Planta Medica Letters 2015; 2(01): e28-e30
DOI: 10.1055/s-0035-1557793
Letter
Georg Thieme Verlag KG Stuttgart · New York

Rottlerin Derivatives and Other Compounds from Mallotus philippinensis Fruits and Their Potential Antimycobactrial Activity

Rima Bharadwaj
1   Division of Organic Chemistry, CSIR-National Chemical Laboratory, Pune, India
,
Ashish A. Chinchansure
1   Division of Organic Chemistry, CSIR-National Chemical Laboratory, Pune, India
,
Roshan R. Kulkarni
1   Division of Organic Chemistry, CSIR-National Chemical Laboratory, Pune, India
,
Manisha Arkile
2   Combi Chem-Bio Resource Centre, CSIR-National Chemical Laboratory, Pune, India
,
Dhiman Sarkar
2   Combi Chem-Bio Resource Centre, CSIR-National Chemical Laboratory, Pune, India
,
Swati P. Joshi
1   Division of Organic Chemistry, CSIR-National Chemical Laboratory, Pune, India
› Author Affiliations
Further Information

Correspondence

Dr. Swati P. Joshi
Division of Organic Chemistry, CSIR-National Chemical Laboratory
Dr. Homi Bhabha Road
Pune-411008, Maharashtra
India
Phone: +91 20 25 90 23 27   
Fax: +91 20 25 90 26 27   

Publication History

received 14 May 2015
revised 30 June 2015

accepted 30 June 2015

Publication Date:
04 August 2015 (online)

 

Abstract

The methanolic extract of the fruits of Mallotus philippinensis afforded 13 compounds, 7,11-diketo-lanost-3-ol (1, as acetate), lanosta-8-ene-3β-ol (2, as acetate), pregnenolone (3, as acetate), trans-chalcone (4), kamalachalcone E (5), oleanolic acid (6), gallic acid (7), kaempferol (8), myricetin (9), 1-(5,7-dihydroxy-2,2,6-trimethyl-2 H-1-benzopyran-8-yl)-3-phenyl-2-propen-1-one (10), 4′-hydroxyisorottlerin (11), rottlerin (12), and shikimic acid (13). Compound 1 was isolated as a new natural product and its structure was elucidated by 1D and 2D nuclear magnetic resonance analyses including heteronuclear single quantum correlation, heteronuclear multiple-bond correlation, correlation spectroscopy, and nuclear Overhauser effect spectroscopy experiments. All of the isolated compounds were evaluated for their antimycobacterium activity against Mycobacterium tuberculosis H37Ra. Compounds 11 and 12 exhibited promising inhibitory activity with IC50 values of 0.89 ± 0.33 µg/mL (MIC 2.06 ± 0.41 µg/mL) and 7.59 ± 0.42 µg/mL (MIC 11.56 ± 0.35 µg/mL), respectively.


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The genus Mallotus is a large genus of trees/shrubs distributed chiefly in the tropical and subtropical regions of the Old World with around 20 species in India [1]. The genus Mallotus is represented by five species in Maharashtra, and Mallotus philippinensis (Lam.) Muell.Arg. (Euphorbiaceae) is a branched tree distributed throughout the state [2]. A red dye called Kamala is secreted on the surface of the fruits and is used medicinally as well as commercially as a dye. It has a purgative property and is also used in the external applications for parasitic infections of the skin. It also has a lithontriptic property that dissolves or destroys stones in the kidneys and it is styptic (antihemorrhagic agent). Earlier work on the chemical analysis of different parts of this tree has revealed the occurrence of several triterpenes [3], [4], flavonoids [5], [6], [7], [8], and dimeric chalcone derivatives [9], [10].

In our earlier work, from the acetone extract of whole uncrushed fruits of M. philippinensis, we isolated chalcones, including one new dimeric chalcone, kamalachalcone E, which was shown to have antifungal activity against the human pathogens Candida albicans, Candida glabrata, and Cryptococcus neoformans [11]. In the continuation of this work, we have examined the methanol extract of the crushed fruits of M. philippinensis to isolate 13 compounds (113) and evaluated them for antimycobacterium activity against Mycobacterium tuberculosis H37Ra.

Compound 1 was isolated as an acetate. Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) showed pseudomolecular peaks at 501.02 [M + 1]+ and 539.01 [M + K]+, corresponding to the molecular formula C32H52O4 with seven degrees of freedom. The 1H NMR (Fig. S1, Supporting Information) spectrum showed five singlet methyls at δ H 0.72, 0.84, 0.92, 1.21, 1.30, one three proton doublet methyl at δ H 0.85 (3 H, d, 6.0 Hz), one six proton doublet methyl at 0.87 (6 H, d, 6.7 Hz), and seven methines at δ H 1.35, (m), 1.53 (sp, 6.9 Hz), 1.60 (m), 1.26 (m), 2.22 (d, 12.9 Hz), 2.67 (d, 12.7 Hz), and 4.51 (dd, 7.6, 5.6 Hz). The 13C NMR (Fig. S2, Supporting Information) and distortionless enhancement by polarization transfer (DEPT, Fig. S3, Supporting Information) spectra showed the presence of eight methyls, nine methylenes, seven methines, and six quaternary carbons, including two carbonyl groups at δ C 209.0 and 209.6. This data suggests that compound 1 was a tetracyclic triterpenoid belonging to the class lanostanol. The complete structure was elucidated by 2D NMR. A proton at δ H 2.59 (H-12) showed an HMBC correlation with the carbonyl carbon at δ C 209.6 (C-11), while a proton at δ H 1.26 (H-5) showed an HMBC correlation with the carbonyl carbon at δ C 209.0 (C-7). The key HMBC correlations are shown in Fig. S4, Supporting Information. NOESY correlations were observed between H3-18 (δ H 0.72) and H-8 (δ H 2.67), between H3-19 (δ H 1.30) and H-8 (δ H 2.67), H3-29 (δ H 0.91), and between H3-21 (δ H 0.85) and H-9 (δ H 2.22), which led to the assignment of the relative stereochemistry (Fig. S4, Supporting Information). Thus, 1 was identified as 7,11-diketo-lanost-3β-ol. It has been prepared synthetically and reported earlier [12]. To the best of our knowledge, this is the first report of the isolation of compound 1 as a new natural product.

Compounds 213 were identified as lanosta-8-ene-3β-ol (2, as acetate) [13], pregnenolone (3, as acetate) [14], trans-chalcone (4), kamalachalcone E (5) [11], oleanolic acid (6) [15], gallic acid (7) [16], kaempferol (8) [17], myricetin (9) [18], 3.3.2 1-(5,7-dihydroxy-2,2,6-trimethyl-2H-1-benzopyran-8-yl)-3-phenyl-2-propen-1-one (10) [11], 4′-hydroxyisorottlerin (11) [10], rottlerin (12) [11], and shikimic acid (13) [19] ([Fig. 1]) by comparison with the literature NMR data and were confirmed by the LC-ESI-MS. An attempt has been made to define the configuration of compound 11 by comparing it with structurally similar flavanone derivatives. Compound 11 was determined to have a negative specific rotation, [α]D 26 − 12.11 (c 0.6, chloroform). Literature analysis suggests that the negative specific rotation was consistent with the α orientation of the group at position 2 [20], [21], [22]. This allowed for the determination of the α orientation of the group at position 2 in compound 11. Compounds 113 were evaluated for their antimycobacterium activity against M. tuberculosis H37Ra. Compounds 11 and 12 exhibited antimycobaterium activity with IC50 values of 0.89 ± 0.33 µg/mL (MIC 2.06 ± 0.41 µg/mL) and 7.59 ± 0.42 µg/mL (MIC 11.56 ± 0.35 µg/mL), respectively, as compared to the positive control rifampicin.

Zoom Image
Fig. 1 Compounds isolated from the methanol extract of M. philippinensis fruits.

Materials and Methods

General experimental procedures

1H and 13C NMR spectra were recorded on a Bruker Avance III Ultra Shield NMR (1H operating frequency: 400 MHz) instrument. LC-ESI-MS was recorded with the Waters Acquity LC-MS instrument. Optical rotations were measured with a JASCO P-1020 polarimeter. The Spectramax plus384 plate reader (Molecular Devices, Inc.) was used. All the solvents used were distilled prior to use. Column chromatography (CC) was performed with silica gel purchased from Thomas Baker Ltd. Preparative thin-layer chromatography (TLC) was carried out using TLC plates supplied by Merck Ltd. The M. tuberculosis H37Ra (ATCC 25 177) strain was obtained from MTCC, Chandigarh, India. Rifampicin (97.0 %), XTT sodium salt, and menadione were purchased from Sigma-Aldrich.


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Plant material

Fruits of M. philippinensis were collected from Bhimashankar Forest area, Pune District in March 2012. A herbarium voucher is deposited at Botanical Survey of India, Western Circle, Pune (No. PCOPAVMA2).


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Extraction and isolation

Whole fruits obtained after acetone extraction were dried under airflow and pulverized. The pulverized fruit powder, 1.2 kg, was extracted with methanol by a cold maceration technique (3 L × 3 × 14 h) at room temperature. After solvent evaporation at a reduced pressure, a red-brown extract (31.0 g, 2.58 %, based on dry weight) was yielded. The methanol extract, 30 g, was separated by repeated CC on silica gel with different elution systems. The final purification of the compounds was achieved by preparative TLC. The detailed extraction and isolation of the compounds 113 as well as LC-ESI-MS, UV, IR, and NMR data of the compounds are available as Supporting Information.


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Antimycobacterium bioassay using the XTT reduction menadione assay

M. tuberculosis H37Ra cells (ATCC 25 177) were grown to the logarithmic phase (O. D.59 5 ~ 1.0) in M. pheli medium. Compounds 1 - 13 were dissolved in dimethyl sulfoxide and stored in aliquots at − 20 °C. A freshly prepared stock solution (1.25 mM) of XTT sodium salt in sterile 1 x PBS and 6 mM menadione in DMSO was used. Compounds were screened for their inhibitory effect on M. tuberculosis by following the XTT aeduction menadione assay (XRMA) protocol [23], [24]. Briefly, in all wells of the assay plate, 200 µM XTT were added and incubated at 37 °C for 20 min. Menadione, 60 µM, was added and incubated at 37 °C for 40 min. The optical density was recorded on a microplate reader at 470 nm (filter) against a blank prepared from cell-free wells. Absorbance given by the cells treated with the vehicle alone was taken as 100 % of cell growth. All the experiments were performed in triplicate and the quantitative values are expressed as the average ± standard deviation, and the IC50 values were calculated from their dose-response curves (Figs. S5 and S6, Supporting Information). Compounds 11 and 12 exhibited promising inhibitory activity with IC50 values of 0.89 ± 0.33 µg/mL (MIC 2.06 ± 0.41 µg/mL) and 7.59 ± 0.42 µg/mL (MIC 11.56 ± 0.35 µg/mL), respectively, as compared to that of positive control rifampicin with an IC50 value of 0.0019 ± 0.0003 µg/mL (MIC 0.02 ± 0.31 µg/mL).


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Supporting information

1H, 13C, DEPT, and 2D correlations of compound 1, dose-response curves for compounds 11 and 12 against M. tuberculosis H37Ra, and the extraction, isolation, NMR, and other characterization data of compounds 113 are available as Supporting Information.


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Acknowledgments

We are thankful to Dr. Sagar Pandit, Division of Biochemical Sciences, CSIR-NCL, Pune for identifying the plant material. We are grateful to the University Grants Commission, New Delhi, India, for providing financial support.


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Conflict of Interest

There is no conflict of interest among all authors.

Supporting Information

  • References

  • 1 Thacker MS, Lala SR, Krishnan MS, Prashad B, Chopra RN, Santapau H, Sastri BN. The wealth of India: A dictionary of Indian raw materials & industrial products. New Dehli: CSIR; 1998: 229
  • 2 Singh NP, Lakshminarasimhan P, Karthikeyan S, Prasanna PV. Flora of Maharashtra State, Dicotyledons. Vol. 2. Calcutta: Botanical Survey of India; 2001: 894
  • 3 Bandopadhyay M, Dhingra VK, Mukerjee SK, Pardeshi NP, Seshadri TR. Triterpenoid and other components of Mallotus Philippinensis . Phytochemistry 1972; 11: 1511
  • 4 Nair SP, Rao JM. Kamaladiol-3-acetate from the stem bark of Mallotus philippinensis . Phytochemistry 1993; 32: 407-409
  • 5 Ahluwalia VK, Sharma ND, Mittal B, Gupta SR. Novel prenylated flavanoids from Mallotus philippensis . Indian J Chem 1988; 27?B: 238-241
  • 6 Lounasmaa M, Widén CJ, Tuuf CM, Huhtikangas A. On the phloroglucinol derivatives of mallotus philippiners . Planta Med 1975; 28: 16-31
  • 7 Crombie L, Green CL, Tuck B, Whiting DA. Constituents of kamala. Isolation and structure of two new components. J Chem Soc C 1968; 2625-2630
  • 8 Widén CJ, Puri HS. Natural occurrence and chemical variability of phloroglucinols in kamala. Planta Med 1980; 40: 284-287
  • 9 Tanaka T, Ito T, Iinuma M, Takahashi Y, Naganawa H. Dimeric chalcone derivatives from Mallotus philippensis . Phytochemistry 1998; 48: 1423-1427
  • 10 Furasawa M, Ido Y, Tanaka T, Ito T, Nakaya K, Ibrahim I, Ohyama M, Iinuma M, Shirataka Y, Takahashi Y. Novel, complex flavonoids from Mallotus philippensis (kamala tree). Helv Chim Acta 2005; 88: 1048-1058
  • 11 Kulkarni RR, Tupe SG, Gample SP, Chandgude MG, Sarkar D, Deshpande MV, Joshi SP. Antifungal dimeric chalcone derivative kamalachalcone E from Mallotus philippinensis . Nat Prod Res 2014; 28: 245-250
  • 12 Brieskorn CH, Dertinger G. Neue Carbonylderivate des Lanosterols aus Wollwachs. Tet Lett 1968; 59: 6237-6239
  • 13 Emmons GT, Wilson WK, Schroepfer jr. G. 1H and 13C NMR assignments for lanostan-3β-ol derivatives: Revised assignments for lanosterol. J Magn Reson Chem 1989; 27: 1012-1024
  • 14 Ishar MPS, Girdhar NK, Kumar K, Kaur S. Investigations on photochemical linking of steroids with amino acids: Irradiation of α, β unsaturated steroidal ketones in the presence of amino acids in aqueous medium. Indian J Chem 1999; 38B: 1253-1261
  • 15 Ragasa CY, Lim K. Secondary metabolites from Schefflera odorata Blanco. Philipp J Sci 2005; 134: 63-67
  • 16 Eldahshan OA. Isolation and structure elucidation of phenolic compounds of carob leaves grown in Egypt. Curr Res J Biol Sci 2011; 3: 52-55
  • 17 Panichayupakaranant P, Kaewsuwan S. Bioassay-guided isolation of the antioxidant constituent from Cassia alata L. leaves. J Sci Technol 2004; 26: 103-107
  • 18 Rashed K, Zhang XJ, Luo MT, Zheng YT. Anti-HIV-1 activity of phenolic compounds isolated from Diospyros lotus fruits. Phytopharmacology 2012; 3: 199-207
  • 19 Bochkov DV, Sysolyatin SV, Kalashnikov AI, Surmacheva IA. Shikimic acid: review of its analytical, isolation, and purification techniques from plant and microbial sources. J Chem Biol 2012; 5: 5-17
  • 20 Borges-Argáez R, Díaz MEP, Waterman PG, Peña-Rodríguez LM. Additional flavonoids from Lonchocarpus yucatanensis and L. xuul . J Braz Chem Soc 2005; 5: 1078-1081
  • 21 Yenesew A, Twinomuhwezi H, Kabaru JM, Akala HM, Kiremire BT, Heydenreich M, Peter MG, Eyase FL, Waters NC, Walsh DS. Antiplasmodial and larvicidal flavonoids from Derristrifoliata . Bull Chem Soc Ethiop 2009; 3: 409-414
  • 22 Yenesew A, Midiwo JO, Miessner M, Heydenreich M, Peter MG. Two prenylated flavanones from stem bark of Erythrina burttii . Phytochemistry 1998; 48: 1439-1443
  • 23 Ciapetti G, Cenni E, Pratelli L, Pizzoferrato A. In vitro evaluation of cell/biomaterial interaction by MTT assay. Biomaterials 1993; 14: 359-364
  • 24 Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983; 65: 55-63

Correspondence

Dr. Swati P. Joshi
Division of Organic Chemistry, CSIR-National Chemical Laboratory
Dr. Homi Bhabha Road
Pune-411008, Maharashtra
India
Phone: +91 20 25 90 23 27   
Fax: +91 20 25 90 26 27   

  • References

  • 1 Thacker MS, Lala SR, Krishnan MS, Prashad B, Chopra RN, Santapau H, Sastri BN. The wealth of India: A dictionary of Indian raw materials & industrial products. New Dehli: CSIR; 1998: 229
  • 2 Singh NP, Lakshminarasimhan P, Karthikeyan S, Prasanna PV. Flora of Maharashtra State, Dicotyledons. Vol. 2. Calcutta: Botanical Survey of India; 2001: 894
  • 3 Bandopadhyay M, Dhingra VK, Mukerjee SK, Pardeshi NP, Seshadri TR. Triterpenoid and other components of Mallotus Philippinensis . Phytochemistry 1972; 11: 1511
  • 4 Nair SP, Rao JM. Kamaladiol-3-acetate from the stem bark of Mallotus philippinensis . Phytochemistry 1993; 32: 407-409
  • 5 Ahluwalia VK, Sharma ND, Mittal B, Gupta SR. Novel prenylated flavanoids from Mallotus philippensis . Indian J Chem 1988; 27?B: 238-241
  • 6 Lounasmaa M, Widén CJ, Tuuf CM, Huhtikangas A. On the phloroglucinol derivatives of mallotus philippiners . Planta Med 1975; 28: 16-31
  • 7 Crombie L, Green CL, Tuck B, Whiting DA. Constituents of kamala. Isolation and structure of two new components. J Chem Soc C 1968; 2625-2630
  • 8 Widén CJ, Puri HS. Natural occurrence and chemical variability of phloroglucinols in kamala. Planta Med 1980; 40: 284-287
  • 9 Tanaka T, Ito T, Iinuma M, Takahashi Y, Naganawa H. Dimeric chalcone derivatives from Mallotus philippensis . Phytochemistry 1998; 48: 1423-1427
  • 10 Furasawa M, Ido Y, Tanaka T, Ito T, Nakaya K, Ibrahim I, Ohyama M, Iinuma M, Shirataka Y, Takahashi Y. Novel, complex flavonoids from Mallotus philippensis (kamala tree). Helv Chim Acta 2005; 88: 1048-1058
  • 11 Kulkarni RR, Tupe SG, Gample SP, Chandgude MG, Sarkar D, Deshpande MV, Joshi SP. Antifungal dimeric chalcone derivative kamalachalcone E from Mallotus philippinensis . Nat Prod Res 2014; 28: 245-250
  • 12 Brieskorn CH, Dertinger G. Neue Carbonylderivate des Lanosterols aus Wollwachs. Tet Lett 1968; 59: 6237-6239
  • 13 Emmons GT, Wilson WK, Schroepfer jr. G. 1H and 13C NMR assignments for lanostan-3β-ol derivatives: Revised assignments for lanosterol. J Magn Reson Chem 1989; 27: 1012-1024
  • 14 Ishar MPS, Girdhar NK, Kumar K, Kaur S. Investigations on photochemical linking of steroids with amino acids: Irradiation of α, β unsaturated steroidal ketones in the presence of amino acids in aqueous medium. Indian J Chem 1999; 38B: 1253-1261
  • 15 Ragasa CY, Lim K. Secondary metabolites from Schefflera odorata Blanco. Philipp J Sci 2005; 134: 63-67
  • 16 Eldahshan OA. Isolation and structure elucidation of phenolic compounds of carob leaves grown in Egypt. Curr Res J Biol Sci 2011; 3: 52-55
  • 17 Panichayupakaranant P, Kaewsuwan S. Bioassay-guided isolation of the antioxidant constituent from Cassia alata L. leaves. J Sci Technol 2004; 26: 103-107
  • 18 Rashed K, Zhang XJ, Luo MT, Zheng YT. Anti-HIV-1 activity of phenolic compounds isolated from Diospyros lotus fruits. Phytopharmacology 2012; 3: 199-207
  • 19 Bochkov DV, Sysolyatin SV, Kalashnikov AI, Surmacheva IA. Shikimic acid: review of its analytical, isolation, and purification techniques from plant and microbial sources. J Chem Biol 2012; 5: 5-17
  • 20 Borges-Argáez R, Díaz MEP, Waterman PG, Peña-Rodríguez LM. Additional flavonoids from Lonchocarpus yucatanensis and L. xuul . J Braz Chem Soc 2005; 5: 1078-1081
  • 21 Yenesew A, Twinomuhwezi H, Kabaru JM, Akala HM, Kiremire BT, Heydenreich M, Peter MG, Eyase FL, Waters NC, Walsh DS. Antiplasmodial and larvicidal flavonoids from Derristrifoliata . Bull Chem Soc Ethiop 2009; 3: 409-414
  • 22 Yenesew A, Midiwo JO, Miessner M, Heydenreich M, Peter MG. Two prenylated flavanones from stem bark of Erythrina burttii . Phytochemistry 1998; 48: 1439-1443
  • 23 Ciapetti G, Cenni E, Pratelli L, Pizzoferrato A. In vitro evaluation of cell/biomaterial interaction by MTT assay. Biomaterials 1993; 14: 359-364
  • 24 Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983; 65: 55-63

Zoom Image
Fig. 1 Compounds isolated from the methanol extract of M. philippinensis fruits.