ISOLATION AND CHARACTERIZATION OF PHYTOSTEROLS FROM DIEFFENBACHIA AMOENA LEAF EXTRACT

ISOLATION AND CHARACTERIZATION OF PHYTOSTEROLS FROM DIEFFENBACHIA AMOENA LEAF EXTRACT

Mohd. Rehan ¹, Shafiullah ^{* 1} and Ompal Singh ²

Department of Chemistry ¹, Chemical Research Unit ², Aligarh Muslim University, Aligarh - 202002, Uttar Pradesh, India.

ABSTRACT: Dieffenbachia amoena is a house plant and known dumb cane. The study was performed based on isolation and structure elucidation of phytosterols from the extract of this plant. The CH₃OH crude extract was loaded over the silica gel (60-120 mesh) column, using the stepwise gradient C₆H₆, CH₃OAc, CH₃OH. The fractions were further purified by preparative TLC (GF254) to yield compound (1). Compound (1) was characterized by using various standard spectroscopic techniques such as IR, 1HNMR, 13CNMR, DEPT-135, COSY, HSQC and HMBC. Based on the spectral analysis, it was confirmed that the compound (1) was a mixture of β-sitosterol and stigmasterol. Phytosterols have high medicinal importance and play a vital role in reducing blood cholesterol, a high level of blood cholesterol can cause a risk of cardiovascular disease. Cardiovascular disease is the main problem of the whole world and increasing day by day. We have isolated β-sitosterol and stigmasterol first time from the leaves of the Dieffenbachia amoena plant.

Dieffenbachia amoena, β-sitosterol, Stigmasterol, HMBC, COSY

INTRODUCTION: Dieffenbachia amoena, commonly known as Besar Putih or Dumb Cane, belongs to family Araceae Fig. 1. Dieffenbachia is distributed in tropical America and grows in shady, moist, low land of tropical America, Brazil, and north to the islands of the West Indies ¹. It has two types of calcium oxalate crystals (druses and raphides) ². Chemicals investigation shows that it has a proteolytic enzyme which possesses poisonous properties ³. When the leaves extract of the plant comes in contact with the skin, it causes itching, swelling, salivation and Potential of speech loses for near about two days ⁴.

Phytosterol is bioactive compounds which are found in cell membranes of all plants ⁵ and have been isolated from various species of many plants such as Odontonema strictum, Rubus suavissimus, Ageratum conyzoides and show high medicinal activities and are an essential component of plant cell biofilm ^{6, 7, 8}.

They are mostly similar in structure and biological function to cholesterol ⁹. Stigmasterol (stigma) and β-sitosterol (β-sito) are common phytosterols Fig. 2, which are primarily used in the human diet and are useful in the treatment of NAFLD (Non-Alcoholic Fatty Liver Disease) ¹⁰. They play a vital role in the regulation of biological processes such as plant growth, modulation of the activity of membrane-bound enzymes, metabolic cycles ^{11, 12, 13}. Animals, including humans, cannot synthesize phytosterols, therefore, they can be assimilated from food. ^{14, 15} Both phytosterols play an essential role in lowering blood cholesterol level and beneficially influence the cardiovascular and immune system in humans and also shows anticancer activity ^{16, 17}. In the United States, cardiovascular disease is the most common cause of death and over 15 million deaths worldwide by the American Heart Association report in 2017 ¹⁸. Both phytosterol were exhibited various biological activities such as anti-depressant ¹⁹, apoptosis ^{20, 21}, uterus ²², anti-cancer ^{23, 24}, anti-Alzheimer’s ²⁵, anti-fungal infection ²⁶, immunomodulatory activity ²⁷, inhibitory action on glucoamylase in-vitro ²⁸, anti-microbial activity ²⁹, ³⁰, anti-tumour ³¹, anti-diabetic ³², anti-bacterial 33, anti-allergic ³⁴, and AChE inhibitory activity ³⁵.

In this study, we describe the isolation and characterization of the two significant phytosterols, namely, β-sitosterol and stigmasterol, based on 1H-NMR, 13C-NMR, DEPT-135, COSY, HSQC, and HMBC.

FIG. 1: THE SPECIES DIEFFENBACHIA AMOENA

FIG. 2: STRUCTURE OF COMPOUND (1): (A) β -SITOSTEROL (B) STIGMASTEROL

MATERIALS AND METHODS:

Plant Materials: The Dieffenbachia amoena leaves were purchased from a nursery, AMU area of district Aligarh, UP, India and authenticated by Professor M. Badruzzaman Siddiqui (Plant Taxonomy and Ethnobotany), Department of Botany, Aligarh Muslim University, Aligarh, India.

General Experimental Procedures: Measurement of the melting point was determined in glass capillaries on Stuart digital melting point apparatus (SMP10), which are uncorrected. Thin-layer chromatography (TLC) was carried out on pre-coated glass plates with silica gel (GF254). Various spectroscopic methods were used in the characterization of the isolated compound (1). The infrared spectrum was recorded in KBr pellets on the Perkin Elmer instrument. 1HNMR, 13CNMR, DEPT, COSY, HSQC, HMBC spectra were determined on a Bruker Avance Neo 500 MHz instrument using CDCl₃ solvent, and the chemical shift was reported in ppm with respect to TMS.

Extraction and Isolation: Shade air-dried and pulverized plant leaves (1 Kg) were extracted with 82% methanol for 15 days at room temperature. The CH₃OH extracts were filtered separately and concentrated using rotary evaporators to yield a dark reddish-brown residue (75 g). The CH₃OH extract was fractionated by using benzene and EtOAc solvent to give the benzene extract (18g) and EtOAc  extract (22g).

The C₆H₆ extract was subjected by glass column chromatography on silica gel (60-120 mesh) using a petroleum-ether to benzene gradient stepwise (100:0/0:100 v/v), to give eight main fractions (P1-P8). Each fraction collected and monitored by TLC. Fraction P-6 was further chromatographed using a glass column packed with silica gel, eluting with petroleum/C₆H₆ (20/80 v/v) to yield five subfractions (P-6(a)-P-6(e). The subfractions P-6(d) was purified by preparative TLC using gradient petroleum/ethyl acetate (75/25 v/v), to give a compound (1) (55mg). The compound was visualized single spot when subjected to TLC using various solvent systems such as petroleum/ethyl acetate (90/10 v/v), petroleum/ethyl acetate (75/25 v/v), hexane/ethyl acetate (75/25 v/v), chloroform/ ethyl acetate (80/20 v/v) and it showed to be homogenous compound. The white amorphous solid (60 mg) with melting point 139 ºC was further characterized by IR, 1H-NMR, 13C-NMR, DEPT-135, and 2D-NMR (summarized in Table 1).

TABLE 1: NMR SPECTROSCOPIC DATA (1H 500 MHZ AND 13C 125 MHZ) OF THE ISOLATED COMPOUND (1) RECORDED IN CDCl₃.a

The data were analyzed by DEPT-135, COSY, HSQC, and HMBC

Compound (1): white solid isolated from petroleum/benzene(20/80) fraction, Melting point 139 ºC, IR ῡ_max (KBr) cm^-1: 3434 (OH), 2934 and 2867 (CH), 1640 (C=C), 1463 (CH2), 1378 (OH def), 1058. 1HNMR (CDCl3, 500 MHz): δH 5.35 (1H, bd-s, H-6), 5.14(1H, m, H-21), 5.0 (1H, m, H-20), 3.53 (1H, m, H-3), 0.92 (3H, d, J= 6.1, H-19), 0.83 (3H, t, J= 7.6, H-24), 0.81 (3H, d, J= 6.9, H-26), 0.77 (3H, d, J= 6.1, H-27), 0.80 (3H, s, H-28), 0.68 (3H, s, H-29). 13C NMR (CDCl₃ 125 MHz): δC 140.72 (C-5), 121.73 (C-6), 71.83, 76.6 (C-3), 56.76, 56.9 (C-14), 56.1, 56.04 (C-17), 50.12, 51.1 (C-9), 45.82 (C-22), 42.31 (C-4), 42.27 (C-13), 39.8, 39.7 (C-12), 37.24 (C-1), 36.5 (C-10), 36.18, 40.5 (C-18), 138.3, 33.94 (C-20), 31.9, 32.8 (C-8), 31.89 (C-7), 31.64 (C-2),  29.14 (C-25), 28.8, 28.3 (C-16), 26.05, 24.4 (C15), 129.3, 24.36 (C-21), 25.4, 23.06 (C-23), 21.08 (C-11), 20.2, 19.8 (C-26), 19.4, 19.0 (C-27), 19.0, 18.3 (C-19), 18.78, 15.4 (C-28), 12.2, 12.0 (C-24), 12.0, 11.9 (C-29). HSQC: C-1 (37.24, 1.83, 1.06; CH₂), C-2 (31.64, 1.95, 1.83; CH₂), C-3 (76.6, 71.83, 3.53; CH), C-4 (42.31, 2.28, 2.23; CH₂), C-6 (121.73, 5.35; CH), C-7 (31.89, 1.99, 1.45; CH2), C-8 (32.8, 31.91, 1.83; CH), C-9 (51.1, 50.12, 0.90; CH), C-11 (21.08, 1.50; CH₂), C-12 (39.77, 1.85, 1.13; CH₂), C-14 (56.8, 56.76, 0.9; CH), C-15 (26.05, 24.4, 1.15; CH₂), C-16 (28.8, 28.25, 1.83, 1.27; CH₂), C-17 (56.04, 1.02; CH), C-18 (40.5, 36.18, 1.20; CH), C-19 (19.03, 81.3, 0.92; CH₃), C-20 (138.3, 33.94, 5.0, 1.28, 1.02; CH₂), C-21 (129.3, 24.36, 5.14, 1.57, 1.06; CH₂), C-22 (45.82, 0.9; CH), C-23 (25.4, 23.06, 1.20, 1.0; CH₂), C-24 (12.2, 12.0, 0.83; CH₃), C-25 (29.14, 1.72; CH), C-26 (20.2, 19.82, 0.81; CH₃), C-27 (19.4, 2.77; CH₃), C-28 (18.78,15.4, 0.80; CH₃), C-29 (12.0, 11.86, 0.68; CH₃).

FIG. 3: 13C-NMR SPECTRA FOR COMPOUND (1)

FIG. 4: HMBC CORRELATIONS FOR COMPOUND (1). β-SITOSTEROL (1) AND STIGMASTEROL (2)

FIG. 5: COSY CORRELATIONS FOR COMPOUND (1). β-SITOSTEROL (A) AND STIGMASTEROL (B)

FIG. 6: CHARACTERISTIC 1H-NMR AND 13C-NMR PEAK ASSIGNMENT OF β-SITOSTEROL

RESULTS AND DISCUSSION: Compound (1) was isolated as a white amorphous solid with melting point 139 ºC. The ¹H-NMR and 13C-NMR spectra of compound 1 shows a broad signal at  δ_H 5.35 (1H) and 13C-NMR at  δ_C 121.7, indicating the presence of a double bond. The other signals were observed at δH 3.53 (1H) and δC 71.83 corresponding hydroxyl groups. A 13C-NMR spectrum of compound 1 is shown in Fig. 3.

In HMBC, the 2J and 3J correlations between δH 0.68 and δC 39.8 (C-12), 42.27 (C-13) and 56.8 (C-14) suggested the presence of a methyl group in position (C-29) and 3J correlations between δH 0.82 and δC 45.8 supporting its placement at C-24 Fig. 4. The correlations observed between δH 1.28 and 1.06 in the COSY spectrum suggested the presence of methylene group at C-20, C-21, and connectivity of methine proton δH 2.0 (1H) to alkene proton δH 5.14 (1H) indicating the location of alkene at C-20 Fig. 5. Thus, compound (1) is a mixture of β-sitosterol and stigmasterol. Spectra have shown that β-sitosterol has a maximum portion.

Isolation of β-sitosterol is very difficult because β-sitosterol and stigmasterol have same R_f value. The difference between two compounds is only at position C-20, and C-21, where β-sitosterol has a single bond and stigmasterol, has a double bond at this position. Furthermore, the literature reveals that it is very difficult to obtain β-sitosterol in pure form ^{36, 37}. Characteristic NMR peak assignment of β-sitosterol summarized in Fig. 6.

CONCLUSION: These compounds were the first time reported from the leaves of Dieffenbachia amoena. Both phytosterols were reduced risk of heart diseases. The various spectroscopic techniques (IR, 1HNMR, 13CNMR, DEPT-135, COSY, HSQC, HMBC) were confirmed that the isolated compound was mixtures of β-sitosterol and stigmasterol.

ACKNOWLEDGEMENT: M Rehan is thankful to UGC, New Delhi, India for the grant. The authors sincerely thank SAIF, PU, Chandigarh for providing NMR spectral facilities. The authors are also grateful to the Chairman, Department of Chemistry, AMU, Aligarh, for providing necessary research facilities to complete this work.

CONFLICT OF INTEREST: The authors declare no conflict of interest.

REFERENCES:

1. Barnes BA and Fox LE: Poisoning with dieffenbachia. Journal of the History of Medicine and Allied Sciences 1955; 10: 173-181.
2. Genua JM and Hillson CJ: The occurrence, type and location of calcium oxalate crystals in the leaves of fourteen species of Araceae. Annals of Botany 1985; 56: 351-61.
3. Walter WG and Khanna PN: Chemistry of the aroids. I. Dieffenbachia seguine, amoena, and picta. Economic Botany 1972; 26: 364-72.
4. Ligon, Richard, Guy and Thomas: A true and exact history of the Island of Barbados. London Publisher and Peter Parker Distributers, 1673.
5. Uddin MS, Ferdosh S, Akanda JH, Ghafoor K, Rukshana AH, Ali ME, Kamaruzzaman BY, Fauzi MB, Hadijah S, Shaarani S and Sarker MZI: Techniques for the extraction of phytosterols and their benefits in human health a review. Separation Science and Technology 2018; 53: 2206-23.
6. Pierre LL and Moses MN: Isolation and characterisation of stigmasterol and β -sitosterol from Odontonema Strictum (Acanthaceae) 2015; 2(1): 88-95.
7. Chaturvedula VSP and Prakash I: Isolation of stigmasterol and β-sitosterol from the dichloromethane extract of rubus suavissimus. International Current Pharmaceutical Journal 2012; 1(9): 239-42.
8. Martínezcámara S, Bahíllo E, Barredo JL and Rodríguezsáiz M: Scale-up of phytosterols bioconversion into androstenedione. Methods Mol Biol 2017; 1645: 199-10.
9. Shahzad N, Khan W, Md S, Ali A, Saluja SS, Sharma S, Al-Allaf FA, Abduljaleel Z, Ibrahim IAA, Abdel-Wahab AF, Afify MA and Al-Ghamdi SS: Phytosterols as a natural anticancer agent current status and future perspective. Biomed Pharmacother 2017; 88: 786-94.
10. Feng S, Gan L, Yang CS, Liu AB, Lu W, Shao P, Dai Z, Sun P,and Luo Z: Effects of stigmasterol and β‑sitosterol on non-alcoholic fatty liver disease in a mouse model a lipidomic analysis. J Agric Food Chem 2018; 66: 3417-25.
11. Hartmann MA: Plant sterols and the membrane environment. Trends in Plant Science 1998; 3: 170-75.
12. Dufourc EJ: Sterols and membrane dynamics. J Chem Biol 2008; 1: 63-77.
13. Piironen V, Lindsay DG, Miettinen TA, Toivo J and Lampi AM: Plant sterols: biosynthesis, biological function and their importance to human nutrition. Journal of the Science of Food and Agriculture 2000; 80: 939-66.
14. Jong D, Plat J, Ronald P and Mensink: Metabolic effects of plant sterols and stanols. Journal of Nutritional Biochemistry 2003; 14: 362-69.
15. Kritchevsky D and Chen SC: Phytosterols health benefits and potensial concern. Nutr Res 2005; 25: 413-28.
16. Moreau RA, Whitaker BD and Hicks KB: Phytosterols, phytostanols and their conjugates in foods structural diversity, quantitative analysis, and health promoting uses. Prog Lipid Res 2002; 41: 457-500.
17. Berger A, Jones JH and Abumweis SS: Plant sterols factors affecting their efficacy and safety as functional food ingredients. Lipids in Health and Disease 2004; 3: 5.
18. Benjamin EJ, Blaha MJ, Chiuve SE, Das SR and Deo R: Heart disease and stroke statistics 2017 update a report from the American Heart Association. Circulation 2017; 135(10): e146–e603.
19. Yin Y, Liu X, Liu J, Cai E, Zhao Y, Li H, Zhang L, Li P and Gao Y: The effect of beta-sitosterol and its derivatives on depression by the modification of 5-HT, DA and GABA-ergic systems in mice. RSC Adv 2018; 8: 671-80.
20. Rajavel T, Packiyaraj P, Suryanarayanan V, Singh SK, Ruckmani K and Devi KP: β-Sitosterol targets Trx/Trx1 reductase to induce apoptosis in A549 cells via ROS mediated mitochondrial dysregulation and p53 activation. Scientific Reports 2018; 8: 2071.
21. Rajavel T, kumar RM, Archunan G, Ruckmani K and Devi KP: Beta sitosterol and Daucosterol (phytosterols identified in Grewia tiliaefolia) perturbs cell cycle and induces apoptotic cell death in A549 cells. Scientific Reports 2017; 7: 3418.
22. Occhiuto C, Trombetta D, Smeriglio A, Sturlese E and Occhiuto F: Effects of beta-sitosterol on isolated human non-pregnant uterus in comparison to prostaglandin E2. Pharmacognosy Magazine 2018; 14: 118-22.
23. Novotny L, Abdel-Hamid ME and Hunakova L: Anticancer potential of β-sitosterol. Int J Clin Pharmacol Pharmacother 2017; 2: 129.
24. Keawsa-ard S, Liawruangrath B and Kongtaweelert S: Bioactive compounds from mesua ferrea Stems. Chiang Mai Journal of Science 2015; 42(1): 185-95.
25. Ayaz M, Junaid M, Ullah F, Subhan F, Sadiq A, Ali G, Ovais M, Shahid M, Ahmad A, Wadood A, El-Shazly M, Ahmad N and Ahmad S: Anti-alzheimer’s studies on β-sitosterol isolated from polygonum hydropiper L. Front Pharmacol 2017; 8: 697.
26. Yinusa I, George NI, Ayo RG and Rufai Y: Evaluation of the pharmacological activities of beta sitosterol Isolated from the bark of Sarcocephalus latifolius (smith bruce). Global Journal of Pure and Applied Chemistry and Research 2015; 3(3): 7-14.
27. Parihar G and Balekar N: Isolation and characterisation of stigmast-5-en-3-ol (β-sitosterol) from Calotropis procera latex ethyl acetate fraction for immunomodulatory activity. IJPSR 2017; 8(3): 1375-80.
28. Meshram GA, Metangale GS, Khamkar S and Kulkarni T: Isolation of sterols from Ocimum sanctum leaves and it’s inhibitory action on glucoamylase in-vitro. European Journal of Pharmaceutical and Medicinal Research 2016; 3(3): 341-45.
29. Yusuf AJ, Abdullahi MI, Aleku GA, Ibrahim IAA, Alebiosu CO, Yahaya M, Adamu HW, Sanusi A, Mailafiya MM and Abubakar H: Anti-microbial activity of stigmasterol from the stem bark of Neocarya macrophylla. Journal of Medicinal Plants for Economic Development 2018; 2(1): a38.
30. Houngbèmè AG, Ganfon HMY, Medegan S, Yèhouénou B, Bambola B, Gandonou C and Gbaguidi FA: Anti-microbial activity of compounds from Acanthospermum hispidum DC and Caesalpinia bonduc (L.) ROXB: Beninese plants used by healers against HIV-associated microbial infections. Journal of Applied Pharmaceutical Science 2015; 5(08): 073-81.
31. Kangsamaksin T, Chaithongyot S, Wootthichairangsan C, Hanchaina R, Tangshewinsirikul C and Svasti J: Lupeol and stigmasterol suppress tumour angiogenesis and inhibit cholangiocarcinoma growth in mice via downregulation of tumor necrosis factor-α. PLoS One 2017; 12(12): e0189628.
32. Wang J, Huang M, Yang J, Ma X, Zheng S, Deng S, Huang Y, Yang X and Zhao P: Anti-diabetic activity of stigmasterol from soybean oil by targeting the GLUT4 glucose transporter. Food and Nutrition Research 2017; 61(1): 1364117.
33. Edilu A, Adane L and Woyessa D: In-vitro antibacterial activities of compounds isolated from roots of Caylusea abyssinica. Annals of Clinical Microbiology and Anti-microbials 2015; 14: 15.
34. Antwi AO, Obiri DD, Osafo N, Essel LB, Forkuo AD and Atobiga C: Stigmasterol alleviates cutaneous allergic responses in rodents. Bio Med Research International 2018; 2018: 3984068.
35. Gade S, Rajamanikyam M, Vadlapud V, Nukala KM, Aluvala R, Giddigari C, Karanam NJ, Barua NC, Pandey R, Upadhyayula VSV, Sripadi P, Amanchy R and Upadhyayula SM: Acetylcholinesterase inhibitory activity of stigmasterol and hexacosanol isresponsible for larvicidal and repellent properties of Chromolaena odorata. Biochimica et Biophysica Acta 2017; 1861: 541-50.
36. Kamboj A, Saluja AK: Isolation of stigmasterol and β-sitosterol from petroleum ether extract of aerial parts of Ageratum conyzoides (Asteraceae). International Journal of Pharmacy and Pharmaceutical Sciences 2011; 3: 94-96.
37. Pateh UU, Haruna AK, Garba M, Iliya I, Sule IM, Abubakar MS and Ambi AA: Isolation of stigmasterol, β-sitosterol and 2-Hydroxyhexadecanoic acid methyl ester from the Rhizomes of Stylochiton lancifolius pyer and Kotchy (Araceae). Nigerian Journal of Pharmaceutical Sciences 2008; 7: 19 -25.
    

    ---

    How to cite this article:

    Rehan M, Shafiullah and Singh O: Isolation and characterization of phytosterols from dieffenbachia amoena leaf extract. Int J Pharm Sci & Res 2020; 11(6): 2875-81. doi: 10.13040/IJPSR.0975-8232.11(6).2875-81.

    ---

    All © 2013 are reserved by the International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.

    ---