GC-MS analysis of n-hexane extract of Fagonia indica Burm.f. with hypoglycaemic potential

Abstract The present study was aimed at gas chromatography-mass spectrometry (GC-MS) analytical investigation of n-hexane extract of the aerial parts of Fagonia indica to identify hypoglycaemic compounds. Also, to investigate this extract for lactase enzyme inhibition responsible for hypoglycaemic activity. Phytochemical screening, GC-MS analysis and lactase inhibition of n-hexane extract was performed by the standard methods. GC-MS analytical study identified 15 compounds in this extract. The maximum percentage of lactase enzyme inhibition of n-hexane extract was 26.21 ± 1.25% (IC50 value of 311.2 ± 16.09 μg/mL) at 100 μg/mL concentration. The standard acarbose showed lactase inhibition of 63.21 ± 0.92% (IC50 value of 32.51 ± 0.85 µg/mL) at the same concentration. n-Hexane extract can be a potential source in the management of diabetes due to the presence of biologically active hypoglycaemic compounds. Graphical Abstract


Introduction
Diabetes mellitus (DM), a chronic metabolic disorder occurs by the abnormality in metabolism of carbohydrates linked mostly with no sensitivity of target organs towards insulin (Deshmukh and Jain 2015).It is commonly characterised by hyperglycaemia in which blood sugar level is increased significantly.This results in the disturbance of normal metabolism of body that is related to the breakdown of fat, protein, and carbohydrate.It proves fatal if not managed or treated properly (Mohan and Nandhakumar 2014).It leads to major complications like retinopathy, diabetic neuropathy and cardiovascular disease (Tahya and Karnelasatri 2021).Worldwide there is an increase in number of cases of DM and this effects all areas of the world.Approximately 170 million of people all over the world had diabetes in 2000 and this number will raise to 366 million in the year 2030 (Loh and Hadira 2011).As per WHO report, approximately 1.6 million deaths were directly and indirectly due to diabetes in 2016 which makes it 17 th leading cause of death worldwide (WHO 2020).There are many control measures on diabetes type 2 including exercise, dietary changes and medications.Recently there is growing interest in the plant-based therapeutic approaches for maintaining normal levels of blood glucose (Feskens et al. 2020).The inhibition of carbohydrate metabolizing enzymes (a-amylase and a-glucosidase) is one of the most significant chemotherapeutic targets for diabetes management.Inhibition of these enzymes slows down the digestion of carbohydrate and decrease the absorption of glucose, thus reduced the postprandial glucose levels of plasma (Oboh et al. 2016).The occurrence of DM is increasing rapidly among the communities of developing countries (Tee and Yap 2017).There is an increase in diabetic patients every year worldwide, especially in the countries of low-middle income (Kumar et al. 2011;Munasaroh et al. 2018).It is one of the serious and leading health concerns all over the world.There is rise in DM due to the prevalence of increasing obesity status in worldwide population (Lee et al. 2010).It is a multifactorial disorder which is characterized by hyperglycemia (increase in glucose levels).It is primarily categorised as type 1 DM (insulin dependent diabetes) and type 2 DM (non-insulin dependent diabetes).Type 2 DM accounts for more than ninety percent of all diabetes cases worldwide.It is a metabolic disorder which is characterized by the resistance in insulin and deficiency of insulin (Kimmel and Inzucchi 2005).There is much contribution of postprandial hyperglycaemia to the overall control on glycaemia in type 2 diabetes patients (Woerle et al. 2004).Oscillating and hyperglycaemic concentrations of blood glucose attribute directly in developing cardiovascular (CV) disease (Ceriello et al. 2008), so a proper control on the level of blood glucose can slow or protect against developing such complications (Kato et al. 2008).Managing postprandial hyperglycaemia is considered to be a main therapeutic strategy in the management of diabetes type 2. It can be achieved by slowing the glucose release by inhibiting carbohydrate metabolising enzyme alpha-glucosidase (lactase) in the digestive tract.Inhibition of this enzyme class slows gastrointestinal (GI) absorption of dietary carbohydrates.This occurs by the restriction in breakdown of branched or linear oligosaccharide units such as maltose, lactose and maltotriose to generate glucose so inhibiting absorption of glucose into the blood stream (Lee et al. 2012).This enzyme inhibition assists in avoiding diabetic complications (Proenc ¸a et al. 2017).Commercial available alphaglucosidase inhibitors are mostly used as mono therapy in addition to combination with other hypoglycaemic agents to slow postprandial raise in level of blood glucose (Fujisawa et al. 2005).A large number of medicinal plants are different in their ability for the management and to cure different human ailments which owe for the contribution of valuable phytoconstituents present in various parts of plant (Yuan et al. 2016;Anand et al. 2019;Semwal et al. 2019).Since ancient times the plants have been the medicinal source and several modern drugs are derived from the natural sources.Botanical remedies are used for the management of human disease because the plants contain novel components of multitude in nature having therapeutic value (Qureshi et al. 2016).Plant-derived compounds or phytomedicines are cheaper, safer, sometimes more effective and are available easily than synthetic ones.Pharmacological and ethnobotanical studies exhibited the hypoglycaemic nature of medicinal plants used traditionally in managing diabetes (Magaji et al. 2020).
Family Zygophyllaceae is famous for 25 genera and 240 species which is confined to subtropical, tropical and warm temperate, mostly in dry areas of the world.All of the plants of this family and especially genus Fagonia are of much interest to pharmacists as previous pharmacological studies have proved that these plants have much medicinal potential for treating health disorders of broad spectrum in nature (Qureshi et al. 2016).Fagonia indica Burm.f. is a significant medicinal plant which is distributed in various parts of Indo-Pakistan sub-continent and westwards to North and East Tropical Africa (Farheen et al. 2015).Several ethnomedicinal knowledge is linked to this plant and is used in the treatment of a wide range of diseases such as urinary discharges, fever, vomiting, asthma, skin diseases, thirst, dysentery, typhoid, toothache, stomach troubles (Gulshan et al. 2012).It is used traditionally in managing diabetes (Yaseen et al. 2015).It has become the subject of intense pharmacological and chemical studies due to the popularity of this plant in folklore medicine.
F. indica had been reported for a number of biological activities.The compounds indicacin and fagonicin isolated from this plant were investigated for cytotoxic activity against H-29 human cancer cell line.Indicacin showed 51.41% cytotoxicity and fagonicin showed 39.3% cytotoxicity at 6.25 mM/mL dose (Farheen et al. 2015).Ethanolic extract of the plant was investigated for in vitro antimicrobial activity against bacteria and fungi by agar diffusion technique.The plant had this effect against some bacterial and fungal strains (Gehlot and Bohra 2000;Jigna et al. 2005;Mandeel and Taha 2005).Ethanol extract of the plant leaves was investigated for anti-inflammatory activity by the method (carrageenan rat paw edema).The plant exhibited a significant (p ˂ 0.05) activity in comparison to the reference (Joseph et al. 2016).Methanolic extract of the plant was investigated for hepatoprotective activity in albino rats induced by carbon tetrachloride and it had significant effect (Bagban et al. 2012).Aqueous and ethanolic extracts of the plant leaves were investigated for analgesic activity in rats.The extracts had significant analgesic activity (Sharma et al. 2009).Hydro-alcoholic extract of the plant was evaluated for antipyretic activity in rabbits.The plant exhibited higher activity compared to control groups (Ahmad et al. 2014).Antioxidant activity of the plant was investigated using electron spin resonance machine and it lowered free radicals effectively (Eman 2011).Ethanol extract of F. indica var.schweinfurthii was investigated for hypoglycemic activity in alloxan-induced hyperglycemic mice.It lowered blood glucose levels on acute basis (Mahdy and Shehab 2015).
Commonly analytical instrumental techniques included liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance spectroscopy (NMR) (Easmin et al. 2017).These techniques provide sensitivity, separation and reproducibility at higher level needed for analysing a complex biological mixture (Wu et al. 2006).Recently fourier-transform infrared and gas chromatography-mass spectrometry techniques have been employed for detecting various functional groups and the identification of bioactive therapeutic compounds present in the medicinal plants (Fan et al. 2018;Satapute et al. 2019).
There is need for the identification and characterisation of chemical compounds present in n-hexane extract of this plant responsible for hypoglycaemic effects.No investigational reports are present about the presence of chemical compounds in this extract having hypoglycaemic effects.So, this study was aimed to investigate the metabolite profile of n-hexane (non-polar) extract of the aerial parts of Fagonia indica (var.indica) using GC-MS based metabolomics.Also, to evaluate its hypoglycaemic effects using in vitro a-glucosidase (lactase) enzyme inhibition assay.This study will be useful for the characterisation purpose of various phytoconstituents.

Results and discussion
Phytochemicals were found to be present in n-hexane extract while alkaloids, anthraquinones, proteins and carbohydrates were absent in this extract.Quantitative analysis of n-hexane extract was not done.Tannins, flavonoids and terpenoids were detected in n-hexane extract by qualitative analysis.Qualitative phytochemical analysis of F. cretica (var.schewenfurthii) showed the presence of flavonoids, terpenoids, alkaloids, tannins, saponins, coumarins, glycosides and sterols in various polar and non-polar extracts of the plant (Eman 2011;Sajid et al. 2011;Dastagir et al. 2012, Hossen et al. 2016).The presence of saponins, amino acids and terpenoid glycosides flavonoids has also been reported in ethanolic and aqueous extracts of this plant (Sharma et al. 2009).The compound fagonilin was isolated from the aerial parts of the plant along with lupeol, b-amyrin and b-sitosterol (Farheen et al. 2014).Also nayabin A-G (sulphated triterpenoid glycosides) and a known triterpenoid glycoside were isolated from this plant (Kanwal et al. 2017).Flavonoid compounds were isolated and then identified in ethanol extract of this plant (Shehab et al. 2011).A known glucopyranosyl ester and flavonoid glycoside was also isolated from the plant (Shaker et al. 2013).Plants show various biological activities due to the phytochemical constituents present in the plants.
The analysis of n-hexane extract was done by one of the most applied techniques gas chromatography-mass spectrometry (GC-MS) which is used for the separation of phytocomponents.The compounds were identified by scrutinising the spectral pattern and by comparing with GC-MS NIST database library which could contribute for the medicinal uses of the plant (Konappa et al. 2020).The compounds identified were primarily plant fatty acids and esters of palmitic acid, octadecatrienoic acid, hexadecatrienoic acid and heptadecatrienoic acid.Gas chromatography which is combined with mass spectroscopy is a significant tool for the analysis of chemical compounds.It is a source of qualitative information of the chemical components and mass spectrum is used to characterize compounds.Fifteen therapeutic important compounds were identified in n-hexane extract of F. indica through GC-MS analysis.Chemical compounds with their molecular formulas and molecular weights are given in supplementary material.Identified compounds in this study were of non-polar in nature which comprised of fatty acids.HPLC analysis of n-hexane fraction of F. indica (var.Schweinfurthii) showed the presence of caffeic acid, ferulic acid, gallic acid, benzoic acid, qercetin and syrinic acid as major compounds (Malik et al. 2017).There is the presence of hydrogen bonding between the hydroxyl (-OH) group of the identified compounds along with the residues having side chains of polar in nature and side chains of electrically charged species.Hydrophobic bond and p-interactions between the structure of compounds and side chains of hydrophobic in nature of the enzyme lactase could be explained by many activities of these compounds of non-polar in nature.It is agreed to the results as reported in literature (You et al. 2016) were exhibited to have potential glucose inhibition capacity.Some of the compounds identified had proved to show a-glucosidase inhibition activity.An investigation was done on the inhibition of enzyme lactase by fatty acids included palmitic acid (Liu et al. 2011).Fatty acids proved to be the active inhibitors of a-glucosidase included hexadecanoic acid (methyl ester), octadecanoic acid, pentadecanoic acid and heptadecanoic acid (Murugesu et al. 2018).It was reported by Marella et al. (2013) that fatty acids in the plants show strong hypoglycaemic effects.
In vitro a-glucosidase (lactase) inhibition for hypoglycaemic activity of n-hexane extract of F. indica is given in supplementary material with IC 50 (half-maximal concentration) values.The highest a-glucosidase (lactase) inhibition observed for n-hexane was 26.21 ± 1.25% while the standard acarbose showed 63.21 ± 0.92% inhibition at 100 mg/mL concentration.GC-MS analysis with hypoglycaemic potential of only n-hexane extract was investigated which is non-polar in nature.a-glucosidase enzyme is the most significant enzyme of chemotherapeutic drug targets in diabetes type 2 management.The levels of blood glucose can be managed by the inhibition of enzymatic properties of this enzyme.Postprandial glucose peaks may be decreased by slowed absorption of glucose.The main benefits which are attributed to the inhibitors of a-glucosidase are reductions in total range of postprandial levels of glucose and postprandial glycemic levels (Li et al. 2005).Intestinal a-glucosidase inhibition is bound to inhibit the digestion of lactose, sucrose and maltose substrates to an absorbable glucose (Priscilla et al. 2014).A large number of medicinal plants are known to have a-glucosidase enzyme inhibition properties (Somtimuang et al. 2018;Yang et al. 2019).The present study showed that n-hexane extract of F. indica possesses strong inhibition property against a-glucosidase (lactase) enzyme.Different solvent extracts of another plant Carica papaya seeds exhibited considerable lactase inhibitory property with IC 50 value ranged from 75.78 to 102.4 mg/ml compared to the standard acarbose (Agada et al. 2020).n-Hexane fraction of another variety showed 31.55 ± 0.84% a-amylase inhibitory activity (Malik et al. 2017).But no earlier report is present about n-hexane extract of F. indica (var.indica) investigated for lactase inhibition activity.The present study for the first time shows n-hexane extract of the plant to be a potential source of antidiabetic agents.The inhibition of enzyme a-glucosidase (lactase) is proved to be an ideal one to prevent hyperglycaemia.It is due to that it can prolong digestion process of carbohydrate in the intestinal tract, which lengthen the time of absorption of glucose, and finally leading to a lesser level of postprandial blood glucose (Sharma et al. 2016).The results suggest that hypoglycaemic effect of n-hexane extract of the plant is shown through inhibition of intestinal a-glucosidase which is a key enzyme of carbohydrate digestion present on the brush borders of intestinal cells surface membrane.This study confirmed the hypoglycaemic nature of the chemical compounds present in n-hexane extract of the plant.So, this study is useful in the discovery of new drugs using the aerial parts of F. indica.

Conclusion
It is concluded from this finding that GC-MS analysis showed the presence of natural chemical hypoglycaemic compounds in n-hexane extract of F. indica responsible for inhibiting alpha glucosidase (lactase) enzyme.It supports the traditional usage of this plant.Further studies are recommended to isolate and characterise these hypoglycaemic compounds.

Disclosure statement
No potential conflict of interest was reported by the author.

Funding
The author(s) reported there is no funding associated with the work featured in this article.