J Nat Med (2011) 65:206211 DOI 10.

1007/s11418-010-0459-9

NOTE

A bioactive sesquiterpene from Bixa orellana

Dennis D. Raga Rafael A. Espiritu Chien-Chang Shen Consolacion Y. Ragasa

Received: 9 February 2010 / Accepted: 23 July 2010 / Published online: 30 September 2010 The Japanese Society of Pharmacognosy and Springer 2010

Abstract A dichloromethane extract of the air-dried leaves of Bixa orellana afforded ishwarane 1, phytol 2, polyprenol 3, and a mixture of stigmasterol 4a and sitosterol 4b by silica gel chromatography. The structure of 1 was elucidated by extensive 1D and 2D NMR spectroscopy. Compound 1 at three doses (25, 50, and 100 mg/kg BW) was tested for prophylactic, gastrointestinal motility, analgesic, hypoglycemic, and antimicrobial potentials. Results of the prophylactic assay demonstrated the antitoxic property of 1 at 100 mg/kg BW. A 50 mg/kg BW dose of 1 resulted in a more propulsive movement of the gastrointestinal tract (88.38 13.59%) compared to the negative control (78.47 10.61%). Tail ick and acetic acid writhing tests indicated that 100 mg/kg BW 1 had minimal analgesic activity. Compound 1 demonstrated no hypoglycemic potential on the animals tested. Compound 1 exhibited moderate antifungal activity against C. albicans, low activity against T. mentagrophytes, and low antibacterial activity against E. coli, S. aureus, and P. aeruginosa. It was inactive against B. subtilis and A. niger. Keywords Bixa orellana Bixaceae Ishwarane Phytol Polyprenol Prophylactic
D. D. Raga Biology Department and Center for Natural Sciences and Ecological Research, De La Salle University, Manila, Philippines R. A. Espiritu C. Y. Ragasa (&) Chemistry Department and Center for Natural Sciences and Ecological Research, De La Salle University, 2401 Taft Avenue, 1004 Manila, Philippines e-mail: consolacion.ragasa@dlsu.edu.ph C.-C. Shen National Research Institute of Chinese Medicine, Taipei, Taiwan

Introduction Bixa orellana seeds, commonly known as achuete, are used for food coloring in the Philippines. The leaves are diuretic, antipyretic, purgative in dysentery; are used in jaundice, headache, and snake bites; and serve as a remedy for gonorrhea and sore throat [1]. A recent study reported that B. orellana lowered the blood glucose levels of C-peptide in dogs with streptozotocin-induced diabetes [2]. Another study reported analgesic and hypoglycemic activities of Bixa orellana, Kyllinga monocephala, and Luffa acutangula [3]. The leaves and seeds of B. orellana also showed a broad spectrum of antimicrobial activity, with the leaves showing higher activity [4]. Twenty-ve components from the essential oil of B. orellana have been identied with the following major components: (Z,E)-farnesyl acetate (11.6%), occidentalol acetate (9.7%), spathulenol (9.6%), and ishwarane (9.1%) [5]. We report here the isolation and identication of ishwarane 1, phytol 2, polyprenol 3, stigmasterol 4a, and sitosterol 4b (Fig. 1) from the dichloromethane extract of the air-dried leaves of Bixa orellana. Results of the prophylactic, gastrointestinal motility, analgesic, and hypoglycemic tests on three doses of 1 (25, 50, and 100 mg/kg BW) and its antimicrobial activity are likewise reported.

Materials and methods General experimental procedures NMR spectra were recorded on a Varian VNMRS spectrometer in CDCl3 at 600 MHz for 1H NMR and 150 MHz for 13C NMR spectra. Column chromatography was performed with silica gel 60 (70230 mesh); TLC was

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15 14

207

H3 C
4 5

H3C

CH 2OH
12 2 1 11 9

13

C H3

]3[
3

]n

OH

Ishwarane 1: colorless oil; 1H-NMR (CDCl3) d: 0.79 (H1), 0.45 (H-2), 1.48, 1.66 (H2-3), 1.62 (H-5), 1.01, 1.31 (H2-6), 1.33, 1.46 (H2-7), 0.94, 1.51 (H2-8), 1.14, 1.45 (H210), 0.99, 2.07 (H2-12), 1.12 (3H, s, H-13), 0.77 (3H, s, H14), 0.72 (3H, d, J = 6.6 Hz, H-15); 13C-NMR (CDCl3) d: 22.99 (C-1), 19.83 (C-2), 34.64 (C-3), 35.65 (C-4), 38.65 (C-5), 30.84 (C-6), 23.95 (C-7), 33.59 (C-8), 43.92 (C-9), 35.84 (C-10), 22.46 (C-11), 39.25 (C-12), 20.31 (C-13), 16.73 (C-14), 16.56 (C-15). Bioassays Experimental animals

H
H

H
H HO H

HO

4a

4b

Fig. 1 The compounds from B. orellana: ishwarane 1, phytol 2, polyprenol 3, stigmasterol 4a, and sitosterol 4b

performed with plastic-backed plates coated with silica gel F254; plates were visualized by spraying with vanillin sulfuric acid and warming. Sample collection Fresh leaves of Bixa orellana L. were collected from General Trias, Cavite, in May 2009. The sample was authenticated at the Institute of Biology of the University of the Philippines-Diliman, and a voucher specimen, #148, is deposited at the Chemistry Department, De La Salle University-Manila. Isolation Air-dried leaves of Bixa orellana (1.102 kg) were ground in an osterizer and then soaked in dichloromethane for 3 days and ltered. The ltrate was concentrated in vacuo to afford a crude extract (66.3 g), which was chromatographed in increasing proportions of acetone in dichloromethane (DCM) at 10% increments. The DCM and 10% acetone in DCM fractions were combined and rechromatographed (59) with petroleum ether as eluent to afford 1 (290.4 mg). The more polar fractions were combined and rechromatographed in 2.5% ethyl acetate in petroleum ether, followed by 5% ethyl acetate in petroleum ether to afford 3 (12 mg). The 20% acetone in the DCM fraction was rechromatographed using 20% ethyl acetate in petroleum ether to afford 2 (15 mg). The 40% acetone in DCM fraction was rechromatographed with 5%, 7.5%, and 10% ethyl acetate in petroleum ether to afford a mixture of 4a:4b in a 3:1 ratio (22 mg).

A total of 100 male albino mice (Mus musculus L.) of an inbred ICR strain (7 weeks old) weighing 19.0 2.0 g were acclimatized for 7 days prior to conducting the bioassay. The animals were procured from the Bureau of Food and Drugs, Muntinlupa City, Philippines, and housed at the animal containment unit of DLSU-Manila under a 12:12 h light regime with free access to food pellets and water. A 16 h fasting period was carried out prior to each treatment procedure. Cervical dislocation was performed at the end of the animal treatment procedure. All procedures involving animal handling were in accordance with the Philippine Association of Laboratory Animal Science (PALAS) Code of Practice for Care and Use of Laboratory Animals and with Administrative Order 40 of the Bureau of Animal Industry relative to Republic Act No. 8485. Prophylactic assay Seven-week-old male ICR mice (22.67 2.24 g) were acclimatized for 7 days prior to the actual assay. Three doses of 1 (25, 50, and 100 mg/kg BW) and polysorbate 80 (P80) were administered orally to mice. After 1 h, the animals were orally given 12.5 mL/kg BW DMSO. Behavioral indicators of toxicity were noted and recorded in video for 5 h after administration of DMSO. GI tract motility assay Gastrointestinal tract motility was tested on 7-week-old male ICR mice (21.49 2.60 g) acclimatized for 7 days prior to the day of the actual assay. Three doses of 1 (25, 50, and 100 mg/kg BW), polysorbate 80, and Buscopan in dH2O (0.3 mg/kg BW) were administered orally as treatment, negative, and positive controls, respectively. After 1 h, 2% charcoal suspension in dH2O (0.5 mL/20 g BW) was orally administered. After 30 min, the animals were killed by cervical dislocation followed by immediate dissection of the peritoneal cavity. The entire length of the gastrointestinal tract was stretched on a plastic board,

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and the total distance traveled by the charcoal was measured. Antinociceptive activity Tail ick assay Inhibition of thermal pain was tested according to the procedure of Grotto and Sulman [6]. Briey, the mice (n = 9) were orally administered with a dose of 1 (25, 50, and 100 mg/kg BW). Diclofenac sodium (7.14 mg/kg BW) and P80 were used as positive and negative controls, respectively. One hour after administration of the treatments, the bottom third of the tail was submerged in a 50C hot water bath. The time at which the mice attempted to remove their tails was noted and presented as percent inhibition = 100 - [(time at which the experimental mouse attempted to remove its tail/average time at which the control mice attempted to remove their tails) 9 100]. Acetic acid test Analgesia was tested in mice (n = 9) 1 h after oral administration of P80 and dichlofenac sodium (7.14 mg/kg BW) as the negative and positive controls, respectively, and 1 (25, 50, and 100 mg/kg BW) as the test drug. An intraperitoneal injection of 1% acetic acid (Mallinckrodt Chemicals, Pittsburg, NJ) followed 1 h after administration of the treatments [7, 8]. The number of abdominal stretches completed within 10 min after injection of 1% glacial acetic acid was counted and presented as the percent maximum analgesic effect. Anti-diabetes assay Oral glucose tolerance test (5 g/kg BW) was performed on normoglycemic mice (n = 9), followed by measurement of blood glucose levels (mg/dL) using OneTouch Horizon Glucometer (Lifescan, Johnson & Johnson, USA). Polysorbate 80 (25 mg/kg BW, AJAX, Finechem, Australia) and glimepiride (Solosa; 16.7 lg/kg BW; Aventis, Italy) dissolved in distilled H2O were orally administered as the negative and positive controls, respectively. Compound 1 dissolved in P80 was given as the test drug at doses of 25, 50, and 100 mg/kg BW. Blood glucose was measured over a 3 h period at 30 min intervals. Blood glucose reduction was computed and was used in the statistical analysis. Antimicrobial test The microorganisms used were obtained from the University of the Philippines Culture Collection (UPCC). They were Pseudomonas aeruginosa (UPCC 1244), Bacillus subtilis (UPCC 1149), Escherichia coli (UPCC 1195), Staphylococcus aureus (UPCC 1143), Candida albicans (UPCC 2168), Trichophyton mentagrophytes (UPCC

4193), and Aspergillus niger (UPCC 3701). The test compound was dissolved in 95% ethanol. The antimicrobial assay reported in the literature was employed [9]. The activity index was computed by subtracting the diameter of the well from the diameter of the clearing zone and dividing by the diameter of the well. Statistical analysis The results were analyzed using SPSS ver. 13 for Windows. One-way analysis of variance was performed to determine the signicant effects of the analgesic, gastrointestinal motility and anti-diabetes potentials of the B. orellana non-polar extracts. The results were considered signicant at P B 0.05. Signicant differences between group variables were determined by post hoc analysis at 95% DMRT. Values are presented as mean SD.

Results and discussion The dichloromethane extract of the air-dried leaves of Bixa orellana afforded ishwarane 1, phytol 2, polyprenol 3, and a mixture of stigmasterol 4a and sitosterol 4b by silica gel chromatography. The structures of 24b were identied by comparison of their 1H and 13C NMR data with those found in the literature for phytol [10], polyprenol [11], and stigmasterol and sitosterol [12]. The structure of 1 was elucidated by extensive 1D and 2D NMR spectroscopy as follows. The 1H NMR spectrum of 1 indicated resonances for a methyl doublet at d 0.72 (J = 6.6 Hz) and two methyl singlets at d 0.77 and 1.12 (see the section Isolation). The rest of the resonances in the shielded region of the spectrum account for the methylene and methine protons in 1. The 13C NMR spectrum of 1 gave resonances for 15 carbons from d 16.56 to d 43.92, indicating a sesquiterpene hydrocarbon. Two isolated spin systems (Fig. 2) were deduced from the COSY spectrum as follows. The methylene protons at d 1.48 and 1.66 (H2-3) were coupled to the methine proton at d 0.45 (H-2), in turn coupled to the methine proton at d 0.79 (H-1), which was nally coupled to the methylene protons at d 0.99 and 2.07 (H2-12). The second isolated spin system showed coupling between the methine proton at d 1.62 (H-5) and the methylene protons at d 1.01 and 1.31 (H2-6), which were in turn coupled to another set of methylene protons at d 1.33 and 1.46 (H2-7), which were nally coupled to the methylene protons at d 0.94 and 1.51 (H2-8). The 1H and 13C connectivities in 1 were veried by HSQC. The structure of 1 was elucidated by analysis of the HMBC 2D NMR data with key HMBC correlations shown in Fig. 2. Thus, the methyl singlet at d 1.12 was attached to

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15 14

H3C
4
5

essential respects, conrming the hypothesis. The relative stereochemistry of 1 is similar to 3-ishwarone [14].
: C OS Y : HMB C

H3C

Prophylactic property A 5 h observation period was performed on mice administered with different oral doses of 1, following oral administration of 50% DMSO. Behavioral observation was performed within the 5 h window period, and notes were made every 30 min until no more behavioral manifestations were observed. During the rst 30 min after DMSO treatment, all animals experienced reduced activity and inquisitiveness, which persisted for up to 3 h post treatment except for those mice administered with 100 mg/kg BW 1, which exhibited signs of early recovery at 2.5 h post treatment. Circling was observed in the negative control (25%) and 25 mg/kg BW 1 treatments (12.5%). Loss of hind limb strength, which resulted in dragging, was mostly dominant in the 50 mg/kg BW 1 (25%) and 25 mg/kg BW 1 (12.5%) treatment groups. One hour (1 h) after DMSO treatment, mice in the negative control group demonstrated excessive toxic manifestations such as jumping (25%) and loss of hind limb strength (12.5%). Mice in the 50 mg/kg BW 1 treatment exhibited increased hind limb dragging (37.5%), whereas the 100 mg/kg BW 1 treatment had the fewest animals (12.5%) demonstrating such behavior. At 1.5 h post treatment, hind limb dragging was persistent in negative controls (25%), and in the 50 and 100 mg/kg BW 1 treatments (25%). At 2 h post treatment, dragging was only observed in the negative controls (12.5%) and in the 50 mg/kg BW 1 treatment (25%). Circling was noticeable in the negative control (12.5%) and persisted for the next 30 min. At 2.5 h post treatment, dragging was quite dominant in the 25 mg/kg BW 1 (12.5%) and the 50 mg/kg BW 1 (37.5%) treatments. The mice given 100 mg/kg BW 1 no longer demonstrated signs of dragging or circling, and early recovery was notable, characterized by normal inquisitiveness and curiosity. Dragging could still be observed in mice administered with the negative control at 3 h post treatment, with an increased frequency of 37.5%, persisting for the next 1.5 h. Dragging was observed in the mice given 25 and 50 mg/kg BW 1 for only the next 30 min. Animals administered with 100 mg/kg BW 1 exhibited the earliest signs of recovery. No animals administered 1 revealed signs of intoxication at 4 h post treatment other than an isolated case of hind limb dragging (12.5%) at 4.5 h post treatment. The mice given 1 fully recovered at a much earlier time compared to the negative controls, which demonstrated full recovery only at 5 h post treatment. The results of the prophylactic assay clearly demonstrated the anti-toxic property of 1, particularly at 100 mg/kg BW, towards inhibiting toxic interactions in the test animals metabolic activities.

12

9 2

11 13

CH3

Fig. 2

H-1H COSY and key 1H-13C long-range correlations of 1

14

H3C

15

H3C

H
1

2 13 CH3

Fig. 3 NOESY correlations of 1

C-11 due to long-range correlations between these protons and C-1, C-2, C-10, and C-11. The second methyl singlet at d 0.77 was attached to C-4 on the basis of long-range correlations between these protons and C-3, C-4, C-5, and C-9. The methyl doublet at d 0.72 was attached to C-5 since long-range correlations were observed between these protons and C-4, C-5, and C-6. All long-range correlations are consistent with the structure of 1. The relative stereochemistry of 1 was deduced by NOESY (Fig. 3). The cyclopropyl proton at d 0.45 (H-2) was close in space to another cyclopropyl proton at d 0.79 (H-1) and the methyl singlet at d 1.12 (H3-13). This suggested that H-1, H-2, and H3-13 were on the same face of the molecule. Another correlation was observed between the methyl singlet at d 0.77 (H3-14) and the methyl doublet at d 0.72 (H3-15), indicating that they were close to each other in space. This was only possible if the methyl at d 0.77 was at the axial position and the methyl at d 0.72 was in the equatorial position. Literature search revealed that 1 is ishwarane. The 13 C NMR data of 1 and ishwarane [13] match in all

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210 Table 1 Percent motility in mice administered with 1 Treatment P80 Buscopan 25 mg/kg BW 1 50 mg/Kg BW 1 100 mg/Kg BW 1 Motility assay (%) 78.47 10.61 77.66 13.70 81.94 12.24 88.38 13.59 81.42 11.24

J Nat Med (2011) 65:206211 Table 2 Percent maximum analgesic effect of 1 on mice Treatment P80 Diclofenac 25 mg/kg BW 1 50 mg/kg BW 1 100 mg/kg BW 1 Tail ick (%) 22.70 26.01 42.53 37.75 35.54 8.51 36.75 11.62 47.93 15.16 Writhing assay (%) 34.58 17.95 56.84 32.60 50.84 50.90 36.27 26.20 60 41.22

GI tract motility Gastrointestinal motility was evaluated on mice orally administered with different doses of 1 (Table 1). Although the differences in the percent distance travelled by the charcoal marker along the gastrointestinal tract did not attain statistical signicance (P [ 0.05), mean differences suggested that 50 mg/kg BW 1 resulted in a more propulsive movement of the gastrointestinal tract (88.38 13.59%) compared to the negative control (78.47 10.61%). Despite insufcient statistical signicance, results suggest that 1 may possess a possible cholinergic activity. The below normal activity obtained with the positive control Buscopan may have been due to differences in marker administration and time of gastrointestinal tract evisceration. It is therefore possible that in future studies, time differences may show more signicance. Antinociceptive assay

Anti-diabetes assay Glucose-challenged mice revealed normal hepatopancreatic response, resulting in an immediate reduction of blood sugar level in all groups tested. Although statistical data indicated signicant differences between the controls and experimental groups, the percent reduction observed from 0.5 to 1.0 h cannot be attributed to the general effects of 1. The observed percent blood glucose reduction (Table 3) is primarily due to the normal action of insulin rush in a glucose-challenged animal. The positive control (glimepiride), however, was observed to have peaked at 1.0 h (31.86 11.09%) and persisted 22.5 h (3.69 11.83 to 2.88 15.63%, respectively), exhibiting a diminishing hypoglycemic activity. The observed data, however, are in keeping with the ndings in our previous report [8]. This further indicates that 1 demonstrated no hypoglycemic potential in the animals tested. Antimicrobial test

Analgesia was tested using two antinociceptive assays designed to determine the relative property of 1 towards peripheral inhibition of pain. Different doses of 1 were found to have analgesic potential in both the tail ick and acetic acid writhing assay. Although the results were not signicant, 100 mg/kg BW of 1 showed a percent inhibition (47.93 15.16%) relatively close to the positive control (42.53 37.75%) in the tail ick assay, which may indicate minimal but potential analgesic activity of 1. The highest dose (100 mg/kg BW 1) showed potential activity (60 41.22%) in the acetic acid writhing assay compared to the positive control (56.84 32.60%), which further supports the results of the tail ick assay (Table 2). The ndings in the two assays generally indicate that 1 had no analgesic property, but we do not discount the possibility that the minimal analgesic potential may have been affected by several factors such as potentiating time and the manner of drug delivery, which were not considered here. The compound may have had an immediate or longer potency not captured during the test window of 1 h. Also, the minimal activity could have been improved if 1 was administered directly into the blood vessel of the test animal via intravenous or intraperitoneal injection.

As part of our continuing search for antimicrobial compounds from Philippine medicinal plants, 1 was tested for possible antimicrobial activities by the agar well method. Results of the study (Table 4) indicated that 1 is moderately active against the fungus C. albicans with an activity index (AI) of 0.3, slightly active against the fungus T. mentagrophytes (AI = 0.3), and slightly active against the bacteria E. coli (AI = 0.1), P. aeruginosa (AI = 0.3), and S. aureus (AI = 0.1). It was inactive against B. subtilis and A. niger. Conclusions The dichloromethane extract of the air-dried leaves of Bixa orellana afforded ishwarane 1, phytol 2, polyprenol 3, and a mixture of stigmasterol 4a and sitosterol 4b by silica gel chromatography. Three doses (25, 50, and 100 mg/kg BW) of the major compound 1 were tested for prophylactic, gastrointestinal motility, analgesic, and hypoglycemic potential. It was also tested for antimicrobial potential. Compound 1 had very little activity in most of the bioassays performed. It is insufcient, however, to conclude that

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J Nat Med (2011) 65:206211 Table 3 Percent blood glucose reduction in mice orally administered with 1 over a 3 h observation period Group Control (P80) Glimepiride 25 mg/kg BW 1 50 mg/kg BW 1 100 mg/kg BW 1 0.5 h 59.08 8.7a 39.07 17.02 57.63 5.94a 62.85 9.47
a b

211

1.0 h 7.80 11.53b 31.86 11.09a 10.79 12.61b 11.86 19.24

b

1.5 h 5.99 11.85 10.49 12.41 3.97 8.63 6.11 14.23 2.0 21.04

2.0 h 7.24 14.45 3.69 11.83 9.18 9.38 10.36 9.95 9.06 15.40

2.5 h -7.89 23.59 2.88 15.63 5.89 11.90 -7.29 24.62 4.62 23.39

41.25 8.86b

19.21 8.99ab

Means followed by the same superscript letters are not signicantly different at a = 0.05 using Tukeys test

Table 4 Antimicrobial test results for 1 Organism E. coli P. aeruginosa S. aureus B. subtilis C. albicans T. mentagrophytes A. niger
a b c

Sample (30 lg) 1 Chloramphenicolb 1 Chloramphenicolb 1 Chloramphenicol 1 Chloramphenicolb 1 Canesten, 0.2gc 1 Canesten, 0.2g 1 Canesten, 0.2gc
c b

Clearing zone (mm)a 11 23 13 14 11 25 20 13 18 13 55 23

Activity index (AI) 0.1 2.8 0.3 1.3 0.1 3.2 2.3 0.3 0.8 0.3 4.5 0 1.3

Average of three trials Chloramphenicol disc, 6 mm diameter Contains 1% chlotrimazile

1 is really inactive for such properties. As demonstrated in our previous studies, the manner in which the compound was delivered should be taken into consideration. Furthermore, the early recovery and minimal manifestations of toxic behavior in mice administered with 1 clearly indicate its potential for prophylactic functions, most especially during treatment with important medicines such as cytotoxic drugs with high toxic readings. In vitro assays should be performed on both normal and immortal cells in both monolayer and mixed culture to determine the specicity and protective action of 1.

2. Russell KR, Omoruyi FO, Pascoe KO, Morrison EY (2008) Hypoglycemic activity of Bixa orellana in the dog. Method Find Exp Clin Pharmacol 30(4):301305 3. Quanico JP, Amor EC, Perez GG (2008) Analgesic and hypoglycemic activities of Bixa orellana, Kyllinga monocephala and Luffa acutangula. Philipp J Sci 137:6972 4. Fleisher TC, Ameade EPK, Mensah MLK, Sawer IK (2003) Antimicrobial activity of the leaves of Bixa orellana. Fitoterapia 74(1):136138 5. Pino JA, Correa MT (2003) Chemical composition of the essential oil from annatto (Bixa orellana L.) seeds. J Essent Oil Res 15(2):6667 6. Grotto M, Sulman F (1967) Modied receptacle method for animal analgesimetry. Arch Int Pharmacodyn Ther 165:152159 7. Hirate K, Uchida A, Ogawa Y, Arai T, Yoda K (2006) Zaltoprofen, a non-steroidal anti-inammatory drug inhibits bradykinin-induced pain responses without blocking bradykinin receptors. Neurosci Res 54:288294 8. Ragasa CY, Alimboyoguen AB, Urban S, Raga DD (2008) A bioactive diterpene from Smallanthus sonchifolius. Nat Prod Commun 3(10):16631666 9. Guevara BQ, Recio BV (1985) Phytochemical microbiological and pharmacological screening of medicinal plants. Acta Manilana Supplements. UST Research Center, Manila 10. Ragasa CY, Javier ESC, Tan IG (2003) Antimutagenic triterpenes from Vitex parviora. Philipp J Sci 132(1):2125 11. Rideout JA, Ragasa C, Ngo HT (2003) Unusual oxygenated cations in electrospray ionisation mass spectroscopy of polyprenols from Jatropa curcas L. ACGC Chem Res Commun 16:3439 12. Cayme JM, Ragasa CY (2004) Structure elucidation of b-stigmasterol and b-sitosterol from Sesbania grandiora [Linn.] Pers. and b-carotene from Heliotropium indicum Linn. by NMR spectroscopy. Kimika 20(12):512 13. Vilar R, Milo B, Tomi F, Casanova J, Ferro EA, Canigueral S (2001) Chemical composition of the essential oil from the leaves of Piper fulvescens, a plant traditionally used in Paraguay. J Ethnopharmacol 76:105107 14. Lago JHG, de Oliveira A, Guimaraes EF, Kato MJ (2007) 3-Ishwarone and 3-ishwarol, rare sesquiterpenes in essential oil from leaves of Peperomia oreophila Hensch. J Braz Chem Soc 18:16

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