Antidiabetic potential of triterpenoid saponin isolated from Primula denticulate

Abstract

Context: Primula denticulate Sm. (Primulaceae), commonly known as drumstick primula, is traditionally used to treat diabetes and urinary disorders. In the present study, a new triterpenoid saponin was isolated. Triterpenoids generally show antidiabetic activity. Considering its traditional use and chemical nature of the molecule, the present study was designed to evaluate the antidiabetic activity.

Objective: Antidiabetic activity of triterpenoid saponin (TTS) isolated from P. denticulate.

Materials and methods: A new TTS was isolated from the leaf of P. denticulate by column chromatography on CHCl₃/MeOH (8.5:1.5) fraction. It was further characterized by using NMR, UV, and IR spectroscopic methods. Ethanol and aqueous extracts of the leaf were also prepared. Antidiabetic study for TTS, ethanol extract, and aqueous extract was carried out in streptozotocin (STZ)-induced diabetic rats at doses of 200, 1000, and 1000 mg/kg body weight, respectively. A toxicity study was also performed.

Results: Isolated new TTS molecule was characterized as 3-O[β-d-xylopyranosyl-(1 → 2)-β-d-glucopyranosyl-(1 → 4)-α-l-arabinopyranosyloxy]-16α-hydroxy-13β,28-epoxy-olean-30-al by NMR, UV, and IR spectroscopic methods. This new TTS was found to be effective in lowering blood-glucose level in the experimental rat model, thus establishing its antidiabetic property (168.8 ± 4.58) when compared with disease control (258.8 ± 0.60). Its LD₅₀ value was found at a dose of 2000 mg/kg. The level of insulin was restored by TTS and ethanol extract up to 31.49 µU/ml and 38.90 µU/ml, respectively, when compared with disease control (18.45 µU/ml).

Discussion and conclusion: In conclusion, 3-O[β-d-xylopyranosyl-(1 → 2)-β-d-glucopyranosyl-(1 → 4)-α-l-arabinopyranosyloxy]-16α-hydroxy-3β,28-epoxy-olean-30-al possesses potential glucose lowering properties, i.e., antidiabetic potential against STZ-induced diabetic rats.

• Characterization
• isolation
• triterpenoid saponin derivative

Introduction

Primula denticulate Sm. (Primulaceae), commonly known as drumstick primula, is grown in shady places and open slopes, along with shrubberies of mountain zones. Leaves are compact basal rosette sessile or narrowed into a winged petiole, elliptic or oblong spathulate, toothed, glabrous or sparsely ciliated, apex obtuse to give its morphological character (Gaur & Raiwani, 1995). The genus Primula L. (Primulaceae) includes about 400 species belonging to six subgenera and 37 sections. It is widely distributed outside of the Asian highlands and in the high altitudes of North America, Europe, and the eastern Sino-Himalayan region, which are considered the primary centre of diversity for this genus. Many Primula species are also cultivated throughout the world as ornamental plants. This plant has been recently studied by many taxonomists, ecologists, geneticists, and gardeners for its medicinal and therapeutic potential (Anderberg & El-Ghazaly, 2000; Kelso, 1991). Many species of Primula have attractive flowers and have proven to be important garden or house plants. The first comprehensive taxonomic study of Primula was completed by Smith and Fletcher (Gang et al., 2002; Richards, 2003; Yan et al., 2010). This plant reported to possess many triterpenoids including pridentigenins A–E, denticin, denticulatin, primulanin, and saxifragifolia B (Ahmad et al., 1980, 1990). It has rich sources of ascorbic acid (Jones & Hughes, 1983). Further it contains flavonoids like 5-hydroxyflavone, 2-hydroxy flavone, 5,2-dihydroxy flavone, and 5,8-dihydroxy flavone (Tokalov et al., 2004) and 5-hydroxy-6,2′-dimethoxyflavone (Wollenweber et al., 1990).

This plant is already known to possess antileukemia activity in cells of HL-60 with little antioxidative property and strong cytostatic properties (Tokalov et al., 2004). Traditionally, this plant is used for the treatment of diabetes and urinary disorder (Bhat et al., 2013).

To date, this plant was not explored for its antidiabetic activity experimentally. Hence, the present study was designed to re-isolate, characterize, and evaluate the novel triterpenoid saponin (TTS) from P. denticulate for antidiabetic activity.

Materials and methods

All the animal studies conducted were approved by the Institutional Animal Ethical Committee Siddhartha Institute of Pharmacy, Dehradun, India (SIP/IAEC/10/2011).

Plant material

The fresh plant leaves of P. denticulate were collected from Mussoorie, Uttarakhand, in the month of February 2012, and authenticated by Mr. Gaurav Upadhyay, Assistant Professor, Siddhartha Institute of Pharmacy, Dehradun, Uttarakhand (SIP/DPP/consult/14-04-12/170/056).

Chemicals

Silica gel (130–270 mesh), Sephadex LH-20 (Nicholas India Pvt. Ltd, Hyderabad, India), and glass column were used for column chromatography. All solvents used for chromatographic isolation were of analytical grade. Streptozotocin (STZ) and glipizide were purchased from Sigma Aldrich (St. Gallen, Switzerland) and Himgiri Chemicals, Dehradun, Uttarakhand.

Modified extractions and isolation procedure

Dried powder of P. denticulate (3.0 kg) was extracted with methanol (MeOH) at 48 °C under reflux. The extract was concentrated to dryness under reduced pressure to obtain a slurry (351 g). The residue was shaken with n-butanol (n-BuOH) and H₂O, then n-BuOH layer was evaporated. The residue was dissolved in the minimum amount of MeOH and diluted with cold diethyl ether (Et₂O) to yield a cream colored precipitate of crude saponin (15 g), 10 g of which was chromatographed on a silica gel column. The slurry was dissolved in the minimum amount of methanol and was adsorbed on the silica gel (60–120 mesh). The slurry was subjected to a silica gel column using a CHCl₃/MeOH gradient system (10:0, 9.5:0.5, 9:1, 8.5:1.5, 8:2, 7.5:2.5, 7:3, 6.5:3.5, 6:4, 5.5:4.5, 5:5, 4.5:5.5, 4:6, 3.5:6.5, 3:7, 2.5:7.5, 2:8, 1.5:8.5, 1:9 0.5:9.5, and 0:10). A TTS (500 mg) from the fraction of CHCl₃/MeOH (8.5:1.5) was isolated, which was further purified by rechromatography on silica gel (230–400 mesh size) and by HPLC using MeOH–H₂O (4:1) as solvent systems at a flow rate of 4 ml/min (Ahmad et al., 1988; Evans, 2009) (Figure 1).

Figure 1. Structure of compound 1.

Spectral data

TTS C₅₂ H₈₄O_22, Mp 238–239, UV λ_Max MeOH (nm): 205; IR (cm⁻¹): 3400–3200 (OH), 2900 (methylene), 1720 (CHO), 1040, and 890 (ether).

¹³C NMR (CDCl₃) (Table 1); ¹H NMR (CD₃OD, 300 MHz): δ O.83 (3H, s, H-25), 0.90 (3H, s, H-24), 0.94 (3H, s, H-29), 1.01 (3H, s, H-23), 1.14 (3H, s, H-26), 1.22 (1H, dd, J = 5.5, 13 Hz, H-18), 1.25 (3H, s, H-27), 3.01 (1H, d, J = 7.5 Hz, H·28), 3.19 (1H, m, H-3), 3.41 (1H, d, J = 7.8 Hz, H-28), 3.5 (1H, m, H-16), 3.0–3.9 (sugar protons), 4.33 (d, J = 3 Hz, H-1′), 4.53 (d, J = 7.6 Hz, H-1″), 4.54 (d, J = 7.4 Hz, H-1″″), 4.65 (d, J = 7.4 Hz, H-1″′), 9.38 (1H, S, H-30); positive FABMS m/z: 1083 [M + Na]⁺, 1060 [M + H]⁺; negative FABMS m/z: 1058 [M − H]⁺, 926 [M–pentose–H]⁺, 896 [M–glucose–H]⁺, 762 [M–glucose–pentose–H]⁺.

Table 1. ¹³C NMR spectral data of new triterpenoid saponin (TTS) (75 MHz, CD₃OD).

Position	^13C NMR	Position	^13C NMR
1	40.3	1′	103.5
2	27.1	2′	80.3
3	91.0	3′	73.7
4	40.3	4′	76.2
5	56.6	5′	66.2
6	18.5	1″	104.2
7	33.0	2″	84.6
8	42.3	3″	77.3
9	53.5	4″	71.5
10	38.1	5″	77.8
11	19.6	6″	63.6
12	30.7	1′″	106.3
13	87.8	2′″	75.7
14	45.8	3′″	77.1
15	34.3	4′″	71.0
16	77.3	5′″	67.4
17	44.7	1″″	105.4
18	50.0	2″″	77.2
19	36.7	3″″	79.1
20	48.6	4″″	71.9
21	34.8	5″″	77.5
22	34.0	6″″	62.2
23	28.1
24	16.5
25	16.5
26	19.1
27	19.5
28	78.3
29	24.6
30	209.3

Acid hydrolysis of TTS

1. TTS (20 mg) was refluxed with 0.1 M HCl in aqueous MeOH (5 ml) for 4 h. The reaction mixture was then concentrated under reduced pressure to remove MeOH. Addition of H₂O gave a white precipitate which was collected by filtration and identified as a mixture of two compounds, cyclamiretin A and D. The aqueous filtrate was adjusted to pH 7 with Ag₂CO₃ and filtered. The supernatant was concentrated under reduced pressure and compared with standard sugars on TLC (cellulose). The sugars were detected by spraying the plate with a standard solution of aniline phthalate in BuOH (Ahmad et al., 1988).

2. TTS (25 mg) refluxed with 10% H₂SO₄ on a boiling-water bath for 4 h the usual work of the reaction mixture confirmed by cyclamiretin A and D. Identification of sugar moiety: the aqueous layer separated after the removal of cyclamiretin and neutralized with barium carbonate, filtered, and concentrated at reduced pressure. The residue obtained was compared with standard sugar.

3. TTS (10 mg) and 1 ml of HCl (dioxane/H₂O) were placed in an ampule. The ampule was sealed and placed in an oven at 90 °C for 2 h. The ampule was cooled; its seal was broken, and its contents were dried in a stream of nitrogen. The contents of the ampule were suspended in H₂O (3 ml), and the aqueous suspension was washed with EtOAc (3 × 3 ml). After collection of the EtOAc fraction and removal of the solvent, the obtained yield was the aglycon. Neutralizing the aqueous hydrolysate with Ag₂CO₃ and centrifugation of the precipitate gave sugar (Woldemichael & Wink, 2001).

Antidiabetic activity

Animal selection

Healthy adult Wister rats of either sex weighing 150–180 g were selected for the study. The study was carried out in accordance with the rules and regulations of the Institutional Animal Ethics Committee. The animals were housed with free access to food and water. The basal food intake and body weights to the nearest gram were noted. Rats were starved 24 h prior to the study. The activity of TTS was evaluated in four groups of six animals in each (Chattopadhyay et al., 1997).

Acute toxicity study

The acute oral toxicity study was carried out in mice as per OECD guidelines. At a dose of 2000 mg/kg, 50% mortality was observed. Hence, 200 mg/kg body weight of TTS was taken as an effective dose for the evaluation of antidiabetic activity (Vogel, 2002).

Preparation of ethanol and aqueous extract

The ethanol and aqueous extract were prepared by a cold maceration method. For the preparation of ethanol extract, 100 g shade-dried leaf powder was macerated in 300 ml of distilled water while for aqueous extract the same weight of leaf powder was macerated in 300 ml of 95% v/v ethanol. Both extracts were allowed to stand overnight, then boiled for 5–10 min until the volume was reduced to half of its original volume, cooled, filtered, concentrated, dried in the vacuum (yield 60 g), and the extract was stored at 2–8 °C.

Preparation of TTS dose

The isolated TTS (200 mg/kg body weight) was suspended in 2% aqueous acacia solution. The standard drug glipizide (5 mg/kg body weight orally) was also given as a suspension in 2% normal saline. The control group received normal saline orally (OECD, 2001).

Induction of diabetes

STZ has been widely used to induce type-2 diabetes in animal models, especially in rats and mice. It has been reported that administration of STZ either by i.v. or by i.p. induced dose-dependent diabetes. I.p. injection of STZ led to physiologic alteration consistent with reports of spontaneous and chemically induced diabetes in other animals.

The rats with a body weight of 150–200 g were selected for the diabetogenic activity. The animals were deprived for food 24 h prior to administration of STZ. STZ was freshly dissolved in freshly prepared 0.01 M citrate buffer (pH 4.5). STZ was given i.p. (50 mg/kg body weight). The STZ solution was injected with 1 ml tuberculin syringe fitted with a 26 no. gauge needle within 15 min of dissolution in a buffer solution. Diabetes was confirmed by the determination of fasting glucose concentration on the third day post administration of STZ. After the seven-day stabilization period, animals having a blood-glucose level ≥250 mg/dl were selected for the study (Sridhar et al., 2011).

Administration of doses

Test substances were administered in a single dose by gavage. Animals were fasted 18 h prior to dosing. During fasting, the animals were weighed and substance was administered. Food was withheld for an additional 3–4 h after the dose administration.

Evaluation of antidiabetic activity

Before starting the experiment, animals were separated according to their body weight. The animals were injected intraperitoneally with STZ (i.p.) at a dose of 50 mg/kg body weight freshly prepared in 0.01 M citrate buffer (pH 4.5) solution.

To the animals, the test extracts (200 mg/kg body weight orally) and standard drug glipizide (5 mg/kg body weight orally) were administered by dissolving 5% aqueous Gum acacia. The animals were segregated into four groups of six rats each for each extract. Blood sample (0.2 ml) was withdrawn through the tail vein puncture technique using a hypodermic needle after administration of single oral dose. For all drugs, normal, diabetic control, and standard groups were kept the same for comparison with alcohol and aqueous extracts of drugs. The mean ± SEM was statistically calculated for each parameter.

Antidiabetic study in STZ-induced diabetes rats

The antidiabetic study was carried out in STZ-induced diabetic rats. The study was carried out for 21 d. The animals were fasted for 18 h before the experiment and blood-glucose levels were checked. It was considered as the day zero reading. The dose of extract was given orally daily to animals for 21 d. The blood-glucose levels were checked at 0, 7, 14, and 21 d period. The blood was collected by snipping the rats’ tail with a sharp razor. The collected blood was centrifuge at 2000 rpm for 15 min and determination of blood-glucose levels were carried out using a GOD-POD assay method by semi-autoanalyser (Nicholas India Pvt. Ltd, Hyderabad, India) (Gandhi & Sasikumar, 2012).

The following groups were formed for the determination of sub-acute study in STZ-induced diabetes rats:

• Group I: normal control received 5% aqueous gum acacia oral.

• Group II: diabetes control received 5% aqueous gum oral.

• Group III: received standard drug glipizide 5 mg/kg body weight oral.

• Group IV: received TTS, 200 mg/kg body weight oral.

• Group V: received ethanol extract, 1000 mg/kg body weight oral.

• Group VI: received extract, 1000 mg/kg body weight oral.

The results are tabulated in Table 2.

Table 2. Effect of isolated compound 1 on blood glucose level in STZ-induced diabetic rats.

	Blood glucose level in mg/dl
Groups	0th day	7th day	14th day	21st day
Normal control (NC)	90 ± 0.57	88.67 ± 0.61	89.17 ± 0.60	89.83 ± 0.60
Diabetes control (DC)	256.2 ± 1.01^###	258.1 ± 0.77^###	258.3 ± 0.76^###	258.8 ± 0.60^###
Standard (STD)	258.2 ± 1.35^ns	216.8 ± 2.96***	156.2 ± 5.81***	101.3 ± 2.55***
Triterpenoid saponin (TTS)	254.5 ± 0.95^ns	229.8 ± 3.38***	190.8 ± 4.23***	168.8 ± 4.58***
Ethanol extract	262.5 ± 1.91**	221.1 ± 2.47*	169.01 ± 1.09***	125.1 ± 3.50**
Aqueous extract	257.1 ± 3.22*	241.3 ± 3.90**	211.67 ± 2.74***	199.2 ± 2.33***

Results are expressed as mean ± SEM. ns, not significant, compared with diabetic control.

***p < 0.001, **p < 0.01, *p < 0.05, ###p < 0.001 compared to normal versus disease group.

Biochemical parameters

Blood was collected from the retro-orbital venous plexus of the rats under chloroform anesthesia using capillary tubes and Eppendorf tubes containing heparin. The plasma was separated by centrifugation (5 min, 5000 rpm) and was analyzed for plasma profiles, plasma insulin, acid phosphatase, alkaline phosphatase, serum glutamate oxaloacetate transaminase (SGOT), and serum glutamate pyruvate transaminase (SGPT) by using commercially available reagent (Kannan et al., 2012; Matsuda et al., 1999). The results are tabulated in Table 3.

Table 3. Effect of isolated compound 1 on plasma insulin and liver enzyme levels in STZ induced diabetic rats.

Parameters	Group I (NC)	Group II (DC)	Group III (STD)	Group IV (TTS)	Group V (EE)	Group VI (AE)
Plasma insulin (µUml^−1)	84.48 ± 0.44	18.45 ± 0.40	36.43 ± 0.53***	31.49 ± 0.54*	38.90 ± 0.45**	21.46 ± 0.81**
Acid phosphatase (IUL^−1)	6.88 ± 0.17	7.66 ± 0.62	7.58 ± 0.46^ns	8.73 ± 0.20^ns	8.01 ± 0.19*	7.91 ± 0.67***
Alkaline phosphatase (IUL^−1)	9.21 ± 0.44	8.04 ± 0.37	15.54 ± 0.31***	9.48 ± 0.30^ns	9.20 ± 0.08	8.71 ± 0.20**
SGOT (IUL^−1)	85.69 ± 0.69	178.70 ± 0.47	93.35 ± 0.37***	101.50 ± 0.44***	81.58 ± 1.06**	114.99 ± 1.81*
SGPT (IUL^−1)	63.36 ± 0.37	128.70 ± 0.52	105.80 ± 0.62***	79.39 ± 0.48***	64.91 ± 1.48**	101.09 ± 1.06**

Results are expressed as mean ± SEM. ns, not significant, compared with diabetic control.

***p < 0.001, **p < 0.01, *p < 0.05 compared to normal versus disease group.

Results and discussion

The present work was aimed to isolate the novel TTS compound from the ethanol extract of P. denticulate and study the antidiabetic activity in STZ-induced diabetic rats. The results from this study revealed that the isolated compound was characterized by spectral data. TTS at a dose of 200 mg/kg body weight and ethanol extract at a dose of 1000 mg/kg body weight significantly normalized the elevated blood-glucose level and restored serum and liver biochemical parameters towards normal values while the aqueous extract was unable to restore blood glucose and serum parameters significantly.

Structure elucidation

TTS was the triterpenoid saponin (3-O[β-d-xylopyranosyl-(1 → 2)-β-d-glucopyranosyl-(1 → 4)-α-l-arabinopyranosyloxy]-16α-hydroxy-13β,28-epoxy-olean-30-al) resulting from chloroform–methanol (8.5:1.5) elutant.

This was isolated from the ethanol extract of P. denticulate and further confirmed hydroxyl (3400–3200 cm⁻¹) and aldehyde (1720 cm⁻¹) groups in it. The end absorption at 205 nm suggested the absence of double bonds. Moreover, on acid hydrolysis, the product was cyclamiretin-A as the aglycone and d-glucose, l-arabinose, and d-xylose as sugar moieties. The negative ion was a molecular ion peak at m/z 1059 [M − H]⁺ which was due to FABMS spectrum and fragment ions at m/z 925, 896, and 762 attributed to the loss of a terminal pentose, a terminal glucose, and of a terminal glucose–pentose disaccharide or terminal pentose and terminal glucose unit, respectively (Ahmad et al., 1990).

The ¹H NMR spectrum of TTS in CD₃OD revealed the presence of six tertiary methyl groups through signals at δ 0.83, 0.90, 0.94, 1.01, 1.14, and 1.22. Moreover, there were peaks at δ 3.01 (1H, d, J = 7.6, H-28), 3.41 (d, J = 7.9 Hz, H-28), and multiplets at δ 3.19 and 3.5 due to H-3 and H-16, respectively. Four anomeric proton signals were also detected at δ 4.33 (d, J = 3 Hz, H-1′), 4.53 (d, J = 7.6 Hz, H-1″), 4.54 (d, J = 7.6 Hz, H-1″″), and 4.65 (d, J = 7.6 Hz, H-1″′) supporting the α-configuration of l-arabinose, and the β-configuration of d-glucose and d-xylose.

The sequence and configuration of the sugar moieties were also verified by the ¹³C NMR spectrum, in which four anomeric signals appeared at δ 103.5, 104.2, 106.3, and 105.4, consistent with the presence of α-l-arabinopyranosyl, β-d-glucopyranosyl, and β-d-xylopyranosyl configurations in a 1:2:1 ratio. Comparison of the ¹³C NMR spectrum (edited DEPT experiment) of 1 with those of the related compound also helped in deciphering the structure. It was confirmed by the ^l3C NMR data that cyclamiretin A was present with the sugar moieties attached in the C-3 position, as the C-3 signal of the aglycone appeared at δ 91.0. Assuming above, the structure of the isolated TTS is supposed to be 3-O{β-d-xylopyranosyl(1 → 2)-β-d-glucopyranosyl-(1 → 4)-[β-d-glucopyranosyl(1 → 2)]-α-l-rabinopyranosyloxy} 16α-hydroxy-13β,28-epoxy-olean-30-al.

The presence of a novel TTSs compound in P. denticulate prompted us to perform an antidiabetic evaluation with the new compound. We evaluated the antidiabetic potential of TTS, ethanol extract, and aqueous extract. A significant change is its sugar-lowering properties in the experimental rats from day 7 to 21 of the experiment (Table 2). The result was comparatively promising when glipizide was used as a standard (Petit et al., 1993). On the 21st day of the experimental protocol, blood sugar in diabetic rats was significantly (p < 0.001) lowered to 168.8 mg/dl by TTS compared to diabetic control (258.8 mg/dl). Ethanol extract also lowered blood glucose significantly (p < 0.01) to 125.1 mg/dl compared to the diabetic control (258.8 mg/dl) while the aqueous extract was unable to lower blood sugar level by a significant level. Comparison of TTS and ethanol extract clearly reflects that antidiabetic activity of the extract is due to the major presence of a triterpenoid. Triterpenoid is well known for its antidiabetic activity (de Melo et al., 2010; Kazmi et al., 2012; Ramirez-Espinosa et al., 2011; Sarabu & Tilley, 2005). Triterpenoids can reduce blood glucose through increased insulin secretion, as evidenced in our experiment by STZ-induced diabetic rats, which is capable of modulating pancreatic secretion. The level of insulin was restored by TTS and ethanol extract up to 31.49 µU/ml and 38.90 µU/ml, respectively, when compared with disease control (18.45 µU/ml). Increase of insulin level by the new TTS and ethanol extract clearly indicated that this new triterpenoid molecule produces an antidiabetic effect by triggering insulin section from pancreatic β-cells.

Other changes and biochemical parameters like acid phosphatase, alkaline phosphatase, SGOT, and SGPT were also measured for checking and comparing associated complications with diabetes. It is clear from Table 3 that there were only little changes in these biochemical parameters, and diabetic animals were safe from other diabetic complications during therapy.

Conclusion

The present study concludes that the isolation of TTS (3-O {β-d-xylopyranosyl (1 → 2)-β-d-glucopyranosyl-(1 → 4)-[β-d-glucopyranosyl (1 → 2)]-α-l-rabinopyranosyloxy} 16α-hydroxy-13β, 28-epoxy-olean-30-al) and ethanol extract tested for antidiabetic activity has shown appreciable results in decreasing the blood glucose level and other complications associated with diabetes. This research supports the inclusion of this plant in traditional antidiabetic preparations and the formulations made using these identified effective compounds of this plant could serve the purpose better than existing formulations.

Declaration of interest

None to declare.