Hypoglycaemic activity of Smilax canellifolia Mill. rhizomes: a bioassay-guided isolation and identification of synergistic compounds

Smilax canellifolia Mill. is a native shrub used in commercial root tonics as an aphrodisiac, stimulant, and pain reliever. Traditional medicine incorporates the rhizomes of S. canellifolia for the treatment of anaemia, rheumatoid arthritis, and diabetes in Jamaica and its diaspora. In particular, the use of this plant in the management of diabetes has yet to receive any scientific evaluation. In this study, the hexane crude extract of S. canellifolia rhizomes (SCH) was investigated to determine its hypoglycaemic activity in normal Sprague-Dawley rats and to identify the compounds contributing to this activity. The hypoglycaemic compounds were isolated using bioactivity-guided purification which involved hypoglycaemic screening using an Oral Glucose Tolerance Test (via intravenous administration of SCH and its fractions). Purification was performed using column chromatography, and the bioactive fractions were elucidated using spectroscopic techniques (IR; GC-MS; 1H NMR and 13C NMR). Administration of SCH at 50 mg/kg body weight (BW) to normal S-D rats produced a reduced glycaemic response, notably from the 90 to the 150-min intervals when compared with the control, dimethyl sulfoxide (p < 0.05). Purification of this extract yielded four main fractions, SCH1 – SCH4, of which SCH3 and SCH4 displayed significant hypoglycaemia. Further purification of both SCH3 and SCH4 led to the isolation of sub-fractions SCH3.6 and SCH4.2, respectively. Using spectroscopic techniques stigmasterol (1) and β-sitosterol (2) from SCH3.6; and the fatty acids palmitic acid (3), oleic acid (4), and stearic acid (5) from SCH4.2 were identified as the major compounds with significant hypoglycaemic activities comparable to that of glibenclamide. This study demonstrates that the rhizomes of Smilax canellifolia contain several bioactive constituents that are responsible for its hypoglycaemic activity and may be beneficial in the management of hyperglycaemia and complications associated with diabetes.


Background
It has been established that traditional and complementary medicine has had an intensified resurgence during the past decade. This has been due to an increase in the accessibility and acceptability of nutraceuticals, as well as a renewed focus on self-healing and preventative therapy [1]. The cost and availability of conventional drugs used to treat diabetes, and the belief in reduced toxicity from plant medicines have also stressed the demand for herbal or natural remedies [2]. Continuous research has therefore been encouraged to validate and integrate herbal alternatives into healthcare systems especially in countries with East Asian and African medicinal influences [1,[3][4][5][6].
Smilax canellifolia Mill. (S. canellifolia) is a member of the Liliaceae family and the Smilax genus, comprising over 350 species. In Jamaica, S. canellifolia is known as Smilax balbisiana, which has recently been identified as the illegitimate synonym [7]. This native shrub is found widely cultivated in the woodlands of the Cockpit Country, Jamaica. The rhizomes of S. canellifolia, commonly known as Chainy Root, are used as a staple ingredient in Jamaica's traditional "Roots" tonics [8,9]. S. canellifolia is a climbing shrub, with brambled and thorny tendrilclimbing vines that can grow up to 50 m long. These woody vines sprout from a thick basal rhizome that is deep red/brown in colour. The basal leaves are broad and ovate, while the terminal leaflets are lanceolate or elliptical; these leaves are often paired tendrils emerging from the base of the petiole. Its flowers are small and green, while the berries range from reddish-purple to black and are 5 -7 mm in diameter [9,10]. The rhizomes are believed to be an aphrodisiac used to improve impotence and increase energy. Chainy Root is also used in folklore medicine to treat anaemia, analgesia, rheumatoid arthritis, and diabetes [11,12]. Other Smilax species have been found to display considerable ethnopharmacological activities including antioxidant, antidiabetic, hypotensive and anti-inflammatory effects [13][14][15][16][17][18]. However, along with the variation in species, the plant parts and solvents used to obtain the extracts that have been investigated, the extract used to produce significant hypoglycaemic effects were different from those used in our research. These reports also failed to explicitly identify the active naturally occurring compounds responsible for the hypoglycaemic or antidiabetic effects observed [14,16,18]. The crude extracts of S. canellifolia rhizomes have previously been reported to possess significant hypoglycaemic potential in animals [19,20]. Considering these findings, this investigation focused on isolating and identifying the bioactive compounds that contributed to the hypoglycaemic activity of S. canellifolia rhizomes in Sprague-Dawley rats. It is anticipated that these findings will expand the potential use of S. canellifolia rhizomes for the management of diabetes and possibly, its inherent complications.

Chemicals and reagents
All reference standards were purchased from Sigma-Aldrich (St. Louis, USA). Dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich (St. Louis, USA). All other solvents were purchased from Pharmco-Aaper (Ontario, Canada) and were of analytical grade.

Plant material
S. canellifolia rhizomes were collected in March 2016 from Accompong, St. Elizabeth, Jamaica. A sample of the rhizome was authenticated at the University of the West Indies (UWI), Mona Herbarium with an assigned Voucher number, 3633.

Screening for Hypoglycaemic activity Animals
Normoglycaemic Sprague-Dawley (S-D) rats (150-200 g; n = 5 per group) of mixed sexes were obtained from the UWI, Basic Medical Sciences Animal House (Mona, Jamaica). Animals were housed in stainless steel cages inside a ventilated room with an alternate 12 h dark/light period. The animals had free access to water and a standard laboratory diet (LabDiet 500, MO, USA The OGTT was performed using a modified method [21]. The rats were weighed then fasted overnight (approximately 12 h) before experimentation, and water was provided ad libitum.
An incision was made at the tip of the animal's tail to obtain an initial fasting blood glucose reading. The preweighed crude or purified hexane extract (10-50 mg/kg body weight/BW 1 ) was dissolved in the vehicle, DMSO (0.3 mL), and administered intravenously (i.v.). Afterwards, blood glucose readings were measured at 30 and 60-min post-administration. Subsequently, a glucose load of 1.75 g/kg BW was administered by oral gavage. Blood glucose readings were taken at 30-min intervals for a further two and a half (2.5) hours using the ACCU-Chek Active Blood Glucose Monitoring System.

Extraction and purification of hexane crude extract (SCH)
The shade-dried (~28°C) and powdered rhizomes (7.5 kg) were extracted with hexane. The plant material was initially saturated with hexane (2 L) at room temperature for two separate eight (8) hour periods and one twentyfour (24) hour period. Fresh solvent (1 L) was added for each treatment period, and the filtrates were collected and concentrated using rotary evaporation. The hexane crude extract was weighed (16.5 g) and the recovery percentage (0.22%) was determined.
The hexane crude extract from the rhizomes of S. canellifolia (SCH) was obtained as a gold oily residue and displayed significant hypoglycaemic activity during an OGTT. TLC analysis showed that several unresolved compounds were present. The extract (SCH, 16.24 g) was subjected to column chromatography (30 × 6.5 cm; 200 -425 mesh) and eluted with gradient Hex -EtOAc (90:10 to 0:100, v/v) solvent system, collecting 150 mL volumes. The eluted fractions were later pooled based on the similarity of their Thin-layer chromatography (TLC) profiles to afford four main fractions designated SCH1 (0.24 g), SCH2 (7.52 g), SCH3 (3.17 g) and SCH4 (1.73 g). The spots were visualized under white light, UV light, and after spraying with 10% sulfuric acid reagent.

Spectroscopic analysis and structural identification of SCH sub-fractions
Column chromatography was carried out using silica gel 60 Å (200 -425 mesh; Sigma-Aldrich, St. Louis, USA).
TLC was carried out on normal phase silica gel 60 F 254 on pre-coated plastic plates (Sigma-Aldrich, St. Louis, USA), using 5% sulfuric acid spray after heating or ultraviolet (UV) light (254 nm and 365 nm) for visualization.
Infrared (IR) spectra were obtained using the Nicolet iS10 FTIR spectrometer with ATR accessories. 13 C NMR and 1 H NMR spectra were recorded on a Bruker DRX-500 MHz and DRX-200 MHz spectrometer (for 1D and 2D respectively) in deuterated chloroform. Chemical shifts (δ) were expressed in parts per million (ppm) with reference to tetramethylsilane (TMS).
Gas Chromatography-Mass Spectroscopy analyses were performed using a DB-1701 GC capillary column (30 mm, 0.25 mm i.d., 0.25 μm film thickness) on an Agilent 6890 N gas chromatograph (GC) equipped with an Aligent 5973 N Mass Selective Detector, using helium carrier gas at a flow rate of 1.2 mL min − 1 . A ramped temperature program was employed for the oven, starting at 80°C at a rate of 2°C min − 1 for 10 min. The samples were reconstituted in chloroform (1 μL) and injected in splitless mode at 250°C. Mass spectra operations were performed in electron impact (EI + ) mode at 70 eV. The chromatogram was acquired in total ion current (TIC), and the data was given in m/z values. Derivatization was done using Bis-(trimethylsilyl)-trifluoroacetamide (BSTFA). All preliminary identifications were obtained using the NIST/EPA/NIH Mass Spectral Database (version NBS75K.L). The compounds identified were then confirmed by comparison of their retention indices and mass spectra with those of the authenticated standards under the same conditions. The relative ratio of the compounds identified was computed from the GC area percentage and excluded correction factors. All chemical structures were constructed using ACD/ ChemSketch software (ACD/Labs version 2019).

Statistical analysis
The results were presented as mean ± standard error of the mean (SEM). Statistical analysis was carried out using a statistical software (SPSS, Version 22, Chicago, IL, USA) to perform the Student's t-test. Values were considered statistically significant at p < 0.05.

Hypoglycaemic screening
The effect of Smilax canellifolia rhizomes hexane crude extract (SCH) and its semi-purified fractions on blood glucose (BG) concentration were evaluated using an Oral Glucose Tolerance Test (OGTT). It was evident that the control (DMSO; vehicle) produced a glucose tolerance curve demonstrative of a typical response to an oral glucose load. Its administration had a characteristic glycaemic peak at the 90-min interval, followed by a general decrease due to the action of insulin (Fig. 1a).

Discussion
A survey that was conducted in Jamaica highlighted the popularity of self-medicating with traditional medicine for the treatment of common ailments and chronic diseases, including diabetes and hypertension [23]. The rhizomes of Smilax canellifolia, though commonly used as a general health tonic and aphrodisiac has been noted in folklore to be beneficial in the treatment of diabetes [8,9]. Smilax species have made significant contributions as alternative interventions for the treatment of hyperglycaemia, hypertension, and oxidative stress, which are all associated with diabetes [13-15, 18, 24]. This study conducted hypoglycaemic screening on the hexane crude extract of S. canellifolia rhizomes in normoglycaemic rats, which highlighted that intravenous administration (50 mg/kg BW) produced significant hypoglycaemic responses (Fig. 1a). The reduced glycaemic peak observed at the 90-min interval indicated that the extract contained compounds that were able to enhance blood glucose clearance. In previous investigations, the hexane crude extract was found to exert the most potent hypoglycaemic effect from a group of three solvent extracts (hexane, ethyl acetate and methanol) that were screened. The prolonged hypoglycaemic effect displayed by the hexane crude extract (SCH) when compared with the other extracts was significant and therefore, it was consequently subjected to bioassay-guided purification [20,25]. In contrast, other Smilax species such as S. aristolochiifolia and S. moranensis indicated that their polar solvent extracts (methanol or ethanolwater) produced the most potent hypoglycaemic effects [13,26,27]. These studies also focused on other parts of the plant (leaves and roots) and indicated that the bioactive compounds were possibly flavonoids, alkaloids, and polyphenolssome of which were absent in our hexane extract from S. canellifolia rhizomes, possibly due to the variation in polarity [25].
Further screening of the semi-purified fractions (25 mg/kg BW; intravenous administration) from SCH, illustrated that SCH3 and SCH4 were significant in their ability to lower the postprandial glycaemic responses during the OGTT (Figure 1b). Studies have found that therapeutics that target postprandial hyperglycaemia were beneficial in the initial treatment of diabetes as they reduce the formation of glycated-end products and oxidative stress [28,29]. Therefore, natural products that can manage this glycaemic spike may have significant potential as alternative medicines used for diabetes.
Chromatography purification of SCH3 and SCH4 led to the isolation of phytosterols (SCH3.6) and fatty acids (SCH4.2), respectively as revealed by spectroscopy analyses. They were identified as the key naturally isolated compounds to which the hypoglycaemic activities were attributed. It has been noted that the various pharmacological activities displayed by Smilax species could, in part, be linked to the presence of phytosterols that enhanced the absorption and bioavailability of other phytochemicals and drugs [30].
Sub-fraction SCH3.6 (10 mg/kg BW; intravenous administration) displayed significant hypoglycaemia when compared with the control (Fig. 2). GC-MS analysis identified the main constituents of this sub-fraction as a mixture of stigmasterol and β-sitosterol, with several fatty acids making up the remaining minority (Fig. 4). Stigmasterol and β-sitosterol are sterols (steroid alcohols) found mainly in plants (phytosterols), with structural similarity to cholesterol [31]. These sterols differ from cholesterol with the addition of an ethyl group in their side chain, while the unsaturation at carbon 20-21 of stigmasterol distinguishes it from β-sitosterol. Phytosterols are utilized as precursors/ intermediate molecules in plant development and cell signaling [32]. Studies have highlighted that stigmasterol and β-sitosterol exhibit antidiabetic and hypoglycaemic effects in both diabetic and healthy animals respectively [33,34]. They also have potential antioxidative, hepatoprotective, and anti-inflammatory effects [35]. In this study, the hypoglycaemic effect observed throughout the OGTT could be linked to the phytosterols' stimulatory effects on insulin secretion [30,36]. This hypoglycaemic effect may also be independent of BG concentration, as observed with the significant lowering in the animals' fasted BG levels (Fig. 2). Jamaluddin et al. suggested that the hypoglycaemic effect of these phytosterols in Parkia speciosa was due to their synergistic action. When orally administered to diabetic rats, each individual phytosterol failed to cause significant hypoglycaemia [33]. In our study these naturally occurring phytosterols, when administered (intravenously) in combination, also produced significant BG concentration reduction in normoglycaemic S-D rats. The effect observed may have been due to the evasion of the first-pass metabolism, which could have contributed to the rapid onset of hypoglycaemia, as observed in the fasted state.
SCH4.2 was found to contain palmitic acid (PA), oleic acid (OA) and stearic acid (SA). Although these fatty acids (FAs) have been identified in Smilax species, their occurrence has never been reported as the components contributing to their hypoglycaemic activity [37]. Fatty acids are carboxylic acids with hydrophobic hydrocarbon chains that are categorized based on the absence (saturated FA) or presence and position of double bonds (unsaturated; cis and trans) along these chains. The length of these FA chains also determines their physiological use as high energy sources and cell membrane components [38]. PA and SA are bothsaturated fatty acids while OA is a monounsaturated fatty acid. A balanced ratio (saturated : unsaturated ) of these three fatty acids will ensure the homeostatic functioning of membrane phospholipids that are vital in regulating intracellular signaling and maintaining membrane structure and fluidity [38]. This balance also reduces the risks of cardiovascular diseases, hyperglycaemia and inflammatory events due to an accumulation of saturated FAs, especially PA [39]. Free fatty acids (FFAs) are known to increase insulin secretion at elevated glucose concentrations [40,41]. While OA and PA can enhance insulin secretion at physiologically low BG levels, this effect is more apparent with monounsaturated FAs [42]. This insulin-independent effect may have accounted for the reduction observed in the fasted state (60-min interval; Fig. 3). Our findings indicated that the administration of SCH4.2, containing primarily PA, OA, and SA, was able to produce a significant reduction in the postprandial BG levels. The FAs were determined to occur at a ratio of 7:5:1 naturally, and their hypoglycaemic effect may have been in response to the synergistic action of the FFAs and their impact on insulin release [40]. Daley-Beckford et al. conducted a similar study using healthy normoglycaemic S-D rats [43]. They concluded that a mixture of PA and OA in the ratio of 2.3:1 from Eucalyptus camaldulensis was able to improve glucose tolerance (postprandial). The glycaemic control produced was associated with an increase in insulin secretion in the animals. Moreover, PA and OA were also found to contribute to the antidiabetic effect observed in Juniperus oxycedrus leaves [44]. Studies have indicated that the hypoglycaemic effect observed with the administration of these fatty acids could also be associated with their ability to trigger GLUT-4 translocation in rat skeletal muscles [45,46]. This fatty acid-stimulated glucose uptake was linked to increased phosphorylation of signaling proteins, which are critical components in GLUT-4 translocalization [45][46][47]. These findings suggest that an increase in GLUT-4 mediated glucose uptake and insulin secretion could be the primary mechanism ofhypoglycaemiafor the fatty acids identified in this study [45].

Conclusion
This study has established that several compounds present in the hexane sub-fractions of Smilax canellifolia rhizomes had a synergistic hypoglycaemic effect. These compounds were identified as stigmasterol, β-sitosterol, and the fatty acids: palmitic acid, oleic acid, and stearic acid. It was evident that these naturally occurring compounds contributed to an improvement in postprandial glucose tolerance, which led to significant hypoglycaemic activity when administered to Sprague-Dawley rats. This is the first report on the purification, isolation and identification of the hypoglycaemic compounds from S. canellifolia rhizomes. Overall, the presence of these bioactive constituents in S. canellifolia rhizomes add credence to its use in folklore medicine. We, therefore, propose to further ascertain the antidiabetic and physiological effects of these constituents in a diabetic animal model. Smilax canellifolia rhizomes have the potential to be used as a phytopharmaceutical or nutraceutical in the management of diabetes.