PHYTOCHEMICAL PURIFICATION OF ACTIVE CONSTITUENTS ISOLATED FROM ROOT OF THE MEDICINAL HERB, CARALLUMA QUADRANGULA

Jamal AN. Al-Mahweety1, Ammer Al-Fadaly2, Waled Abdo Ahmed3

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

       2School of Chemical Sciences, Faculty of  Science, Sana'a University,  Yemen.

3School of Chemical Sciences, Faculty of Education, Thamar University, Yemen.

ABSTRACT

Objective: Present study aim for the purification of quantitative phytochemical compounds from roots of  Caralluma quadrangula belongs to the family Asclepiadaceae. This type of plants can be use as folk medicine to take care of wide diversity of health and diseases situation.

Methods: Preliminary phytochemical analysis for different type of chemical compounds by using various 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 four pure compounds,  hydroxyoplopan-4-one (4.5 mg), dihydroxyeudesm-4(15)-ene (5.0 mg), and quercetin- rhamnopyranosyl-D-glucopyranose (Rutin) (7.0 mg).

Conclusion: From this study, it can be concluded that the species found four pure compounds from C. quadrangula.

Keywords: Caralluma  quadrangula, hydroxyoplopan-4-one, dihydroxyeudesm-4(15)-ene, quercetin- rhamnopyranosyl- D-glucopyranose (Rutin). 

INTRODUCTION

Medically significant genus Caralluma is widely studied for its stem and fruits. It is belong to the family Asclepiadaceae, which comprises 200 genera and 2500 species1. About 200 species belong to genus Caralluma distributed throughout Africa and Asia. The greater part of species are native in Indian sub-continent and Arabian Peninsula2. A number of Caralluma species use as anti-hyperglycemic goings-on of their crude extracts or their corresponding fractions3,4. The investigation  of the chemical and biological  members of genus Caralluma3,5 the anti-hyperglycemic activity of the extracts, fractions of  the major pregnane glycoside of the aerial parts of C. quadrangula was investigated in Kingdom Saudi Arabia as  novel. We use the extract of C. quadrangula as herbal medicine in Saudi, for the treatment of freckles, diabetes, vitiligo and melasma and for thirst, hunger5,6. Several countries the species of Caralluma are fit to be eaten and variety division for the traditional medicine organization7. These plants can be use as folk medicine as remedies to health situation and treat large multiplicity of diseases8. The species of C. arabica use as traditionally for an emollient and diuretic In United Arab of Emirates. Also used to care for diabetes, hypertension and liver diseases. The C. Arabica flower  used for wounds and cuts, while the juice of the stem is given to sick people to speed convalescence of burns, itchy skin and sunburns9,10. The C. attenuate species in Indian (Andhra Pradesh) use for eaten raw as an anti-diabetic agent, although the juice of the plant beside the black pepper is suggested in the treatment of migraine11. The different applications of Caralluma plants in folk medicine have prompted the phytochemical and biological investigations of their constituents12. The pregnane glycosides, flavone glycosides, megastigm-ane glycosides, bitter principles, triterpenes and saponins isolated from Caralluma13,14,15,16.

 

MATERIALS AND METHODS

Purified every one of chemical constituent  by subsequent standard procedures17,18 and all chemicals used systematic Reagent evaluation.

Plant material:-

Roots of Caralluma quadrangula (Asclepiadaceae) were collected from Sana'a 2014. The plant identified by Dr. Hessen Ibrahim and was deposited voucher sampling of plant in Herbarium, Department of Phytochemistry (Sana'a University).

Extraction and Isolation:-

Shade dried roots were crushed and sieved. Next powder was stored in air closing container. Than weighed and extracted with soxhlet extractor by using solvents Chloroform with consecutive solvent extraction. To concentrate the extracts and removal of final traces of solvent than vapor19,20. After that, recrystallization was done to purify the crude extracts. Melting point was taken by using Fisher-John apparatus. The 1H NMR and 13C NMR spectra were taken on Bruker 100 MHz and 400 MHz, spectrometer, using an internal standard like TMS. 

Extraction and isolation

Extracted by using  Soxhlet (2 Kg) of  C. quadrangula roots powder  with solvents (3X, 8 hours each) and then  evaporated collective extracts to give a brown gummy residue (8 g) after than  separation and purified by silica gel flash column chromatography (FCC) with CHCl3 containing increasing percentages of MeOH as eluent and collected  20 ml for each fraction. Fractions 3-10 were combined and rechromatographed by C.C. with CHCl3-MeOH  (8:2) to afford  JA1 (4.5 mg) identified as 10α- hydroxyoplopan-4-one (1), CHCl3-MeOH  (7:3) to afford  JA3 (5.0 mg) identified as 1β, 6α-dihydroxyeudesm-4(15)-ene (2), CHCl3-MeOH  (6:4) to afford  JA4 (5.0 mg) identified  as  and  CHCl3-MeOH  (3:7) to afford    JA4  (7.0 mg) identified as  quercetin- rhamnopyranosyl- D-glucopyranose (Rutin) (4). NMR data used to identified for each pure compounds.

 

Figure 1: 10α-Hydroxyoplopan-4-one (1)

 

 1H-NMR (100 MHz, CDCl3) δ: 2.75 (1H, m, H-3), 2.30 (3H, s, H-15), 1.50 (3H, s, H-13), 1.10 (3H, d,  H-11), 0.87 (3H, d,  H-12)；13CNMR (MHz, CDCl3) δ: 209.4 (C-14), 73.0 (C-8), 56.0 (C-3), 54.6 (C-9), 48.2 (C-5), 45.8 (C-4), 41.0 (C-7), 28.6 (C-10), 27.5 (C-1), 24.4 (C-2), 21.9 (C-6), 21.0 (C-11), 19.2 (C-13), 18.5  (C-15), 15.0 (C-12).

Dihydroxyeudesm-4(15)-ene (2). 1H-NMR (100 MHz, CDCl3) δ: 5.10 (1H, brs, H-15), 5.01 (1H, brs, H-15), 3.79 (1H, t,  H-6β), 3.42 (1H, dd,  H-1α), 2.33 (1H, ddd,  H-3α), 2.24 (1H, sept,  H-11), 2.07 (1H, ddd, H-3β), 1.91 (1H, s, H-8), 1.85 (1H, ddd, H-2α), 1.75 (1H, brd, H-5α), 1.53 (1H, m, H-2β), 1.53 (1H, m, H-8), 1.43 (1H, brs, 1-OH), 1.28 (1H, m, H-7α), 1.20 (1H, m, H-9a), 1.18 (1H, m, H-9b), 1.02 (3H, d,  H-13), 0.87 (3H, d,  H-12), 0.72 (3H, s, H-14)；13C-NMR (MHz, CDCl3) δ: 147.4 (C-4), 108.2 (C-15), 79.1 (C-1), 67.8 (C-6), 56.4 (C-5), 50.1 (C-7), 42.1 (C-10), 36.9 (C-9), 36.1 (C-3), 32.2 (C-2), 26.5 (C-11), 21.8 (C-13), 19.1 (C-8), 16.6  (C-12), 12.0 (C-14).

Figure 2: dihydroxyeudesm-4(15)-ene (2)

 

Quercetin-L-rhamnopyranosyl-(1→6)-Dglucopyra-nose (3). 1H NMR ( 100 MHz, CDCl3): δ 6.22 (1H, d, H-6), 6.40 (1H, d, H-8), 7.68 (1H, s, H-2'), 6.90 (1H, d, H-5'), 7.59 (1H, d, H-6' ), 5.10 ( 1H, d, Hglc-1), 3.49 (1H, m, Hglc-2), 3.43,(1H,  m, Hglc-3), 3.50 (1H,  m, Hglc 4), 3.58 (1H, m, Hglc-5),  3.30 (2H, m, Hglc -6), 4.51 (1H, br, HRha-1), 3.10 (1H, m, HRha-2), 3.43 (1H, m, HRha-3), 3.54 (1H, m, HRha-4), 3.31 (1H, m, HRha-5), 1.17 (3H, d, HRha-6). 13C NMR (CDCl3): δ 158.1 (C-2), 135.1 (C-3), 180.0 (C-4), 160.0 (C-5), 100.0(C-6), 166.1 (C-7), 95.1  (C-8), 163.0 (C-9), 104.7 (C-10), 123.0 (C-1'), 118.2 (C-2'), 146.1 (C-3'), 150.1 (C-4'), 115.8 (C-5'), 123.4 (C-6'),  105.0 (CGlc-1), 75.6  (CGlc-2), 77.8 (CGlc-3), 74.9 (CGlc-4), 77.2 (CGlc-5), 69.0 (CGlc-6), 103.1 (CRha-1), 71.2 (CRha-2),  72.1 (CRha-3), 74.1 (CRha-4), 70.4 (CRha-5), 19.2 (CRha-6). 

    

Figure 3: Quercetin -rhamnopyranosyl-D-glucopyranose

Figure 4: H1 NMR of 10α- hydroxyoplopan-4-one (1)

Figure 5: 13C NMR of 10α- hydroxyoplopan-4-one (1)

Figure 6: H1 NMR Of dihydroxyeudesm-4(15)-ene (2)

Figure 7: 13C NMR of dihydroxyeudesm-4(15)-ene (2)

RESULTS AND DISCUSSION

Compound 1: The 1H NMR showed  one multiplet proton at δH 2.75 (1H, m), 5.46, High intensity Peaks at δ 2.30, 1.50, 1.10 and 0.88 are corresponding to methyl groups (Me- (15, 13, 14 and 12). 4 methyl, 4 methylene, 5 methine and 2 quaternary carbons presence in  13C NMR spectrum . Carboxylic group signals become visible at δ 209.5. In addition of β-hydroxyl group to C8 is visible from a peak at δ 73.1. hydroxyoplopan-4-one,  it has never been isolated before from Caralluma  quadrangula, it reported from Cassia buds21.

Compound 2:  The 1H NMR showed signals for three angular methyl singlet's at δH 0.95, 0.85 and 0.71.  proton of H-6 and H-1  appeared at δ 3.79 and 3.42.  Olefinic protons present at δ 5.10 and 4.95 for H-15. 13C NMR showed fifty carbon signal including three CH3, five CH2, five CH and two quaternary carbons. The double bond carbons appeared at δ 147.4 and 108.1. The significant signal for the 1β, 6α-dihydroxyeudesm-4(15)-ene would be the signals for two carbon attached to hydroxyl group, which is C-1 and C-6 that appeared at δ79.2 and 67.822,23.

Figure 8: H1 NMR of quercetin -rhamnopyranosyl-D-glucopyranose (3)

Figure 9: 13C NMR of quercetin -rhamnopyranosyl-D-glucopyranose (3)

Compound 3: The 1H NMR spectrum exhibited signals which were typical of a flavone compound. In addition to the presence of five aromatic protons; one was represented by two meta-coupled protons at δH 6.23 (d, H-6) and 6.42 (d, H-8). 13C NMR experiments showed one methyl, 15 methines, one methylene and 10 quaternary carbon atoms, one being the flavone carbonyl (C 180.0)24. NMR spectral data confirmed the sugar part assigned as glucose and rhamnose. A significant downfield shift of the methylene carbon appearing at C 69.1 and assigned to C-6 of glucose, indicated a (1 to 6) type of interglycosidic linkage to the rhamnose moiety. Qercetin-rhamnopyranosyl-D-glucopyranose isolated from C. quadrangula  for first time , it was reported in many plants as Taverniera aegyptiaca25,26.

 

CONCLUSION

The isolation and identification 10α- hydroxyoplopan-4-one, dihydroxyeudesm-4(15)-ene, and quercetin-rhamnopyranosyl- D-glucopyranose (Rutin),    from  the roots of  C.  quadrangula. The work was carried out by means of various physical (solvent extraction, column chromatography, radial chromatography, preparative thin layer chromatography and melting 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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