Pinus roxburghii Sarg. is a traditional plant used in the treatment of diabetes mellitus ethnopharmacology in India and Africa. In this written report, we looked into the antidiabetic activity using α-amylase inhibitory assay on extracts from the bark of Pinus roxburghii Sarg. by bioassay guided fractionation. The ethanol extract (43.4% inhibition) and the isolated compound (49.6% inhibition) exhibited significant enzyme inhibitory activity against α-amylase. This is reported, from this plant, for the first time. The 1H and 13C NMR, IR and mass spectral studies of isolated compound suggested it as quercetin. Our study revealed, for the first time, the isolation and α-amylase inhibitory activity of quercetin from Pinus roxburghii Sarg. bark.
Keywords: Pinus roxburghii Sarg.; α-amylase; Quercetin; Diabetes; Ethanolic extract; Bioassay guided isolation
Diabetes mellitus is a metabolic issue portrayed by perpetual hyperglycemia or expanded blood glucose levels with unsettling influences in fat, starch and protein digestion system coming about because of total or relative absence of insulin secretion . Postprandial hyperglycemia is a noticeable and early imperfection in diabetes  which can thus prompt different auxiliary complexities including danger component for cardiovascular ailments . One helpful way to diminishing the hyperglycemia, particularly after a feast, is to retard and lessen the assimilation and retention of ingested carbohydrates through the hindrance of carbohydrate hydrolyzing enzymes (α-amylase) in the digestive system. Accordingly, these inhibitors could diminish the postprandial climb in blood glucose concentration . α-Amylase is one of the enzymes in the digestive framework that catalyzes the breakdown of starch to maltose lastly to glucose, which is the main sugar that can be used by the body . Enzyme inhibitors can be a potential focus in numerous ranges of ailment control and treatment, as enzymes catalyze the most vital biochemical pathways .
The greater part of the monetarily accessible amylase and glucosidase inhibitors are of microbial origin. Their utilization has been restricted because of their symptoms, for example, fast and loose bowels because of colonic maturation of undigested sugar [7,8]. At the point when contrasted with the microbial partners, amylase inhibitors from therapeutic plant are extensively sheltered and compelling. Among 1200 plants which have hypoglycemic property, just 30% of the opposition to diabetic plants have been pharmacologically tried and explored . In this manner, α-amylase inhibitors can be utilized to treat disease such as diabetes, obesity and hyperlipaemia .
Pinus roxburghii Sarg. (Family: Pinacae) is a pine inhabitant to the Himalaya . Pinus roxburghii Sarg. has reported to exhibit different pharmacological activities such as anti-inflammatory, analgesic  anticonvulsant , antimicrobial  and anticancer  activities. Indian and African healers are using bark and leaf of Pinus roxburghii Sarg. to treat diabetes . Our recent in silico studies demonstrated that secoisoresinol and different phytoconstituents from bark of Pinus roxburghii Sarg. is compelling against aldose reductase,  which is mainly responsible for secondary complications of diabetes . Therefore, in the present study bioassay guided fractionation of ethanolic extract and α-amylase inhibitory activity of isolated flavonoid was evaluated.
General experimental procedures
The IR spectrum was obtained from a Perkin Elmer, Model: Spectrum-100. NMR experiments were carried out on a Bruker 300 MHz spectrometer using tetramethylsilane (TMS) as internal standard. The ESI-MS were recorded on an Agilent Chem station Gas Chromatograph equipped and coupled to a mass detector with a polar column.
Collection of plant material
The bark of Pinus roxburghii Sarg. were gathered from the hilly region of Morni, District Panchkula, Haryana, and was authenticated by Dr. A.K Sharma, Sr. Scientist at Department of Natural Product, FRI, Dehradun, Uttarakhand, India, where a voucher specimen no. 129 FHH was deposited for future reference.
Extraction, isolation and chromatography
Shade dried coarse powdered bark of Pinus roxburghii Sarg. in a quantity sufficient as per the volume of the extractor was packed in a thimble (made of filter paper sheet) and sequentially extracted with petroleum ether, chloroform, ethyl acetate and ethanol. A sufficient volume of solvent was added to the reservoir, and hot continuous extraction process in a soxhlet extractor was started. This extraction process was continued for about 48 hours or until alcohol coming down the siphoning tube became colorless. The over abundance of solvent was distilled under reduced pressure using a rotatory vacuum evaporator. (Heidolph Laborota 4011, digital). All the extracts were evaluated for their potential to inhibit the enzyme α-amylase. As ethanolic extract proved to be most promising extract was further subjected to portioning between n-butanol and water. The n-butanol soluble fraction thus obtained was subjected to column chromatography over silica gel (number 60–120) and eluted gradually with chloroform:ethyl acetate:methanol (5:4:1). Fractions were pooled according to their similarity in behavior on thin layer chromatography. The fractions were concentrated to finally obtain compound 1 (21 mg). The compound 1 was dissolved either in methanol, chloroform, or dimethyl sulfoxide depending on its solubility for analysis. The structure of the isolated compound was elucidated from the data obtained from IR, 1H-NMR and 13C-NMR spectra.
Alpha amylase inhibitory activity
The alpha amylase inhibitory activity was carried out by the method devised by . Briefly, the total assay mixture containing 200 μl of 0.02M sodium phosphate buffer, 20 μl of enzyme (α-amylase) and the plant extracts at a concentration of 100 μg/ml were incubated for 10 min at room temperature followed by addition of 200 μl of 1% starch in all the test tubes. The reaction was terminated with addition of 400 μl of di-nitro salicylic acid color reagent, placed in boiling water bath for 5 min, cooled to room temperature and diluted with 15 ml of distilled water and the absorbance measured at 540 nm (Systronic- UV-VIS spectrophotometer). The control samples were also prepared accordingly without any plant extracts and were compared with the test samples containing the plant extracts prepared with different solvents. The results were expressed as % inhibition calculated using the formula:
Data are expressed as mean ± S.E.M. The data was analyzed by one-way analysis of variance (ANOVA) followed by Dunett’s t test to ascertain the level of significance using GraphPad InStat version 3.05 for Windows. Values of p<0.05 were considered statistically significant.
Alpha amylase inhibitory activity
Acarbose (at a concentrations 100 μg/mL) showed 56.7% inhibitory effects on the α-amylase activity (Table 1). The Compound 1 (at a concentration 100 μg/mL) exhibited 49.6% of α-amylase inhibitory activity. The ethanol, ethylacetate and chloroform extracts of Pinus roxburghii Sarg. (at a concentration 100 μg/mL) exhibited 43.4%, 40.1% and 30.5% of α-amylase inhibitory activity respectively. However, the pet-ether extract did not show α-amylase inhibitory activity. Both ethanol extracts and compound 1 showed appreciable α-amylase inhibitory effects when compared with acarbose (56.7 %).
|Extract||Concentration||Absorbance||a-amylase (% inhibition)|
|Compound 1||100 µg/ml||0.443±0.02||51.1|
|Ethyl acetate||100 µg/ml||0.550±0.02||40.1|
|Pet ether||100 µg/ml||---||No activity|
Table 1: Enzyme inhibition activity of isolated constituents.
Identification of the chemical structure of the isolated compound
The compound was characterized by comparison of their spectroscopic data with those reported in literature. From these data and those presented in Table 2, compound 1 was identified as quercetin.
|Carbon no||literature data||Isolated compound data|
|d13C||d 1H||d13C||d 1H|
|C-6||98.3||6.18 (d, J = 2.0 Hz)||97.69||6.17 (s)|
|C-8||93.5||6.37 (d, J = 2.0 Hz)||92.84||6.38 (s)|
|C-2’||115.0||7.73 (d, J = 2 Hz)||114.51||7.7 (s)|
|C-5’||115.8||6.87 (d, J = 8.0 Hz)||114.97||6.88 (d, J=8.4 Hz)|
|C-6’||120.1||7.62 (dd, J = 2.0, 8.0 Hz)||119.51||7.55 (d, J=8.4 Hz)|
|3-OH||12.49 (s)||12.43 (s, 5-OH)|
|5-OH||9,6 (br s)||9.27 (br s)|
br s-broad singlet, j=coupling contant, s-singlet, d-doublet, dd-doublet of doublet
Table 2: 1H NMR and 13C NMR data of isolated compound.
Compound 1: IR (cm-1): 3433 (-OH), 1651 (-C=O), 1026 (-C-O); 1H-NMR (300 MHz, CDCl3,δ): 6.17 (s, 1H, 6-H), 6.38 (s, 1H, 8-H), 6.88 (d, J=8.4Hz, 1H, 5’-H), 7.55 (d, J=8.4Hz, 1H, 6’-H), 7.7 (s, 1H, 2’-H), 9.27 (bs, 4H, 5, 6, 3’,4’-OH), 12.43 (s, 1H, 3-OH); 13C-NMR (75 MHz, CDCl3,δ): 92.84, 97.69, 102.53, 114.51, 114.97, 119.51, 121.56, 135.20, 144.45, 146.19, 147.06, 155.69, 160.34, 163.34, 175.26.
Anal. Calcd. for : C15H10O7; C, 59.61; H, 3.33; Found: C, 59.48; H, 3.11; O, MS (EI, m/z): 303.14 (M+1)+. The spectral data of compound 1 (Figure 1) closely matched that of 3,3', 4',5,7-pentahydroxyflavone (quercetin) reported in the literature [20,21].
In this study, quercetin, showed the highest α-amylase inhibitory activity. The % inhibition values for α-amylase inhibition by quercetin and acarbose (as the positive control) were 49.6 and 56.7 respectively. This is the first report of the α-amylase inhibitory activity of quercetin, the isolated compound from Pinus roxburghii Sarg. Also, previous studies have reported the isolation of sitosterol , tannins , hexacosyferrulate  from Pinus roxburghii Sarg., but the isolation of quercetin from the plant has not previously been reported. Moreover quercetin isolated from other plants has shown prominent antidiabetic activity . This study shows the possibility of using ethanol extract of Pinus roxburghii Sarg. and quercetin to decrease postprandial hyperglycaemia. Also, the study justifies the use of Pinus roxburghii Sarg. in the management of diabetes mellitus by Indian and African healers.