Lupane-Type Triterpenoids and Sterol from Zizyphus Spina Christi Grown in Sudan

Introduction

Throughout human history, natural products have been widely used as remedies^1-5 to cure and treat various illnesses. Humans are continuously learning more about the indispensible therapeutic properties of natural products as well as becoming conscious of the importance of a healthy life style-to gain life quality.

An impressive amount of natural substances has been high-lighted by the media^3,6 due to their wide-ranging properties. Among these substances are the following lupane- type pentacyclic triterpenoids: Lupeol (1), a significant lupane- type pentacyclic triterpenoid is a common constituent of grape, bazel nut, olive oil, mango pulp and it was isolated from the stem bark of Cralaeva nurvalis⁶ [capparidacaea]. Lupeol was reported to be a constituent of the white part of birch bark [Betula papyfera] and red alder [Alnus rubra]. ^7-9

Figure 1: Structure of compound 1 20(29)-lupen-3-Ol (lupeol)   Click here to View figure

 

Lupeol exhibits a broad spectrum of biological activities ⁷ and can be used as a chemo- preventive substance to avoid several diseases.

Betulinaldehyde (2) was isolated from many plant species¹⁰ and it was the most intensively studied constituent of the birch bark triterpenoids because of its unique anti- cancer and anti HIV bioactivity.^11,12

Figure 2: Structure of compound 2 20(29)-lupen-3-ol -28-al (betulinaldehyde)   Click here to View figure

 

Betulinic acid (3) together with lupeol, betulinaldehyde and betulin were isolated from the stem bark of Tectona grandis¹⁰, Betulinic acid was also isolated from the root bark of Z. spina Christi.^13-16

Figure 3: Structure of compound 3 3-hydroxy-20(29)-lupen-28-oic acid (betulinic acid) Click here to View figure

 

Betulinic acid was synthesized from betulin by selective oxidation.¹⁷The reaction required the choice of the proper oxidizing reagent and conditions so that the oxidation reaction takes place predominantly at the primary C-28 hydroxyl group leaving the secondary hydroxyl group at C-3 in fact. Betulinic acid can then be easily obtained from the resulting betulinaldehyde.

Betulin(4), a pentacyclic triterpene was obtained for the first time in 1788 from birch bark [Betula papyfera]⁸ Betulin occurs in large amounts in the free form in the outer bark of white birch ⁹and it was isolated from alnus species, Trochoden dron, Vicia faba and many other plants.¹⁸

Figure 4: Structure of compound 4 20(29)-lupen-3,28-diol (betulin) Click here to View figure

 

Since the 1970s, intensive and extensive research has been committed to explore more about the medicinal application of betulin and its derivatives, in particular in their anti-tumor and anti-HIV biological activities.^19,20

Betulin is commonly used in medicine, pharmaceutical, perfumes manufacturing and in food and chemical industries.²¹

β-sitosterol (5)  the commonest sterol of higher plants which is widely distributed in plants and also occurs in marine organisms.^22-24

β-sitosterol is an important anti-hyper cholesterol, an estrogenic and a hypolipidemic agent. It shows antibacterial and antifungal properties.^23-25

Figure 5: Structure of compound 5 24-ethyl-5(6)-cholestene-3-ol (β-sitosterol)   Click here to View figure

 

Materials and Methods

The plant Material

The plant Z. spina Christi was collected from Kordofan area (Sudan) and identified by the Botany department, Faculty of science University of Khartoum. The root bark was carefully peeled, shade-dried and finally powdered.

Chemicals

The following either analytical grade or carefully purified and perfectly dried chemicals were used. Petroleum ether (pet.ether)[60-80ºC], ethanol, methanol, acetone, dichloromethane (DCM), silica gel for column chromatography [100-200 mesh], silica gel for thin-layer chromatography (TLC) GF254 and anhydrous sodium sulphate.

Instruments

The following spectroscopic techniques were used wherever needed:

1. Ultra Violet and Visible absorption Spectroscopy [UV\Vis].
2. Infrared Spectroscopy [IR].
3. Mass Spectroscopy[ Electron- Impact Mass Spectrometry EI-MS].
4. Nuclear Magnetic Resonance Spectroscopy [NMR].

(a) One Dimensional NMR Spectroscopy, Proton Nuclear Magnetic Resonance[¹HNMR], carbon-13 Nuclear Magnetic Resonance [¹³CNMR] and Destortionless Enhancement through Polarization Transfer [DEPT].

(b) Two Dimensional NMR Spectroscopy, Proton Correlated Spectroscopy [COSY], Nuclear Over-houser Effect Spectroscopy [NOESY], Hetronuclear Single Quantum Coherence Spectroscopy [HSQC] and Hetronuclear Multiple bond Correlation Spectroscopy [HMBC].

Methods

Extraction

The fine-dried powder of the root bark of Z. spina christiwas exhaustly extracted with 80% ethanol for 72 hours at room temperature with frequent shaking. The aqueous ethanol extract was filtered through Watman No. 42 filter paper. The solvent was carefully removed, as completely as possible; under reduce pressure using a rotatory evaporator.

The wet crude residue was then dissolved in ethanol and perfectly dried with anhydrous sodium sulphate. The ethanol solution of the crude extract was filtered as before and then centrifuged. The solvent was carefully removed and a mean percentage yield of 28 was obtained.

Column Chromatography

A glass column [120x8cm] well packed with silica gel [100- 200 mesh] in petroleum ether was used for the separation of the components of the crude extract. The crude residue was completely dissolved in ethanol and thoroughly mixed with silica gel. Ethanol was completely removed and the silica gel containing the crude extract was carefully packed on the top of the column.

The column was patiently eluted first with pet.ether followed by successive elution with [5:1], [2:3] and [1:3] pet.ether\DCM solvent mixtures. All fractions obtained from each of the above three successive elutions were carefully monitored with thin-layer chromatography and similar fractions from each successive elution were combined. Three, A, B and D combined fractions were obtained from the above successive elutions. Each of the combined fractions was perfectly dried and filtered. Careful removal of the solvent mixtures from each resulted in the formation of a yellow oily substance from A, a white grey solid from B and a pale green sticky material from D.

Isolation and purification of components

A glass column [40 x1 cm], packed with the same silica gel in pet.ether was used for the separation of the components of the yellow oily substance. The crude substance was dissolved in ethanol and thoroughly mixed with silica gel. After complete removal of ethanol the silica gel containing fraction A was carefully packed on the top of the column.

The column was eluted first with pet. ether\acetone [9:1] and finally with pet. ether\acetone[ 1:1]. Thin-layer chromatography monitoring of all fractions obtained led to the accumulation of two combined fractions [A₁ and A₂].

Removal of solvent mixture from each gave a white solid in both cases. Both white solids [A₁ and A₂] were crystallized from pet.ether\acetone [1:1]. A₁ is white needles, m.p 216-220ºC whereas A₂ is white crystals, m.p 188-190ºC.

The white grey solid was similarly chromatographedusing a glass column [80 X 2cm], packed with the same stationary phase in pet.ether. The column was carefully eluted first with pet.ether\acetone [9:1] and finally with pet.ether\acetone [3:7] solvent mixtures. Thin-layer monitoring of all fractions obtained resulted in the accumulation of similar fractions in two combined fractions, B₁ and B₂. The two combined fractions [B₁ and B₂] were perfectly dried separately, filtered and centrifuged.

When the solvent mixture was completely removed from each combined fraction, the resulting solids B₁ and B₂ were both separately crystallized from DCM and pet.ether\acetone[2:3], B₁ gave white needles, m.p. 282-284 ºC whereas B₂ gave a white solid with pleasant smell, m.p. 140-143ºC.

A glass column [60X2cm] packed with the same silica gel in pet.ether was used for the separation of the components of the pale green sticky material [D]. The column was successively eluted with pet.ether\acetone[9:2] and [3:8]and finally with acetone\methanol [1:1] and [1:9]. TLC monitoring of all fractions led to the accumulation of only one main combined fraction. Perfect drying of this combined fraction, filtration, centrifuge and complete removal of the solvent mixture, gave a white solid D₂ which was crystallized from acetone\ methanol [2:3], m.p. 250-252⁰C.

Spectroscopic Studies

The isolated and purified components [ A₁, A₂, B₁, B₂ and D₂ ] were separately subjected to the following spectroscopic investigations using the relevant technique accompanied by the proper interpretation.

Component A₁

UV  λ_max (CHCl₃) 242nm.EI-MS, M/Z. 426.3, M⁺: 411.4, 315.3, 297.2, 218.2, 207.2, 189.2, 175.1, 147.1, 135.1, 121.1, 95.1, 81.1, 69.1, 55.0, 43.0.IR v_max. (KBr) (cm^-1), 3346 (OH), 2925 (C-H), 1452 (C=C), 1381 (CH₃-C), 1038 (C-O), 880 (C-H).

¹H NMR (300MHZ, CDCl₃), ppm); shows the protons chemical shifts at   4.66 (1H,d), at  4.54 (1H,d), at  3.17 (1H,dd), at  2.36 (1H,td) and seven singlet methyl protons at  0.74, 0.76, 0.81, 0.92, 0.94, 1.02 and 1.67. ¹H – ¹³C HSQC (400MHZ, CDCl₃) δ(ppm);  shows that the two protons at  4.66 and  4.54 are bonded to the same carbon atom at δC 109.0) ppm and the proton at δH 3.17 is bonded   to a carbon atom at δC 79.0) δ ppm.¹H-¹H COSY (400MHZ, CDCl₃) δ(ppm);  shows that H₂₉  is correlated to H₃₀. H_29(a&b)are correlated to each other. H₂ is correlated to H₁and H_3, and H₂₁ is correlated toH₁₉ and H₂₂.¹H – ¹³C HMBC (400MHZ, CDCl₃) δ(ppm); shows that H₃₀ is correlated to C_29, C₂₀& C₁₉. H₂₇ is correlated to C_13, C₁₄ & C₁₅.H₂₆ is correlated to C₉ &C₁₄. H₂₅ is correlated to C₁,C₈ , C_9, C₁₀&C₁₁. H₂₄ is correlated to C_3, C_4, C₅ &C_10. H₂₃ is correlated to C_3, C₄ &C₅and H₁₈ is correlated to C_13, C_17, C_19, & C_20.¹H-¹H NOESY (400MHZ, CDCl₃) ppm); shows correlation between H₃ and Me_(23&24).

Component A₂

UVλ_max  (CHCl₃): 241.0nm. El-MS M/Z. 440.1, M⁺:426.1, 411.1, 379.1, 315.1, 207.1, 189.1, 175.1, 161.1, 135.1, 121.0, 95.1, 81.1 and 55.0. IR v_max. (KBr) (cm^-1): 3411 (OH), 2939 (C-H), 1724 (C=O), 1641 (C=C), 1453-1379 (CH₃-C), 1039 (C-O)and 885 (C-H).

¹HNMR (300 MHZ,CDCl₃) δ(ppm); shows the protons chemical shifts at  δH4.73 (1H,s),  at  4.61 (1H,s),  at  δH3.16 (1H,dd), at  δH2.98 (1H,m) and six singlet methyl protons at  0.73, 0.86, 0.83, 0.94, 0.95 and 1.67. ¹H – ¹³C HSQC (400MHZ, CDCl₃) δ(ppm); shows that the two protons at   δH 4.73 and  δH 4.61 are bonded to δC110.0) carbon, the proton at  δH 3.16 is bonded to δC77.2) carbon. ¹H-¹H COSY (400MHZ, CDCl₃) δ(ppm); shows that H₂₉ is correlated to H30. H29(a&b)are correlated to each other. H9 is correlated to H11and H21 is correlated toH19 &H22.¹H–¹³C HMBC (400MHZ, CDCl3) δ (ppm); shows that H₃₀ is correlated to C_29, C₂₀& C₁₈. H₂₇ is correlated to C_13, C₁₄ & C₁₅. H₂₆is correlated to C₉ &C₁₄. H₂₅is correlated to C_1, C₈ , C₉&C₁₀. H₂₄is correlated to C_3, C_4, C₅ &C_10. H₂₃is correlated to C_3, C₄ &C₅. H₁₈is correlated to C_17, C₂₀ & C_29.¹H-¹H NOESY (400MHZ, CDCl₃) ppm); shows the correlation between H₃ and Me_23&24.

Component B₁

UVλ_max CHCl₃) 209.0 nm.E1-MS M/Z. 456.2, M⁺: 438.2, 423.2, 410.3, 395.2, 316.3, 302.3, 259.3, 248.3, 207.3, 189.3, 175.4, 161.3, 135.4, 95.4, 81.4, 69.4, 55.4, 43.4.

IR v_max.(KBr) (cm^-1): 3450 (OH), 2937 (C-H), 1686 (C=O), 1449 (C=C), 1378 (CH₃-C),1189 (C-O), 883 (C-H).¹H NMR (400 MHZ, CDCl₃) δ (ppm); shows the chemical shifts of protons at δH 4.71 (1H,s), at δH 4.58 (1H,s), at  δH3.17 (1H,dd),  at  δH2.98 (1H,m) and six singlet methyl protons at  δH 0.73, 0.80, 0.92, 0.94,0.96 and 1.68.

¹H – ¹³C HSQC of component B₁ (400 MHZ, CDCl₃) δ(ppm); shows that  the two protons at  δH 4.71 and  δH 4.58 are  bonded to δH 110.0) carbon, the protonat  δH 2.98 is bonded to δC 47.0) carbon  and the proton at  δH3.17 (1H,s) is bonded to δC79.0) carbon.¹H-¹H COSY (400 MHZ, CDCl₃) δ(ppm); shows that H₂₉ is correlated to H_30. H_29(a&b)are correlated to each other. H₃ is correlated to H₂. H₂₁ is correlated toH_{19 &}H₂₂  and H₁₅ is correlated toH_16.¹H – ¹³C HMBC (400 MHZ, CDCl₃) (ppm); shows that H₃₀ is correlated to C_29, C₂₀& C₁₉. H₂₇ is correlated to C_13, C₁₄ & C₁₅. H₂₆ is correlated to C₉ &C₁₄. H₂₅ is correlated to C_1, C₈ , C₉&C₁₀. H₂₄ is correlated to C_3, C_4, C_5, C_10,  &C_23. H₂₃ is correlated to C_3, C₄ C_5, C_6, & C₂₄  and H₁₈ is correlated to C_13, C_17, C_20, C₂₈ & C_29.

¹H-¹H NOESY (400 MHZ, CDCl₃) δ(ppm); shows the correlation between H₁₆ and (δH2.2) H₂₂ (δH1.9).

Component B₂

EI-MS M/Z 414.3: M⁺396.3, 381.3, 351.2, 329.2, 303.2, 289.2, 273.2, 255.2, 199.1, 173.1, 159.1, 145.0, 119.0, 107.0, 81.0, 69.0, 55.0.IR v_max (KBr) cm^-1 3363 (OH), 2925 (C-H), 1687.6 (C=O) , 1458.0 (C=C), 1377 (CH₃– C), 1230.5 (C-O), 1037.6 (C-O) and 883.3 (C-H).

¹H NMR (300 MHZ, CDCl₃ + CD₃OD) δ(ppm); shows the protons chemical shifts at δH 5.23 (1H,s), at δH 3.4(1H,d), at δH 3.16 (1H,d), and six singlet methyl protons at  δH 0.57, 0.68, 0.70, 0.72, 0.80 and 0.91.¹³C NMR (500 MHZ, CDCl₃ + CD₃OD) δ(ppm), shows the ¹³C chemical shifts of  C₃ δCat  (70.5 ppm), of C₅ δC (140.8ppm) and of C₆ at  (121.7 ppm).¹H – ¹³C HSQC (500 MHZ, CDCl₃ + CD₃OD) δ(ppm),  shows that the proton at  δH 5.23 is bonded to δC 121.8 carbon and the two protons at δH 3.4  and at δH 3.16 are bonded to  70.5 carbon.¹H-¹H COSY (500 MHZ, CDCl₃ + CD₃OD) ppm); shows that H₆ is correlated to H₇. H_3(a&b)are correlated to each other, H₃ is correlated to H₂&H₄. H₂₀ is correlated to H₂₁ &H_22, and H₂₄ is correlated to H_25.¹H – ¹³C HMBC (500 MHZ, CDCl₃ + CD₃OD) ppm);  shows that H₂₈ is correlated to C_23, C_24,C₂₅& C₂₉. H₂₁ is correlated to C_17, C₂₀ & C₂₂. H₂₀ is correlated to C₂₃ &C₂₄. H₁₉ is correlated to C_1, C₅ , C₉&C₁₀. H₁₈ is correlated to C_11, C_12, C_13, C_14,&C₁₇. H₈ is correlated to C₉. H₇ is correlated toC_9, C_11,C_13,&C₁₄. H₆  is correlated toC_4, C_7,  C_8,&C_10, andH₄  is correlated to C_1, C_2, C_3,C₅ &C_6.¹H-¹H NOESY (500 MHZ, CDCl₃ + CD₃OD) δ(ppm); shows the correlation between H₆(δH 5.23)_, H₄ (δH2.25) and H₇ (δH1.87),between H_4-a (δH2.25) and H_4-b (δH 2.11) between H₇ (δH 1.87) and H₈ (δH 1.35) and between H₃ (δH 3.4) H₄ (δH 2.25) and H₁₉ (δH0.91).

Component D₂

UV λ_max (CHCL₃) 442.0 nm. EI-MS M/Z. 442.3, M⁺: 424.3, 411.3, 302.2, 288.2, 248.1, 207.7, 189.1, 175.1, 147.1, 135.1, 95.0, 81.0, 55.0. IR v_max (KBr) (cm^-1): 3417.8 (OH), 2927.4 (C-H), 1447.9 (C=C), 1378.3 (CH₃-C), 1032.2 (C-O) and 882.9 (C-H).¹HNMR (300 MHZ, (CHCL₃) δ(ppm); shows protons chemical shifts at δH 4.66 (1H,s), at δH 4.71 (1H,s), at δH 3.71 (1H,dd), three alcoholic protons at δH 4.58 (1H,t) δH 3.78 (1H,d), δH 4.71 (1Hs), at δH 3.71 (1H,dd), three alcoholic protons at δH 4.58 (1H,t) δH 3.78 (1H,d), δH 3.3(1H,d) one proton at δH 2.36 (1H,dt) and six singlet methyl protons at δH 0.74, 0.80, 0.92, 0.94, 1.0 and 1.68.

¹H – ¹³C  HSQC (400 MHZ, CDCl₃)δ (ppm); shows that the  two protons at δH 4.66 & 4.71 are  bonded to (δC 110.0) ppm carbon,  a proton at δH 3.17 is bonded to δC 79.0 ppm carbon and two protons at δH 3.3 & δH3.78 are bonded  (δC 60.5 ppm) carbon.

¹H-¹H COSY (400 MHZ, CDCl₃)  (ppm);  shows that H₂₉ is correlated to H₃₀. H_29(a&b)are correlated to each other. H₃ is correlated to H₂. H₂₁ is correlated toH_{19 &}H₂₂ and H₁₅ is correlated toH_16.¹H – ¹³C HMBC (400 MHZ, CDCl₃) δ (ppm);  shows that H₃₀ is correlated to C_29, C₂₀& C₁₉. H₂₇ is correlated to C_13, C₁₄ & C₁₅. H₂₆ is correlated to C₉ &C₁₄. H₂₅ is correlated to C_1, C₅, C₉&C₁₀. H₂₄ is correlated to C_3, C_4, C₅ &C₂₃. H₂₃ is correlated to C_3, C_4, C₅ &C_24, and H₁₉is correlated to C_18, C_20, C_21, C₂₉ & C_30. 

¹H-¹H NOESY (400 MHZ, CDCl₃) δ (ppm); shows a correlation between H₃ (δH 3.17) and Me_23&24 (δH 0.94 & 0.74 ) and between H₁₈ (δH 1.6)  and H_28(a&b) (δH 3.3 & 3.78).

Results and Discussion

Z. spina christi family Rhamnaceae is widely distributed in the tropical and subtropical regions of the World. The tree is a multipurpose plant,widely used in folk medicine ²¹ and its delicious fruit²² has a limited commercial value.

The fine powder of the shade-dried root bark was exhaustively extracted with aqueous ethanol. The dry crude extract [28% mean yield], was carefully and patiently subjected to intensive and extensive fractionation using column chromatography.

Subsequent monitoring with thin-layer chromatography and refractionation of the resulting combined similar fractions led to the isolation of five components referred to as A₁, A₂, B₁, B₂ and D₂. Which were vigorously purified.

Characterization and structure elucidation of the isolated and purified above components were based on different spectroscopic data including 1D and 2D NMR spectroscopy.

Careful study of the proton nuclear magnetic resonance spectroscopic data for each of the components A₁, A₂, B₁, B₂ and D₂ revealed that these components have triterpene skeletons. The HSQC and COSY spectroscopic investigations showed that the above triterpene skeletons are of lupane-type.

Proper interpretation of the infrared analytical spectroscopy and the Hetronuclear Multiple bond Correlation Spectroscopy led to the possible suggestions that the structures of the above components might be: 20(29)-lupen-3-ol, 20(29)-lupen-3-ol-28-al, 20(29)-lupen-3-ol-28-oic acid, 20(29)-lupen-3,28-diol respectively. Their NOESY spectroscopic correlations data confidently confirm the above suggested structures. Identification of these components was based on comparing their spectroscopic data with literature data given for triterpenes isolated from plant materials. These components are lupeol, betulinaldehyde, betulinic acid and betulin respectively.

Study of the ¹HNMR, HSQC and COSY of spectroscopic data indicate that component B₂ is Sterol. Careful interpretation of the infrared resonance spectra and the HMBC correlation spectra showed that the structure of B₂ might be 24-ethyl-5(6)-cholestene-3-ol. The NOESY correlation spectra of B₂ confirmed the determined structure and comparison with literature data given for steroids isolated from plant species proved that B₂ is β-sitosterol. Thus all the isolated components are known compounds previously obtained from other plants.

To the best of the author’s knowledge, lupeol, betulinaldehyde, betulin and β-sitosterol were isolated for the first time from the root bark of Z. spina Christi grown in Sudan.

Acknowledgement

We are highly indebted to Prof. Mohammed Igbal Choudhary and Dr. Achyut Adhikari and all members of Hussin Ebrahim Jamal (HEJ) research institute of chemistry, International Center for Chemical and Biological Sciences (ICCBS), University of Karachi, Karachi-Pakistan, for their indispensible help and constructive advices.

References

1. Sofowora, Medicinal plants and Traditional Medicine in Africa, John Wiley and Sons limited. (1982)
2. Abdel wahhab, M. A.; Omara, E. A; Abdel Gali, M. M.; Hassan, N. S.; Nada, S. A.; Saeed, A.; Elsayed M. M. African Journal of Traditional, Complementary and Alternative Medicines. 2007, 4, 248-256.
3. Sofowora, A. Medicinal Plants and Traditional Medicines in Africa. Chichester John, Willey & Sons New York; 256. (1993).
4.  Steven, M.; Russell, J. C.; Molyneux, J.; Bioactive Natural Components of the petroleum ether fraction from the root bark ethanol extract using GC\MS   l Products Detection, Isolation and Structural Determination Taylor & Francis, USA, (2008).
5. New man, D.J; Cragg G.M. Journal of Natural Products. 2007, 70, 461-477.
6. Natural Products Reports, New York. 1985, 26, 299- 306.
7. International Journal of Biochemical and Pharmaceutical sciences. Global Science Books  (2009).
8. Basa Vara, J.a. ; H. S.  J. Pharm. Science and Res., 2010, 3, 13 – 15.
9. Agar Wal, R. B. ; and Rangari, V. D. Indian Journal of Phamacology. 2003, 35,284 -287.
10. Indian Journal of Pharmaceutical Sciences. 1988,  50,124 -125,.
11. Slinivasan, T. BioOrganic and Medicinal Chemistry Letters.  2002, 12, 2803 -2806.
    CrossRef
12. Shah, A. H; Pandey, V.B ; Eckhard, G. , Tschesche, R. J. Nat. Prod. 1985 48, 555-558.
    CrossRef
13. Jan Sarek; J. Med. Chemistry. 2003, 71, 5402 -5415.
    CrossRef
14. Shah ,A.H. ; Ageel, A.M.; Tarig, M.; Mossa, J.S.; Al-Yahya, M.A. Chemical constituents of the stem bark of Zizyphus spina Christi. Fitoterapia. 1986  ,57,452-454,.
15. Anthony, C.; Dweck, F.L.S . Personal Care Magazine . 2005, 2-5.
16. Adzu, B.;  Haruna, A.K.;  Ilyas M. ; Pateh, U.U; Tarfa, F.D.;  Chindo, B.A ;  Gamaniel, A .S .Journal of Pharmacy and Nutrition Sciences. 2011, 1, 48 -53.
    CrossRef
17. Hongyan Liu; Synthesis of Betulinic acid from Betulin; M.Sc of science; Natural library of Canada; Acquisitions and Bibiographic Servicas; 2001.
18. Yunusoy, M. S. Russion Plant, 6, pp. CODEN, RUXX, 67, RU 2270202 C1 20060220.
19. EL Znhmer, D. V. XLI 2Q. Drugs. 2007, 7,359 – 373.
20. Fujioka T.; Kashiwada Y.; Kilkuskie R. E.; Consentino L. M.; Ballas L. M.; Jiang J. B.; Janzen W. P.  Journal of Natural Products. 1994, 57, 243 – 247.
    CrossRef
21. www/Natural News. Com., Natural health, Natural living, Natural News.
22. Sudhersan C.; Hussain J. Turk J Bot. 2003, 27,167-171.
23.  Steele, J. A. JOC. 1963, 28, 571-572.
    CrossRef
24. Mastomato, T.  Phytochemistry. 1983, 22,2622-24.
    CrossRef
25. Aizawa, K. Org Mass Spectrom. 1974, 9, 470 -79.
    CrossRef

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