ISOLATION OF β-SITOSTEROL & STIGMASTEROL AS ACTIVE IMMUNOMODULATORY CONSTITUENTS FROM FRUITS OF SOLANUM XANTHOCARPUM (SOLANACEAE)

ISOLATION OF β-SITOSTEROL & STIGMASTEROL AS ACTIVE IMMUNOMODULATORY CONSTITUENTS FROM FRUITS OF SOLANUM XANTHOCARPUM (SOLANACEAE)

S. Khanam* and R. Sultana

Department of Pharmacognosy, Al Ameen College of pharmacy, Hosur Road, Bangalore-560027, Karnataka, India

ABSTRACT

General phytochemical screening of the aqueous extract of fruits of Solanum xanthocarpum (Solanaceae) revealed the presence of steroids, terpenes, phenolic compounds, saponins, fatty acids, alkaloids. Fractionation of aqueous  extract  and activity guided  isolation  resulted in isolation of two white crystalline powder which were subjected to physical, chemical and spectral identification using IR, ¹H‐NMR, ¹³C‐NMR and LC‐MS. On the basis of the spectral data analysis and chemical reactions, the compounds have been established as β-sitosterol and Stigmasterol.

Stigmasterol

INTRODUCTION:Solanum xanthocarpum belonging to family Solanaceae commonly known as Indian night shade or yellow berried night shade is a perennial herbaceous weed. Different parts of this plant have been used traditionally for curing various ailments such as fever, cough, asthma and diabetes in Indian traditional medicines ¹. The hot aqueous extract of dried fruits is used for treating cough, fever and heart diseases ². The plant is extensively studied for the various pharmacological activities like antiasthmatic ³, hepatoprotective ⁴, cardiovascular ⁵, hypoglycemic ⁶,antiulcer ⁷ and other properties.

Scientific evidence in favor of the traditional use of the fruits of Solanum xanthocarpum for the treatment of diabetes mellitus has been reported ⁸. The total extracts and steroidal saponins of Solanum xanthocarpum, have been reported to exhibit potent antistress-adaptogenic effects ⁹.

To prove traditional claim of this plant preliminary immunomodulatory evaluation of aqueous extract of fruits of Solanum xanthocarpum on cyclophosphamide induced immunosuppression was carried out and the results indicated good protection by showing increase in all the hematological parameters ¹⁰.  In continuation of the work, it was proposed to isolate active immunomodulating agents from the extract. The fruits are reported to contain several steroidal alkaloids like solanacarpine ¹¹, solanacarpidine, solancarpine, solasodine, solasonine ¹² and solamargine ¹³. Other constituents like caffeic acid ¹⁴ coumarins like aesculetin and aesculin ¹⁵, steroids carpesterol, diosgenin, campesterol, daucosterol and triterpenes like cycloartanol and cycloartenol are also reported from the fruits ¹⁶.

In this paper, we report the isolation and characterization of bioactive principles namely β-sitosterol and stigmasterol from aqueous extracts of fruits of Solanum xanthocarpum.

MATERIAL AND METHODS:

Plant material. Fruits of the plant, Solanum xanthocarpum were collected from APRC Lab Chennai and authentication no APRC/78/22/08-09. The sample was shade dried and powdered to #22.

Extraction: Powdered plant material was defatted with petroleum ether (60-80^oC) in a soxhelet extractor. The marc was dried and refluxed with water for 8 hrs. The aqueous extract was filtered and concentrated using rotary vacuum evaporator and the dried extract was stored in an air tight container.

Chromatographic Separation:

TLC: The aqueous extract of fruits was subjected to thin layer chromatography using silica gel as stationary phase and petroleum ether: methanol (1:1) and petroleum ether: chloroform: methanol (5:2:1) as mobile phase. The chromatograms when developed yielded seven and eight spots respectively that showed zones for steroidal nucleus with Liebermann – Buchard visualizing reagent.

Isolation: Column chromatography of fruit aqueous extract (10g) was conducted using silica gel (Mesh 100-200) by wet packing method. The column was run using petroleum ether, ethyl acetate, methanol and water successive by gradient elution technique. TLC was used to monitor the eluates. A total of 200 eluates were collected. Similar fractions were pooled together to yield fifteen fractions. Eluates A, and G were worked upon to yield Sx5, and Sx9 respectively.

Sx5 and Sx9 yielded single spots when subjected to TLC using several solvent systems including petroleum ether: ethyl acetate (70:30), petroleum ether: methylene chloride (50:20) and Petroleum ether: Chloroform: Methanol (60:30:10) and it showed it to be homogenous compound.

Sx5 a white amorphous powder (8.3mg) with a melting point (139^oC) was  subjected to thin layer chromato-graphy using various solvent systems such as petroleum ether: chloroform: methanol (5:3:1), methylene chloride: petroleum ether (50:50) indicated it to be homogenous compound. Sx5 was further subjected to IR, ¹HNMR, ¹³CNMR and LCMS to ascertain the chemical structure.

Sx9 a white crystalline powder (9mg) with a melting point (137^oC) was subjected to TLC using various solvent systems such as petroleum ether: chloroform: methanol (5:3:1), methylene chloride: petroleum ether (50:50) indicated it to be homogenous compound. Sx9 was further subjected to ¹HNMR, ¹³CNMR and LCMS to ascertain the chemical structure.

The structures were simulated using ACD/NMR program to obtain the chemical shifts of both proton and carbon.

Phytochemical analysis (Salkowski’s test and Lieberman-Burchard test) of both the compounds confirms its steroidal nature.

Spectroscopic Characterization: Different spectro-scopic methods were used to elucidate the structure of isolated compounds. Among the spectroscopic techniques IR, ¹H‐NMR, ¹³C‐NMR and LCMS were carried out. The infra red spectrum was recorded on FTIR (model Shimadzu 8700), ¹H‐NMR and ¹³C‐NMR spectra were recorded using CDCl₃ as solvent on Bruker Advance II 400 NMR spectrometer and LCMS spectra were recorded at high resolution on a mass spectrometer (model Shimadzu) at Sophisticated Instrumentation centre and the data are given in m/z values.

Characterization of Sx5 and Sx9: The exact molecular mass for the Sx5 and Sx9 was found to be 414.7 and 412 respectively.  Based on this the proposed molecular formula of the compounds could be tentatively: C₂₉H₅₀O and C₂₉H₄₈O. From ¹³CNMR and ¹HNMR the number of C and H were found to be near to the formula C₂₉H₅₀O and C₂₉H₄₈O. Since, the compounds give positive test for steroids so all of the other structures other than steroids were rejected.

Based upon the functional group analysis it was found that the nature of oxygen was hydroxyl, also supported by IR spectroscopy. This implies presence of one double bond in the structure. So, the steroids with other functional groups were rejected. Also on considering the nature of oxygen as hydroxyl and presence of one double bond, the general formula for the compounds were C_nH_2n-6.

Therefore, they must be tetracyclic compounds. Based on the melting point and other related data (IR, NMR and Mass) the structure of the isolated compounds Sx5 and Sx9 were proposed as (fig. 1);

FIG. 1: Sx5 and Sx9

The compound Sx5 is a white amorphous powder, m.p. 137^oC. IR absorptions bands appeared at 3549.99 cm-1 (OH), 2935.73 cm-1 (CH₂), 2867.38 cm-1 (CH), 1637.63 cm-1 (C=C), 1063.34cm-1 (C-O). Mass  spectra of this compound suggested that its molecular mass is 414 (M.F. C₂₉H₅₀O) having characteristic fragments observed at m/z: 414, 396, 381, 329, 303, 289, 273, 255, 231, 213, 199, 173, 159, 119, 95, 81, 69. NMR spectrum of this compound resembled data published in previous studies ^16-17.

This compound is having six methyl, eleven methylene and three quaternary carbons with a hydroxyl group. The carbons of alkenes conjugated are at 140.78 ppm (C5) and 121.72 ppm (C6) which was confirmed from the ¹³CNMR.  The above IR, ¹HNMR, ¹³CNMR, LCMS spectal data and a comparison of the ¹³CNMR signal with those described in the literatures ^18-20 showed the structure of Sx5 to be the β-Sitosterol.

The compound Sx9 is a white crystalline substance with a melting point of 139^oC. IR absorptions bands appeared at 3384cm^-1 (OH), 3218 cm^-1 (cyclic olefinic –HC= CH- str), 3025 (=CH str) and 2868 cm^-1 assigned to C-H str. Other absorption frequencies include 1665 cm^-1 as a result of C=C absorption, however, this band is weak. 1462 cm^-1 is a bending frequency for cyclic (CH2)n and 1382 cm^-1 for –CH2 (CH3)2γ. The absorption frequency at 1332 cm^-1 can be attributed to OH def. and at 1046 cm^-1 signifies cycloalkane. These absorption frequencies resemble the absorption frequencies observed for Stigmasterol ²⁰.

The Proton NMR has revealed the existence of signals for olefinic proton at 5.358 (br.,s.), Angular methyl proton at 0.68 (s), 0.699 (s) and 1.01 (s) corresponding to C₁₈ and C₁₉ proton respectively. The ¹³CNMR has shown recognizable signals 140.8 and 121.7ppm, which are assigned C5 and C6 double bonds respectively as in Δ5 spirostene ¹⁹. The δ value at 71.0 ppm is due to C3 β- hydroxyl group ²⁰. The signals at δ 19.4 and 11.9 ppm corresponds to angular carbon atom (C₁₈ and C₁₉ respectively). The above IR, ¹HNMR, ¹³CNMR, LCMS spectal data and a comparison of the ¹³CNMR signal with those described in the literatures ^20-23 showed the structure of Sx9 to be the Stigmasterol.

The structures were simulated using ACD/NMR program to obtain the chemical shifts of both proton and carbon. On comparison the experimental data matched with the simulated data which supports the proposed structure of this compound.

RESULTS AND DISCUSSION: Solanum xanthocarpum is widely recognized in Ayurvedic system of Indian medicine for treatment of respiratory problems. It has been reported as immunomodulatory agent. Preliminary immunomodulatory screening indicated the protective effect of the aqueous extract of fruits of Solanum xanthocarpum against Cyclophosphamide induced immunosuppression in mice. The study affirmed that aqueous extract of the fruits of Solanum xanthocarpum is effective immunomodulatory agent.

The extract potentiatedthe non- specific immune response by increasing the haematological parameter and neutrophil adhesion test, which may attributed to different phytoconstituents. Hence, to isolate the phytoconstituent it was subjected to fractionation and characterizations of isolated compounds.

Two compounds Sx5 and Sx9 showing positive tests for steroids and alcohols were isolated. The Sx5 is white amorphous powder like substance with melting point 137^oC. Whereas, the Sx9 is white crystalline substance with melting point 139^oC on subjection to IR spectroscopic analysis, the observed absorption frequencies resemble the absorption frequencies observed for β- sitosterol and Stigmasterol. The proton NMR showed the proton environment resembles the protons of β- sitosterol and Stigmasterol.

The ¹³C‐NMR has shown recognizable signals of β sitosterol and Stigmasterol.

The weak molecular ions were given at m/z 414 and 412.7. The molecular weight and fragmentation pattern indicate that the isolated compounds are β-sitosterol and stigmasterol.

The above I.R., ¹HNMR, ¹³C‐NMR and LCMS spectral data and their comparison with those described in the literatures showed the structure of isolated compounds are to be the β-sitosterol   and stigmasterol ²⁴.

CONCLUSION: β- sitosterol and Stigmasterol isolated from the aqueous extract of fruits of Solanum xanthocarpum these compounds may be the responsible for immunomodulatory activity.

ACKNOWLEDGEMENT: Authors are thankful to the Principal and Manager of Al-Ameen College of Pharmacy for providing the necessary facility to carry out the research work. One of the authors, Rokeya Sultana is thankful to IPA, for providing financial support.

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