Isolation and Characterization of Chemical Constituents from the Fruits of Terminalia bellirica towards Achieving their Chemical Standardization

 

Mr. Mejo Joseph¹*, Dr. S. Alaxander²

¹Asst Prof. Department of Pharmaceutical Chemistry, Nehru College of Pharmacy, Kerala, India. 680588.

²Associate Prof. Department of Pharmacy, Vinayaka Mission College of Pharmacy, Tamilnadu, India. 636008.

 

ABSTRACT:

Terminalia bellirica fruits were extracted successively with solvents such as chloroform, ethyl acetate, methanol and water first on lab scale to find out the yield. The yield was found to be chloroform extract- 0.4%, ethyl acetate extract - 4%, methanolic extract- 30%, water extract - 23%. The extracts were separately dissolved in methanol and then spotted on the silica gel G 254 plates along with β-sitosterol and gallic acid with the help of capillary tubes. TLC plates were developed and scanned at 254 and 366nm. The Anisaldehyde Sulphuric acid reagent (ANS) was sprayed and the chromatogram was observed for separation. The chloroform extract showed different R_f values than the standard compounds. Ethyl acetate extract showed the presence of both gallic acid and β-sitosterol. Methanolic and aqueous extract shown the presence of gallic acid. Depending upon the TLC pattern and yield ethyl acetate and methanolic extraction was carried out in large scale. Both methanolic and ethyl acetate extracts were subjected for different columns and eluted with solvents in increasing polarity (pet ether, ethyl acetate methanol) and total seven compounds were isolated by column chromatography.

 

 

 

INTRODUCTION:

Terminalia bellirica is a large handsome deciduous¹ tree. It is an important drug of Indian System of Medicine and is used in medicines since time immemorial. During the last two decades, the drug has been subjected to extensive phytochemical, pharmacological and clinical investigations and many interesting findings² have been reported in various fields. The plant is found throughout the forests of India.

 

Phytochemical Constituents of Terminalia bellirica

Fruit is very hard and broken surface is yellow. Four lignans including termilignan, thannilignan, hydroxy-3', 4’-[methylenedioxy] flavan, and anolignan B identified. Fruit contains tannins about 23.60%~37.36%, tannins composed of Chebulinic acid Chebulagicacid; 1,3,6-Trigalloylglucose and 1,2,3,4, 6-pentagalloylglucose; Corilagin (C27 H22); Terchebin(C₄₁H₃₀O₂₆); Glucogallin(C13 H16 O10); Ellagic acid and Gallic acid, etc.

 

 

β-sitosterol

 

 

Gallic Acid

 

        

Ellagic acid                                             Ethyl gallate

 

Medicinal uses of Terminalia bellirica:

Extract of Terminalia bellirica fruits has been reported for antidepressant activity, hepatoprotective activity antimicrobial activity, spermicidal³ activity, hypotensive effect, stimulate bile secretion, antiHIV, antimalarial, antifungal.

 

MATERIALS AND METHOIDS:

Collection of plant material.:

The dried and authenticated fruits of Terminalia bellirica were obtained from Nehru College of Pampady, Kerala

 

Extraction of plant material:

The powdered plant material was extracted with chloroform, ethyl acetate, methanol and water in an extractor successively. The process was first performed in small scale to find out the yield and taken for pilot scale.⁴

 

Chemicals:

Ethanol, Chloroform, Methanol, Sodium bicarbonate, Hydrochloric acid, Formic acid, Ethylacetate, Toluene, Silicagel. Etc.

 

Lab scale extraction:

The powdered fruits of Terminalia bellirica (100gm) was refluxed with Chloroform (400ml) for 1 ½ hrs. The same process was repeated for three times, then the filtration was carried out and after concentrating the solvent the % yield was calculated which was found to be 0.4%. The powdered fruits of Terminalia bellirica (100gm) were refluxed with Ethyl acetate (400ml) for 2 hrs. The same process was repeated for four times, then the filtration was carried out and after concentrating the solvent the % yield was calculated which was found to be 4%. The spent obtained after chloroform extraction was refluxed with methanol (300ml) for 1½ hrs. The same process was repeated for three times, then the filtration was carried out and after concentrating the solvent the % yield was calculated which was found to be 30%. The spent obtained after methanolic extraction was refluxed with water (300ml) for 1½ hrs⁵. The same process was repeated for three times, then the filtration was carried out and after concentrating the solvent the % yield was calculated which was found to be 23%. On the basis of this the quantity of the raw material to be taken for pilot scale extraction was decided.

 

Optimization of TLC System:

Different solvent systems were tried to develop a TLC system for identification of constituents in the extracts and the one showing maximum separation was selected as mobile phase for the study. The following solvents were used for the development of the TLC system: Toluene: Ethyl acetate: Acetic acid: Formic acid (2.2: 1.1: 1.1:0.1). Toluene: Ethyl acetate: Acetic acid: Formic acid (1.1: 2.2: 1.1:0.1). Butanol: Acetic acid: Water (20: 5: 25). The extract was dissolved in methanol and then spotted on the silica gel G 254 plates along with β-sitosterol and gallic acid with the help of capillary tubes. TLC plates were developed and scanned at 254 and 366nm. The ANS reagent was sprayed and the chromatogram was observed for separation.

 

Column Chromatography:

Methanolic extract was subjected to systematic column chromatography using different solvent systems. The extract which was adsorbed in silica gel was then poured on to the bed of silica, a layer of cotton covered it again and more amount of solvent was poured over it, the column was then run by gradient elution technique⁶.

 

Procedure:

The column was first eluted with 100% pet ether. The polarity of mobile phase was gradually increased with ethyl acetate and methanol. The fractions collected were dried in rotavapour and weighed. The fractions collected from the column. Methanol wash given further to purify it. TLC of the ppt showed a sharp single spot which was sensitive at UV 254nm and also after spraying with FeCl₃ reagent. It was a pure compound. TLC of all the fractions collected from Column done with various solvent systems. It is shown in the following table and the TLC images are shown in the Depending upon the TLC pattern of all the fractions from column-1, three fractions were taken for the next column chromatography to isolate the targeted compound. (Fractionation and Isolation) Requirements: Stationary phase: Silica gel (60-200 mesh). Mobile phase: Pet ether, Ethyl acetate. Methanol. Volume of each fraction: 500ml.

 

TLC Analysis of Isolated Compounds:

TLC was done for all the isolated compounds with various suitable solvent systems. Stationary phase: Precoated silica gel G plates., Mobile phase: Ethyl acetate: Toluene: Formic acid -2.2:1.1:1.1. Visualization: UV at 254nm, after spraying with FeCl₃ reagent. TLC of compounds (Stationary phase: Pre-coated silica gel G plates. Mobile phase: Ethyl acetate: Toluene: Formic acid -2.2:1.1:1.1. Visualization: UV at 254nm, after spraying with FeCl₃ reagent.

 

Estimation of Isolated Compounds in Methanolic And Ethylacetate Extract HPLC method:

Sample preparation (extract):

11.34mg and 11.05mg of methanolic extract and ethyl acetate extract of the fruits of T. bellirica was weighed and dissolved separately in 2ml of HPLC grade methanol with the help of sonicator⁹. For standard solutions were used as standards for the estimation of the acetone fraction⁷. The procedure was same as described in their sample preparation and chromatogram was recorded using the same protocol respectively.

 

The estimated amount was calculated by using the formula =

 

Peak area of sample × standard dilution × purity of compound

––––––––––––––––––––––––––––––––––––––––––––

Peak area of standard       sample dilution

 

(Standard = HI-01 and HI-004, sample=Acetone extract).

 

Spectroscopic analysis of the isolated compounds:

As the purity of the isolated compounds were up to the mark, they were taken for further characterization by IR, NMR Mass spectrometry analysis etc. using CDCl₃ and pyridine as the solvent. These were done at Sophisticated Instrument Facility, QUEST Research and Training Institute, Bangalore and IISc, Bangalore.

 

 

RESULTS:

Optimization of TLC Method:

Optimization of TLC method was done using different mobile phases. The solvent system (Toluene: ethylacetate: formic acid in 2.2:4.4:2.2 ratio) and ANS and FeCl₃ as visualizing agent showed best resolution.⁸ Gallic acid was found in the extracts (lab scale and pilot scale) corresponding to the R_f values as follows:

Gallic acid= 0.66, β-sitosteol= 0.789

 

TLC Of Lab Scale, Pilot Scale Extracts with Some Standards

 

Solvent system =toluene: ethyl acetate: formic acid. (1.1: 2.2: 1.1)

 

 

Fig No. 1(Visualization UV at 254 nm.).

Solvent system = ethyl acetate: toluene: formic acid. (1.1: 2.2: 1.1)

 

Fig No. 2 TB-04 to TB-07 (Visualization UV at 254 nm.)

Solvent system Ethyl acetate: toluene: Formic acid- 2.2: 1.1: 1.

 

Isolation of Phytoconstituents:

The present study aimed at phytochemical analysis of Terminalia bellirica to isolate the major chemical constituents. The Methanolic extract was first subjected for isolation of phytoconstituents using column chromatography.¹⁰ Systematic column chromatography of the methanolic extract was performed using 2 columns.

 

TLC (All Fractions Together):

Solvent system: Butanol: Acetic acid: Water- 4:1:5.

 

Isolation /Fractionation using column-2(CC-2):

7 fractions were collected from column-2 using fractions CC-1/F15, CC-1/F16 and CC-1/F17 as starting material These fractions were concentrated under vacuum. Solvent system Ethyl acetate: toluene: Formic acid- 2.2: 1.1: 1.1) By processing the ppt obtained in CC-3/F-1, a pure compound was isolated and coded as TB-03. Ppt obtained from CC-3/F-4 was processed to get a pure compound and coded as TB-04. Ppt obtained from CC-3/F-7 was processed to get another isolated pure compound which was coded as TB-05. By processing the ppt obtained from CC-3/F-9 a pure compound was obtained which was found same as TB-02. By processing the ppt obtained in CC-3/F-12, a pure compound was isolated and coded as TB-06. Ppt obtained from CC-3/F-14 was processed to get a pure compound and coded as TB-07.

 

CONCLUSION:

Terminalia bellirica is an important drug of Indian System of Medicine and used in medicines since time immemorial. Various pharmacological activities have been reported such as antidepressant, hepatoprotective, antimicrobial, antifungal, hypolipidemic, antimalarial. In the present study an attempt had been made to isolate and standardize the major chemical constituents which can serve as a powerful tool for standardization. The methanolic and ethyl acetate fruit extract of Terminalia bellirica were fractionated using silica gel¹¹ column chromatography (60-200 mesh). Totally three different column chromatography has been done with different solvent systems which resulted in the isolation¹² of seven different compounds. These were given codes as TB-01, TB-02, TB-03 and TB-04, TB-05, TB-06 and TB-07.Determination of Melting point and solubility profile were done for the isolated compounds. New HPLC methods were developed to analyze the purity of the compounds.¹³ The HPLC method indicated that the compound TB-01 was 99.41 % pure, TB-02 was 100 % pure, TB-04 was 100% pure TB--06 was 96.3470% pure and TB-07 was 99.84 % pure. These compounds were taken for further characterization¹⁴.

 

Isolated Compounds:

Altogether seven compounds were isolated from methanolic extract and ethylacetate extract namely TB-01, TB-02, TB-03 TB-04, TB-05 TB-06 and TB-07. The TLC of isolated compounds are shown as like this. TB-01=0. (Visualized after spraying with ANS reagent). TB-O2 = 0.7568. (Visualized at UV 254 nm) TB-03 = 0. 4091. (Visualized after spraying with ANS reagent). TB-04 = 0.5952. (Visualized after spraying with ANS reagent). TB-05 = 0.3778. (Visualized after spraying with ANS reagent). TB-06 = 0.6582. (Visualized at UV 254 nm). TB-07 = 0.7468. (Visualized at UV 254 nm)

 

The yields of isolated compounds are shown in Table No. 3 and the physical properties of these compounds are shown in the Table No. 4.

 

TLC OF TB-O1 toTB-07:

 

Fig. no.3 Visualized at UV 254 nm.

Solvent system: Ethyl acetate: Touene: Formic acid- 2.2: 1.1: 1.1.

 

 

Spectroscopic Analysis of The Isolated Compounds.

All the spectroscopic data obtained for the compound TB-01 suggested that the structure of the compound may be the following:

 

 

The supporting integral values (¹H NMR) for this structure were as follows:

δ = 3.22 – m – 11H (at a, b, 1, c, d)., δ = 1.40 – s – 4H (at e and f).,δ = 0.30 – s – 1H (at g)., δ = 1.18 – d – 4H (at h and i)., δ = 0.35 – s – 1H (at j)., δ = 1.51 – s – 4H (at k and l)., δ = 0.31 – s – 1H (at m)., δ = 0.32 – s – 1H (at n)., δ = 2.63 – m – 6H (at 2 and 3)., δ = 2.92 - m – 12H (at 4, 5, 7 and 8).,δ = 1.00 – m – 3H (at 6)., δ = 4.58 –s – 2H (at =CH₂).

 

The supporting IR values were as follows:

2940.61 = Aliphatic C-H of -CH₃, =CH₂.,1736.97 = C=O.,875.72 = Aliphatic C-C.1366.62 = Aliphatic C-H.

 

 

 

The supporting integral values (¹H NMR) for this structure were as follows:

δ = 3.86 – s – 3H (Aromatic O- CH₃ at 4). δ = 6.43 – s – 1H (Aromatic H at 3). δ = 6.52 – d – 2H (Aromatic H at 5).δ = 7.40 – d – 2H (Aromatic H at 6).δ = 9.41 – s – 1H (Aromatic CHO at 1).δ = 11.48 – s – 1H (Aromatic OH at 2).

 

The supporting IR values were as follows:

3050 = Aromatic –OH., 1633 = Aldehydic C=O., 1326.12 = Aromatic C=C., 2850 = Aldehydic C-H., 1505.51 = Aromatic C-O., 2902 = C-H of O- CH₃ at 4.

 

ACKNOWLEDGMENTS:

I would like to express my gratitude to Nehru College of Pharmacy for their support to conduct this study.

AUTHOR’S CONTRIBUTIONS:

The author declares that this work was done by the author named in this article.

 

CONFLICTS OF INTEREST:

The author declares that he has no conflict of interest.

 

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Accepted on 16.06.2020           © RJPT All right reserved

Research J. Pharm. and Tech 2021; 14(3):1680-1684.