chemical characterization of a commercial commiphora wightii resin...

Abstract!

The gum resin of Commiphora wightii [(Hook. exStocks) Engl.] is an ayurvedic medicine for thetreatment of arthritis, inflammation, obesity, lipiddisorders, and cardiovascular diseases and isknown as guggul. Morphologically, it is not easytodistinguishguggul fromclosely relatedgumres-ins of other plants. Reliability of the commerciallyavailable guggul is critical due to the high risk ofadulteration. To check authenticity, a commercialguggul sample was investigated for its chemicalmarkers and 17 metabolites were identified, in-cluding three new, 20(S),21-epoxy-3-oxocholest-

4-ene (1), 8β-hydroxy-3,20-dioxopregn-4,6-diene(2), and 5-(13′Z-nonadecenyl)resorcinol (17) fromthe ethyl acetate soluble part. During the currentstudy, compounds 14–17 were identified as con-stituents ofMangifera indicagum, as an adulterantin the commercial guggul sample. This discoveryhighlighted the commonmalpractices in the tradeofmedicinal rawmaterial in the developingworld.The structures of the compoundswere deducedbythe spectroscopic technique and chemical meth-ods, as well as by comparison with the reporteddata. The structure of 20(S),21-epoxy-3-oxochol-est-4-ene (1) was also unambiguously deduced bysingle-crystal X‑ray diffraction technique.

Chemical Characterization of a CommercialCommiphora wightii Resin Sample andChemical Profiling to Assess for Authenticity

Authors Rida Ahmed1,2, Zulfiqar Ali1,2, Yunshan Wu3, Swapnil Kulkarni3, Mitchell A. Avery3,Muhammed Iqbal Choudhary2, Atta-ur-Rahman2, Ikhlas A. Khan1,4

Affiliations The affiliations are listed at the end of the article

Key wordsl" Commiphora wightiil" guggull" Burseraceael" Mangifera indical" single‑crystal X‑ray diffrac-

tion

received October 7, 2010revised Nov. 24, 2010accepted Dec. 7, 2010

BibliographyDOI http://dx.doi.org/10.1055/s-0030-1250674Published online January 14,2011Planta Med 2011; 77: 945–950© Georg Thieme Verlag KGStuttgart · New York ·ISSN 0032‑0943

CorrespondenceProf. Ikhlas A. KhanNational Centerfor Natural Products ResearchSchool of Pharmacy,University of MississippiUniversity, MS 38677USAPhone: + 16629157821Fax: + [email protected]

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Introduction!

Commiphora wightii (Arnott.) Bhanol. [syn = Com-miphora mukul (Hook. ex Stocks) Engl.] belongs tothe family Burseraceae. The arid and semi-aridareas of the Indian subcontinent are the habitatof C. wightii. Its gum resin, known as guggul, is re-ported as a folk remedy in the ayurvedic systemof medicine. The pharmacological properties as-sociated with guggul include anti-inflammatory,antibacterial, anticoagulant, antirheumatic, COXinhibitory, and hypolipidemic activities aremostly due to the presence of steroids [1–3]. Pre-vious phytochemical studies reported the pres-ence of steroids, di/triterpenoids, and lignans inguggul [1–3]. Owing to the pharmacological sig-nificance of guggul, its legitimacy is vital, as mor-phologically guggul resembles the gum resins ofother species of the genus, within and outside.Therefore, risk of adulteration is high in commer-cial samples, either deliberately to get more profitor accidentally. To assess authenticity, phyto-chemical investigation of a commercial specimenof guggul for its chemical markers was undertak-en to address the issues relevant to the safety andefficacy of botanicals as a part of our program.

Ahmed R et al. Ch

Seventeen compounds were purified from theethyl acetate fraction of the commercial sampleof guggul, including three new ones, 20(S),21-ep-oxy-3-oxocholest-4-ene (1), 8β-hydroxy-3,20-di-oxopregn-4,6-diene (2), and 5-(13′Z-nonadece-nyl)resorcinol (17), together with four not pre-viously identified from this source, diasesartemin(3), 6α-hydroxycholest-4-ene-3-one (4), cholest-4-ene-3,6-dione (5), and epieudesmin (6)(l" Fig. 1). Compounds 14–16 [mangiferolic acid,cycloartenol, and 5-(11′Z-heptadecenyl)resorci-nol], earlier isolated as metabolites of Mangiferaindica gum, were also present as adulterants inthe commercial guggul sample. Compound 17most likely belongs to Mangifera indica gum be-cause of its structural resemblance with 16. Thebasic skeleton and the positions of different sub-stituents were assigned by spectroscopic tech-niques including HRESI‑MS and extensive NMRexperiments together with chemical methodsand comparison with the reported spectroscopicdata. The structure of 1 was further supported bysingle-crystal X‑ray diffraction technique. To thebest of our knowledge this is the first report ofthe NMR spectroscopic data of 3.

emical Characterization of… Planta Med 2011; 77: 945–950

Fig. 1 Structures of compounds 1–17.

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Materials and Methods!

General experimental proceduresNMR spectrawere recorded in CDCl3 on a Varian AS 400 or VarianUnity Inova 600 NMR and in DMSO-d6 on Bruker AV 300MHz and600MHz NMR spectrometers. IR spectra were recorded on aBruker Tensor 27 spectrophotometer. UV spectra were measuredon a Varian Cary 50 Bio UV-visible spectrophotometer. Specificrotations were measured at an ambient temperature by using aRudolph Research Analytical Autopol IV automatic polarimeter.HRESI‑MS data were obtained on an Agilent Series 1100 SL massspectrometer. TLC analysis was on aluminum-backed plates pre-coated with silica gel F254 (20 × 20 cm, 200 µm, 60 Å; Merck). Vi-sualization was made by spraying with p-anisaldehyde [1.0mL inglacial acetic acid (100mL)] spray reagent followed by heating.Gravity column chromatography was performed using silica gel(40 µm for flash chromatography, 60 Å; J.T. Baker) and reversed-

Ahmed R et al. Chemical Characterization of… Planta Med 2011; 77: 945–950

phase silica (RP-18, Polarbond; JT Baker). HPLC brand was WaterLC Module 1, equipped withWaters 486 variable wavelength UV/VIS detector, Millennium32 version 3.05 software, and Phenom-enex Gemini C-18 column (250 × 10mm, 5 µm). A Triogen ozonegenerator was used for ozonolysis. The single-crystal X‑ray dif-fraction data was collected on a Bruker Smart Apex II system, us-ing CuKα radiation with a graphite monochromator. The solvents(Fisher brand) used for HPLC and other chromatographic proce-dures were of HPLC and analytical grades, respectively.

Plant materialCommiphora wightii (Arnott.) Bhanol. [syn = C. mukul (Hook. exStocks) Engl.] gum resin was purchased from a herbal market inKarachi, Pakistan in November 2008. The authenticated gum res-in samples of Commiphora wightii andMangifera indicawere ob-tained from different locations of Pakistan and India and identi-fied by Dr. Joshi, a plant taxonomist at the National Center for

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Natural Products Research, School of Pharmacy, University ofMississippi, USA (voucher specimen numbers # 4985, 4997,7764, 2567 for C. wightii and 6517, 7761, 7762 for Mangifera in-dica). The voucher specimens were preserved at the NationalCenter for Natural Products Research, University of Mississippi.

Extraction and purificationCommiphora wightii gum resin (1.5 kg) was extracted with ethylacetate (5.0 L × 72 h × 1) at room temperature. The solution wasevaporated under reduced pressure to yield a yellowish browngummy extract (245.1 g); of which 230 g was subjected to silicagel column chromatography (CC) 60–200 µm (63 × 7.5 cm) andeluted with hexanes/EtOAc [19:1 (3.0 L), 9 :1 (5.0 L), 4 :1 (5.0 L),7 :3 (5.0 L), 1 :1 (3.0 L), 0 :1 (2.0 L)] and acetone (2.0 L) to yield 24parts (A–X) [A (4.1 g), B (0.7 g), C (9.2 g), D (7.3 g), E (13.2 g), F(9.7 g), G (6.4 g), H (4.4 g), I (10.1 g), J (6.3 g), K (10.6 g), L (11.3 g),M (25.4 g), N (12.6 g), O (4.3 g), P (4.2 g), Q (18.7 g), R (2.3 g), S(5.7 g), T (12.5 g), U (5.3 g), V (2.6 g), W (35.2 g), and X (10.1 g)]. Aportion of F (2.16 g) was fractionated by CC [silica gel (107 ×4 cm), CHCl3 (3.5 L)] into 6 fractions [nothing was significant inearly 2.0 L of eluent, F1 (22mL), F2 (330mL), F3 (630mL), F4(125mL), F5 (125mL), and F6 (125mL)]. Compound 16 (55.9mg)was obtained from F2 (670mg) by repeated CC [RP-18 silica gel(15 × 3 cm), MeOH (2.0 L) and silica gel (96 × 2.5 cm), hexanes/acetone (9:1 2.5 L), respectively]. A part of G (2.15 g) was sub-jected to CC [silica gel (105 × 4 cm), hexanes/acetone (9:1 3.0 L,4 :1 2.0 L, 7 :3 1.0 L)] to afford six fractions [nothing was signifi-cant in early 1.0 L elution, G1 (176mL), G2 (900mL), G3 (110mL,compound 12, 157.5mg), G4 (400mL), G5 (1.72 L), and G6(1.82 L)]. Fraction G1 (617.6mg)was sectioned by CC [RP-18 silica(15 × 3 cm), MeOH/H2O (9:1 2.0 L, 1 :0 1.0 L)] into G1a-G1f (0.5 Leach). Compounds 1 (12.2mg) and 13 (77.5mg) were isolatedfrom G1a (460.1mg) by repeated CC [RP-18 silica (32 × 2.5 cm),MeOH (400mL) and silica gel (82 × 2.5 cm), hexanes/EtOAc (7:3700mL), respectively]. A segment of M (2.25 g) was subjected tovacuum liquid chromatography (VLC) [RP-18 silica (19 × 6.5 cm)MeOH/H2O (4:1 3.0 L, 17:3 3.0 L, 9 :1 2.0 L, 1 :0 1.0 L)] to give 9fractions [M1(1.0 L), M2 (1.5 L), M3 (1.0 L), M4 (1.0 L), M5 (0.5 L),M6 (1.0 L), M7 (0.5 L), M8 (1.0 L), and M9 (1.5 L)]. Compounds 7(86.0mg) and 8 (59.6mg) were purified from fraction M1 (1.5 g)by CC [silica gel (75 × 2.5 cm), hexanes/EtOAc (3:1 4.6 L)]. Com-pounds 14 (29.2mg) from fraction M6 (77.7mg) and 17(25.0mg) fromM8 (209.3mg) were obtained by repeated CC [sil-ica gel (45 × 2.5 cm), CHCl3/MeOH (49:1 1.4 L) and silica gel(71 × 2.5 cm), hexanes/EtOAc (13:7 700mL), respectively]. Frac-tion M9 (219.5mg) afforded 4 (2.7mg) and 15 (6.3mg) by CC [sil-ica gel (84 × 2.5 cm), CHCl3/MeOH (99:1 2.2 L)]. A fraction of Q(5.1 g) was applied to VLC [RP-18 silica (20 × 6.5 cm), MeOH/H2O(3:2 1.5 L, 13:7 1.5 L, 7 :3 1.0 L, 4 :1 2.0 L, 9 :1 3.0 L, and 1:01.0 L), and acetone (1.0 L)] to give 19 fractions [Q1 (1.5 L), Q2(0.9 L), Q3 (0.6 L), Q4 (0.5 L), Q5 (0.75 L), Q6 (0.5 L), Q7-Q11(0.25 L each), Q12 (0.75 L), Q13, Q14 (0.25 L each), Q15 (0.5 L),Q16 (1.0 L), Q17 (0.25 L), Q18 (1.0 L), and Q19 (1.0 L)]. FractionQ2 (852.3mg) was fractionated by CC [silica gel (63 × 2.5 cm),hexanes/CH2Cl2/MeOH (79:120:1 5.8 L)] into 8 subfractions[nothing was eluted in early 2.7 L elution, Q2a (44mL), Q2b (260mL), Q2c (154mL), Q2d (176mL), Q2e (264mL), Q2f (88mL), Q2g(1.0 L), and Q2h (2.5 L)]. The methanol soluble portion (284.5mg)of subfraction Q2b (364.6mg) was subjected to CC [silica gel(74 × 2.5 cm), hexanes/acetone (4:1 2.7 L)], followed by semi-preparative HPLC [Phenomenex Gemini C-18 column (250 × 10mm, 5 µm), MeCN/H2O (3:2), flow rate (3mL/min)] to give com-

pounds 3 (15.7mg, ret. time 14.14min) and 6 (4.4mg, ret. time11.73min). Compounds 2 (8.0mg) from Q2g (54.6mg) and 9(18.3mg) from fraction Q14 (1.4 g) were purified by repeated CC[silica gel (82 × 2.5 cm), hexanes/CH2Cl2/acetone (7:2:1 3.6 L)and silica gel (57 × 4 cm), hexanes/CH2Cl2/acetone (4:1:1 5.2 L),respectively]. Compound 5 (6.0mg) was purified from fractionQ9 (290.1mg) by preparative thin-layer chromatography (PTLC)[CHCl3/MeOH (97:3)]. Compounds 10 (10.1mg) from fractionQ6 (691.3mg) and 11 (118.5mg) from part H were obtained asmethanol insoluble material.20(S),21-Epoxy-3-oxocholest-4-ene (1): White powder; mp: 99–100°C; [α]D25 + 112 (c 1.0, CHCl3); UV (MeOH): λmax nm (log ε) =239.9 (4.5); IR (NaCl): νmax = 2946, 1680 cm−1; 1H and 13C NMRspectroscopic data: see l" Table 1; HRESI‑MS (positive-ionmode): m/z = 399.3264 [M + H]+ (C27H42O2 + H, requires399.3263); X‑ray crystallography: CCDC (Deposit No: 755746)contains the supplementary crystallographic data.21-Hydroxy-3-oxocholest-4,20(22)-diene (1b): Transparent gum;[α]D21 + 3, (c 1.0, CHCl3); UV (EtOH): λmax nm (log ε) = 240 (3.5);IR (NaCl): νmax = 2924, 1676 cm−1; 1H and 13C NMR spectroscopicdata: see l" Table 1; HRESI‑MS (positive-ion mode): m/z =399.3283 [M + H]+ (C27H42O2 + H, requires 399.3263).8β-Hydroxy-3,20-dioxopregn-4,6-diene (2): Light yellow powder;mp: 169–170°C; [α]D25 + 372, (c 1.0, CHCl3); UV (MeOH): λmax nm(log ε) = 280 (4.6); IR (NaCl): νmax = 3412, 1702, 1650 cm−1; 1H and13C NMR spectroscopic data: see l" Table 1; HRESI‑MS (positive-ion mode): m/z = 329.2141 [M + H]+ (C21H28O3 + H, requires329.2117).Diasesartemin (3): [α]D25 + 331 (c 1.0, CHCl3); 1H NMR (CDCl3,400MHz): δ 6.59 (2H, s, H-2′, H-6′), 6.58 (1H, s, H-6′′), 6.56 (1H,brs, H-2′′), 5.98 (2H, s, OCH2O), 4.89 (2H, dd, J = 6.8, 6.0 Hz, H-1,H-4), 3.92 (3H, s, 5′′-OCH3), 3.89 (6H, s, 3′-OCH3, 5′-OCH3), 3.86(3H, s, 4′-OCH3), 3.74 (2H, dd, J = 9.8, 2.0 Hz, H-3), 3.57 (2H, m,H‑6), 3.17 (2H, overlapped, H-3a, H-6a); 13C NMR (CDCl3,100MHz): δ 153.4 (C-3′, C-5′), 149.1 (C-3′′), 143.7 (C-5′′), 137.1(C-4′), 134.8 (C-1′), 134.4 (C-4′′) 133.7 (C-1′′), 105.7 (C-6′′), 103.3(C-2′, C-6′), 101.7 (OCH2O), 100.7 (C-2′′), 84.3 (C-1), 84.2 (C-4),69.1 (C-3), 69.0 (C-6), 61.1 (4′-OCH3), 56.8 (5′′-OCH3), 56.3 (3′-OCH3, 5′-OCH3), 49.7 (C-3a), 49.6 (C-6a).5-(13′Z-Nonadecenyl)resorcinol (17): Light brown gum; UV(MeOH): λmax nm (log ε) = 280 (4.1); 1H NMR (CDCl3, 400MHz):δ 6.22 (2H, brs, H-4, H-6), 6.15 (1H, brs, H-2), 5.33 (2H, over-lapped, H-13′, H-14′), 2.46 (2H, brt, J = 7.6 Hz, H-1′), 2.00 (4H,overlapped, H2-12′, H2-15′), 1.53 (2H, m, H-2′), 1.30–1.23 (24H,overlapped, remaining methylenes), 0.87 (3H, brt, J = 6.6 Hz, H3-19′); 13C NMR (CDCl3, 100MHz): δ 156.7 (C-1, C-3), 146.5 (C-5),130.1 (C-13′), 130.2 (C-14′), 108.3 (C-4, C-6), 101.3 (C-2), 36.1 (C-1′), 32.2 (C-17′), 31.3 (C-2′), 30.0–29.5 (C-3′, C-4′, C-5′, C-6′, C-7′, C-8′, C-9′, C-10′, C-11′, C-16′), 27.2 (C-13′), 27.4 (C-15′), 22.6 (C-18′),14.3 (19′); HRESI‑MS (negative-ion mode): m/z = 409.2845 [M +Cl]− (C25H42O2 + Cl, requires 409.2873).

Acetylation of 5-(13′Z-nonadecenyl)resorcinol (17)and ozonolysis of acetylated analogue (17a)Compound 17 (5.0mg) was dissolved in a mixture of pyridine/acetic anhydride (0.5mL each) and kept overnight. The reactionmixture was then dried under nitrogen to get the acetylatedproduct 17a (5.7 g). The acetylated analogue 17a (0.013mmol)was dissolved in 0.5mL of CH2Cl2. A stream of ozonewas bubbledthrough the solution at − 78°C by using a Triogen ozone genera-tor, until the solution acquired a peculiar blue color. Excess ozonewas removed by a stream of argon. The reaction mixture was


Table 1 1H and 13C NMR spectroscopic data for 1, 1b, and 2.

Position 1a 1ba 2a 2b

δC δHc δC δHc δC δHc δC δHc

1 36.2 t 1.67, 2.00m 35.9 t 1.69, 2.03m 35.3 t 1.69, 2.11 34.7 t 1.60, 2.05

2 34.5 t 2.35, 2.40 34.2 t 2.34m, 2.47 33.8 t 2.45, 2.56m 33.4 t 2.25 br. dd(4.2, 18), 2.54

3 200.0 s – 199.9 s – 199.8 s – 198.4 s –

4 124.4 d 5.70 s 124.0 d 5.72 s 125.6 d 5.70 s 124.1 d 5.67 s

5 171.8 s – 171.8 s – 163.2 s – 163.6 s –

6 32.4 t 1.01m, 1.81m 32.1 t 1.06m, 1.86m 128.6 d 6.08 d (9.6) 126.8 d 6.15 d (9.6)

7 33.4 t 2.20, 2.32 33.1 t 2.23m, 2.44 139.0 d 6.15 d (9.6) 140.2 d 6.18 d (9.6)

8 35.7 d 1.48m 36.2 d 1.50 71.6 s – 69.7 s –

9 54.2 d 0.94m 54.2 d 0.95m 53.3 d 1.45 dd (2.0, 12.4) 52.6 d 1.36 dd (2.4, 12.4)

10 39.1 s – 38.8 s – 36.3 s – 35.7 s –

11 21.4 t 1.54, 1.57 21.2 t 1.52, 1.59 18.1 t 1.70, 1.88m 17.6 t 1.55, 1.79m

12 39.2 t 1.23, 1.95m 38.2 t 1.72, 1.78 40.2 t 1.52m, 2.14 39.3 t 1.50, 1.99

13 43.2 s – 43.7 s – 45.6 s – 44.9 s –

14 56.5 d 1.09 55.7 d 1.20 57.3 d 1.48 56.7 d 1.41 dd (7.2, 12.6)

15 22.8 t 1.11 24.3 t 1.22m, 1.70 19.4 t 1.70 18.8 t 1.66m

16 24.3 t 1.09, 1.63 25.7 t 1.70, 1.78 22.4 t 1.66, 2.24m 21.7 t 1.51, 2.04

17 51.5 d 2.04 t (9.0) 55.2 d 2.23 64.2 d 2.47 63.0 d 2.53

18 13.8 q 0.68 s 13.0 q 0.57 s 16.3 q 0.91 s 16.1 q 0.82 s

19 17.9 q 1.15 s 17.6 q 1.17 s 19.1 q 1.30 s 18.6 q 1.27 s

20 59.6 s – 137.7 s – 209.1 s – 208.4 s –

21 50.3 t 2.55 d (4.2),2.84 d (4.2)

61.4 t 4.02 d (11.4)4.20 d (11.4)

31.6 q 2.08 s 31.1 q 2.03 s

22 37.2 t 1.33, 1.75m 131.6 d 5.40 t (7.2)

23 22.5 t 1.25m, 1.34 26.0 t 2.15 q-like (7.5)

24 39.7 t 1.11, 1.22 39.8 t 1.24, 1.32m

25 28.5 d 1.52 28.0 d 1.52

26 23.1 q 0.82 d (6.0) 22.7 q 0.88 d (6.6)

27 23.2 q 0.82 d (6.0) 22.7 q 0.88 d (6.6)

a In CDCl3; b in DMSO-d6; c multiplicity is not clear; δ in ppm; J in Hz in parentheses

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stirred at − 78°C for 3 h and the reactionwas quenched by the ad-dition of dimethyl sulfide (0.027mmol) at − 78°C. The resultingsolution was warmed up to room temperature and concentratedunder reduced pressure. The resulting aldehyde adduct 17b wasanalyzed by using HRESI‑MS.

Single-crystal X-ray diffractionThe single-crystal X‑ray diffraction data of 1 was collected on aBruker Smart Apex II system, using CuKα radiation with a graph-ite monochromator. The crystal was kept at 100(2) K under astream of cooled nitrogen gas from a KRYO-FLEX cooling device.Crystal data: C27H42 O2, orthorhombic, space group P 212121,a = 10.9024(2) Å, b = 14.3505(2) Å, c = 15.1248(2) Å, volume =2366.35(7) Å3, Z = 4, the final R1 = 0.0372 for 4010 independentdata. The crystal diffraction data can be obtained from CCDC-755746, available free of charge. Most hydrogen atoms exceptthose at chiral centers are not shown for clarity. The side chain isshowing a great disorder due to its free rotation and flexibility.

Results and Discussion!

Repeated silica gel (normal and reverse phase) column chroma-tography and high pressure liquid chromatography were used topurify the compounds from the ethyl acetate extract of the com-mercial sample of guggul.Compound 1was obtained as a white powder. An [M + H]+ at m/z399.3264 in the HRESI‑MS, along with the 13C NMR spectroscopic


data facilitated the establishment of its molecular formula ofC27H42O2. The characteristic IR and UV absorption bands for α,β-unsaturated ketone functionality were detected at 1680 cm−1 and240 nm, respectively. The 27 signals in the broadband-decoupled13C NMR spectrum were resolved as four methyl, twelve methy-lene, six methine, and five quaternary carbons. The 1H NMR spec-trum displayed resonances for two methyl singlets each inte-grated for three protons assigned to Me-18 (δH 0.68) and Me-19(δH 1.15) and a 6H doublet assigned tomethyls Me-26 andMe-27at δH 0.82, J = 6.0 Hz. The 13C NMR resonances observed at δC200.0, 124.4, and 171.8 were assigned to the α,β-unsaturated car-bonyl carbons C-3, C-4, and C-5, respectively. The resonances at δC59.6 (C-20) and δC 50.3 (C-21), assignable to an oxygen containingquaternary and oxygenated methylene carbons, respectively, andfor a pair of isolated doublets at δH 2.55 (J = 4.2 Hz, H-21a) and δH2.84 (J = 4.2 Hz, H-21b), assignable to the oxygenated methyleneprotons, revealed C-20–C-21–O–C-20 epoxide functionality. TheNMR (l" Table 1) and HRESI‑MS spectroscopic data indicated asteroidal skeleton. The chemical shift assignment of 1 was basedon COSY, HMQC, and HMBC spectra (l" Fig. 2) and was in agree-ment with the literature [4,5]. The long-range 1H-13C (HMBC)correlations of H2-21 with C-20 and C-22, H-17 with C-20 andC-21, and H2-1 and H2-2 with C-3, H2-1, H2-7, and H3-19 withC-5, as well as H-4 with C-6 and C-10 further support the epoxyand α,β-unsaturated carbonyl functionalities (l" Fig. 2).The X‑ray diffraction data supported the S configuration at C-20(l" Fig. 3). Finally, compound 1 was elucidated as 20(S),21-ep-oxy-3-oxocholest-4-ene.

Fig. 4 Conversion of epoxide into allylic alcohol 1b, possibly an isolationartefact.

Fig. 3 ORTEP drawing of final X‑ray structure of 20(S),21-epoxy-3-oxo-cholest-4-ene (1).

Fig. 2 HMBC correlations for 1 and 2.

Fig. 5 Important NOE correlations of compound 2.

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Compound1was found to convert into1bwith thepassageof time(l" Fig. 4), which was fully characterized as 21-hydroxy-3-oxo-cholest-4,20(22)-diene with the help of 1D/2DNMR (l" Table 1)and HRESI‑MS (m/z 399.3283) and found to be new to the litera-ture. Compound 1b is likely an isolation artefact which arosefrom an intermediate and decomposes into allylic alcohol in thepresence of CHCl3 and traces of acid [6]. When the NMR spectro-scopic data of 1b was compared with those of 1, the resonancesfor an epoxy ring and amethylene adjacent to an epoxy unit werefound missing in 1b due to the oxirane ring opening resulting ina C-20/C-22 double bond and C-21 hydroxymethylene [δC 137.7(C-20), δC/H 61.4 (C-21)/4.02 (d, J = 11.4 Hz, H-21a) and 4.20 (d,J = 11.4 Hz, H-21b), δC/H 131.6 (C-22)/5.40 (t, J = 7.2 Hz, H-22)].Compound 2was obtained as a light yellow powder. The molecu-lar formula C21H28O3 was deduced from an [M + H]+ ion at m/z329.2141 in the HRESI‑MS. The IR absorptions at 1702 and1650 cm−1 showed two carbonyl functionalities. The characteris-tic absorption of an α,β-unsaturated ketone with an extendedconjugation was observed at 280 nm in the UV spectrum. Thebroadband-decoupled 13C NMR spectrum displayed 21 resonan-ces, which were resolved as three methyl, six methylene, sixmethine, and six quaternary carbons by DEPT experiment. The1H/13C‑NMR spectra possessed resonances for three quaternarymethyls [δH/δC 0.91/16.3 (Me-18), 1.30/19.1 (Me-19), 2.08/31.6(Me-21)], two olefinic bonds [δH/δC 5.70 (brs, H-4)/125.6 (C-4),6.08 (d, J = 9.6 Hz, (H-6)/128.6 (C-6), 6.15 (d, J = 9.6 Hz, (H-7)/

139.0 (C-7) and δC 163.2 (C-5)], two oxo groups [δC 199.8 (C-3)and 209.1 (C-20)] and an oxygen containing quaternary carbon[δC 71.6 (C-8)] (l" Table 1). The 1H/13C‑NMR resonances were as-signed by observing the 1H-1H (COSY), 1H-13C (HMQC), and long-range 1H-13C (HMBC) correlations (l" Fig. 2), together with thespectroscopic data reported in literature [7]. The HMBC correla-tion of H-2 with C-3 supported the ketone functionality at C-3.Similarly, the long-range 1H and 13C interaction of the olefinicproton H-4 with C-6/C-10 and H-6 with C-4/C-5 and C-10 indi-cated the presence of olefinic bonds at C-4/C-6 (l" Fig. 2). Hydrox-yl and oxo functionalities at C-8 and C-20, respectively, were sup-ported by the HMBC correlations of H-6 with C-8 and H2-16, H-17, H3-21 with C-20. The β-orientation of the hydroxyl was as-signed due to the absence of Me-18 and Me-19 correlation inthe ROESY spectrum, which is obvious in case of α-orientationof the hydroxy group. The 1H‑NMR experiment was carried outin DMSO-d6 to assign the hydroxyl proton chemical shift (δH4.44). The hydroxyl proton showed strong NOESY (in DMSO-d6)cross-peaks with H3-19 (δH 1.27) and H3-18 (δH 0.82), which un-ambiguously confirmed the β-orientation of the hydroxyl group(l" Fig. 5). Ultimately, compound 2 was elucidated as 8β-hy-droxy-3,20-dioxopregn-4,6-diene.Compound 17 was obtained as a light brown gum. An [M + Cl]−

ion observed atm/z 409.2845 in the HRESI‑MSwas in accordancewith the formula of C25H42O2. The 1H and 13C NMR spectroscopicdata (see experimental part) supported a 1,3,5-trisubstitutedphenyl ring with two hydroxyl and one alkenyl substituents [δC156.7 (s, C-1, C-3), 146.5 (s, C-5), 108.3 (d, C-4, C-6), and 101.3 (d,C-2); and δH 6.22 (2H, brs, H-4, H-6), 6.15 (1H, brs, H-2)]. The res-onances for an olefinic bond were at δH 5.33 (2H, overlapped, H-13′, H-14′) and δC 130.1 (C-13′) and 130.2 (C-14′). The length ofthe aliphatic side chain substituted at C-5 was disclosed fromthe HRESI‑MS data, whereas the chemical shift of the methylenesadjacent to the double bond (27–28 ppm) [8] indicated the Z-configuration of the olefinic bond (otherwise 32–33 ppm). The


Fig. 6 Aldehyde adduct formation by ozonolysis ofan acetylated analogue of 17.

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mass spectrometric data analysis of an aldehyde adduct (17b,m/z390 forC23H34O5),obtainedbyozonolysis [9]of theacetylatedana-logue of 17 (l" Fig. 6), was used to determine the position of theolefinic bond in the side chain. The chemical shift assignment wasconfirmedbyCOSY,HMQC, andHMBCspectra. Thus compound17was identified as 5-(13′Z-nonadecenyl)resorcinol.Known compounds were identified as diasesartemin (3) [10], 6α-hydroxycholest-4-ene-3-one (4) [11], cholest-4-ene-3,6-dione(5) [5], epieudesmin (6) [12], Z-guggulsterone (7), E-guggulster-one (8) [13], guggulsterol III (9) [4,13], 20(S)-acetyloxy-4-preg-nene-3,16-dione (10) [14], sesamin (11) [10], (13E,17E,21E)-8-hydroxypolypodo-13,17,21-trien-3-one (12), (13E,17E,21E)-polypodo-13,17,21-trien-3,8-diol (13) [3], mangiferolic acid (14)[15,16], cycloartenol (15) [17], and 5-(11′Z-heptadecenyl)resor-cinol (16) [18] through comparison of spectral and physical data.The presence of mangiferolic acid (14), a known metabolite ofMangifera indica [15,16], raised suspicion of adulteration. Thus,genuine gum resin samples of Commiphora wightii andMangiferaindica were obtained from different sources from Pakistan andIndia, and their ethyl acetate extracts were subjected to co-TLC(thin-layer chromatography) together with the isolates. Com-pounds 14–17, present in the commercial sample, were not de-tected in any authentic guggul material; however they werefound in theMangifera indica specimens. This study revealed thatthe commercial guggul sample was adulterated. It also empha-sizes the need for care, as well as regulatory control, to ensurethat consumers and manufacturers of herbal products get un-adulterated raw material from commercial vendors, along within-house methods for checking the authenticity of raw material.The metabolites obtained as a result of detailed phytochemicalinvestigation of C. wightii can be useful in its chemical finger-printing. The study also emphasizes the risk of adulteration andsupport dialogue and debate on ensuring the authenticity of theraw plant material used in dietary supplements or folk medicinesall over the world.

Acknowledgements!

This research was co-funded by the Pak-USAID project (Linkagesof Centers for Chemical Sciences, Grant No. 1-5/ILS‑US/HEC/2004) and The United States Food and Drug Administration(FDA) Specific Cooperative Research Agreement Number5U01FD002071-08. The authors are thankful to Dr. B. Avula forrecording the MS data.

Affiliations1 National Center for Natural Products Research, School of Pharmacy,University of Mississippi, University, MS, USA

2 H.E. J. Research Institute of Chemistry, International Centerfor Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan

3 Department of Medicinal Chemistry, School of Pharmacy,University of Mississippi, University, MS, USA

4 Department of Pharmacognosy, School of Pharmacy,University of Mississippi, University, MS, USA


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chemical characterization of a commercial commiphora wightii resin...

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