ANTI-DIABETIC ACTIVITIES OF ISOLATED COMPOUND BETA-SITOSTEROL FROM THE ETHANOLIC EXTRACT OF STEM OF ANDROGRAPHIS ECHIOIDES

ANTI-DIABETIC ACTIVITIES OF ISOLATED COMPOUND BETA-SITOSTEROL FROM THE ETHANOLIC EXTRACT OF STEM OF ANDROGRAPHIS ECHIOIDES

Manigandan * and V. Subramaniyan

PG and Research Department of Biotechnology, J.J. College of Arts and Science, J.J Nagar, Sivapuram Post, Pudukkottai, Tamil Nadu, India.

ABSTRACT: To study the anti-oxidant and anti-diabetic activities of isolated compound beta-sitosterol from the ethanolic extract of stem of Andrographis echioides under the in-vitro model. The isolation was done using column chromatography using gradient elution with different mobile phases. Structural elucidation was carried out on the basis of spectral analysis. Anti-oxidant activity was determined by 2,2-diphenyl-1-picrylhydrazyl scavenging assay. The anti-diabetic activity was evaluated by the inhibitory potential of isolated compound beta-sitosterol against alpha-amylase and alpha-glucosidase assays. The study revealed that the beta-sitosterol exhibited significant α-amylase (58.57±0.29) and α-glucosidase (43.85±0.032) inhibitory activities respectively and well compared with standard acarbose drug. The beta-sitosterol showed the best scavenging activity (74.46±0.036) against the tested radicals like 1,1-diphenyl-2-picrylhydrazyl, The infra-red spectrum specific absorption bands for beta-sitosterol viz:  3427.78 cm^‐1 (O‐H stretching.); 2937.40 cm^‐1 (aliphatic C‐Hstretching); 1640.58 cm^‐1 (C=C absorption peak); other absorption peaks includes 1464.31cm^‐1 (CH₂); 1381.56 cm^‐1 (OH def), 1054.11 cm^‐1 (cycloalkane) and 800.97 cm^‐1. Structural elucidation of beta-sitosterol was done by spectrum analysis such as 13C and 1H depth nuclear magnetic resources. Therefore, it is concluded that beta-sitosterol is a potential source for natural anti-oxidant and anti-diabetic compounds and could have potential use in the management of diabetes mellitus.

Keywords: Andrographis echioides stem, betasitosterol, Anti-oxidant activity, alpha amylase and alpha glucosidase inhibitory activity

INTRODUCTION: Diabetes mellitus is a chronic metabolic disorder, and it also affects the metabolism of carbohydrates, protein, and fat. The main reason is the production of the low amount of insulin by pancreas ¹. Type I diabetes occurs due to low amount of insulin production by ? cells, while type II diabetes occurs due to ? cell dysfunction ².

Diabetes mellitus type 1 and type 2 are associated with microvascular complications and macro-vascular complications. Microvascular compli-cations: Hyperglycemia and hypertension is the major reason for microvascular complications. Diabetic nephropathy is the leading cause of mortality ^{3, 4}.

The enzymes alpha-glucosidase are responsible for the breakdown of oligo- and disaccharides to monosaccharides. α -amylase and α- glucosidase inhibitors is useful for lowering the process of glucose absorption and decreases glucose level in blood ^{5, 6}. Diabetes mellitus patients suffer from a high blood sugar level, unusual thirst, frequent urination, extreme hunger, loss of weight, blurred vision, nausea and vomiting, extreme weakness and irritability, tiredness and mood change ⁷. Inhibitors of amylase and α glucosidase are responsible for the high amount of glucose in the blood ⁸. Now day’s herbal medicines are more effective than synthetic medicines. There is no side effect while using herbal medicine ^{9, 10}.

Andrographis echioides belong to the Acanthaceae family, and its Tamil name is Gopuramthangi ^{11, 12}. It is an ayurvedic herb plant used in the treatment of many ailments such as anti-inflammatory, anti-arthritic, antimicrobial, anti-ulcer, anti-oxidant activity, hair problems, etc., ^13-14.

Their phytochemical constituents like flavonoids, tannins, phenol, glycosides, terpenoids, saponins, steroids, etc., Andrographis echioides is a medicinally valuable species widely distributed in the tropical region in South Asian countries ^15-19.

Therefore, in the present study, the anti-oxidant and anti-diabetic activities of beta-sitosterol isolated from the ethanolic extract of stem of Andrographis echioides were evaluated employing in-vitro assay methods.

MATERIALS AND METHODS:

Collection of Plant Material: The stem of Andrographis echioides was collected from Manarkkudi, Mayiladurai, Nagapattinam district, Tamil Nadu, India, in December 2019. It was authenticated by Dr. N. Ravichandran, Research associate, Centre for advanced research in Indian system of Medicine, (CARISM) and SRC; SASTRA Deemed to be University, Tirumalaisamuthiram, Thanjavur (Voucher no. SRC SASTRA 0005).

Chemicals and Reagents: All the chemicals, including solvents such as ethyl acetate, n-hexane, chloroform, methanol, anisaldehyde sulphuric acid reagents (0.5 ml p-anisaldehyde in 50 ml glacial acetic acid and 1 ml conc. sulfuric acid was of analytical grade and was procured from E. Merck, India. Alpha (α)-Glucosidase, porcine pancreas alpha (α)-amylase, p-nitrophenyl-α-D-gluco-pyranose (p-NPG), 3,5-dinitrosalicylic acid (DNS), 1,1-Diphenyl-2-picrylhydrazyl (DPPH), ascorbic acid and acarbose were purchased from Sigma Chemical Co. (St. Louis, MO, USA) was purchased from Sigma‐Aldrich, New Delhi.

Preparation of Ethanol extracts: The stems of Andrographis echioides were washed in running water, cut into small pieces and then shade dried for a week at 35-40 ºC, after which it was grinded to a uniform powder of 40 mesh size. The ethanol extracts were prepared by soaking 1.5 kg of the dried powder plant materials in 1 L of ethanol by continuously using a soxhlet extractor for 10 hr. The extracts were filtered through Whatman filter paper No. 42 (125mm). The filtered extract was concentrated and dried by using a rotary evaporator under reduced pressure. The final dried samples (998 g) were stored in labeled sterile bottles and kept at -20 ºC. The filtrate obtained was used as a sample solution for further isolation ²⁰.

Isolation of Beta-sterol by Column Chromatography: The condensed methanol extract of the stem (500 g) of the sample was subjected to column chromatography over TLC grade silica gel. Elution of the column first with n-hexane, an increasing amount of ethyl acetate in n-hexane and finally with methanol yielded a number of fractions. The preparation of solvent systems used to obtain beta-sitosterol (234 mg/500 g) was ethyl acetate-methanol (60:40) from fraction 7. The compounds were detected on TLC plates by spraying with Libermann Burchard reagent and heated at 100 °C for 10 min ²¹.

Purification of Isolated Compounds Beta-sitosterol by HPTLC and High-Performance Liquid Chromatography:

High-Performance Thin-layer Chromatography (HPTLC): The isolated pure compound was dissolved in appropriate solvents. 5 μl of isolated compounds (beta-sitosterol) were applied to silica gel plates, Merck (Germany) 20 × 20 cm, 0.25 mm in thickness. Plates were developed using the solvent system n-hexane: ethyl acetate (8:2 v/v). The separated zones were visualized with freshly prepared Libermann Burchard reagent and heated at 100 °C for 10 min. Chromatograms were then examined under daylight within 10 min ²².

High-performance Liquid Chromatography (HPLC): The analytical HPLC system (Shimadzu) was equipped with a diode array detector, a 20 µl loop, 200 × 4.6 mm C18 column, methanol (HPLC grade, 0.2 mm filtered) used as a mobile phase. The isolated beta-sitosterol compounds were separated using a mobile phase of n-hexane: ethyl acetate (8:2 v/v) at a flow rate of 1.0 ml/min, column temperature 30 °C. Injection volume was 40 μl, and detection was carried out at 346 nm ²³.

Structural Elucidation Study of Isolated Compound: Different spectroscopic methods including U.V., FTIR, 1H NMR, 13C NMR were used to elucidate the structure of isolated compounds. The U.V.–visible spectrum of the isolated compounds in methanol was recorded using a Shimadzu 160A U.V.–visible spectro-photometer. The Fourier Transform Infrared (FTIR) spectra were recorded with a nominal resolution of 4 cm^-1 and a wavenumber range from 400 to 4000 cm^-1 using the KBr pellet technique.

1H and 13C NMR spectra were acquired on Bruker WP 200 SY and AM 200 SY instruments (1H, 200.13 MHz; 13C, 50.32 MHz) using TMS as internal standard and CDCL₃ as solvent. GC-MS analysis of the extract was performed using a Perkin-Elmer G.C. Clarus 500 system and Gas chromatograph interfaced to a Mass spectrometer (GC-MS) equipped with an Elite-I, fused silica capillary column (30 mm × 0.25 mm 1D × 1 μMdf, composed of 100% Dimethylpolysiloxane)  ^{24, 25}.

Anti-oxidant Activity (DPPH Free Radical Scavenging Activity) Determination: The anti-oxidant activity of the isolated compound beta-sitosterol was examined on the basis of the scavenging effect ²⁶. Ethanolic solution of DPPH (0.05 mM) (300 μl) was added to 40 μl of isolated compound with different concentrations (20 – 100 μg/ml). The freshly prepared DPPH solution was kept in the dark at 4 °C. 96% (2.7 ml) of ethanol was added and shaken vigorously. The mixture was kept constant for 5 min, and absorbance was measured at 517 nm spectrophotometrically. Ethanol was used to set the absorbance at zero. A blank sample was also prepared, which contains the same amount of ethanol and DPPH. All the determinations were performed in triplicate. The radical scavenging activities of the tested samples expressed are calculated as a percentage of inhibition according to the equation,

DPPH activity (% inhibition) = [(A – B) / A] × 100

Where A and B are the absorbance value for the test and blank sample, respectively.

In-vitro Antidiabetic Activity of Beta-sitosterol from the Stem of Andrographis echnoides:

Alpha-Amylase Inhibitory Assay: The alpha-amylase inhibitory assay was carried out by using the isolated compounds beta-sitosterol from the stem of Andrographis echioides. A total of 250 μL of beta-sitosterol compound (20-100 μg/ml) was placed in a tube and 250 μL of 0.02M sodium phosphate buffer (pH 6.9) containing α-amylase solution (0.5 mg/mL) was added. This solution was pre-incubated at 25 ºC for 10 min, after which 250 μL of 1% starch solution in 0.02M sodium phosphate buffer (pH 6.9) was added at regular time intervals and then further incubated at for 25 ºC for 10 min. The reaction gets terminated by the addition of 500 μL of dinitrosalicylic acid (DNS) reagent. The tubes were then incubated in boiling water for 5 min and cooled at room temperature. The reaction mixture was diluted by adding 5 mL distilled water, and the absorbance was measured at 540 nm using a spectrophotometer. A control was prepared during the same procedure by the replacement of the extract with distilled water. The α-amylase inhibitory activity was calculated in terms of percentage inhibition ²⁷.

%Inhibition = [(Abs control – Abs beta-sitosterol) / Abs control] × 100

Alpha-glucosidase Inhibitory Assay: The activity of isolated compound beta-sitosterol on α-glucosidase was determined by using α-glucosidase from Saccharomyces cerevisiae. P-nitro phenyl glucopyranoside (p-NPG) was prepared in 20mM phosphate buffer as a substrate solution and pH 6.9. 100 μL of α- glucosidase (1.0 U/mL) was pre-incubated with 50 μL of the different concentrations (20-100 μg/ml) of the isolated compound for 10 min. Then 50 μL of 3.0 mM (pNPG) substrate was dissolved in 20 mM phosphate buffer (pH 6.9) were added to start the reaction. The reaction mixture was incubated at 37 ºC for 20min and stopped by adding 2 mL of 0.1 M sodium carbonate. The α-glucosidase activity was determined by measuring the yellow-colored p-nitrophenol released from pNPG at 405 nm. The results were expressed in the percentage of the blank control. The α-glucosidase inhibitory activity was calculated by percentage inhibition ²⁸.

% Inhibition = [(Abs control – Abs beta-sitosterol) / Abs control] × 100

Statistical Analysis: All assays were conducted in triplicate. Statistical analyses were performed with SPSS 16.0 for an analysis of variance (ANOVA) followed by Duncan's test. Differences at P < 0.05 were considered to be significant.

RESULTS AND DISCUSSION:

Structural Elucidation of Isolated Compounds: Beta-sitosterol melting point 136 °C, M.W.: 414.7 g/mol, which corresponds to the molecular formulae C₂₉H₅₀O. The U.V. λ_max value of compound beta-sitosterol was 257 nm Fig. 1.

FIG. 1: UV SPECTRA OF THE ISOLATED COMPOUND BETA-SITOSTEROL

The IR absorption spectrum showed absorption peaks at 3427.78 cm^‐1 (O‐H stretching.); 2937.40 cm^‐1 (aliphatic C‐Hstretching); 1640.58 cm^‐1 (C=C absorption peak); other absorption peaks includes 1464.31 cm^‐1 (CH2); 1381.56 cm^‐1 (OH def), 1054.11 cm^‐1 (cycloalkane) and 800.97 cm^‐1 Fig. 2.

FIG. 2: IR SPECTRA OF THE ISOLATED COMPOUND BETA-SITOSTEROL

In the proton 1H NMR spectra of beta-sitosterol Fig. 3 δ 2.28 (1H, m, H‐3), 5.36 (1H, m, H‐6), 5.34 (1H, m, H‐23), 5.34 (1H, m, H‐22), 2.28 (1H, m, H‐3), 2.27(1H, m, H‐20), 1.8‐2.0 (5H, m) ppm. Other peaks are observed at δ 0.76‐0.89 (m, 9H), 0.91‐1.07 (m, 5H), 1.28‐1.43 (m, 4H), 0.69‐0.79 (m, 3H), 1.81‐2.00 (m, 5H), 1.07‐1.12 (m, 3H), 1.28‐1.58 (m, 9H) ppm.

FIG. 3: 1H NMR SPECTRA OF THE ISOLATED COMPOUND BETA-SITOSTEROL

FIG. 4: 13C NMR SPECTRA OF THE ISOLATED COMPOUND BETA-SITOSTEROL

In the proton 13C NMR spectra of beta-sitosterol Fig. 4 showed 140.7 (C‐5), 138.3 (C‐22), 121.7, 129.2 (C‐6), 77.4 (C‐3), 55.9(C‐14), 55.9(C‐17), 50.1 (C‐9), 45.8 (C‐9), 40.5 (C‐20), 39.7(C‐12), 39.6 (C‐13), 38.8 (C‐4), 38.8 (C‐12), 37.2 (C‐1), 37.2 (C‐10), 36.5(C‐8), 35.8(C‐20), 33.9 (C‐22), 33.9(C‐7), 32.4 (C‐8), 29.1 (C‐25), 28.9 (C‐16), 28.26 (C‐2), 28.2 (C‐15), 26.0 (C‐28), 26.0 (C‐11,26), 21.2 (C‐27), 19.8 (C‐19), 17.5 (C‐21), 15.4 (C‐18, 29). The structure was confirmed by comparison with literature data.

FIG. 5: MASS SPECTRA OF THE ISOLATED COMPOUND BETA-SITOSTEROL

Mass spectrum of isolated compound beta-sitosterol showed parent molecular ion [M+] peak at mlz 414, which corresponds to the molecular formula C₂₉H₅₀O.

The GCMS spectra of these isolated compounds revealed the characteristic fragments m/z with % abundance 414.2, 396.5, 381.3, 329.6, 303.4, 289.5, 273.2, 255.1, 231.0, 213.4, 199.2, 173.5, 159.1, 145.1, 119.3, 95.7, 81.6, 69.3, 55.4.

The molecular weight and fragmentation pattern indicate that the compounds were presenting beta-sitosterol, respectively Fig. 5.

Purification of Isolated Compound by HPTLC and HPLC: HPTLC fingerprint patterns have been therefore evolved to check the purity of isolated compound from methanolic extract of the sample.

The R_f value of standard beta-sitosterol 0.70 was matched with the R_f value of isolated compound beta-sitosterol was about 0.70 was shown in peak Fig. 6.

FIG. 6: HPTLC CHROMATOGRAM OF PURITY OF THE ISOLATED COMPOUND (A) STANDARD BETA-SITOSTEROL (B) ISOLATED BETA-SITOSTEROL IN ETHANOLIC EXTRACTS OF STEM OF
ANDROGRAPHISECHIOIDES

The Retention time of beta-sitosterol isolated from the ethanolic extracts of stem of Andro-graphisechioides. was about 8.274 was shown by HPLC peak Fig. 7.

FIG. 7: HPLC SPECTRA OF PURITY OF THE ISOLATED COMPOUND BETA-SITOSTEROL

Previous studies suggested that the beta-sitosterol, are the main bioactive compound, has been reported to possess anti-inflammatory, anti-oxidant, anticancer, hepatoprotective, and hypoglycaemic activities ²⁹. On subjection to I.R. spectroscopic analysis, absorptions bands appeared at 3426 cm^-1 that is characteristic of O‐H stretching, 2868 cm^-1 is due to aliphatics or C‐H stretching or (CH₃), 1540 cm^-1 due to double (C=C) stretching, 1054 cm^-1 due to (C-O).

The absorption frequency at 738 cm^‐1 signifies cycloalkane. The out-of-plane C‐H vibration of the unsaturated part was observed at 591 cm^‐1. These absorption frequencies resemble the absorption frequencies observed for β-sitosterol as resembled data published ^{30, 31}.

The 1H NMR spectrum (300MHz, CDCl₃) of compound Fig. 7 has revealed a one proton multiplet at δ 2.26, the position and multiplicity of which was indicative of 3H of the steroid nucleus.

The typical 6H of the steroidal skeleton was evident as a multiplet at δ 5.36 that integrated for one proton. The spectrum further revealed signals at δ 1.49 and δ 1.19 (3H each) assignable to two tertiary methyl groups at C- 18 and C-19, respectively.

The 1 HNMR spectrum showed two doublets centered at δ 0.90 (J = 6.7Hz) and δ 0.91 (J = 6.7Hz), which could be attributed to two methyl groups at C-26 and C -27 respectively. The doublet at δ 1.65 (J = 6.5Hz) was demonstrative of a methyl group at C-21. On the other hand, the triplet of three proton intensities at δ 0.86 could be assigned to the primary methyl group at C- ²⁹. This compound is having six methyl, eleven methylene, and three quaternary carbons with a hydroxyl group.

The above spectral features are in close agreement with those observed for β – Sitosterol (Manoharan et al., 2005 and Escudero et al., 1985) ^{29, 30}. The 13C‐NMR has shown recognizable signals of 140.76 and 129.27 ppm, assigned C5 and C6 double bonds, respectively.

The value at 24.31 ppm corresponds to the angular carbon atom (C19). Spectra show twenty-nine carbon signals, including six methyls, nine methylenes, eleven methane, and three quaternary carbons. The alkene carbons appeared at 140.76 and 129.27 ppm. In comparison, the standard data matched with the simulated data, which supports the proposed structure of this compound as β – Sitosterol ^32-35.

Anti-oxidant Activity of Isolated Compound Beta-sitosterol by DPPH Method: The isolated compound beta-sitosterol contains the best anti-oxidant activity at high concentrations when compared with ascorbic acid Fig. 8. The compound showed 74.46% activity at 100 µl/ml at the same time ascorbic acid gave 95.03 at the same concentration Table 1.

The previous studies concluded that the free radical scavenging activity was showed by β-sitosterol, which implies its concentration-dependent antioxidant activity.

Low percent inhibition was observed as 11% at a concentration of 12.5 µg/ml, while high inhibition was observed at a maximum concentration of 1000 µg/ml ³⁶.

TABLE 1: IN-VITRO ANTI-OXIDANT ACTIVITY OF THE BETA-SITOSTEROL USING DPPH METHOD AND COMPARISON WITH STANDARD DRUG ASCORBIC ACID

Each value was obtained by calculating the average of four experiments, and data are presented as mean ± SEM

FIG. 8: GRAPHICAL REPRESENTATION OF DPPH INHIBITORY ACTIVITY OF ACARBOSE VS. BETA-SITOSTEROL ISOLATED FROM ANDROGRAPHIS ECHIOIDES STEM

In-vitro Alpha-Amylase Inhibitory Assay:  In this study, in-vitro alpha-amylase inhibitory activities of the beta-sitosterol isolated from ethanolic extract of Andrographis echioides stem was examined. The isolated compound 48.57 0.365 to 58.57 0.29 showed inhibitory activity at a concentration of 100 μg/ml Table 2.

TABLE 2: IN-VITRO ANTI-DIABETIC ACTIVITY OF THE BETA-SITOSTEROL USING ALPHA-AMYLASE METHOD AND COMPARISON WITH STANDARD DRUG ACARBOSE

Each value was obtained by calculating the average of four experiments and data are presented as mean± SEM

FIG. 9: GRAPHICAL REPRESENTATION OF Α-AMYLASE INHIBITORY ACTIVITY OF ACARBOSE VS. BETA-SITOSTEROL ISOLATED FROM ANDROGRAPHIS ECHIOIDES STEM

Acarbose is a standard drug for α-amylase inhibitors. Acarbose at a concentration of (20-100 μg/ml) showed α-amylase inhibitory activity from 60 0.28 to 77.14 0.16% at the same concentrations 100 μg/ml. A comparison of α–amylase inhibitory activity between the standard drug is shown in Fig. 9. Gurupriya et al., 2018 suggested that lupeol is a potential source for natural anti-diabetic and anti-oxidant compounds and could have potential use in the Management of diabetes mellitus ³⁷.

In-vitro α-glucosidase Inhibitory Assay: The result of ? glucosidase inhibitory assay of anti-diabetic activity of beta-sitosterol isolated from ethanolic extract of Andrographis echioides stem are shown in Table 3.

TABLE 3: IN-VITRO ANTI-DIABETIC ACTIVITY OF THE BETA-SITOSTEROL USING ALPHA GLYCOSIDASE METHOD AND COMPARISON WITH STANDARD DRUG ACARBOSE

Each value was obtained by calculating the average of four experiments and data are presented as mean± SEM

FIG. 10: GRAPHICAL REPRESENTATION OF Α- GLYCOSIDASE INHIBITORY ACTIVITY OF ACARBOSE VS. BETA-SITOSTEROL ISOLATED FROM ANDROGRAPHIS ECHIOIDES STEM

The percentage inhibition various from 21.05 0.0455 to 43.85 0.032 for the high concentration to the lowest concentration. A comparison of ? glucosidase activity between the standard and isolated compound beta-sitosterol showed in Fig. 10. A previous study suggested that anti-oxidant activity of the plant extract occurs due to the presence of phenolic compounds and their redox properties, hydrogen donor capacity, and singlet oxygen quenching ^38-41.

CONCLUSION: From the present study, beta-sitosterol isolated and characterized from the ethanolic extract of Andrographis echioides stem showed maximum inhibitory activity of anti-oxidant and anti-diabetic activities under in-vitro conditions. α-amylase inhibitory action decreases the digestion of carbohydrates, and α-glucosidase reduces glucose levels in blood. An isolated compound, beta-sitosterol, showed inhibitory action of free radical scavenging activity. The beta-sitosterol has inhibitory activity against α-amylase and α-glucosidase, and this therapeutic potentiality could be exploited in the management of postprandial hyperglycemia in treating type 2 diabetes mellitus.

ACKNOWLEDGEMENT: The author extends their thanks to the management, Principal of J.J. College of arts and science, (Autonomous) Pudukkottai, Tamil Nadu, India, for providing the facilities to do the preliminary research work in the Department of Biotechnology. Dr. G. Manigandan acknowledges Dr. K. Manjula, Managing Director, Bio Techno Solutions Training and Research Institute, Tiruchirapalli, for providing me the infrastructure to carry out the proposed research work. Dr. G. Manigandan acknowledges Dr. N. Ravichandran, Research associate, Centre for advanced research in Indian system of Medicine (CARISM), for identifying and authenticating the plants.

AUTHOR CONTRIBUTION: All authors contributed equally to this manuscript.

CONFLICTS OF INTEREST: The authors declare that they have no conflict of interest. It has not been published elsewhere. That it has not been simultaneously submitted for publication elsewhere. All authors agree to the submission to the Journal.

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    How to cite this article:

    Manigandan G and Subramaniyan V: Antidiabetic activities of isolated compound beta-sitoterol from the ethanolic extract of stem of Andrographis echioides. Int J Pharm Sci & Res 2021; 12(9): 5125-33. doi: 10.13040/IJPSR.0975-8232.12(9).5125-33.

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