Isolation and Characterization of two New Flavonoids Derived from Leaves of Jatropha gossypifolia Linn.

 

Sachin K Jain²*_, Gajendra Pratap Choudhary², Dinesh Kumar Jain¹

¹College of Pharmacy, IPS Academy, Indore MP- 452012India

²School of Pharmacy, Devi Ahilya Vishwavidyalaya, Indore- 452012MP India

 

ABSTRACT:

Jatropha gossypifolia L. (Euphorbiaceae), widely known as “bellyache bush,” is a medicinal plant largely used throughout Africa and America for treatment of inflammatory analgesic antidiarrheal diseases. In spite of some published data on its efficiency, there are still few toxicological data unfolding the safety of this plant. The plant was extracted with many solvents (pet. ether, ethanol, and water). The preliminary phytochemical outcomes revealed that flavonoids and other glycosides as active constituents in ethanol extract of Jatropha gossypifolia. The ethanol extract of Jatropha gossypifolia was undertaken column chromatography with dissimilar solvent fractions. Two compounds were isolated from ethanol extract of Jatropha gossypifolia with the compound 1 was eluted with benzene: Chloroform 90:10 v/v and compound 2 were eluted with eluted with ethyl acetate: ethanol 80:20 v/v. The structures of the two isolated compounds were characterized by using FT-IR, NMR and Mass spectrophotometric methods. Thus, the compound 1 was characterized as 2-8 -4,5 –dihydroxy -6- (hydroxymethyl) – tetrahydro - 2H - pyran-2-yl) -2,5,6-trimethoxy -4-oxo-3,4 -dihydro-2H-chromen-7-yl) -6- (3,4-dihydroxyphenyl) -7- ethoxy -5-hydroxy-8-3-hydroxy-6-(hydroxymethyl) - 4,5 dimethoxy-tetrahydro-2H-pyran-2-yl)-4H-chromen-4-one(C₄₃H₅₀O₂₀) and compound 2 was characterized as 2 -(2, 4-diethoxy-5-hydroxyphenyl)-7-(5-ethoxy-4-oxo-2-phenylchroman-7-yloxy)-5-hydroxy-8-6-(hydroxymethyl)-5-methoxy-tetrahydro-2H-pyran-2-yl)-4H-chromen-4-one(C₄₃H₄₄O₁₃). Consequently, additional biological investigations essential to be carried out isolated compounds present in this plant.

 

 

INTRODUCTION:

Jatropha gossypifolia, generally known as bellyache bush, black physicnut or cotton-leaf physicnut, is a species of flowering plant in the spurge family, Euphorbiaceae ^[1]. The species is native to Mexico, South America, Gujarat State in India and the Caribbean islands. It is a professed noxious weed in Puerto Rico and is established in northern Australia, including Queensland where it is listed as a Class 2 declared pest plant ^{[1, 2]}.

 

It cultivates to 2.5–4 m (8.2–13.1 feet) high. The three lobed leaves are purple and gluey when young and develop bright green with age. The small red flowers with yellow middles appear in clusters ^[3-4]. These are shadowed by cherry-sized seed pods that are poisonous ^[2]. Powdery mildew fungal disease was recounted ^[3]. It is a small deciduous shrub with luscious stem, 1-1.5 m tall. Leaves palmately 3-5 lobed, purple; petiole clothed with many stipitate glands. Flowers small, red in terminal corymbose cymes ^[5-6]. Fruit a capsule, about 1.3 cm across. A decoction of the bark is used as an emmenagogue. The leaves are laxative; applied to boils, carbuncles, eczema and itches. Sap exudates taken from leaf petiole is assorted with syrup and given to cure dysentery. Seeds are severe purgative and emetic, but they are said to cause foolishness ^[7]. Seed oil is used in skin diseases and as an external restorative in rheumatism and paralytic affections. Consistent brushing with the twigs keeps the teeth and gum disease free and remedies tooth-ache. Leaves contain flavonoids, a saponin, a resin, tannin and triterpenes ^[8]. They likewise contain flavonoids, vitexin, isovitexin and apigenin. Seeds encompass fatty oil. Roots contain antileukemic and tumour-inhibitor macrocyclic diterpene, jatrophone and jatropholones A and B. Bark contain β-sitosterol^[9-13]. Roots, stems and seeds contain arylnaphthalene lignan and the lignan prasanthaline. Cyclogossine, a cyclic heptapeptide, had been isolated from the latex of the plant. Stem contains a novel lignan, jatrodien.

 

MATERIALS AND METHODS:

Plant Material:

The leaves of JG were collected from Indore, Madhya Pradesh. The verification was done by Prof. S. R. Upadhayaya, Division of Botany, Govt. PG College Indore. A specimen no. (COPIPS/T/098/2010) submitted in the department.

 

Preparation of extracts:

Extracts were prepared according to successive solvent extraction method extracts were collected dried under vacuum and stored in containers. The percentage yield of petroleum ether, ethanolic and aqueous extracts was found to be 2.8%, 48% and 6% W/W correspondingly.  Phytochemical investigation shows the presence of glycosides, alkaloids, tannins, phenolics, flavonoids and other phytoconstituents ^{[14, 15]}.

 

Isolation of ethanolic extract of Jatropha gossypifolia by Column Chromatography:

The 20 gms of ethanolic extract of Jatropha gossypifolia was mixed with 20 gms silica gel (60/120 meshes) to get even mixing. 200 gms of silica gel (70/325 meshes) was reserved in a appropriate column and packed very cautiously without air bubbles by hexane as filling solvent. The column was reserved aside for 1 hour and permissible for close packing. Admixture was then added at the topmost of the stationary phase and ongoing separation of compounds by the eluting with numerous solvent mixtures with cumulative order of polarity. All the column fractions were collected distinctly and concentrated under reduced pressure. Finally the column was eroded with ethanol.

 

TLC characterization of Ethanolic extract of Jatropha gossypifolia:

The principle of portioning is either partition or adsorption. The constituent which is having more attraction for mobile phase moves with it, while the constituent which is having more attraction for stationary phase gets adsorbed on it. This technique various compounds appear as a band on the TLC plate, having dissimilar R_f values. The ethanolic extract of Jatropha gossypifolia was exposed to thin layer chromatographic studies for the parting and identification of their components.

 

Preparation of plates:

100 g of silica gel G was considered and made into a similar suspension with 200 ml of distilled water to form slurry. The slurry was decanted into a TLC applicator, which was attuned to 0.25 mm thickness on flat glass plate of different dimensions (10x2, 10x5, 30x5, 20x10 cm etc.). The coated plates were allowable to dry in air, shadowed by heating at 100-105⁰C for 1 hour, cooled and threatened from moisture. Before using, the plates were triggered at 110⁰C for 10 minutes.

 

Separation of components:

The ethanolic extracts of Jatropha gossypifolia was dissolved in ethanol separately and spotted using a capillary tube on TLC plates 2 cm above from the bottom of the plate. The selection of solvent systems was based on increasing the order of polarity. The different spots developed in each system were detected by means of iodine staining.

 

Characterization of isolated Compounds:

FT-IR

IR spectra of the compounds isolated from the ethanolic extract of Jatropha gossypifolia were chronicled using a Nicolet 170SX. The spectral determination for the Nicolet 170SX was 0.25cm^-1, and the spectral data were stowed in the database at interludes of 0.5 cm^-1 at 4000-2000 cm^-1, and of 0.25 cm^-1 at 2000-400 cm^-1. Liquid samples were unrushed with liquid film method, and solid samples were unrushed by using KBr disc methods.

 

¹HNMR

¹HNMR spectra of the compounds isolated from the ethanolic extract of Jatropha gossypifolia was documented using a Brukar Avance II 400 NMR spectrometer. The measuring circumstances for the most of the ranges were as shadows flip angle of 22.5-30.0 degrees, pulse replication time of 30s. The long pulse replication time and small flip angle is used to guarantee precise relative strengths. The ¹H NMR chemical shifts were referred to TMS in organic solvents and TSP in D₂O ^[5].

 

¹³CNMR

¹³CNMR spectra of the compounds isolated from the ethanolic extract Jatropha gossypifolia was documented with a BRUKAR AVANCE II 400 NMR spectrometer. The calculating conditions for the most of the spectra were as follows: a pulse flips angle of 22.45-45 degrees, a pulse replication time of 4-7 seconds, and a determination of 0.025-0.045 ppm. The spectra whose spectral codes started with “CDS” were reassembled from peak positions, strengths, and line widths by arrogant all resonance peaks were Lorenz lines. The chemical shift was referred to a TMS for all solvents.

 

Mass Spectrum

Mass spectra of the compounds isolated from the ethanolic extract of Jatropha gossypifolia was recorded with WATERS, Q-TOFMICROMASS by the electron impact method where an electron is accelerating voltage 75eV and an ion accelerating voltage of 8-10nV. The reservoir inlet systems were used. The dynamic range for the peak intensities were 3 digits and the accuracy of the mass number was 0.5 ^[7].

 

RESULT AND DISCUSSION:

Compound 1:

The compound was obtained as a brown solid m. p. 170°C, willingly soluble in chloroform, ethyl acetate and methanol. The molecular mass and molecular formula was found to 886.85 and C₄₃H₅₀O₂₀. The purity of the compound was confirmed from TLC experiments using different solvent systems.

 

2-8-4,5-dihydroxy-6-(hydroxymethyl)-tetrahydro-2H-pyran-2-yl)-2,5,6-trimethoxy-4-oxo-3,4-dihydro-2H-chromen-7-yl)-6-(3,4-dihydroxyphenyl)-7-ethoxy-5-hydroxy-8-3-hydroxy-6-(hydroxymethyl)-4,5-dimethoxy-tetrahydro-2H-pyran-2-yl)-4H-chromen-4-one

 

Spectral Characterization:

The structure of the compound was recognized from comprehensive analysis of its IR, ^l H-NMR, and ¹³C-NMR and mass spectral data. The IR spectrum (Figure 1) displayed absorption bands at V_max3500, 3025, 1736, 2925 and 2859 showing the presence of – OH, aromatic, keto, methyl, and methylene groups correspondingly from the ¹H-NMR (Figure 2): (Table 1) exposed the presence of OH group (δ 7.26, 1H, s), aromatic (δ 5.34, 1H, s), five methyl groups (δ 1.92, 3H, d), (δ 1.48, 3H, s), (δ 1.12, 3H d), (δ 1.02, 3H, s) and (δ 0.86, 3H, s). The ¹³C NMR (Figure 3): (Table 1) exhibited the signals for aromatic groups δ124, C-2, δ77, C-1, δ76, C-2, δ46, C-3, δ39, C-4, δ29, δ27,δ25,δ24 represents the methyl and methylene carbon correspondingly and mass spectrum, which exhibited peak at m/z 887 denotes molecular ion peak and fragmented ion peaks 613, 585, 543, 371 and 159 correspondingly. The IUPAC name of compound 1 found to be 2-8-4, 5-dihydroxy-6-(hydroxymethyl)-tetrahydro-2H-pyran-2-yl)-2,5,6-trimethoxy-4-oxo-3,4-dihydro-2H-chromen-7-yl)-6-(3,4-dihydroxyphenyl)-7-ethoxy-5-hydroxy-8-3-hydroxy-6-(hydroxymethyl)-4,5-methoxy-tetrahydro-2H-pyran-2-yl)-4H-chromen-4-one.

 

Fig. 1 IR spectrum of compound 1

 

Compound 2:

It was obtained as colorless crystals, m.p.196-197°C. It was readily soluble in DMSO (Dimethyl sulfoxide).The molecular weight and molecular formula was found to 768.8 and C₄₃H₄₄O₁₃. The purity of the compound was ensured from TLC experiments using different solvent system.

 

2-(2,4-diethoxy-5-hydroxyphenyl)-7-(5-ethoxy-4-oxo-2-phenylchroman-7-yloxy)-5-hydroxy-8-6-(hydroxymethyl)-5-methoxy-tetrahydro-2H-pyran-2-yl)-4H-chromen-4-one

 

Fig. 2 IR spectrum of compound 2

Table 1 Functional groups detection by IR

 

 

Spectral Characterization:

The structure of the compound was recognized from comprehensive analysis of its IR, ^l H-NMR, and ¹³C-NMR and mass spectral data. The IR spectrum (Figure 4) showed absorption bands at V_max3649, 3392, 3136, 2921 and 1772 showing the presence of – OH, - NH, aromatic, keto and methyl groups correspondingly from the ¹H-NMR (Figure 5): (Table 2) exposed the presence of one OH group (δ 7.26, 1H, s), two aromatic (δ 5.37, 1H, s), (δ5.09, 1H, s) and six methyl groups (δ 2.07, 3H, s), (δ 2.04, 3H, d), (δ 1.9, 2H d), (δ 1.8, 3H, d), (δ 1.2, 3H, s) and (δ 1.0, 2H, d) and two methylene groups (δ 0.96, 3H, d) and (δ 0.87, 3H, d). The ¹³C NMR (Figure 6): (Table 2) exhibited the signals for aromatic group’s δ77, C-1, δ78, C-2, δ48, C-3, δ38, C-4 for methyl and methylene carbon δ29, δ26, δ24, δ23 and signal for δ124, C-2 denotes for keto group. Mass spectrum, which exhibited peak at m/z 754 represents molecular ion peak (Figure 7-8). The IUPAC name of compound 2 was found to be 2-(2,4-diethoxy-5-hydroxyphenyl)-7-(5-ethoxy-4-oxo-2-phenylchroman-7-yloxy)-5-hydroxy-8-6-(hydroxymethyl)-5-methoxy-tetrahydro-2H-pyran-2-yl)-4H-chromen-4-one.

 

The chemical examination of the leaves of Jatropha gossypifolia has been carried out and thin layer chromatography was performed of selected extract for the identification of active constituents present in the extract. Column was eluted with benzene, chloroform, ethyl acetate and ethanol. Column chromatography afforded 2 unknown compounds 2-8-4, 5-dihydroxy-6-(hydroxymethyl)-tetrahydro-2H-pyran-2-yl)-2,5,6-trimethoxy-4-oxo-3,4-dihydro-2H-chromen-7-yl)-6-(3,4-dihydroxyphenyl)-7-ethoxy-5-hydroxy-8-3-hydroxy-6-(hydroxymethyl)-4,5-methoxy-tetrahydro-2H-pyran-2-yl)-4H-chromen-4-one and 2-(2,4-diethoxy-5-hydroxyphenyl)-7-(5-ethoxy-4-oxo-2-phenylchroman-7-yloxy)-5-hydroxy-8-6-(hydroxymethyl)-5-methoxy- trahydro-2H-pyran-2-yl)-4H-chromen-4-one.

 

Fig. 3 ¹HNMR spectrum of compound 1

 

 

Fig. 4 ¹H NMR spectrum of unknown compound 2

 

Fig. 5 ¹³C NMR spectrum of compound 1

 

Fig. 6 ¹³C NMR spectrum of compound 2

 

 

Fig. 7 Mass spectrum of compound 1

 

Fig. 8 Mass spectrum of compound 2

 

Table 2 ¹H and ¹³C NMR Chemical Shifts for unknown compound 1 and 2

 

CONCLUSION:

The above research work describes the collection, identification and chemical examination of plant material Jatropha gossypifolia. The chemical examination affords two new compounds and it was confirmed by spectral analysis such as IR, ¹H-NMR, ¹³C-NMR and Mass spectra.

 

ACKNOWLEDGEMENT:

Authors are acknowledge to very thankful for this research work to College of Pharmacy, IPS Academy, Indore and Sophisticated analytical instrument laboratory Punjab University, Chandigarh to successful completion of my research work.

 

REFERENCES:

1.     Agrawal PK. Carbon-13 NMR of Flavonoids. 1989; New York: Elsevier.

2.     Aitken RJ, Baker GF, Doncel MM, Matzuk CK, Harper MJK. As the world grows. Contraception in the 21^st century. Journal of Clinical Investigation. 2008; 118: 1330-1343.

3.     Arvind BN. Benzophenous, Naphthophenones and related compounds as spermicidal agents. Indian Journal of Pharmaceutical Sciences. 1994; 56(3): 105-08.

4.     Bagul MS, Niranjan SK, Rajani M. Evaluation of free radical scavenging properties of two classical polyherbal formulations. Indian Journal of Experimental Biology. 2005; 43: 732-36.

5.     Breitmaier E, Voelter W. Carbon-13 NMR Spectroscopy. New York: 1989; VCH Verlagsgesells ChaftmbH.

6.     Chauhan A, Agarwal M, Kushwaha S, Mutreja A. Antifertility studies of Aegle marmelos Corr., an Indian medicinal plant on male albino rats. Egyptian Journal of Biolog. 2008; 10: 28-35.

7.     Duffield AM, Aplin RT, Budzikiewicz H, Djerassi C, Murphy CF, Wildman WC. Mass spectrometry in structural and stereo chemical problems. A study of the fragmentation of some Amaryllidaceous alkaloids. J. Am. Chem. Soc. 1965; 87: 4902– 4912.

8.     Ghosh K, Bhattacharya TK. Preliminary study on the anti-implantation activity of compounds from the extract of seeds of Thespesia populnea. Indian Journal of Pharmacology. 2004; 38: 288-291.

9.     Igoli JO, Ogeji OG, Tor-Anyiin TA, Igoli NP. Traditional medical practice amongst the igede people of Nigeria. Afr J Trad CAM. 2005; 2: 134-152.

10.  Khandelwal KR. Practical Pharmacognosy techniques and experiments. Nirali Prakashan. 2000; Pune. 22-38.

11.  Kokate CK. Practical pharmacognosy.Vallabha Prakashan. 2001; New Delhi.

12.  Muller JC, Botelho GG, Bufalo AC, Boaereto AC, Rattman YD, Martins ES. Morinda citrifolia Linn (Noni): In-vivo and in-vitro reproductive toxicology. J Ethnopharmacol. 2009; 121: 229-233.

13.  Orihuela PA, Rios M, Croxatto HB. disparate effects of estradiol on egg transport and oviductal protein synthesis in mated and cyclic rats. Biol Reprod. 2001; 65: 1232-1237.

14.  Pilon AC, Carneiro RL, Carnevale Neto F, Bolzani VS, Castro-Gamboa I. Interval multivariate curve resolution in the dereplication of HPLC-DAD data from Jatropha gossypifolia. Phytochemical Analysis. 2013; 24(4): 401–406.

15.  Sankara S, Subramanian S, Sulochana N. Flavonoids of the leaves of Jatropha gossypifolia. Phytochemistry. 1971; 10: 7 1690.

 

 

Accepted on 10.02.2017           © RJPT All right reserved