Structural Elucidation and Toxicity Evaluation of Bioactive Compounds from the Leaves and Stem woods of Synadenium glaucescens Pax.

The leaves and stem woods of Synadenium glaucescens Pax. are traditionally used for treatment of various human diseases in Tanzania. However, there is limited information on the structural elucidation and toxicity properties of bioactive compounds responsible for their pharmacological properties. Thus, this study was aimed to elucidate and evaluate toxicity properties of bioactive compounds from these parts. Separation of bioactive compounds was achieved by using column chromatographic method, whereas their structures were determined by GC-MS and NMR data and by comparing with literature spectroscopic data. Toxicity properties of isolated compounds was done by brine shrimp lethality test (BSLT) method. In this study, column chromatographic separation led to isolation of tetracosane (1), hexadecyl palmitate (2), octacosane (3), tetracosanol (4) and β-sitosterol (5) from the leaves and 1-hexacosene (6), campesterol (7), octadecanoic acid (8) and tetracosanoic acid (9) from the stem woods. BSLT of compounds isolated in sufficient amount (1, 2, 3, 4, 5 and 7) were revealed to be non-toxic to brine shrimp larvae (LC 50 >100 µg/ml). Except compound 5, this study reports the isolation of other eight (8) compounds for the first time from this plant. Therefore, it can be reasonably concluded that S. glaucescens is a good source of bioactive compounds which justify its traditional uses in treatment of various ailments. Additionally, observed non-toxic effect of the tested compounds indicates safety and it is expected that its traditional use has no toxicity effect.


INTRODUCTION
In Tanzania, Synadenium glaucescens Pax (family Euphorbiaceae) is one of the useful medicinal plants traditionally used for treatment of various diseases both in humans and animals (Mabiki et al., 2013a). For instance, in Njombe region-Southern Highlands of Tanzania, its leaves and stem woods are traditionally used for treatment of human immunodeficiency virus (HIV), cough or tuberculosis (TB), asthma, leprosy, sores, wounds and worms in humans and healing of east coast fever (ECF) in cattle and Newcastle disease in poultry (Mabiki et al., 2013;Nyigo et al., 2016;Nyigo et al., 2015). Previous pharmacological studies have demonstrated that, several parts of this plant including the leaves and stem woods have antimicrobial activities (Mabiki et al., 2013a(Mabiki et al., , 2013bMax et al., 2014).
Despite the vast traditional uses of this plant, both externally and internally, in the treatment of both humans and animals diseases, it has been previously implicated for causing mild to moderate dermal irritancy (Nyigo et al., 2015).
Pharmacological and toxicological properties of medicinal plants, including S. glaucescens, are attributed by the presence of phytochemical compounds they produce (Faridha et al., 2016). Previous phytochemical screening of crude extracts from various parts of S. glaucescens revealed to possess several classes of phytochemical compounds such as terpenoids, steroids, tannins, coumarins, glycosides and phenolics (Mabiki et al., 2013).
Despite the possession of many phytochemical compounds and its usefulness in traditional medicine as described above, information about structural elucidation/identification and toxicity evaluation of its phytochemical compounds are limited.

General
All chemicals used in this study were of analytical grade. They were obtained from either Loba Chemie, Mumbai-India, i.e. dichloromethane (DCM) and petroleum ether (PE), or Finar Chemical, Gujarat-India, i.e. methanol (MeOH). The silica gel 60 (70-230 mesh, 60 angstrom pore size) and precoated thin layer chromatography aluminium sheets (TLC, silica gel 60 F 254 ) were obtained from Merck KGaA group, Darmstadt, Germany. The brine shrimps (Artemia franciscana) eggs and sea salt were obtained in the Department of Chemistry and Physics, Sokoine University of Agriculture-Tanzania.

Materials
Identification of plant species was done by a botanist from the Department of Botany, University of Dar es Salaam (UDSM). Its leaves and stem woods were collected from Njombe district (08°34′ to 08°49′ S and 034°55′ to 035°10′ E), Njombe region, Tanzania in December 2018 and the voucher no. 3672 was stored in the herbarium of the Department of Botany-UDSM. Plant materials were air dried in an open space under shade at room temperature, then pulverized into fine powder by using a milling machine type Y (Hangyu ® , China).

Extraction of Plant Materials
1 kg of each powdered part of S. glaucescens was extracted exhaustively with 100% dichloromethane (DCM) by soxhlet method (Azwanida, 2015). The filtrates obtained were evaporated using a rotary evaporator (Büchi Labortechnik, Flawil, Switzerland) at 40°C to obtain DCM extracts. The residues of each plant part obtained was dried and further exhaustively extracted with 100% MeOH by maceration method. The filtrates obtained were evaporated to obtain MeOH extracts.
Isolation and Purification of Bioactive Compounds 40 g of 100% DCM extract from the leaves was chromatographed on silica gel by gradient elution using PE, DCM and MeOH. By using TLC to monitor separations, the column was eluted by solvent containing 25% DCM in PE to obtain 30 fractions (Fr. 1-30), 50 ml each. Solvent polarity was further increased to 50% of DCM in PE to give 50 fractions . Furthermore, the solvent polarity was increased to 75% of DCM in PE to yield 63 fractions (Fr. 81-143) followed by 100% DCM to give 8 fractions . Then, solvent polarity was increased to 10% of MeOH in DCM giving 14 fractions , finally increased to 20% of MeOH in DCM affording 5 fractions .
Similarly, 40 g of 100% MeOH extract from the leaves was also chromatographed on silica gel by gradient elution using PE, DCM and MeOH. The column was eluted by 25% of DCM in PE to afford 14 fractions (Fr. 1-14), and then increased to 50% of DCM in PE to give 3 fractions (Fr. 15-17). Furthermore, polarity was increased to 75% of DCM in PE to yield 17 fractions (Fr. 18-34) followed by 100% DCM to obtain 25 fractions . Then, solvent polarity was further increased to 10% of MeOH in DCM to give 5 fractions (Fr. 60-64) and finally to 20% of MeOH in DCM to give 15 fractions (Fr. 65-79).
On the other hand, 20 g of 100% DCM extract from the stem woods was chromatographed on silica gel by gradient elution with PE, DCM and MeOH. Column was eluted with 25% of DCM in PE to afford 20 fractions (Fr. 1-20), then to 50% of DCM in PE to give 16 fractions (Fr. 21-36). Solvent polarity was further increased to 75% of DCM in PE to afford 10 fractions (Fr. 37-46) followed by 100% DCM to give 13 fractions (Fr. 47-59). Then increased to 10% of MeOH in DCM to give 10 fractions (Fr. 60-69) and finally to 20% of MeOH in DCM to give 9 fractions (Fr. 70-78).
Finally, 50 g of 100% MeOH extract from the stem woods was chromatographed on silica gel by gradient elution with PE, DCM and MeOH. Column was eluted with 25% of DCM in PE to give 14 fractions  and then increased to 50% of DCM in PE to give10 fractions (Fr. 15-24). Furthermore, solvent polarity was increased to 75% of DCM in PE to obtain 19 fractions (Fr. 25-43) followed by 100% DCM to afford 7 fractions (Fr. 44-50) and finally to 10% of MeOH in DCM to give 14 fractions (Fr. 51-64).
All fraction combinations which did not form precipitates were few in amount (less than 0.5 mg), hence, not subjected to further purification.

Spectroscopic Analysis
Mass spectrum having molecular ion peak, [M] + of each compound was recorded in electron impact (EI) mode by an Agilent 5973 Triple Quadrupole GC-MS system (from California, USA) with the National Institute of Standards and Technology (NIST) spectral library (version 2002). Based on the value of mass to charge (m/z) ratio of [M] + , the structure of each compound was suggested by the NIST spectral library.
The 1 H and 13 C NMR data of isolated compounds were recorded on a 600 Megahertz Bruker Avance III HD 600 (UltraShield TM ) NMR spectrometer (from Massachusetts, USA) with tetramethylsilane (TMS) as internal standard. The acquired data were processed by using Topspin software version 3.6.3.

Brine Shrimp Lethality Test (BSLT)
With some minor modifications, toxicity study of compound 1, 2, 3, 4, 5 and 7 was done by BSLT method according to previous described procedure (Magadula et al., 2012). In brief, 40 mg/ml stock solution of each compound dissolved in 20% dimethylsulphoxide (DMSO) were made. Five concentration levels (1000, 800, 600, 400 and 200 µg/mL) were made by drawing different volumes from the stock solutions and then added into vials, each having ten brine shrimp larvae. Then, volume of vial was adjusted to 5 mL with artificial seawater prepared by dissolving 3.8 g of sea salt in 1 L of distilled water. Each concentration was tested in duplicate. The negative control containing artificial seawater, brine shrimp and 20% DMSO was prepared. The vials were then incubated under light for 24 h. The number of died brine shrimps for each vial after incubation were counted and their mean at each concentration was determined.
Then, mean percentage of mortality at each concentration was determined using the equation: (1) The mean percentage mortality was subjected to regression analysis using Microsoft Excel (version 2016). The mean percentage mortality was plotted against the logarithm of concentrations. The regression equations obtained from the graphs were used to determine the lethal concentrations for 50% mortality of the larvae (LC 50 ) and the 95% confidence interval (95% CI) values as per previous described procedure (Magadula et al., 2012;Nondo et al., 2015). The results of brine shrimp toxicity were interpreted as follows: LC 50 ˂ 1.0 μg/ml as highly toxic; LC 50 1.0-10.0 μg/ml as toxic; LC 50 10.0-30.0 µg/ml as moderately toxic; LC 50 >30-100 ml as mildly toxic and LC 50 > 100 μg/ml as non-toxic (Nondo et al., 2015).

Isolation of Bioactive Compounds
Compounds 1-4 were isolated from 100% DCM leaves extract of S. glaucescens; while compound 1 was white crystals with 1729 mg, and compound 2 was colorless waxy with 1249 mg. Compound 3 and 4 were white amorphous powder with 260 and 91 mg respectively. Compound 5 was isolated from MeOH leaves extract as white amorphous powder with 84 mg.
Moreover, compounds 6-8 were isolated from DCM stem woods extract; compound 6 was white waxy with 12 mg while compound 7 and 8 were white amorphous powder with 88 and 25 mg respectively. Finally, compound 9 was isolated from MeOH stem woods extract as white amorphous powder with 23 mg. All isolated compounds showed single spot on TLC analysis to confirm their purity.

Brine Shrimp Lethality Test
BSLT assay was done to predict toxicity properties of bioactive compounds from the leaves and stem woods of S. glaucescens. The results revealed that all tested compounds were non-toxic to brine shrimp larvae (LC 50 > 100 µg/ml) as shown in Table 1.
BSLT is a convenient method for screening bioactive natural products. Additionally, it is a rapid, cheap and simple bioassay for testing plant extracts bioactivity, which greatly correlates with cytotoxic and antitumor properties (Lavilla et al., 2014). In this study, compound 6, 8 and 9 were not assessed for toxicity since they were obtained in inadequate amount to perform the test.

CONCLUSION
In conclusion, this study demonstrates that S. glaucescens is a good source of bioactive compounds, which is a justification for its traditional uses in treatment of several diseases. Furthermore, observed non-toxic effect of its tested compounds indicates safety and perhaps its traditional use has no toxicity effect. However, further studies on toxicity properties of these compounds are recommended.

CONFLICT OF INTEREST
The authors declare no conflict of interests.  Credo, et al. Pharm Sci Res, Vol 9 No 2, 2022 Waghulde