Isolation of Betulinic Acid from Byttneria herbacea (Malvaceae)

 

Sanjeeb K. Kar¹*, Uma S. Mishra², Prasanna K. Dixit³, Shubhrata Nayak⁴

¹Faculty of Pharmacy, Gopal Narayan Singh University, Jamuhar, Sasaram - 821305, Bihar, India

²Department of Pharmacy, Royal College of Pharmacy and Health Sciences, Berhampur - 760002, Odisha, India

³Department of Zoology, Berhampur University, Berhampur - 760007, Odisha, India

⁴Senior QC Officer, Acme Generics LLP, Davni, Solan -174101, Himachal Pradesh, India

 

ABSTRACT:

During the phytochemical analysis, flavonoids, saponins, tannins, glycosides, alkaloids, and terpenoids were identified in Byttneria herbacea (Malvaceae) among other things. The goal of this research is to identify and characterize the plant's bioactive principle. It has a wide range of folk medicinal applications. The gradient fractionation method was used to isolate and characterize phytoconstituents from ethanol extract. Physical, chemical, and spectroscopic data were used to determine the structure of the isolated substance (IR, UV, ¹HNMR, MS). The ethanol extract of the plant yielded a triterpenoid structure. Various spectroscopical approaches were used to identify the odorless, colorless chemical that solubilizes in ethyl acetate and has a melting point of 315°C - 318°C and an R_f value of 0.62 in the mobile phase toluene: ethyl acetate (85:15). As a result of this research, the isolated component has been identified as Betulinic acid, which is accountable for the plant's diverse pharmacological properties.

 

 

 

INTRODUCTION:  

For a large proportion of the world's population, traditional plants provide an infinite source of life-saving remedies¹. The bioactive segment and extracts of these plants provide a wide range of bioactive chemicals that are important in the fight against different diseases². An in-depth investigation of phytochemicals found in medicinal plants could aid in the finding of prototypes during the drug development process^3,4. Genus Byttneria comprises many plants demonstrating much ethnomedicinal potency. Byttneria herbacea belongs to the family Malvaceae and contain many important phytoconstituent which are responsible for various type of ethnomedicinal activity in the Indian indigenous systems of medicine and are considered to cure cholera, looseness of the bowels, dysentery, leprosy, limb fractures, tuberculosis, analgesic and relieve asthma and inflammation.

 

 

 

 

The root paste is used on the wound and is taken orally to alleviate bodily pain,the leaf is used for dysentery and impaction, and the root is used to control swelling^5–7. Byttneria is a genus of flowering plants of the family Malvaceae. There are approximately 135 species in this pantropic genus. It is indigenous to India and distributed and found in Uttar Pradesh, Assam, Andhra Pradesh, Odisha, Tamil Nadu, West Bengal, and Nagaland^8,9. The plant's phytochemical profile revealed the presence of flavonoids, saponins, tannins, glycosides, alkaloids, and terpenoids, among other things^5,6. Different parts of this plant have exhibited antihistaminic activity, free radical scavenging, antioxidant activity, and anti-inflammatory activity^9,10. Literature review shows that no special attention has been provided to the isolation and characterization of phytoconstituents. So in the current study, the objective is to identify and characterize the plant's bioactive principle. Recently, one new triterpenoid has been isolated from plant extract. The present constituents have not been reported previously.

 

MATERIAL AND METHODS:

Chemicals:

Sephadex LH-20 was acquired from Life Technologies (India) Pvt. Ltd. Silica gel-60-120 mesh, Aluminium TLC silica gel plates 60 F 254 (20 × 20cm), HPTLC pre-coated plates were from Merck Life Science Pvt. Ltd. Mumbai.

 

Preparation of Reagents:

TLC study was carried out by exposing the plates to both short and long-wavelength ultraviolet light. Derivatization was performed on the plates by immersing them in Anisaldehyde Sulphuric acid reagent and heating for 5 minutes at 120 degrees Celsius.

 

Isolation of phytoconstituents¹¹:

Vacuum Liquid Chromatography was used to separate the components of the ethanol extract of the areal parts of Byttneria herbacea into 21 fractions using the solvent mixture of petroleum ether, ethyl acetate, and methanol in different proportions of increasing polarity. On the TLC plate, the occurrences of similar spots were discovered by TLC screening of fractions 11-14. By paring through the Sephadex column, these five fractions were combined and fractionated again and analyzed using the ethyl acetate: toluene (15:85) eluent in preparative thin-layer chromatography (PTLC). Six spots were identified under a UV light at 254nm on the prepared PTLC plates that showed purplish color on heating in the oven after spraying with vanillin-sulphuric acid. Bands of sport 2 from the PTLC plate were scraped and filtered with ethyl acetate. To ensure purity, the dried mass was tested to Mass, ¹H NMR spectroscopy. The compound weighed around 1.6mg.

 

Identification Test of Triterpenoids:

Foam Test:

A dilute aqueous solution of the extracts was agitated for 5 minutes in a test tube. The presence of saponins is indicated by the development of foam^12,13.

 

Sedimentation Test:

To the extract, a few drops of 10% Lead acetate solution were added. The formation of a precipitate indicates the presence of triterpenoids^12,14.

 

Color Test:

To the extract, one drop of 10% Copper sulfate solution and 1 ml of Sulphuric acid were added. The appearance of the blue-green color on heating indicates the presence of triterpenoids^12,15.

 

Characterization of Isolated Compound:

1H NMR, Mass, and IR studies, and comparisons with previously reported results were used to determine the structure of the isolated compound (compound-1)^15–17.

 

RESULTS AND DISCUSSION:

The existence of a methyl group (allylic) at δ1.84 (3H, s) terminal methylene protons at δ 4.75 (1H, s) and δ 4.78 (1H, s) was found in the ¹H NMR spectra of compound-1 (1H, s). There were additionally five methyl singlets at δ0.80, 0.91, 0.99, 1.04, and 1.049, as well as one hydroxyl group (secondary) singlet at δ1.70. At δ 3.5 and δ 3.4, two multiplates were identified, which were attributed to H-3 and H-19, respectively. The isolated compound (Compound-1) was recognized as Betulinic acid by comparing the ¹H NMR spectra to the published one. A strong ion peak of m/z (+1) 478.52 is visible in the mass spectral analysis, indicating a compound peak with a single sodium ion attached. This reveals that the compound is Betulinic acid, molecular formula C₃₀H₄₈O₃ with a molecular weight of 456.7.

 

 

Figure 1. Structure of Betulinic acid

 

 

Figure 2. ESI-MS spectra of Compound 1

 

Figure 3. ¹H NMR Spectra of Compound 1

 

Figure 4. FT-IR spectra of Compound 1

 

 

The FT-IR spectra show strong bands of IR absorption at 3433 cm^-1 and 2941 cm^-1 in the high wave region, which are accredited to stretching vibrations of -OH,
-CH₃, and -CH₂,  both asymmetric and symmetric respectively. The major bands in the FT-IR spectra are 1686, 1454, 1369, 1037, 884, and 756 cm-1 in the fingerprint region, as well as numerous other bands with medium to low intensity. The C=O stretching of the functional group –COOH is responsible for the observed band at 1686 cm^-1. Other bands in the spectrum are assigned to bending vibrations of -CH₃, -CH_2, and –OH groups as well as skeleton bending bonds. The band at 1369 cm^-1 is caused by C-O stretching. An intense band at 884 cm^-1 is caused by the CH₂ vibration of the alkene group.

 

 

Figure 5. TLC of isolated Betulinic Acid

 

CONCLUSION:

We isolated one phytoconstituent Betulinic acid from the areal parts of Byttneria herbacea. For the first time, a component has been separated and reported from Byttneria herbacea (Malvaceae).

 

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Accepted on 04.06.2022        © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(2):535-538.