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Cashmirins A and B, new antifungal and urease inhibitory prenylated coumarins from Sorbus cashmiriana

Abstract

Cashmirins A (1) and B (2), new prenylated coumarins, have been isolated from the EtOAc- soluble fraction of the whole plant of Sorbus cashmiriana Hedlung, Monog. along with seselin (3), scopoletin (4), 3-hydroxyxanthyletin (5) and luteolin (6), reported for the first time from this species. Their structures were elucidated by spectroscopic techniques including MS, 1D and 2D NMR spectroscopy. Both new compounds 1 and 2 were investigated for biological activities and showed significant antifungal and urease inhibitory activities. Compounds 1 and 2 exhibited significant activity against Aspergillus flavus, Macrophomina phaseolina, Trichophyton simii, Trichophyton schoenleinii, and Pseudallescheria boydri. Both compounds also exhibited significant inhibitory activity against Jack bean urease with IC50 values of 28.2±0.12 µM and 30.3±0.18 µM, respectively compared to thiourea used as positive control.

Keywords:
Sorbus cashmiriana; Prenylated coumarins; Cashmirins; Antifungal activity; Urease inhibition

INTRODUCTION

The genus Sorbus (Rosaceae) comprises of 200 species which are commonly grown in Asia, Africa and South America. Plants of the genus Sorbus are found in customary and local medicines that are used as anti- diarrhea, diuretic, anti-inflammatory, anti-diabetic, vaso- protective, broncho- and vasorelaxant, along with potent antioxidative qualities. In Pakistan, it is represented by seven species. One of these is Sorbus cashmiriana Hedlung, Monog, which is a tree of two seasons, one in the spring with lovely pink-tinted flowers and one in the autumn when the leaves are gone, and glorious white fruits shine out. It is distributed in Kashmir and the western Himalayas. A tea made from its bark is used to treat nausea. The bark preparation is also used to treat heart diseases. Berries are used to cure scurvy (Bhattacharjee, 2003Bhattacharjee SK. Hand Book of Medicinal Plants, Pointer Publisher, Jaipur India, 2003. 30 p.; Perry, Metzger, 1980Perry LM, Metzger J. Medicinal Plants of East and South Asia, The MIT Press: Cambridge England, 1980. 341 p.; Krishna, 1972Krishna A. The Wealth of India, Vol. IX. CSIR: New Delhi, 1972. 435 p.; Jayaweera, 1982Jayaweera DMA. Medicinal Plants, the National Science Council of Sri Lanka, Vol. IV. Colombo. 1982. 257 p.; Krachmal, 1980Krachmal AC. A Guide to the Medicinal Plants of the United States, Vol. IX. Quadrangle New York Times Book Co., 1980. 207 p.). Previously, six triterpenes (Kazmi et al., 2007Kazmi MH, Ahmed E, Hameed S, Malik A, Ashraf M. Isolation and structural determination of sorbinols A and B, new triterpenes from Sorbus cashmariana, by 1D and 2D NMR spectroscopy. Magn Reson Chem. 2007;45(5):416-419.; Kazmi et al., 2009Kazmi MH, Ahmed E, Hameed S, Malik A, Fatima I, Ashraf M. Cashmirols A and B, new lipoxygenase inhibiting triterpenes from Sorbus cashmiriana. Chem Biodiversity . 2009;6(9):1471-1476.; Kazmi et al., 2011Kazmi MH, Fatima I, Malik A, Iqbal L, Latif M, Afza N. Sorbicins A and B, new urease and serine protease inhibitory triterpenes from Sorbus cashmiriana. J Asian Nat Prod Res. 2011;13(12):1081-1086.) and two coumarins (Khan et al., 2015Khan S, Fatima I, Kazmi MH, Malik A, Afza N, Iqbal L, et al. Cashmins A and B, potent antioxidant coumarins from Sorbus cashmiriana. Chem Nat Comp. 2015;51(4):626-629.) have been reported by us from this species. The ethnopharmacologic and chemotaxonomic importance of the genus Sorbus prompted us to carry out further phytochemical studies on S. cashmiriana. Herein we report the isolation and structural elucidation of two new prenylated coumarins named as cashmirins A (1) and B (2) (Figure 1) along with seselin (3) (Cazal et al., 2009Cazal CM, Domingues VC, Batalhão JR, Bueno OC, Rodrigues-Filho E, Forim MR, et al. Isolation of xanthyletin, an inhibitor of ants’ symbiotic fungus, by high- speed counter-current chromatography. J Chromatogr A. 2009;1216(19):4307-4312.), scopoletin (4) (Darmawan et al., 2012Darmawan A, Kosela S, Kardono LBS, Syah YM. Scopoletin, a coumarin derivative compound isolated from Macaranga gigantifolia Merr. J Appl Pharm Sci. 2012;2(12):175-177.), 3-hydroxyxanthyletin (5) (Chen et al., 2010Chen LW, Cheng MJ, Peng CF, Chen IS. Secondary metabolites and antimycobacterial activities from the roots of Ficus nervosa. Chem Biodiversity. 2010;7(7):1814-1821.) and luteolin (6) (Nissler, Gebhardt, Berger, 2004Nissler L, Gebhardt R, Berger S. Flavonoid binding to a multi-drug-resistance transporter protein: an STD-NMR study. Anal Bioanal Chem. 2004;379(7-8):1045-1049.), reported for the first time from this species.

FIGURE 1
Structures of cashmirins A (1) and B (2).

The plant kingdom has provided a number of therapeutic compounds, such as antifungal, antibacterial, antiparasitic, antihistamine, analgesics, anti-inflammatory, asthma medications and many more (Ficker et al., 2003Ficker CE, Arnason JT, Vindas PS, Alvarez LP, Akpagana K, Gbeassor M. Inhibition of human pathogenic fungi by ethnobotanically selected plant extracts. Mycoses. 2003;46(1-2):29-37.; Islam et al., 2001Islam A, Sayeed A, Bhuiyan MSA, Mosaddik MA, Islam MAU, Astaq GRM, et al. Antimicrobial activity and cytotoxicity of Zanthoxylum budrunga. Fitoterapia. 2001;72(4):428-430.; Jones et al., 2000Jones NP, Arnason JT, Abou-Zaid M, Akpagana K, Vindas PS, Smith ML. Antifungal activity of extracts from medicinal plants used by First Nations Peoples of eastern Canada. J Ethnopharmacol. 2000;73(1-2):191-198.; Omar et al., 2000Omar S, Lemonnier B, Jones N, Ficker C, Smith ML, Neema C, et al. Antimicrobial activity of extracts of eastern North American hardwood trees and relation to traditional medicine. J Ethnopharmacol . 2000;73(1-2):161-170.). Some of the traditionally used plants may lead to the development of new antifungal agents that are in increasing demand due to their resistance to traditional medicines (White et al., 1998White TC, Marr KA, Bowden RA. Clinical, cellular, and molecular factors that contribute to antifungal drug resistance. Clin Microbiol Rev . 1998;11(2):382-402.).

Urease (EC.3.5.1.5) has been identified as a key virulence determinant in the pathogenesis of a variety of clinical conditions, with implications for human and animal health, as well as agriculture. It leads to the pathogenesis of urolithiasus, pyelonephritis, hepatic coma, and urinary catheter encrustation by being specifically involved in the formation of infection stones (Mobley et al., 1989Mobley HLT, Hausinger RP. Microbial ureases: Significance, regulation and molecular characterisation. Microbiol Rev. 1989;53(1):85-108.; Mobley et al., 1995Mobley HLT, Island MD, Hausinger RP. Molecular-biology of microbial ureases. Microbiol Rev . 1995;59(3):451-480.).

In the current study, we have described the antifungal and urease inhibitory activity of the new prenylated coumarins named as cashmirins A (1) and B (2).

MATERIAL AND METHODS

General experimental procedure

The UV and IR spectra were recorded on a Hitachi UV-3200 and JASCO 320-A spectrometers, respectively. NMR data were recorded on a Bruker AV-500MHz spectrometer (500 MHz for 1H and 125 MHz for 13C) in CDCl3 with tetramethylsilane (TMS) as an internal standard. The chemical shift values are reported in ppm (δ) and the coupling constant (J) is in Hz. EI- and HREI-MS were recorded on Finnigan MAT 12 and MAT 312 spectrometers in m/z and %. Thin layer chromatography (TLC) was executed on precoated with silica gel F254 plates (E. Merck, Darmstadt, Germany): detection at 254 nm and by spraying with ceric sulfate in 10% H2SO4 solution. Silica gel (230-400 mesh, E. Merck, Darmstadt, Germany) was used for column chromatography.

Plant material

The whole plant of Sorbus cashmiriana Hedlung, Monog was collected from Kashmir and identified by Sher Wali Khan, Department of Biological Science, Karakoram International University Gilgit. A voucher specimen (No. KUH 73/67 760) has been deposited with the Herbarium of the Department of Botany, University of Karachi, Pakistan.

Extraction and isolation

The whole plant S. cashmiriana (16 kg) was shade dried ground and extracted with MeOH (3 × 40 L). The MeOH extract was evaporated under reduced pressure to yield a residue (300 g), which was divided into n-hexane (70 g), CHCl3 (60 g), EtOAc (78 g), and H2O (80 g) soluble sub-fractions. The EtOAc sub-fraction was subjected to column chromatography over silica gel eluting with n-hexane/CHCl3 in increasing order of polarity to obtain five fractions F1-F5. The fraction F4 which eluted with n-hexane/CHCl3 (30:70) was further purified through preparative TLC using n-hexane/ CHCl3 (45:55) to afford compound 6 (18 mg). The fraction F3 which eluted with n-hexane/CHCl3 (40:60) was further chromatographed, eluting with n-hexane/ CHCl3 (55:45) to obtain compounds 4 (35 mg) and 5 (30 mg) from the top and the tail fractions, respectively. The fraction F2 which eluted with n-hexane/CHCl3 (50:50) was further chromatographed and successively eluted with n-hexane/CHCl3 (60:40) and (55:45) to obtain compounds 1 (5 mg) and 2 (5.5 mg), respectively. The fraction F1 which eluted with n-hexane/CHCl3 (60:40) furnished compound 3 (25 mg).

Cashmirin A (1)

White amorphous solid; m.p. 107-108 °C; UV λmax (MeOH) 339, 264, 235, 220 nm; IR υmax (KBr) 1730, 1720, 1635, 1602, 1518 cm-1; EIMS m/z (rel. int.) 354 (10), 339 (32), 300 (21), 296 (100), 285 (45), 162 (72); HR-EIMS (m/z) 354.1463 [M]+ (calcd for C21H22O5 , 354.1467); 1H and 13C NMR data, see Table I.

TABLE I
1H and 13C NMR data (500 and 125 MHz) for 1 and 2 (CDCl3, δ in ppm, J in Hz)

CashmirinB (2)

White amorphous solid; m.p. 107-108 °C; UV λmax (MeOH) 338, 264, 233, 222 nm; IR υmax (KBr) 1728, 1722, 1632, 1602, 1522 cm-1; EIMS m/z (rel. int.) 384 (9), 369 (28), 330 (18), 316 (20), 302 (32), 296 (100), 285 (52), 244 (40), 162 (75); HR-EIMS (m/z) 384.1576 [M]+ (calcd for C22 H24 O6 , 354.1572); 1H and 13C NMR data, see Table I.

Antifungal activity assay

The antifungal assay was performed on human, animals and plants pathogens by agar tube dilution method (Ahmad et al., 2007Ahmad B, Hassan Shah SM, Bashir S, Nisar M, Chaudry MI. Antibacterial and antifungal activities of Andrachne cordifolia muell. J Enzyme Inhib Med Chem. 2007;22(6):726-729.) using ten pathogenic fungi which are illustrated in Table II. The crude extracts, compounds 1, 2 and the standard drugs (each 200 µg/ml of Sabourd Dextose Agar) were subjected to antifungal activity against Aspergillus flavus ATCC 32611, Aspergillus niger ATCC 1015, Macrophomina phaseolina ATCC 53789, Trichophyton simii ATCC 25923, Microsporum canis ATCC 36299, Trichophyton schoenleinii ATCC 22775, Fusarium solani ATCC 36031, Pseudallescheria boydri ATCC 44330, Candida glabrata ATCC 90030, and Rhizoctonia solani ATCC 76131.

TABLE II
In vitro fungicidal bioassay of crude extract and cashmirins A (1) and B (2)

Urease inhibitory activity

Reaction mixtures comprising 25 µL of enzyme (Jack bean urease) solution and 55 µL of buffers containing 100 mM urea were incubated with 5 µL of test compounds at 30 °C for 15 min in 96-well plates. Urease activity was determined by measuring ammonia production using the indophenol method (Weatherburn, 1967Weatherburn MW. Phenol-hypochlorite reaction for determination of ammonia. Anal Chem. 1967;39(8):971-974.). 45 µL each of phenol reagent (1% w/v phenol and 0.005% w/v sodium nitroprusside) and 70 µL of alkali reagent (0.5% w/v NaOH and 0.1% active chloride NaOCl) were added to each well. The increasing absorbance at 630 nm was measured after 50 min, using a microplate reader (Molecular Device, USA). In a final volume of 200 µL, all reactions were performed in triplicate. The results were processed by using Soft Max Pro software (Molecular Device, USA). Assays were performed at pH 8.2 potassium phosphate buffer (0.01 M K2HPO4.3H2O, 1 mM EDTA and 0.01 M LiCl). Percentage inhibitions were calculated from the formula 100 − (ODtest well/ODcontrol) × 100). Thiourea was used as the standard inhibitor of urease.

RESULTS AND DISCUSSION

Cashmirin A (1) was obtained as a white amorphous solid. The molecular formula was established as C21H22O5 through HR-EIMS showing an [M]+ peak at m/z 354.1463 (calcd 354.1467). The IR spectrum showed the presence of an α, β-unsaturated lactone (1720 cm-1), ester carbonyl (1730 cm-1), aromatic C=C (1602 and 1518 cm-1) and olefinic (1635 cm-1) functionalities. The UV spectrum showed absorption maxima at λmax 220, 235, 264, and 339 nm, characteristic of 6-substituted-7-oxygenated-pyranocoumarins (Seiji et al., 1989Seiji Y, Ryozo M, Shinobu Y, Yoshiyuki K. The synthesis of some dimethylpyranocoumarins and isopropenyldihydrofuranocoumarins. Bull Chem Soc Jpn. 1989;62(11):3593-3597.).

The 13C NMR (BB and DEPT) spectra of 1 showed 21 carbon signals, including five methyl, five methine, one methylene and ten quaternary carbons (Table I). The downfield signal at δc 164.0 and 161.4 ppm were assigned to the carbonyl carbons of the ester and α, β-unsaturated lactone moieties, respectively. The olefinic carbons of the coumarin nucleus were observed at δc 139.2 and 128.2 ppm. The oxygenated aromatic carbons appeared at δc 166.7 and 155 ppm. The signals of dimethylchromene moiety were observed at δC 129.4, 123.0 and 28.0 ppm (2 × Me), respectively. The signals of tri-substituted olefinic carbons at δc 133.2 and 121.7 ppm, methyl groups at δc 25.0 and 18.9 ppm together with methylene carbon at δc 30.2 ppm could be attributed to γ,γ-dimethylallyl moiety. In the EIMS, the γ,γ-dimethylallyl moiety was further confirmed by a base peak at m/z 285 [M-C5H9]+. The signal at δc 56.4 ppm could be assigned to the methoxyl carbon of the methyl ester.

The 1H NMR spectrum (Table I) showed the signals of γ,γ-dimethylallyl moiety at δ H 1.75 and 1.62 (3H each, br. s, Me), 3.23 (d, J = 7.0 Hz, 2H) and 5.25 ppm (m, CH). The notable up-field shift of the H-4 signal to δ H 7.29 ppm instead of 7.65‒8.03 ppm and the lack of its coupling with H-3, allowed us to assign the γ,γ-dimethylallyl moiety to C-3 (Cordova, Garelli, 1974Cordova HEB, Garelli LN. A new coumarin in Amyris simplicifolia. Phytochemistry. 1974;13(4):758-760.). It further showed characteristic signals of dimethylchromene ring [δ H 1.38 ppm (6’’-Me × 2) and olefinic protons at δ H 5.49 and 6.41 ppm (d, J = 9.5 Hz)]. The spectrum further showed an aromatic proton as singlet at δ H 7.02 ppm and carbomethoxy protons at δ H 3.78 ppm. One of the major fragment at m/z 296 [M- 59]+ in EIMS resulted from the loss of methyl carboxylate group. The collective data showed close resemblance to those of previously reported 3-prenylxanthyletin (Cordova, Garelli, 1974Cordova HEB, Garelli LN. A new coumarin in Amyris simplicifolia. Phytochemistry. 1974;13(4):758-760.), the only notable difference being the presence of methyl carboxylate group at C-8. The location of various substituents was finally confirmed by HMBC correlations (Figure 2). The presence of γ,γ-dimethylallyl moiety at C-3 could be confirmed by 3 J correlations of H-1’ with C-4 (δ C 139.2 ppm), C-2 (δ C 161.4 ppm) and C-3’ (δC 133.2 ppm) as well as 2 J correlations with C-3 (δ C 128.2 ppm) and C-2’ (δ C 121.7 ppm). The attachment of 2, 2-dimethylchromene ring at C-6 and C-7 positions of the aromatic ring could be inferred by 2 J correlation of H-4’’ with C-6 (δ 116.7 ppm) and 3 J correlations with C-7 (δ C 158.7 ppm) and C-5 (δ C 120.7 ppm). The presence of the methyl ester group was evident by the 3 J correlation of methoxyl protons (δ C 3.78 ppm) with ester carbonyl (δ C 164.0 ppm). Its presence at C-8 was authenticated by aromatic proton H-5 which showed 3 J correlations with C-4 (δ C 139.2 ppm), C-7 (δ C 158.7 ppm), C-10 (δ C 155.0 ppm) and C-4’’ (δ C 123.0 ppm), revealing substitution at C-8. All these data were in complete agreement with the assigned structure of cashmirin A (1) as 3-(γ,γ-dimethylallyl)- 6’’,6’’-dimethylpyrano-8-(methylcarboxylate)-8H- pyrano[2’’,3’’,7,8]-chromen-2-one.

FIGURE 2
Important HMBC correlations of cashmirin A (1).

Cashmirin B (2) was obtained as a white amorphous solid. The molecular formula was established as C22H24O6 through HR-EIMS, showing an [M]+ peak at m/z 384.1576 (calcd 384.1572). The IR and UV spectra were similar to that of 1. The 1H and 13C NMR spectra (Table I) were also similar to those of 1 except for an additional signal due to a methoxyl group at δ H 3.89 ppm and δ C 62.2 ppm. The absence of aromatic signal and the downfield shift of C-5 allowed us to assign it to C-5. It was confirmed by HMBC and NOESY spectra. The methoxyl protons at δ H 3.89 ppm showed 3 J correlation with C-5 (δ C 151.8 ppm) as well as NOESY interactions of 5-OCH3 with both H-4 and H-4’’, respectively. The structure of cashmirin B (2) could thus be assigned as 5-methoxy-3-(γ,γ-dimethylallyl)- 6’’,6’’-dimethylpyrano-8-(methylcarboxylate)-8H- pyrano[2’’,3’’,7,8]-chromen-2-one.

The antifungal activity of both 1 and 2 was determined by the agar tube dilution method using ten pathogenic fungi. Both of these showed significant activity against A. flavus, M. phaseolina, T. simii, T. schoenleinii, and P. boydri; moderate activity against A. niger, F. solani, and C. glabrata and weak activity against M. canis, and R. solani (Table II). It is important to note that compound 2 showed slightly more potency than 1 which is probably is due to the presence of additional methoxyl group at C-5.

The inhibitory activity of compounds 1 and 2 against Jack bean urease was determined by the method described in the experimental section. The IC50 values of compounds 1 and 2 were found to be 28.2±0.12 µM and 30.3±0.18 µM, respectively, as against IC50 value of 22.4±0.15 µM, which was observed for thiourea that was used as a positive control (Table III).

TABLE III
In vitro inhibition of urease of cashmirins A (1) and B (2)

CONCLUSION

In conclusion, two new prenylated coumarins, named as cashmirins A (1) and B (2) have been isolated from Sorbus cashmiriana Hedlung, Monog. along with four known compounds. Their structures were elucidated by spectroscopic techniques including MS, 1D and 2D NMR spectroscopy. Both new compounds showed significant antifungal activity against A. flavus, M. phaseolina, T. simii, T. schoenleinii, and P. boydri. The results showed compound 2 was slightly more antifungal than 1. Both of these compounds also exhibited significant inhibitory activity against the enzyme urease.

REFERENCES

  • Bhattacharjee SK. Hand Book of Medicinal Plants, Pointer Publisher, Jaipur India, 2003. 30 p.
  • Cazal CM, Domingues VC, Batalhão JR, Bueno OC, Rodrigues-Filho E, Forim MR, et al. Isolation of xanthyletin, an inhibitor of ants’ symbiotic fungus, by high- speed counter-current chromatography. J Chromatogr A. 2009;1216(19):4307-4312.
  • Chen LW, Cheng MJ, Peng CF, Chen IS. Secondary metabolites and antimycobacterial activities from the roots of Ficus nervosa Chem Biodiversity. 2010;7(7):1814-1821.
  • Ahmad B, Hassan Shah SM, Bashir S, Nisar M, Chaudry MI. Antibacterial and antifungal activities of Andrachne cordifolia muell. J Enzyme Inhib Med Chem. 2007;22(6):726-729.
  • Cordova HEB, Garelli LN. A new coumarin in Amyris simplicifolia Phytochemistry. 1974;13(4):758-760.
  • Darmawan A, Kosela S, Kardono LBS, Syah YM. Scopoletin, a coumarin derivative compound isolated from Macaranga gigantifolia Merr. J Appl Pharm Sci. 2012;2(12):175-177.
  • Ficker CE, Arnason JT, Vindas PS, Alvarez LP, Akpagana K, Gbeassor M. Inhibition of human pathogenic fungi by ethnobotanically selected plant extracts. Mycoses. 2003;46(1-2):29-37.
  • Islam A, Sayeed A, Bhuiyan MSA, Mosaddik MA, Islam MAU, Astaq GRM, et al. Antimicrobial activity and cytotoxicity of Zanthoxylum budrunga Fitoterapia. 2001;72(4):428-430.
  • Jayaweera DMA. Medicinal Plants, the National Science Council of Sri Lanka, Vol. IV. Colombo. 1982. 257 p.
  • Jones NP, Arnason JT, Abou-Zaid M, Akpagana K, Vindas PS, Smith ML. Antifungal activity of extracts from medicinal plants used by First Nations Peoples of eastern Canada. J Ethnopharmacol. 2000;73(1-2):191-198.
  • Kazmi MH, Ahmed E, Hameed S, Malik A, Ashraf M. Isolation and structural determination of sorbinols A and B, new triterpenes from Sorbus cashmariana, by 1D and 2D NMR spectroscopy. Magn Reson Chem. 2007;45(5):416-419.
  • Kazmi MH, Ahmed E, Hameed S, Malik A, Fatima I, Ashraf M. Cashmirols A and B, new lipoxygenase inhibiting triterpenes from Sorbus cashmiriana Chem Biodiversity . 2009;6(9):1471-1476.
  • Kazmi MH, Fatima I, Malik A, Iqbal L, Latif M, Afza N. Sorbicins A and B, new urease and serine protease inhibitory triterpenes from Sorbus cashmiriana J Asian Nat Prod Res. 2011;13(12):1081-1086.
  • Khan S, Fatima I, Kazmi MH, Malik A, Afza N, Iqbal L, et al. Cashmins A and B, potent antioxidant coumarins from Sorbus cashmiriana Chem Nat Comp. 2015;51(4):626-629.
  • Krachmal AC. A Guide to the Medicinal Plants of the United States, Vol. IX. Quadrangle New York Times Book Co., 1980. 207 p.
  • Krishna A. The Wealth of India, Vol. IX. CSIR: New Delhi, 1972. 435 p.
  • Mobley HLT, Hausinger RP. Microbial ureases: Significance, regulation and molecular characterisation. Microbiol Rev. 1989;53(1):85-108.
  • Mobley HLT, Island MD, Hausinger RP. Molecular-biology of microbial ureases. Microbiol Rev . 1995;59(3):451-480.
  • Nissler L, Gebhardt R, Berger S. Flavonoid binding to a multi-drug-resistance transporter protein: an STD-NMR study. Anal Bioanal Chem. 2004;379(7-8):1045-1049.
  • Omar S, Lemonnier B, Jones N, Ficker C, Smith ML, Neema C, et al. Antimicrobial activity of extracts of eastern North American hardwood trees and relation to traditional medicine. J Ethnopharmacol . 2000;73(1-2):161-170.
  • Perry LM, Metzger J. Medicinal Plants of East and South Asia, The MIT Press: Cambridge England, 1980. 341 p.
  • Seiji Y, Ryozo M, Shinobu Y, Yoshiyuki K. The synthesis of some dimethylpyranocoumarins and isopropenyldihydrofuranocoumarins. Bull Chem Soc Jpn. 1989;62(11):3593-3597.
  • Weatherburn MW. Phenol-hypochlorite reaction for determination of ammonia. Anal Chem. 1967;39(8):971-974.
  • White TC, Marr KA, Bowden RA. Clinical, cellular, and molecular factors that contribute to antifungal drug resistance. Clin Microbiol Rev . 1998;11(2):382-402.

Publication Dates

  • Publication in this collection
    16 Jan 2023
  • Date of issue
    2022

History

  • Received
    07 June 2021
  • Accepted
    20 Nov 2021
Universidade de São Paulo, Faculdade de Ciências Farmacêuticas Av. Prof. Lineu Prestes, n. 580, 05508-000 S. Paulo/SP Brasil, Tel.: (55 11) 3091-3824 - São Paulo - SP - Brazil
E-mail: bjps@usp.br