INTRODUCTION:

Momordica is a genus of annual or perennial climbers is essentially a native of tropical regions on Asia distributed extensively in China, Japan, South East Asia, Polynesia besides tropical Africa and South America. Many species of Momordica genus have been found to grow wildly in India, Bangladesh, Srilanka, Myanmar, Malay etc¹.

Momordica balsamina also refers to as balsam fruit or apple belongs to the family of Cucurbitaceae. It is a plant commonly used by local poultry farmers in Plateau State, Nigeria for general well being of birds. The Balsam apple is a climber or trailer with annuals stems attaining 4-5 m length. The fruit is orange yellow, beaked, 21/2 inches in length bursting and exposing red brown seeds. The species is closely related to Momordicacharantia (Bitter mellon) which occurs in areas of greater rainfall and whose properties and actions includes antibacterial, anti-inflammatory, anti-oxidant, antiviral, immunostilmulant, hypoglycemic etc^2-5. Some Momordica species are already established as medicine and their marketed preparations are available and used in the treatment of different diseases. The rationale behind the study is to isolate, characterize and quantitatively determine the constituents present in petroleum ether extract of Momordica balsamina fruits.

MATERIALS AND METHODS:

Plant material:

The fruits of Momordica balsamina was collected from rural area of Surat. The herbarium of the Momordica balsamina was authenticated by Veer Narmad South Gujrat University, Surat voucher specimen (MA 01) was deposited in library.

Preparation of Extracts:

Fruits of Momordica balsamina was extracted by Soxhlet extractor with Pet ether and macerated with ethanol and water successively.

Storage of Extracts:

All the extracts were stored in tightly closed glass bottles in refrigerator at 2-8 ⁰C.

Preliminary Phytochemical analysis of extracts:

All the extracts were tested for the presence of various chemical constituents^[6,7].

Isolation of Phytoconstituents from Pet ether extract of M. balsamina fruits

A) Separation of unsaponifiable fraction from Pet ether (PE) extract:

PE extract was taken in a flask and to it 0.5 N methanolic KOH was added and kept overnight at room temperature. Next day mixture was refluxed for 6 h and cooled. Twice of its volume distill water was added, extracted with diethyl ether and allowed to stand for few minutes. Ethereal layer was separated, the procedure was repeated twice. Ethereal layer was combined and washed with distill water till neutral to litmus paper. Ether was evaporated to obtained unsaponifiable fraction^[8,9].

B) Isolation of phytoconstituents from unsaponifiable fraction of PE extract:

The unsaponifiable fraction was loaded on silica gel column. Elution of column was carried out by PE (100%) with increasing concentration of ethyl acetate (EA). Elution of the column with PE (100%) was carried out to afford 60-125 fractions (each fraction 250 ml). All fractions were monitored by TLC. All fractions were collected, mixed and concentrated to obtained white solid powder (Compound 1). Elution of the column with 3% EA was carried out to afford fractions 170-240. All fractions were collected, mixed and concentrated to obtained white solid powder (Compound 2). Fraction 290-350 eluted with 5 % EA afforded white crystals (Compound 3). Elution of the column with 8% EA was carried out to afford fractions 390-450. All fractions were collected, mixed and concentrated to obtained white solid powder (Compound 4).

C) Spectroscopical characterization:

UV-visible spectrum was measured on a JASCO V-530 UV-Visible spectrophotometer using methanol as solvent. IR spectra of isolated compounds were taken on a FT-IR spectrophotometer (Perkin Elmer) at room temperature. ¹H and ¹³C NMR spectra were taken on a Mercury Plus 300MHz NMR Spectrometer (Varian, USA). ¹H and ¹³C chemical shifts (δ, ppm) are relative to the solvent signals used as references. Mass spectrum was recorded on AccuTOF mass spectrometer (JEOL).

HPTLC instrumentation and chromatographic conditions:

A) Preparation of stock solution of lupeol, -amyrin and ursolic acid:

Standard lupeol, -amyrin and ursolic acid were obtained from YUCCA enterprises, Mumbai. 10 mg of lupeol was transferred to volumetric flask. 5 ml of petroleum ether solution was added into the 10 ml volumetric flask and sonicated in ultrasonicator for 5 min for complete dissolution of lupeol. The volume was then made up to the mark with petroleum ether. A stock solution of lupeol with concentration of 1000 ug/ml was prepared. Similarly stock solutions of-amyrin and ursolic acid were prepared.

B) Preparation of sample solution:

200 mg Petroleum ether extract of Momordica balsamina was transferred to 50 ml volumetric flask. 25 ml of petroleum ether was added into the volumetric flask and sonicated in ultasonicator for 20 min. Sample was filtered through whatman filter paper no. 1. The filtrate was then transferred to 50 ml volumetric flask and final volume was made up with petroleum ether. Similarly the sample solution of petroleum ether extract of Momordica balsamina was prepared.

C) Chromatographic conditions:

The sample solutions were spotted in the form of bands of width 6.0 mm with a Camag microliter syringe on precoated silica gel aluminum plate 60F₂₅₄ (20 cm × 10 cm with 250 μm thickness; E. Merck, Darmstadt, Germany, supplied by Anchrom Technologists, Mumbai) using a Camag Linomat V (Switzerland). The plates were activated at 120°C for 20 minutes prior to chromatography. A constant application rate of 1.0 μl/s was employed, and space between two bands was 13.2 mm. The slit dimension was kept at 5.0 mm × 0.45 mm and 10 mm/second scanning speed was employed. The slit bandwidth was set at 20 nm, each track was scanned thrice and baseline correction was used. The mobile phase for fingerprinting of lupeol, β-amyrin and ursolic acid consisted of Pet Ether: Ethyl acetate: Toulene (7:1:2), Toulene: Ethyl actate: Glacial acetic acid (8:2: 0.2) and Toulene: Ethyl actate: Glacial acetic acid (7:3: 0.2) respectively and anisaldehyde sulfuric acid was used for derivatization, 20 ml of mobile phase was used per chromatography. Linear ascending development was carried out in 20 cm × 10 cm twin trough glass chamber (Camag, Muttenz, Switzerland) saturated in the mobile phase. The optimized chamber saturation time for mobile phase was 30 minutes at room temperature (25°C ± 2) at relative humidity of 60% ± 5. The length of the chromatogram run was 8.0 cm. Subsequent to the scanning; thin layer chromatography (TLC) plates were dried in a current of air with the help of an air dryer. Subsequent to the development; TLC plate was dipped in anisaldehyde sulfuric acid reagent followed by drying in the oven at 110°C. Post derivatization, densitometric scanning was performed with Camag TLC scanner IV in the reflectance absorbance mode at at 580 nm, 550 nm and 530 nm for lupeol, β-amyrin and ursolic acid respectively and operated by Win CATS software (1.4.6 Camag) with the help of tungsten lamp. Concentrations of the compound chromatographed were determined from the intensity of diffusely reflected light. Evaluation was carried out by comparing peak areas with linear regression^10,11,12,13.

RESULT AND DISCUSSION:

The results of preliminary phytochemical screening of various extracts of fruits of Momordica balsamina revealed the presence of glycosides, flavonoids, steroids, phenolic compounds, saponins, carotenoids and carbohydrates (Table 1).

Table 1: Preliminary Phytochemical Screening of M. balsamina

S.no	Constituents	*Momordica balsamina*
S.no	Constituents	Pet Ether Extract (PEMB)	Ethanolic Extract (AlcMB)	Aqueous Extract (AqMB)
1	Phytosterols	++	–	–
2	Glycosides	–	+	–
3	Carbohydrates	–	–	++
4	Flavonoids	–	+	–
5	Alkaloids	–	–	–
6	Tannins	–	+	–
7	Proteins	–	–	–
8	Saponins	–	+	+
9	Carotenoids	+	–	–
10	Phenolic compounds	–	+	–

+Presence, – Absence

Compound 1:

Elution of the column with PE (100%) furnished white coloured compound 1. It gave pink colour with Libermann- Burchard reagent indicating the triterpenoid nature of the molecule. It was soluble in PE and CHCl₃ indicating non polar nature of compound. Melting point of compound was found to be in the range of 215-217^oC. IR spectrum showed characteristic absorption bands for hydroxyl groups (3386 cm^-1), absorption band of C-O stretching (1595 cm^-1), C-H stretching (2945 cm^-1 ) and C=C bending (1460 cm^-1). The mass spectrum of compound1 exhibited a molecular ion peak at m/z 426 consistent with the molecular formula C₃₀H₅₀O.

The ¹H NMR spectrum of 1 showed two one-proton multiplets at δ value 0.78 to 1.07 ppm assign to methyl protons. One multiplet proton peak at δ 4.68 ppm, 4.57 ppm attributes to alkene protons. ¹³C NMR spectrum exhibited signals between δ value 14.08 - 55.2 ppm indicating the presence of several methyl and methylene proton in the structure. The ¹³C NMR spectrum of 1 exhibited signals for isopropene group attached to cyclic ring carbon at δ value 151.1 ppm (C-20), 109.5 ppm (C-29); Signal at δ 79.1 ppm (C-3) indicates the presence of electronegative group like OH.

On the basis of physical characteristics ¹H NMR, ¹³C NMR, Mass, and IR spectral evidence, the structure of compound 1 was elucidated as lupeol (Fig 1).

Fig 1: Chemical structure of compound 1(Lupeol)

Compound 2:

Elution of the column with PE: EA (97: 03) the PE extract of fruits of M. balsamina furnished white coloured compound 2. It gave pink colour with Vanillin sulphuric acid reagent indicating the triterpenoid nature of the molecule. It was slightly soluble in ether, chloroform and PE indicating non polar nature of compound. Melting point of compound was found to be in the range of 285-288⁰C. IR spectrum showed characteristic absorption bands for hydroxyl groups (3510 cm-1), carboxylic C=O group (1697 cm-1) and olefinic groups (1635 cm-1). The mass spectrum of compound 2 exhibited a molecular ion peak at m/z 456.3597 consistent with the molecular formula C30H48O3.

The 1H NMR spectrum of compound 5 showed multiple peaks proton signals (two secondary of the α –type triterpene) at δ 0.81 (s, 6H, H-25, H-26) , d 0.91-0.97 (s, 6H, H-23, H-24), δ 0.83 (3H, d, J = 6.4 Hz H-29), and d 0.92 (d, 3H, J = 4.4Hz, H-30) and δ 1.06 (3H, s, H-27). This is further supported by a doublet at d 2.18 (d, 1H, J = 15 Hz, H18) indicating that H-18 and H-19 are trans to one another. The signal at δ 3.19 d 3.19 (dd, 1H, J = 10.8, 4.4 Hz, H-3) was assigned to H-3 of the 3β – equatorial hydrogen and the olefinic signal at δ 5.24 (d, 1H, J = 12 Hz, H-12) of H-12 which coupled to H-11.On the basis of physical characteristics 1H NMR, Mass, and IR spectral evidence, the structure of compound 2 was elucidated as ursolic acid (Fig 2).

Fig 2: Chemical structure of compound 5 (Ursolic acid)

Compound 3:

Elution of the column with PE: EA (97: 03) furnished white coloured compound 3. It gave bluish colour with AS reagent indicating the triterpenoid nature of the molecule. It was soluble in PE indicating non polar nature of compound. Melting point of compound was found to be in the range of 197-199^oC. IR spectrum showed characteristic absorption bands for hydroxyl groups (3427 cm^-1), absorption band of C=C stretching (1655 cm^-1) and C-H stretching (2990 cm^-1). The mass spectrum of compound 3 exhibited a molecular ion peak at m/z 426 consistent with the molecular formula C₃₀H₅₀O.

The ¹H NMR spectrum of compound 3 showed multiple peaks at δ values from 0.75 to 1.14 ppm assign to methyl protons. One multiplet proton peak at δ value 3.80 ppm attributes to alkene protons. The ¹³C NMR spectrum exhibited signals between δ value 14.08 - 55.2 ppm indicating the presence of several methyl and methylene proton in the structure. ¹³C NMR spectrum also exhibited signal at δ 79.1 (C-3) indicates the presence of electronegative group like OH. ¹³C NMR spectrum showed signal at δ 134.8 ppm (C-13) indicates the presence of quaternary carbon.On the basis of physical characteristics ¹H NMR, ¹³C NMR, Mass, and IR spectral evidence, the structure of compound 3 was elucidated as - amyrin (Fig 3).

Fig 3: Chemical structure of compound 2 (- amyrin)

Compound 4:

Elution of the column with PE: EA (95: 05) furnished colorless crystals compound 3. It gave blue colour with AS reagent indicating the triterpenoid nature of the molecule. It was soluble in PE indicating non polar nature of compound. Melting point of compound was found to be in the range of 137-139^oC. IR spectrum showed characteristic absorption bands for hydroxyl groups (3427 cm^-1), absorption band of C=C stretching (1655 cm^-1) and C-H stretching (2930 cm^-1). The mass spectrum of compound 4 exhibited a molecular ion peak at m/z 414 consistent with the molecular formula C₂₉H₅₀O.

The ¹H NMR spectrum of 4 showed several singlet peaks at δ values from 0.91 to 1.21ppm assign to methyl protons. One multiplet proton peak at δ value 3.56 ppm attributes to alkene protons. The ¹³C NMR spectrum exhibited signals between δ value 12.08 - 51.3 ppm indicating the presence of several methyl and methylene proton in the structure. ¹³C NMR spectrum also exhibited signal at δ 71.1 (C-3) indicates the presence of electronegative group like OH. ¹³C NMR spectrum showed signal at δ 138.2 ppm (C-5) indicates the presence of quaternary carbon.On the basis of physical characteristics ¹H NMR, ¹³C NMR, Mass, and IR spectral evidence, the structure of compound 4 was elucidated as - sitosterol (Fig 4).

Fig 3: Chemical structure of compound 3 (- Sitosterol)

From literature survey, reported mobile phases with some modifications for separating and determining lupeol, β-amyrin and ursolic acid by HPTLC were used.

The calibration curve was plotted and Rf values for lupeol, β-amyrin and ursolic acid were 0.53, 0.78, and 0.79, respectively. Fig 5, 6 and 7 shows typical HPTLC chromatograms of standard lupeol, β-amyrin and ursolic acid with PE extracts of Momordica balsamina respectively.

Fig 5: 3D display of all tracks of lupeol

Fig 6: 3D display of all tracks of β-amyrin

Fig 7: 3D display of all tracks of ursolic acid

From calibration curve and equation of slope (Fig 8, 9 and 10) the concentration of lupeol, β-amyrin and ursolic acid was determined. In Momordica balsamina lupeol 1.15%, 𝛽- amyrin 2.8% and ursolic acid 0.6% were found (Table 2).

Fig 8: Calibration curve for Lupeol

Fig 9: Calibration curve for β-amyrin

Fig 10: Calibration curve for Ursolic acid

Table 2: Quantitative determination of isolated compounds by HPTLC

Compound	Slope Equation	Correlation coefficient	Area and amount
Lupeol	y = 2576.5x + 359.6	R² = 0.9846	Area = 6426.5 Conc. = 2.3 μg/200 μg
β-amyrin	y = 771.65x + 323.31	R² = 0.9813	Area = 4711.7 Conc. = 5.6 μg/200 μg
Ursolic acid	y = 2354x - 554.55	R² = 0.9873	Area = 3599.7 Conc. = 1.2 μg/200 μg

CONCLUSION:

Petroleum ether extract of Momordica balsamina was subjected for isolation by column chromatography. From the physical, chemical and spectral evidences compound 1, 2 3 and 4were confirmed as Lupeol, ursolic acid,- amyrin and - sitosterol. The isolated compounds were quantitative determined by HPTLC densitometric method. It is thus concluded that the plant contains appreciable amount of these compounds which may be responsible for its pharmacological action.

ACKNOWLEDGEMENT:

The authors are highly thankful to MVP’s college of Pharmacy, Nashik for providing all the facilities for the present study. The authors are also thankful to Principal, Dr. D. G. Baheti Sitabai Thite College of Pharmacy, Shirur for continuous his support.

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Received on 09.07.2020 Modified on 21.07.2020

Res. J. Pharmacognosy and Phytochem. 2020; 12(3):156-161.

DOI: 10.5958/0975-4385.2020.00026.6