A STUDY OF PHYTOCHEMICAL CONSTITUENTS IN CARALLUMA  QUADRANGULA

Jamal A. N. Al-Mahweety1image,  Sadeq HS Azzam1image, Ali Alyahawi2image

1School of Chemical Sciences, Faculty of Science, Sana'a University, Yemen

 2Faculty of Medical Sciences, Department of Pharmacy Al-Razi  University, Yemen

ABSTRACT 

Objectives:  Caralluma belongs to the family Asclepiadaceae, native to the Indian sub-continent have different medicinal properties and used in folk medicine as remedies to treat wide variety of diseases and health conditions.

Objective of present study was to isolate, analyze and identify the phytochemicals composition from steam of Caralluma  quadrangula.

Methods:  Extractions of the chemical components was carried out by different chromatographic techniques. The phytochemical characterizations were evaluated by nuclear magnetic resonance and mass spectrometry.

Results:  The quantitative phytochemical analysis of this species exhibited the presence of three compounds, Glochidonol (5gm), Dihydroxy-14-pregn-5-en-20-one (9.8gm), Hydroxystigmast-5-en-7-one (7.8gm) and Stigmasterol (5.7 mg). Hydroxystigmast-5-en-7-one, report from Caralluma  quadrangula stem as first time.    

Conclusion:  The present study is strengthen for the discovery three pure chemical compounds from C. quadrangula. 

Keywords: Caralluma  quadrangula ,  Glochidonol, 3,14-dihydroxy-14-pregn-5-en-20-one , 3-Hydroxystigmast-5-en-7-one,  Stigmasterol.


INTRODUCTION

Thousands of years, medicinal plants have an important role during the world in treating and preventing a diversity of diseases.  Caralluma is belongs to the family Asclepiadaceae, medically important genus widely studied for its stem and fruits. Caralluma classification to 200 genera and 2500 species1. Caralluma genus have  about 200 species spread in Africa and Asia. The common of these species are native to the Indian sub-continent and Arabian peninsula2. Most species of Caralluma are edible and form part of the traditional medicine system in the word3. Usually used in folk medicine as remedies to treat wide variety of diseases and health conditions4. Also used to treat liver diseases, diabetes and hypertension. The Caralluma flowers  are useful superficially for wounds and cuts, while the juice of the stem is given to sick people to speed convalescence of burns, itchy skin and sunburns5,6. In the Indian state Andhra Pradesh, C. attenuata (Wight) is eaten raw as an antidiabetic agent, while the juice of the plant along with black pepper is recommended in the treatment of migraine7. The diverse applications of Caralluma plants in folk medicine have prompted the phytochemical and biological investigations of their constituents8. The key phytochemical ingredients in Caralluma are pregnane glycosides, flavone glycosides, megastigmane glycosides, bitter principles, triterpenes and saponins9-12.

 

MATERIALS AND METHODS

General experimental procedures:- 

All active constituent were purified by following standard procedures13,14 and all chemicals used Analytical Reagent grade.

Plant material:-

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Figure 1: Glochidonol

Stems a of Caralluma quadrangula  (Asclepiadaceae) from Sana'a were collected (2014). The plant identified by Dr. Hessen Ibrahim. Deposited the sample of plant in Herbarium, Department of Phytochemistry.

Extraction and Isolation:- 

Shade dried stems were powdered. The powder was stored in air close container. The powder was weighed and extracted with soxhlet extractor by using solvents (Hexane, Chloroform, and ethanol). To concentrate the extracts and removal of solvent, rotary evaporate was used15,16. Then, purify the crude extracts. By using Fisher-John apparatus melting point was taken. The 1H NMR and 13C NMR spectra done by Bruker 100 MHz and 300 MHz, spectrometer, using an internal standard like TMS. Mass spectra were recorded by using ZAB-HS mass spectrometer. 

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Figure 2: 1HNMR of Glochidonol (1)

 

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Figure 3:13CNMR of Glochidonol (1)

Test for alcohol 

Dissolved little amount of crude extract was in 0.5 ml of dioxane. The solution was added to 0.5 ml of ceric ammonium nitrate reagent. Then one ml of dioxane was added and stunned. Yellow to red color formation gives an alcoholic hydroxyl group17. 

Libermann-Burchard test 

Acetic anhydride drops were added to the extract and boiled. Concentrated sulphuric acid was added to the above cooled solution. Presence of sterols give a brown ring at joint of two layers and green color in upper layer17.

General extraction and isolation

The air-dried powder (1100g) of stems of Caralluma  quadrangula  was Soxhlet with solvents (3X, 8 hours each) and evaporated the combined extracts to give a dark brown  residue (6 g) subjected the extract to silica gel flash column chromatography (FCC) with chloroform containing increasing percentages of ethanol as eluent.  Fractions 1-5 were combined and purification  by C.C. to yield JJ1 (5.0 mg) known as Glochidonol, (1),  (hexane-EtOAc), JJ2 (9.8 mg) known as 3,14-dihydroxy-14-pregn-5-en-20-one (2),  JJ3 (7.78 mg) known as 3-Hydroxystigmast-5-en-7-one (3) and JJ5 (5.7 mg) known as Stigmasterol (4) . All the isolated compounds were recognized by relationship with spectroscopy data.

Glochidonol (1): White powder (4.9 mg), mp 226-230 0C. 1H NMR (CDCl3, 300 MHz): δ 4.71, 4,60 (2H, s, H-29a, b), 3.26 (1H, dd, J= 4.76, 11.00 Hz), 0.61, 0.63, 0.81, 0.92, 0.93, 1.01, 1.10 (each 3H, s, Me×7).  13C NMR (CDCl3, 100 MHz):δ 214.68 (C-3), 149.08 (C-20), 105.46 (C-29), 80.09 (C-1), 55.51 (C-5), 50.62 (C-9), 48.54 (C-18), 48.10 (C-19), 43.12 (C-17), 43.02 (C-14), 41.08 (C-8), 40.24 (C-22), 39.10 (C-13), 38.29 (C-4), 37.30 (C-10), 35.85 (C-16), 34.53 (C-7), 30.01 (C-21), 28.25 (C-23), 27.63 (C-15), 27.54 (C-12), 25.27 (C-2), 21.06 (C-11), 19.52 (C-30), 18.48 (C-6), 18.21 (C-28), 16.26 (C-25), 16.20 (C-26), 15.61 (C-24), 14.66 (C-27).

3,14-dihydroxy-14-pregn-5-en-20-one (2): Mp: 200 -201 0C. 1H-NMR (300 MHz, CDCl3): δ 1.05 (3H s),  1.07 (3H s),  1.31 (3H, s),  1.45 (2H m), 1.50 (2H, m),  1.54 (1H, m),  1.60 (1H m),  1.75, 1.80  (2H), 1.97 (2H, dd J1 =12.5; J2 = 5.5 Hz), 2.27 (1H,q, J = 7.8Hz), 2.42 (2H, m), 3.54 (H, m, H-3), 4.12 (H, s, H-14) 5.17 (1H, s, H- 6). 13C-NMR (75 MHz, CDCl3): δ 36.64  (C-1), 28.01 (C-2),  75.16 (C-3), 36.62 (C-4), 143.06 (C-5),  121.86 (C-6),  36.10 (C-7), 53.19 (C-8),  50.41 (C-9), 36.48 (C-10), 25.34 (C-11), 25.21 (C-12),  43.65 s, C-13),  78.30 ( s, C-14),  32.81  (C-15),  25.43 (C-16),  53.6 (s, C-17), 15.45 (C-18), 15.32 (C-19), 121.47 (s, C-20),  30.07 (C-21).

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Figure 4: 3β,14β-dihydroxy-14β-pregn-5-en-20-one

3-Hydroxystigmast-5-en-7-one (3): Colorless granules; mp: 127.5 0C. EIMS m/z (rel. int.): 428 [M]+ (100), 410 (19), 395 (31), 287 (17), 247 (09), 192 (31).  1H-NMR (300 MHz, CDCl3): δ 0.66 (3H, s, H-18), 1.15 (3H, s, H-19), 3.68 (1H, m, H-3α), 5.67 (1H, d, J = 1.8 Hz, H-6). 13C-NMR (150 MHz, CDCl3): δ 36.27 (C-1), 31.15 (C-2), 70.51 (C-3), 41.77 (C-4), 166.12 (C-5), 124.20 (C-6), 202.83 (C-7), 45.55 (C-8), 49.92 (C-9), 38.32 (C-10), 21.21 (C-11), 38.82 (C-12), 43.08 (C-13), 49.93 (C-14), 26.54 (C-15), 28.11 (C-16), 54.48 (C-17), 11.94 (C- 18), 17.30 (C-19), 36.07 (C-20), 18.91 (C-21), 33.93 (C-22), 26.34 (C-23), 45.87 (C-24), 29.68 (C-25), 18.75 (C-26), 19.78 (C-27), 23.02 (C-28), 11.93 (C-29). 

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Figure 5: 3β-Hydroxystigmast-5-en-7-one

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Figure 6: 1HNMR of dihydroxy-14β-pregn-5-en-20-one (2)

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Figure 7:13CNMR OF dihydroxy-14β-pregn-5-en-20-one (2)

Stigmasterol (4): 1H NMR ( 100 MHz, CDCl3): δ 0.85 (10H, q, J=7.19 Hz, Me-26, Me-27, Me-29), 0.91 (1H, t, J=5.40 Hz Me-21), 5.34 (1H, d, J=4.62 Hz, H-22), 1.09 (8H, d, J=6.57 Hz , H-1, Me-18, Me-19, Me-21), 1.18 (6H, m, J=6.95 Hz,  H-12, H-14, H-15, Me-19, H-28), 5.10 (1H, q, J=7.86 Hz, H-23), 1.48 (8H, m, J=5.91 Hz,  H-2, H-8, H-9, H-11, H-15, H-16), 1.65 (3H, d, J=10.35 Hz, H-17, H-25), 1.80 (2H, d, J=10.05 Hz, H7),  2.12 (3H, m, J=8.07 Hz, 3-OH, H-20, H-24), 2.29 (2H, t, J=8.13 Hz, H-4), 3.51 (1H, m, J=5.09 Hz, H-3), 4.97 (1H, q, J=7.85 Hz, H-6). 

13C NMR (CDCl3): δ 12.21 (C-29), 12.32 (C-21), 19.09 (C-27), 20.07 (C-26), 21.30 (C-19), 21.35 (C-18), 21.55 (C-11), 24.46 (C-16), 25.52 (C-15), 29.03 (C-28), 31.74 (C-8), 32.00 (C-7), 32.01 (C-25), 32.04 (C-12), 36.61 (C-1), 37.36 (C-20), 39.83 (C-2), 40.61 (C-10), 42.32 (C-13), 42.38 (C-9), 50.26 (C-4), 51.30 (C-14), 56.04 (C-24), 56.97 (C-17), 71.81 (C-3), 121.82 (C-6), 129.36 (C-22), 138.40 (C-23), 140.81 (C-5). 

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Figure 8: Stigmasterol

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Figure 9: 1HNMR of Hydroxystigmast-5-en-7-one (3)


Figure 10: 13CNMR of Hydroxystigmast-5-en-7-one (3)

RESULTS AND DISCUSSION 

Compound 1: White powder. 1H NMR scale showed seven tertiary methyl groups and one secondary hydroxyl group. Olefinic  protons appeared  at δ 4.71 and 4.60. 13C NMR of the Glochidonol showed hydroxyl group C-1 appeared at δ 80.09, 30 signals for the terpenoid of lupine skeleton which have seven methyl groups. The carbon double bonded of alkenic carbons appeared at δ 149.08 and 105.46.

Compound 2: Was white needle-like crystals, mp 200-2010C, molecular formula C21H32O, m/z 412 M+. 1H-NMR for three methyl singlet's at δ 1.05, 1.07 and 1.31. H-3 proton appeared as a multiple at δ 3.53 and singlet at δ 4.12. Also have olefinic protons at δ 5.17. The  13C NMR showed alkenes carbons appeared at δ 143.06 and 121.86 and have  twenty one carbon signal with three methyles, nine methylenes, five methins and five quaternary carbons. Carbon bond to hydroxyl group is C-3 and C-14 that appeared at δ75.2 and 78.3.  

Compound 3: was colorless granules, MS data of the compound gave a m/z 428. 1H NMR  spectrum showed  tow methyl groups  appeared at δ 0.66 and  1.15.The  H-3 proton appeared at δ 3.68 as amultiplet and showed olefinic protons at δ 5.67. 13C NMR  showed twenty one carbon signal with three methyles, ten methylenes, nine methins and four quaternary carbons. Double bond carbons appeared at δ 166.12 and 124.20.

Compound 4: 1H NMR data showed c proton of sterol part at δ 3.51 as multiple. Signals at δ 5.34 , 5.10  and 4.97 correspond to two and one ethylene protons correspondingly present on C22, C23 and C6. Also have Peaks at δ 1.09 are corresponding to methyl groups (Me-18, Me-19, Me-21) and 0.85 are consequent to methyl groups (Me-26, Me-27 and Me-29). 13C NMR spectrum have six methyl, nine  methylene, eleven  methine and three  quaternary carbons.  Double bond Signals appeared at δ 140.81 and 121.82. Proton additional of β-hydroxyl group to C3 showed a peak at δ 71.81.


Figure 11: 1HNMR of Stigmasterol (4)



Figure 12:13CNMR of Stigmasterol (4)

 

CONCLUSION 

The isolation and identification Glochidonol, 3,14-dihydroxy-14-pregn-5-en-20-one, 3-Hydroxystigmast-5-en-7-one  and Stigmasterol from the stems of Caralluma  quadrangula. The isolation, purification and analysis carried out by means of various physical (solvent extraction, column chromatography, radial chromatography, preparative TLC and malting points) and spectral techniques.

 

AUTHOR’S CONTRIBUTION

The manuscript was carried out, written, and approved in collaboration with all authors.

 

CONFLICT OF INTEREST 

No conflict of interest associated with this work. 

 

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