractor. The marc left were extract using suitable solvents. Extract were reduced by vacuum distillation and then kept for drying in open air to produce dry extracts³⁰.

Figure 1: Extraction by Soxhlet Apparatus.

2.6 Preliminary phytochemical screening:

Initially phytochemical screening of various extracts was carried out using the standard procedure^31-33.

Table no.1

Sr. No.		Test	Inference
1	Carbohydrate Tests	Molish test: The extracts solution mixed with 15% ethanolic Alpha-napthol solution in a test tube and sulphuric acid was added carefully along the side of tubes.	Reddish violet colour, carbohydrate present.
1	Carbohydrate Tests	Fehling’s test: 5ml of extract solution was mixed with 5ml Fehling’s solution (equal mixture of Fehling’s solution A and B) and boiled.	Brick red precipitate, Carbohydrate present.
2	Proteins Test	Biuret test: Extract was treated with 1ml of 10% sodium hydroxide solution and heated. A drop of 0.7% copper sulphate solution was added to the above mixture.	Purple violet colour, proteins present.
2	Proteins Test	Millon’s test: Extract was treated with 2ml of Millon’s reagents.	White precipitate, proteins and amino acids present.
3	Amino acids Test	Ninhydrin test: Extract was treated with Ninhydrin reagent at Ph range of 4-8 and boiled.	Purple colour, amino acid present.
4	Steroids Test	Salkowski test: 1ml of concentrated sulphuric acid was added to 10mg of extract dissolved in 1ml of chloroform.	Reddish brown colour, steroids present.
		Liebermann-Buchard test: The extract was dissolved in chloroform, acetic anhydride, concentrated sulphuric acid from the side of the tube	Reddish violet colour, steroids present.
		Liebermann’s test: The residue mixed with acetic anhydride and gentle heated and added concentrated sulphuric acid	Blue colour, steroids present.
5	Terpenoid Test	2ml of the organic extract was dissolved in 2ml of chloroform and evaporated to dryness. 2ml of concentrated sulphuric acid was then added and heated for about 2 min	Grayish colour, terpenoids present.
6	Glycosides Test	Anthraquinone glycosides- Borntragers test: 3ml extract dilute sulphuric acid was added, boiled and filtered. To the cold filtrate, equal volume of benzene was added and shaked well. Organic layer was separated and to that ammonia solution was added.	Ammonia layer turns pink or red, glycoside present.
6	Glycosides Test	Cardiac Glycoside: Keller-Killani test: 2ml of extract, glacial acetic acid, one drop 5 % ferric chloride and conc. Sulphuric acid was added.	Bluish green, glycoside present.
7	Saponins Test	Foam formation test: 1ml solution of the extract was diluted with distilled water to 20ml and shaken in a graduated cylinder for 15 minutes.	Development stable foam, Saponins present.
8	Alkaloids Test	Dragendroffs test: 0.1ml dilute hydrochloric acid and 0.1ml Dragendroffs reagent was added in 2ml of extract in a test tube.	Orange brown precipitate, alkaloids present.
		Mayers test: 2ml of extracts, 0.2ml of dilute hydrochloric acid and 0.1ml of Mayer’s reagent was added.	Yellowish buff precipitate, alkaloids present.
		Hagers test: 2ml of extract was allowed to react with 0.2ml dilute hydrochloric acid and 0.1ml of Hager’s reagents.	Yellowish precipitate, alkaloids present.
		Wagners test: 2ml of extract was treated with 0.2ml dilute hydrochloric acid and 0.1ml of Wagner’s reagent.	Reddish brow precipitate, alkaloids present.
9	Tannins and Phenolic compounds Test	Ferric chloride test: 5ml of extract solution was allowed to react with 1 ml of 5 % ferric chloride solution.	Greenish black coloration, tannins Present.
9	Tannins and Phenolic compounds Test	Dilute nitric acid test: 2ml of extract solution was allowed to react with few drops of dilute HNO₃ solution.	Reddish to yellow colour, tannins present.
10	Flavonoids Test	Shinoda test: the extract, 5ml (95%) ethanol, few drops of conc. HCl and 0.5gm of magnesium turning was added	Pink colour, flavonoids present.
		Lead acetate test: Few drops of 10 % lead acetate were added to the extract.	Yellow colour precipitate, flavonoids present.
		Sodium Hydroxide test: the extract increasing amount of sodium hydroxide was added.	Yellow colour was produced which disappeared after addition of acid, flavonoids present.

2.7 Thin layer chromatography:

The extracts obtain from leaves of H. cannabinus were subjected for TLC with appropriate solvent system for separation of the phytoconstituents. The R_f values of the separated phytoconstituents were recorded³⁴.

3. RESULTS AND DISCUSSIONS:

3.1 Pharmacognostic Studies:

Macroscopic Studies: The macroscopic characters were served as important keys in proper identification of plant material and also acts as an important quality control parameter. The macroscopic features of leaves Hisbiscus cannabinus were described here figure no.1. The organoleptic evaluation was discussed in table no.2.

Fig.1- Hibiscus cannabinus Plant

Table 2: Macroscopic characteristics

Sr. No	Organoleptic Character	Observations
1.	Colour	Green
2.	Odour	Characteristic
3.	Taste	Sour
4.	Size	5-10cm in length and 4 to 10cm in width.
5.	Shape	3-5 lobed

Microscopic studies:

Microscopically powder characters were the leaves has 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. Trichomes were rarely seen and were glandular in nature. Palisade layer was present below the upper epidermis and contains beam shaped cells. Arc shaped vascular bundle was present which contains lignified xylem and unlignified phloem. Collenchyma was present below the upper epidermis. Calcium oxalate crystals were present in parenchymatous region and mucilage-present. Figure no.2.

Table No: 3 Microscopic studies of Leaves of Hibiscus cannabinus Linn

Sr. No.	Name of Microchemical Reagent	Observation	Inference
1	T. S. + Phloroglucinol + Conc.HCl (1:1)	Pink Colour	Lignified Cells, Pericyclic Fibers, Stone Cells
2	T.S. + Sudan Red III	Red Colour	Cuticles
3	T.S. + Dil. Hydrochloric acid	Soluble	Calcium Oxalate Crystals
4	T.S. + Dil. Acetic Acid	Insoluble	Calcium Oxalate Crystals


T. S. + Phloroglucinol + Conc.HCl (1:1)	T.S. + Sudan Red III	T.S. + Dil. Hydrochloric acid	T.S. + Dil. Acetic Acid

Fig 2: Transverse sections of Hibiscus cannabinus Leaves

Leaf constants:

Table 4: Leaf Constants of Hibiscus cannabinus Leaves.

Sr. No.	Leaf Constant	Result
1	Vein islet numbers	27
2	Vein termination	13
3	Stomatal numbers	157
4	Stomatal index	35

Fig 3: Stomatal number of H.cannabinus.

3.2 Physical-chemical Properties: Physical constant viz. Ash values, Extractive values and Loss on drying at 110⁰C were determined and results Table no.5.

Table 5: Physicochemical Parameters of Hibiscus cannabinus Leaves.

Sr. No.	Evaluation specification	Value (% w/w)
1	% of Moisture contents	8.6 %
2	Total Ash Values	7.1 %
3	Acid Insoluble Ash Values	4.2 %
4	Water Soluble Ash Values	3.5 %
5	Hexane Soluble extractive Values	2.1%
6	Ethanol Soluble extractive Values	6.2 %
7	Water Soluble extractive Values	3.3 %

3.3 Preliminary Phytochemical tests:

The preliminary phytochemical tests of different extracts were performed identified with using specific reagents and results Table no.6.

Table 6: Preliminary phytochemical Screening of Leaf extract from different solvents.

Sr. No.	Chemical constituent	Chemical Tests	Observations
Sr. No.	Chemical constituent	Chemical Tests	Ethanol extract	Hydro-Alcoholic extract	Aqueous extract
1.	Tests for carbohydrates	Molish Test	+	+	-
		Fehling Test	+	+	+
		Benedict Test	+	+	-
2.	Test for Proteins	Biuret test	+	-	+
2.	Test for Proteins	Millions test	+	+	-
3	Test for amino acid	Ninhydrin test	-	-	-
4.	Tests for Steroids	Salkowaski test	+	+	-
4.	Tests for Steroids	Libermann Burchard test	+	+	-
5.	Tests for Terpenoids	-	+	-	-
6.	Test for Glycosides	Borntrager’s Test	+	+	+
6.	Test for Glycosides	Killer- Killani Test	+	-	+
7.	Test for Saponin	Foam test	+	+	-
8.	Tests for Flavonoids	Shinoda test	+	+	+
		Lead acetate Test	+	+	+
		Sod-hydroxide Test	+	+	+
9.	Tests for Alkaloids	Mayers Test	+	+	-
		Wagner’s Test	+	+	-
		Hager’s Test	+	+	-
		Dragendorff Test	+	+	+
10	Test for Tannins and Phenolic compounds	FeCl3	+	+	+
10	Test for Tannins and Phenolic compounds	Lead acetate	+	+	+

(+ Sign indicates presence of phytoconstituents, - Sign indicates absence of phytoconstituents)

The preliminary phytochemical shows leaves extract mainly revealed the presence of carbohydrate, alkaloids, flavonoids, steroids, tannins and terpenoids in hydroalcoholic extract.

Table 7: Extraction/Isolation of Leaf extract from different solvents

*Hibiscus cannabinus* Leaves Extract	Ethanolic extract	Hydro-alcoholic extract	Aqueous extract
Weight of drug (wt, gm)	50	50	50
Weight of glassware without Extract (w1,gm)	44.27	45.35	44.52
Weight of glassware with Extract (w2,gm)	51.07	53.85	52.47
Extract (wt) x=w2-w1 (gm)	6.8	8.5	7.9
Color	Dark Green	Dark Green	Dark Green
Percentage (%)	13.60%	17.00%	15.80%

The 50gms of Hibiscus cannabinus shed dried leaves were powdered was Soxhlet extracted with Ethanol (60-70˚C), Hydro- alcohol and aqueous every single homogenate was filtered and then extracts were cooled and concentrated individually, each extract was weighted and examined. The weight of ethanolic extract was 6.8 gm (13.60%) having dark green color, hydro alcoholic extract was weighted 8.5gm (17.00%) with dark green color and aqueous extract weight was 7.9gm (15.80%) having dark green color, the extracts were stored in tube for further use.

3.4 Thin layer chromatography:

The R_f values and color helps in determining the numbers of related type of compounds present in the plant extracts. The results show presence of alkaloids, steroids, glycosides, flavonoids and tannins. Table no 8.

Table no. 8: Thin layer chromatography of various chemical constituents of H. Cannabinus L.,

Sr. No	Chemical constituent	Mobile Phase	Visualization Spraying reagent	Color of spot	R_f- value
1.	Alkaloids	Toluene : Ethyl acetate: Formic acid (50:40:10)	10% H₂SO₄ in ethanol	Violet -blue	Ethanol : 0.49 Hydroalcoholic : 0.45 Aqueous: 0.43
2.	Glycoside	Ethyl acetate : Methanol : Water (100 : 16.5:13.5 )	Under UV -365	Violet -blue	Ethanol:0.86 Hydroalcoholic : 0.79 Aqueous: 0.82
3.	Flavonoid	Toluene: Ethyl acetate : Glacial acetic acid : Water (100:11:11:26)	Anisaldehyde – Sulfuric acid.	Yellowish green	Ethanol:0.59 Hydroalcoholic : 0.53 Aqueous: 0.56
4.	Tannin	Ethyl acetate: Formic acid : Acetic acid: Water (100:11:11:26)	5 % FeCl₃in 0.1N HCl	Black	Ethanol : 0.6 Hydroalcoholic : 0.9 Aqueous: 0.11
5.	Steroids	Ethyl acetate : Methanol : Acetic acid (70 : 20 : 10)	Vanillin – Sulfuric acid.	Pink	Ethanol : 0.67 Hydroalcoholic : 0.68 Aqueous: 0.64

Hibisus cannabinus L. belong to the family Malvaceae. The macroscopical evaluation i.e.; colour, size, shape, taste of the plants was performed by identification. Microscopically evaluation was also performed by transverse section of leaves. By this method parenchyma, collenchyma, vascular bundles, palisade, upper and lower epidermis, trichomes were identified. Leaf constants like vein islet numbers: 27, vein termination numbers: 13, stomatal numbers: 157, stomatal index-35 of the plants. The pharmacognostical parameters like extractive values, loss on drying, crude fiber content were performed. The solvent was selected by using the order of polarity; ethanol showed 6.2% w/w of extractive value where as Hexane showed 2.1% w/w for Hibiscus cannabinus. Extraction was performed by using Soxhlet extraction apparatus by using ethanol, hydroalcoholic and aqueous as solvent and the crude extract was found to 6.8g, 8.5g and 7.9g for Hibiscus cannabinus L., respectively. The ethanolic, hydro-alcoholic and aqueous extract were analyzed by using coloured reactions based on chemical tests for identification of alkaloids, flavonoids, glycosides, steroid, terpenoid, saponins, proteins, tannin, phenol, carbohydrates, amino acid, etc. respectively for plants. Drug powder with different microchemical reagents was used and observed under compound microscope at 10X. Finally we conclude that in hydro alcoholic extracts of Hibiscus cannabinus L., has more secondary metabolites were present as compare to ethanolic and aqueous extracts.

4. CONCLUSION:

The present work deals with evaluation of quality control parameters of Hibiscus cannabinus leaves. The plants were evaluated by using various pharmacognostical, physicochemical and phytochemical parameters. Pharmacognostical screening includes macroscopical parameters. Microscopically parameters (transverse section), physiological parameters were performed and values were reported. The extraction and identification of phytochemical screening. Hydroalcoholic extract was more secondary metabolites were present as compare to ethanolic and aqueous extracts. The above research work will be useful to conduct the further research in future studies. Now a day developed countries are also following herbal medicines because of herbal medicine are safe, less toxic and have good pharmacological action compared to allopathic system of medicine. Herbal medicines are economical so, for a developing country like India herbal medicine can be used for treatment of disease.

CONFLICTS OF INTEREST:

The authors declare no competing interests.

ACKNOWLEDGEMENTS:

It my privilege to express my sincere thanks to Pravara Institute of Medical Sciences (Deemed University), Loni, Tal-Rahata, Dist- Ahmednagar (MS).

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Received on 04.11.2023 Modified on 09.02.2024

Res. J. Pharmacognosy and Phytochem. 2024; 16(2):89-94.

DOI: 10.52711/0975-4385.2024.00017