Isolation and Characterization of Quercetin from Hydroalcoholic extract of Hibiscus cannabinus Linn Leaves

 

Dattatraya B Thorat¹*, Sandeep Narwane², Rahul Kunkulol³, Sanjay B Bhawar⁴

¹Pravara Rural Education Society, Institute of Pharmacy, Loni, Tal - Rahata, Dist - Ahmednagar.

²Institute of Medical Sciences (Deemed University), Loni, Tal - Rahata, Dist - Ahmednagar.

³Pravara Institute of Medical Sciences (Deemed University), Loni, Tal - Rahata, Dist - Ahmednagar.

⁴Pravara Rural College of Pharmacy, Loni, Tal Rahata, Dist-Ahmednagar.

 

Abstract:

Herbal medicine is making a dramatic comeback as the side effects of synthetic medicine are daunting and the therapeutic approach is drifting towards substituent medicine. Quercetin is a plant pigment of flavonoid. It's found in many plants and foods, such as red wine, onions, green tea, apples, and berries. Quercetin has antioxidant and anti-inflammatory effects that might help reduce swelling, kill cancer cells, control blood sugar, and help prevent heart disease. Hibiscus cannabinus Linn., Leaves (Malvaceae) is an endangered plant species occurring in the Ahmednagar district (Maharashtra), India. Leaves are used in the treatment of antibilious, antioxidant, aphrodisiac, poultice and purgative. Fraction of hydroalcoholic extract of Hibiscus cannabinus Linn., Leaves by column chromatography was done. During the column elution process, the fractions 15 - 30 have a single banding pattern which was confirmed by TLC study. Phytochemical screening showed the presence of Flavonoids, Phenolic and Tannins. Physical and chemical tests of isolated compound were conducted. By using HPTLC, FTIR, 1H-NMR and GC-MS spectra of the isolated compound was confirmed as Quercetin.

 

 

 

INTRODUCTION:

The use of traditional medicines and medicinal plants as therapeutic agents for the maintenance of good health has been widely observed. Interest in medicinal plants as a re-emerging health aid has been fueled by the rising costs of prescription drugs^1-2. Lots of Industry has shown interest in investment for medicinal herbs and novel chemical compounds^3-4. The various parts of plants such as seeds, berries, roots, leaves, bark, or flowers are used for medicinal purposes^5-7. Herbal medicines are gaining interest because they were cost effective and eco-friendly nature⁸. All ancient medicine, including herbal medicine, provides their knowledge and practice of prevention, diagnosis and elimination of physical, mental or social imbalance⁹. Herbs and different plants were various chemicals for their metabolic activities and to protect them from various disease and predators also^10-11. A balance between herbal industry and regulation is needed to protect public health^12-13.

 

Hibiscus cannabinus Linn., belongs to the family Malvaceae the plant extract shows presence of primary and secondary compounds such as carbohydrates, fatty acids, steroids, alkaloids, flavonoids, saponin, glycosides and tannins¹⁴. It is an endangered plant species occurring in the Ahmednagar district Maharashtra, India. Leaves are used in the treatment of antibilious, antioxidant, aphrodisiac, poultice and purgative. The secondary metabolites, Flavonoids, a pigment that color most leaves, flowers, and seeds are widely distributed in plants with different metabolic functions^15-16. These polyphenolic compounds are ubiquitous group characterized by the flavan nucleus and available as a group of bioactive compounds in leaves¹⁷. The basic structure of flavonoids is diphenyl propane skeleton, namely, two benzene rings (ring A and B– Fig. 1)^18-19

 

Fig.:1 Structure of Quercetin

 

MATERIALS AND METHODS:

Preparation of extracts: The fresh plant parts leaves of Hibiscus cannabinus Linn., was shadow dried and powdered. Powdered material was passed through sieve No.150.Then extracted separately using aqueous, hydroalcoholic and ethanol by the Soxhlet extraction method. The extracts were concentrated using a rotary vacuum evaporator further study²⁰.

 

Qualitative phytochemical Screening: Various qualitative tests were performed on the various extracts of Hibiscus cannabinus Linn., (leaves) for the identification of phytoconstituents²¹.

 

Table-1.

S. 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 resent.
2	Proteins Test	Biuret test: extract was treated with 1 ml 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.
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: 1 ml of concentrated sulphuric acid was added to 10 mg of extract dissolved in 1 ml of chloroform.	Reddish brown colour, steroids present.
5	Terpenoid Test	2 ml of the organic extract was dissolved in 2 ml of chloroform and evaporated to dryness. 2 ml of concentrated sulphuric acid was then added and heated for about 2 min	Grayish          colour, terpenoids   present.
6	Glycosides Test	Anthraquinone glycosides- Borntragers test: 3 ml 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.
7	Saponins Test	Foam formation test: 1 ml solution of the extract was diluted with distilled water to 20 ml and shaken in a graduated cylinder for 15 minutes.	Development stable foam, Saponins present.
8	Alkaloids Test	Dragendroffs test: 0.1 ml dilute hydrochloric acid and 0.1 ml Dragendroffs reagent was added in 2 ml of extract in a test tube.	Orange     brown precipitate, alkaloids present.
		Mayers test: 2 ml of extracts, 0.2 ml of dilute hydrochloric acid and 0.1 ml of Mayer’s reagent was added.	Yellowish buff precipitate, alkaloids present.
9	Tannins and Phenolic compounds Test	Ferric chloride test: 5 ml of extract solution was allowed to react with 1 ml of 5 % ferric chloride solution.	Greenish  black coloration, tannins Present.
10	Flavonoids Test	Shinoda test: the extract, 5 ml (95%) ethanol, few drops of conc. HCl and 0.5 gm 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.

 

Phytochemical tests: The phytochemical tests of different extracts were performed identified with using specific reagents and results Table no.2.

 

Table 2: Phytochemical Screening of leaves extract from different solvents.

Sr. No.	Chemical constituent	Chemical Tests	Observations
			Ethanol extract	Hydro-Alcoholic extract	Aqueous extract
1.	Tests for carbohydrates	Molish Test	+	+	-
2.	Test for Proteins	Biuret test	+	-	+
3	Test for amino acid	Ninhydrin test	-	-	-
4.	Tests for Steroids	Salkowaski test	+	+	-
5.	Tests for Terpenoids	-	+	-	-
6.	Test for Glycosides	Borntrager’s 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	+	+	-
10	Test for Tannins and Phenolic compounds	FeCl3	+	+	+

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

 

The Hibiscus cannabinus Linn. Hydroalcoholic and ethanol were having maximum number of phytoconstituents. Isolation and characterization of phytoconstituents of Hibiscus cannabinus Linn., leaves hydroalcoholic extract. (Table 2).

 

During the column elution process, the fraction 15- 30 has a single banding pattern which was confirmed by TLC study. So, the fractions are combined and kept for evaporation to dry at room temperature. After drying the dried residue was scrapped off once again checked for its purity.

 

The remaining fractions were not worked out because of lower yield as well as impure. The compound with R_f value 0.52 was subjected for Phytochemical screening, Physical properties and spectral analysis, i.e., HPTLC, FTIR, 1HNMR, GC-MS and structural elucidation^22-24.

 

Physical properties and other details of isolated compound:

Color: White crystalline powder

M.P: 200-204˚C

TLC: Single spot

R_f value by TLC: 0.52

λ max: 254nm

 

Fig.:2 Shows the presence of Quercetin

 

Interpretation and Observation:

HPTLC- (High performance thin layer chromatography)

Table 3: Development System for HPTLC

1.	Stationary phase	Silica gel G 60 F254
2.	Mobile phase	Toluene: Ethyl Acetate: Formic Acid (5:5:0.3 % v/v)
3.	Development	Development chamber pre-saturated (20 min) with the mobile phase
4.	Source of radiation	deuterium lamp 200-400 nm
5.	Detection	A. at 254 nm B. Visible Light

               

Fig.3: Visualization of Developed Plate A. at 254 nm B. Visible Light.

 

In the figure no A and B shows the following observation,

Quercetin - 254 nm

R_f - 0.52

Both the compound run parallel, the blue spectra show the graph of reference substance and green spectra shows the isolated compound of quercetin, which is identified above HPTLC method (Graph 1).

 

7.6.3 FTIR absorptions bands appeared at 3375.20 cm-1 (OH), 2927.74 cm-1 (CH), 1747.39 cm-1 (C=C), 1639.38cm-1 (C=O), 1491.98 cm-1 (C-H), 1380.94 cm-1 (OH),1095.49, 1037.63 cm-1 (C-O-C), 883.34 cm-1 (C-H), (Graph 2).

 

7.6.4 1H NMR Spectrum of this compound having  three aromatic protons doublets (δ 6.895, δ 7.546 and δ 7.684)  and two flavonone singlet (δ 6.198 and δ 6.417) in the ¹H-NMR spectrum (and the carbons of alkenes conjugated are at 176.4 ppm (C₄) which was confirmed from the ¹³C-NMR. The structure was simulated using ACD/NMR program to obtain the chemical shifts of both proton and carbon. (Graph 3).

 

7.6.5 GC-MS Mass spectra of this compound suggested that its molecular mass is 303 (Molecular formula C₁₅H₁₀O₇) having characteristic fragments observed at m/z: 303, 275, 247, 229, 199, 185, 152, 135, 108 (Graph 4). On comparison the experimental data matched with the simulated data which supports the proposed structure of this compound is quercetin. (Graph 4).

 

Spectral analysis:

HPTLC-

 

Graph.1: Spectra Comparison HPTLC of Quercetin.

Blue –Spectra of RS Quercetin, Green – Spectra of isolated Quercetin from extract

 

Graph. 2- Fourier-Transform Infrared spectra of isolated Compound

 

 

Graph 3- ¹HNMR spectra of isolated Compound

 

 

Graph.4-Mass spectrum of isolated Compound

Table 4: IR Interpretation of isolated Compound

 

Table 5:  Interpretation of ¹ H-NMR spectra of isolated Compound

 

Table 6: Interpretation of Mass spectra

 

Table 7: Interpretation of Mass spectra (Mass Fragmentation)

 

The compound, a white crystalline solid, resolved at R_f 0.52 upon TLC of fraction 15-30 of the hydroalcoholic extract using the mobile phase toluene: ethyl acetate: methanol (4.4: 5: 0.6). The IR spectrum of this compound showed the presence of a hydroxyl function (OH) which showed intense bands in the spectrum at 3375.20 and 1380.94 cm⁻¹. The olefinic moiety showed its presence in the spectrum at 1639.38 cm⁻¹. The stretching and bending of methyl group was observed as an intense band at 2927.74 cm⁻¹ and as a medium intensity band at 1491.98 cm⁻¹. The presence of three aromatic protons doublets and two flavonone singlet (δ 7.684 and δ 6.198) in the ¹H-NMR spectrum revealed that the compound may be a flavonoid. The GC-MS spectra indicated the molecular weight of the compound to be 303 with base peak at 69. The physical and spectral data and the comparison of ¹ H-NMR chemical shifts with that of the reported data of similar type of compounds led to the conclusion that the compound is quercetin.

 

2-(3, 4-dihydroxyphenyl)-3, 5, 7-trihydroxy-4H-chromen-4-one

 

Characterization of the Compound:

Phytochemical analysis (Shinoda test, Lead acetate Test and Sod-hydroxide Test) of the compound confirms its Flavonoid nature. The elemental analysis (Elementar, Vario EL III) revealed that the compound contains 47.13% of C, 31.63% of H and 21.84% of O. The N % was found to be nil. Now based on the number of O (6 or 7) in the proposed compound the molecular weight could be: 302.23 or 274.22 respectively. So, Based upon the number of O the formula could be tentatively:  C₁₅H₁₀O₇ or C₁₄H₁₀O₆. From ¹³C-NMR and ¹H NMR the number of C and H was found to be near to the first formula i.e. C₁₅H₁₀O₇. The exact molecular mass for the formula was found to be 302.23. This formula produces 302.23 hits when searched in http://www.chemspider.com/Search.aspx.andhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC7790550/. Since, the compound gives positive test for Flavonoid so all of the other structures  other than Flavonoid were rejected. Based upon the functional group analysis it was found that the nature of oxygen was hydroxyl, also supported by FTIR spectroscopy (Shimadzu, IR Prestige-21). This implies presence of one double bond in the structure. So, the Flavonoid with other functional groups was rejected. Also on considering the ketocarbonyl group in its molecule, and the oxygen atom on the first carbon is basic and can generate salts with strong acids. Its molecular structure contains four active groups, namely, a dihydroxy group between the A ring, O-dihydroxy group B, C ring C2, C3 double bond, and 4-carbonyl. The presence of a phenolic hydroxyl group and double bonds endows. Therefore it must be a heterocyclic compound. Based on the melting point and other related data (HPTLC, FTIR, 1HNMR and GC-MS) the structure of the isolated compound is confirmed as Quercetin.

 

Fig. 4: Structure of Quercetin

 

Needle shaped crystals of quercetin were obtained. The structure of the isolated compound was established on the basis of elemental analysis and spectroscopic evidences (HPTLC, FTIR, UV, ¹H-NMR, GC-MS). The structure was simulated using ACD/NMR program to obtain the chemical shifts of both proton and carbon.

 

RESULTS AND DISCUSSION:

Hibiscus cannabinus Linn. Leaves are used in the treatment of antibilious, antioxidant, aphrodisiac, poultice and purgative. From the literature, it was found that no other study regarding the wound healing activity of the plant, has Hibiscus cannabinus Linn. been conducted and as the plant was used to treat antioxidant and other ailments which are a caused due to we decide to go for wound healing activity.

 

The hydroalcoholic extract of Hibiscus cannabinus Linn.  Leaves showed the presence of more phytoconstituents, alkaloids, flavonoids, carbohydrate, Phenolic and Tannins. So further study was conducted with the extract. Column chromatogtaphy by gradient elution technique was performed and the fractions 15 – 30 has a single banding pattern which was confirmed by TLC study. So the fractions are combined and kept for evaporation to dry at room temperature. After drying the dried residue was scrapped off once again checked for its purity. The compound with R_f value 0.52 was subjected for phytochemical screening, Physical properties and spectral analysis, i.e., HPTLC, FTIR, 1HNMR and GC-MS for structural elucidation. Based on the study the column isolate was confirmed as quercetin.

 

CONCLUSION:

Despite the recent interest in molecular modeling, combinatorial chemistry and other synthetic chemistry techniques by pharmaceutical companies and funding organizations, natural products, particularly medicinal plants, remains an important source of new drugs, new drug leads and new chemical entities. It is evident that, natural products have played a vital role in drug discovery, by contributing to a wide variety of phytochemicals for the treatment of cancer, cardiovascular diseases, infections related with viral and microbial origin and other health disorders. After collection and authentication of plant material, fresh plant of Hibiscus cannabinus Linn. leaves were shadow dried and powdered. Powdered material was passed through sieve No.150. Then extracted using aqueous, hydroalcoholic and ethanol by the Soxhlet extraction method. The extracts were concentrated using a rotary vacuum evaporator.

 

The qualitative chemical tests were performed on the plant extracts to detect the various phytoconstituents present in them as per the standard procedure and findings were recorded. From the qualitative chemical tests, it was found that the hydroalcoholic and ethanol extracts of Hibiscus cannabinus Linn. Leaves were having the maximum number of constituents. So, the hydroalcoholic extract was subjected to column chromatography, by means of gradient elution technique. The fractions 15- 30, gave one compounds. Later by spectral analysis they were found that the component is quercetin. Based on the literature quercetin is a well-known antioxidant present in plant. So we concluded that hydroalcoholic extract of Hibiscus cannabinus Linn. leaves containing quercetin and the scientific data presented in this study demonstrate that hydroalcoholic extract of Hibiscus cannabinus Linn. leaves used for isolation and characterization of other bioactive components.

 

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Received on 14.11.2024      Revised on 17.02.2025 Accepted on 21.04.2025      Published on 15.05.2025 Available online from May 17, 2025 Research J. Science and Tech. 2025; 17(2):139-147. DOI: 10.52711/2349-2988.2025.00020
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