Int J Pharm Pharm Sci, Vol 7, Issue 12, 25-29Original Article


ISOLATION AND CHARACTERIZATION OF STIGMASTEROL FROM CHLOROFORM FRACTION OF AERIAL PART OF ARGEMONE MEXICANA L

PRAVEEN S. NAYAK1*, D. M. KAR1, SHWETA P. NAYAK2

1School of Pharmaceutical Sciences, SOA University Bhubaneswar Odisha, 2GRY Institute of Pharmacy, Borawan Khargone M. P
Email: praveen_nayak2000@yahoo.com    

 Received: 30 Sep 2015 Revised and Accepted: 27 Oct 2015

ABSTRACT

Objective: Phytosterols are group of steroidal alcohol play important roles in structural component in the cell membrane and play a role in membrane stability. There are almost 22 different sterols are found yet and the major phytosterols include ß-sitosterol, campesterol and stigmasterol. The objective of this study was to isolate and characterize the bioactive principles from the chloroform fraction ofArgemone Mexicana L.

Methods: The isolation was done using column chromatography using gradient elution with different mobile phases. The isolated compound was subjected to spectral analysis. Structure elucidation was carried out on basis of spectral analysis.

Results: The chemical investigation of the chloroform fraction of aerial parts of belonging to the family Papaveraceae led to the isolation of stigmasterol. The isolated compounds were characterized using various spectroscopic data as well as chemical studies.

Conclusion: From the spectral characteristics, the isolated compound from the chloroform fraction of aerial parts was confirmed to be stigmasterol.This is the first ever report of these stigmasterol compound from the chloroform fraction of aerial parts of Argemone mexicana.

Keywords: Phytosterols, Argemone mexicana, Stigmasterol, Isolation, Papaveraceae.

© 2016 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

INTRODUCTION

Plants have shaped the basis of sophisticated traditional medicine systems that have been used for thousands of years in countries, such as China [1] and India [2]. The use of plants in the traditional remedy of many other cultures has been widely documented. These plant-based systems continue to play an significant role in health care and it has been projected by the World Health Organization that around 80% of the world’s inhabitants rely mainly on traditional medicines for their primary health care, although plant products also play an main role in the health care systems of the remaining 20% of the population mostly residing in developed countries [3].

Argemone mexicana L., known as Ghamoya (family: Papaveraceae) is an exotic weed indigenous in South America and has widespread distribution in many tropical and sub-tropical countries including West Africa [4]. This plant is common every whereby roadsides and fields in India also [5]. The plant is an erect prickly annual herb of about 1 m high; leaves are usually 5 to 11 cm long, and more or less blotched with green and white, glaucous broad at the base, half-clasping the stem prominently sinuate-lobed, and spiny [6]. The flowers become 4 to 5 cm in diameter, and are terminal, yellow, and scentless. The capsule is spiny, obovate or elliptic-oblong, and about 3 cm in length. The seeds are spherical, shining, black and pitted. A. mexicana is considered as an important medicinal plant in India; the yellow juice, which exudes when the plant is injured, has long been used in India as traditional medicine for dropsy, jaundice, opthalmia, scabies and cutaneous affections [6-8]. Different parts of this plant are used in chronic skin diseases, and also as emetic, expectorant, demulcent and diuretic; the seeds and seed oil are employed as a remedy for dysentery, ulcers, asthma and other intestinal affections [6, 7, 9-11]. Leaves and seeds are also reported to find application in maintaining normal blood circulation and cholesterol level in human body [12].

The plant Argemone mexicana is the source of a different kind of chemical constituents having mostly abundant al­kaloids as berberine [13], protopine, nor-san­guinarine [14], sanguinarine [15], Angeline [15], chelerythrine [16] etc. Other active constituents which are found are terpenoids {trans-phytol [16], β-amyrin [17]}, steroids {β-sitostero [18]}, long-chain alcohols {myristic acid, palmitic acid, stearic acid, arachidic acid, oleic acid and linoleic acid [19], argemonic acid [20]}, fatty acids from seed oil {9-and 11-oxo-octacos­anoic and 11-oxotriacontanoic acids}, amino acids {cysteine and phenylalanine}[20], flavonoids {luteolin, eriodyctiol [21], isorhamnetin-3-O-β-Dglucopyanoside [16-22], iso rhamnetin [22, 23] isorhamnetin-7-O-β-Ddiglucopyanoside [23], isorhamnetin-3,7-O-β-Ddiglucopyranoside, quercetin, rutin, mexitin [24, 25]}, aromatic acids {5,7-dihydroxy chromone-7-neohesperi­doside [26], tannic acid, caffeic acid, ferulic acid [27], vanillic acid [22]}, miscellaneous compounds {α-tocopherol, adenosine, adenine}[16].

In the present study, first time we have isolated and characterized stigmasterol, a phytosterol, from the chloroform fraction of aerial parts of Argemone mexicana L. Stigmasterol is reported to exhibit a spectrum of pharmacological activities against various disease conditions. These include conditions such as inflammation, arthritis, diabetes, cardiovascular ailments, renal disorder, hepatic toxicity, microbial infections and cancer.

MATERIALS AND METHODS

Collection and authentification of plant

The plant material used in this study was aerial parts of A. mexicana, collected from Kasrawad dist.Khargone M. P., India, during spring (mid-March to mid-April) and was authenticated by the Former Taxonomist Dr. S. K. Mahajan, department of Botany, Government P G CollegeKhargone M. P. The plant materials were initially rinsed withdistilled water and dried on paper towel in laboratory at (37±1) oC for 24 h.

Extraction and fractionation  

The coarse powder of A. mexicana was submerged in alcohol and water (50:50) and allowed to stand for continuous hot extraction. After extraction the solvents were allowed to evaporate using rotary evaporator at temperature 40-45 °C. Thus the highly concentrated crude hydroalcoholic extract were obtained. They were then fractionated using Petroleum ether, Chloroform and water. The chloroform fractions obtained from A. mexicana was then stored in a refrigerator at 4 °C for further use for phytochemical investigation.

Isolation of compounds from chloroform fraction of A. mexicana

The dried chloroform fraction of A. mexicana(20 gm) was mixed with 80 gm silica gel (60-120 mesh) to make the material to get adsorbed in the silica gel. The column was eluted with solvent increasingly initial from 100 % n-hexane, then increasing order of ethyl acetate in n-hexane (0, to 100% ethyl acetate in n-hexane) and total 108 fractions were collected. After evaporating the solvents on water bath all the collected fractions were subjected to TLC analysis. On the basis of Rf values, same fractions were pooled together. The pools which gave single spot in iodine exposure were 8-19, 28-35, and 74-86. On the basis of high quantity, 74-86 pool of fractions was taken for purification.

Purification of the isolated compound from A. mexicana

Fractions from column chromatography were subjected to preparative TLC as required to obtain pure compounds. Mixed fractions of 74-86, after evaporating solvent, was subjected for thin layer chromatographic study using various solvent systems. Among them the Toluene: Acetone (8:2) gave good resolution this solvent system was further selected for preparative TLC. The concentrated pool was dissolved in chloroform and the sample was spotted in the preparative TLC plates. The sample applied plates were kept in completely saturated chamber of selected mobile system. After development of chromatogram, the plates were put in iodine vapour and the band (Rf =0.64) was identified and scrapped out from the plates. The scrapped material was then dissolved in pet ether and filtered through whatman filter paper. The filtrate was concentrated and the isolated product was obtained as white solid crystals named as compound AM (64 mg).

Test for steroid

Salkowski reaction

A few crystals of compounds AM were dissolved in chloroform and a few drops of concentrated sulphuric acid were added to the solution, compounds AM formed a reddish color in the upper chloroform layer [28] indicating presence of steroids.

Liebermann-Burchard reaction

A few crystals of compounds AM were dissolved in chloroform and few drops of concentrated sulfuric acid were added to it followed by the addition of 2-3 drops of acetic anhydride. In this case compounds AM turned to violet blue and finally formed green color which indicates the presence of steroids [28].

Spectroscopic characterization

UV spectra of the isolated compounds were recorded in methanol over a scanning range of 200-400 nm and λmax of compounds were determined. Spectra were recorded with a Shimadzu 1700 double beam-UV-VIS spectrophotometer. EIMS (electron impact mass spectrum) in positive mode were recorded on Waters Micromass Q-Tof Micro mass spectrometer instrument at SAIF, Chandigarh. The isolate was mixed with 200 mg KBr (FT-IR grade) and pressed into a pellet. The sample pellet was placed into the sample holder and FT-IR spectra were recorded in the range 375-7500 cm-1 in FT-IR spectroscopy (Model RZX (Perkin Elmer) at SAIF, Chandigarh. 1H and 13C-NMR spectra were recorded on a FT-NMR Cryomagnet Spectrometer 400 MHz (Bruker) using TMS as an internal standard at SAIF, Chandigarh, India. The solvents used were methanol and DMSO. Chemical shifts ware shown in δ values (ppm) with TMS as an internal reference. For column chro­matography silica gel 60 (70-230 mesh, Merck, Darmstadt, Germany) was used. Solvents for chromatography were distilled before use. Thin layer chromatography (TLC) was performed using TLC plates (Silica Gel G-60).

RESULTS

The melting point of compound AM was 169 °C; the UV λmax value of compound AM was 257 nm. Mass spectrum of isolated compound AM showed parent molecular ion [M+] peak at mlz 412 which corresponds to the molecular formula C29H48O (fig. 1).

In the IR spectrum of isolated compound a very intensely broad peak at 3428 cm-1 and moderately intense peak at 1192 and 699 cm-1were observed for the O-H bond vibrations of hydroxyl group. In the 1H-NMR spectrum of isolated compound, H-3 proton appeared as a triplet of a double doublet (tdd) at δ3.20 and, H-6 olefinic proton showed a multiplet at δ5.24. Two olefenic protons appeared downfield at δ 4.57 m and δ 4.14 m. Six methyl protons also appeared at δ1.23, δ1.19, δ1.06, δ 1.00, δ0.98 and δ0.91 (3H each, s, CH3) (table 1).

The 13C-NMR has shown recognizable signals at 140.8 and 121 ppm, which corresponds to double bond at C-22 and C-6 double bonds respectively as well as it also represent signals at 130.1 and 129.1 ppm, which shows one more double bond in between C-5 and C-23. The δ value at 71.6 ppm is due to C-2 β-hydroxyl group. The signal at δ 31.7 and δ 12.8 ppm corresponds to angular carbon atom at C-25 and C-27 respectively (table 1). From the above observations, isolated compound was found to be stigmasterol.

Fig. 1: Structure of stigmasterol isolated from chloroform fraction of A. Mexicana.

Table 1: Spectroscopic data of isolated compound am from chloroform fraction of A. mexicana

Spectroscopic techniques

Data

UV λ max

257 nm

IR (ranges in cm-1) (CHCl3)

3428 (O-H stretching), 2937(C-H stretching), 2852(C-H stretching), 1642 (C=C stretching),

1465 (C-H bend.), 1460 (C-H bend.), 1192 (O-H bend.), 1053 (C-C str.), 739 (CH2 rocking), 699 (O-H bend.).

1HNMR (DMSO)

δ 5.24 (m, 1H, H-6), δ 4.57 (s, 1H), δ 4.14 (s, 1H), 3.20 (tdd,OH,H-3),

δ 1.23 (s, 3H), δ 1.19 (s, 3H), δ 1.06 (s, 3H), δ 0.98 (s, 3H), δ 0.91 (s, 3H).

13CNMR (DMSO)

δ 140.8 (C-22), δ 130.1 (C-5), δ 129.1 (C-23), δ 121(C-6), δ 71.6 (C-3), δ

56.1 (C-4), δ 55.1 (C-5), δ 52.2(C-24), δ 0.10 (C-17), δ 43.8 (C-9), δ

41.2 (C-13), δ 39.4 (C-10), δ 37.7 (C-10), δ 33.4 (C-20), δ 31.7 (C-25),

δ 29.1 (C-21), δ 28.1 (C-23), δ 25.1 (C-12), δ 21.8 (C-11, C-25, C-26), δ 15.1 (C-29), δ 12.8(C-27).

EIMS (70 ev): m/z with % abundance

412 [M+, C29H48O] 355(101), 311 (49), 301 (49), 279 (71), 219 (60), 200 (65), 175 (95)

DISCUSSION

The isolated phytochemical was found as a white amorphous solid compound with melting point of 169-170 °C. The UV λmax value was 257 nm. In IR spectrum of AM, a very intensely broad peak at 3428 cm-1 and moderately intense peak at 1192 and 699 cm-1were observed for the O-H bond vibrations of hydroxyl group. The out of plane C-H vibrations of the unsaturated part was observed at 881 cm-1. The corresponding C=C vibrations was shown around 1642 cm-1 as weakly intense peak. The stretching and bending vibrations of methyl part were noticed by the intense peak 2937 cm-1and medium intensity peak at 1465 cm-1. The vibration of the methylene part was shown by the peak at 2852 cm-1 and the medium peak at 1460 cm-1. The moderately intense peak at 739 cm-1was attributed to the rocking movement of methylene part. The corresponding C-C vibration was shown as weak intense peak at 1053 cm-1.

Fig. 2: IR spectra of the isolated lead compound

In lH-NMR spectrum of AM, H-3 proton appeared as a triplet of a double doublet (tdd) at δ3.20 (J = 4.5 and 1.1 MHZ) and H-6 olefinic proton showed a multiplet at S 5.24. Two olefenic protons appeared downfield at δ4.57 (m) and δ4.14 (m) which were identical with the chemical shift of H-22 and H-23, respectively of stigmasterol [29]. Six methyl protons also appeared at δ1.23, δ1.19, δ1.06, δ 1.00, δ0.98 and δ0.91 (3H each, s, CH3).

Fig. 3: 1H-NMR spectra of the isolated lead compound

Fig. 4: 13C-NMR spectra of the isolated lead compound

Fig. 5: Mass spectra of the isolated lead compound

The 13C-NMR has shown recognizable signals at 140.8 and 121. ppm, which corresponds to double bond at C-22 and C-6 double bonds respectively as well as it also represent signals at 130.1 and 129.1 ppm, which shows one more double bond in between C-5 and C-23. The δ value at 71.6 ppm is due to C-2 β-hydroxyl group. The signal at δ 31.7 and δ 12.8 ppm corresponds to angular carbon atom at C-25 and C-27 respectively. Mass spectrum of compound AM showed parent molecular ion [M+] peak at mlz 412 which corresponds to the molecular formula C29H48O. This assignment is overall in good agreement for the structure of Stigmasterol as described by [30-33].

CONCLUSION

The phytochemical examination of the chloroform fraction of the aerial parts of A. mexicana belonging to the family Papaveraceae was effectively carried out. From these physical, chemical and spectral evidences compound AM was confirmed as Stigmasterol (fig. 1). The stigmasterol isolated from this fraction must account for the biological activities exhibited by the chloroform fraction of the plant. Consequently, it is now turn of the pharmacologists/biologists to investigate the plant more thoroughly by carrying out individual bioactivity of the stigmasterol. So, the present work will enhance the scientific communities to do more work on this important medicinal plant in near future.

ACKNOWLEDGEMENT

The authors are grateful to SPS, SOA University, Bhubaneswar for providing necessary facilities to carry out the research work in the faculty of pharmacy, SOA University. Thanks are also to sophisticated analytical instrumentation facility (SAIF), Chandigarh for providing spectroscopic analytical data.

CONFLICT OF INTERESTS

Declared None

REFERENCES

  1. Chang HM, But PH. Pharmacology and applications of Chinese materia medica. Vol. 1-2. Singapore: World Scientific Publishing; 1986.
  2. Kapoor LD. CRC Handbook of ayurvedic medicinal plants. Boca Raton, Florida: CRC Press; 1990. p. 416.
  3. Farnsworth NR, Akerele O, Bingel AS, Soejarto DD, Guo Z. Medicinal plants in therapy. Bull W H O 1985;63:965-81.
  4. Ibrahim HA, Ibrahim H. Phytochemical screening and toxicity evaluation on the leaves of Argemone mexicana Linn. (Papaveraceae). Int J Appl Sci 2009;3:39-43.
  5. Bhalke RD, Gosavi SA. Anti-stress and antiallergic effect of Argemone mexicana stems in asthma. Arch Pharm Sci Res 2009;1:127–9.
  6. Chopra RN, Nayar SL, Chopra IC. Glossary of Indian medicinal plants. New Delhi: NISCOM, CSIR; 1956. p. 23.
  7. Ambasta SP. The useful plants of India New Delhi: PID, CSIR; 1986. p. 51.
  8. Sharma J, Gairola S, Gaur RD, Painuli RM. The treatment of jaundice with medicinal plants in indigenous communities of the Sub-Himalayan region of Uttarakhand, India. J Ethnopharmacol 2012;143:262-91.
  9. Bose BC, Vijayvargiya R, Saifi AQ, Sharma SK. Chemical and pharmacological studies on Argemone mexicana. J Pharm Sci 1963;52:1172-5.
  10. Prajapati ND, Purohit SS, Sharma AK, Kumar T. A handbook of medicinal plants. Jodhpur, India: Agrobios; 2003. p. 59-60.
  11. Savithramma N, Sulochana CH, Rao KN. Ethnobotanical survey of plants used to treat asthma in Andhra Pradesh, India. J Ethnopharmacol 2007;113:54-61.
  12. Albuquerque UP, Monteiro JM, Ramosa MA, Amorim ELC. Medicinal and magic plants from a public market in northeastern Brazil. J Ethnopharmacol 2007;110:76-91.
  13. Israilov IA, Yuhusov MS. Alkaloids of four species of Argemone. Chem Nat Compd 1986;22:189-92.
  14. Tripathi PN, Tripathi M, Pandey VB, Singh D. Alkaloids of Argemone mexicana. Orient J Chem 1999;15:185-6.
  15. Chang YC, Hsieh PW, Chang FR, Wu RR, Liaw CC, Lee KH, et al. Two new protopines argemexicaines A and B and the anti-HIV alkaloid 6-acetonyl dihydro chelerythrine from formazan Argemone mexicana. Planta Med 2003;69:148-52.
  16. Chang YC, Chang FR, Khalil AT, Hsieh PW, Wu YC. Cytotoxic benzophenanthridine and benzylisoquinoline alkaloids from Argemone mexicana. Zeitschrift für Naturforschung 2003;58:521-6.
  17. Sukumar D, Nambi RA, Sulochana N. Studies on the leaves of Agremone mexicana. Fitoterapia 1984;55:325-53.
  18. Usman H, Abdulrahman FI, Usman A. Qualitative phytochemical screening and in vitro antimicrobial effects of methanol stem bark extract of Ficus thonningii (moraceae). Afr J Tradit Complementary Altern Med 2009;6:289-95.
  19. Badami RC, Gunstone FD. Vegetable oils. Examination of component acids of Argemone mexicana seed oil by reversed-phase chromatography. J Sci Food Agric 1962;13:255-7.
  20. Rukmini C. New, unusual long chain fatty acid (argemonic acid) from Argemone mexicana. J Am Oil Chem Soc 1975;52:171-3.
  21. Harborne JB, Williams CA. Flavonoids in the seeds of Argemone mexicana:a reappraisal. Phytochemistry 1983;22:1520-1.
  22. Pathak NKR, Biswas M, Seth KK, Dwivedi SPD, Pandey VB. Chemical investigation of Argemone mexicana. Die Pharmazie 1985;40:202.
  23. Rahman W, Ilyas M. Flower Pigments. Flavonoids from Argemone mexicana L. (Papaveraceae). J Org Chem 1962;27:153-5.
  24. Singh S, Singh TD, Pandey VB. Constituents of argemone species. Indian Chem Soc 2011;88:275-6.
  25. Singh S, Pandey VB, Singh TD. Alkaloids and flavonoids of Argemone mexicana. Nat Prod Res 2012;26:16-21.
  26. Bhardwaj DK, Bisht MS, Jain RK, Munyal A. Phenolics from the seeds of Argemone mexicana. Phytochemistry 1982;21:2154-6.
  27. Singh S, Singh A, Jaiswal J, Singh TD, Singh VP, Pandey VB, et al. Antifungal activity of the mixture of quaternary alkaloids isolated from Argemone mexicana against some phytopathogenic fungi. Arch Phytopathol Pflanzenschutz 2010;43:769-74.
  28. Harborne JB. Phytochemical methods: a guide to modern techniques of plant analysis. 3rd Edn. Chapman and Hall: London; 1998. p. 302.
  29. Li C, Bu PB, Yue DK, Sun YF. Chemical constituents from roots of ficus hirta. China J Chin Mater Med 2006;31:131-3.
  30. Fleischer TC, Sarkodie JA, KOmlaga G, Kuffour G, Dickson A, Mensah MLK. Hypoglycemic and antioxidant activites of the stem bark of Morinda lucida benth in streptozocin induced diabetic rats. Pharmacogn Commun 2011;1:23-9.
  31. Muhit MA, Tareq SM, Apu AS, Basak D, Islam MS. Isolation and identification of compounds from the leaf extract of Dillenia indica linn. Bangladesh Pharm J 2010;13:49-53.
  32. Habib MR, Nikkon F, Rahman M, Haque ME, Karim MR. Isolation of stigmasterol and beta-sitosterol form methanolic extract of root of bark of Calotropis gigantean (Linn.). Pak J Biol Sci 2007;10:4174-6.
  33. Jain PS, Bari SB. Isolation of stigmasterol and gamma sitosterol form petroleum ether extract of woody stem of Abelmsochus manihot. Asian J Biol Sci 2009;2:112-7.