Discussion
Phytochemical components in the plant extracts are considered to be active biologically and are accountable for various actions like antidiabetic, anticancer, antifungal, anti-inflammatory and antibacterial [
36,
37]. Two types of metabolites are produced by a plant, primary metabolites i.e. lipids, carbohydrates and proteins and secondary metabolites which include alkaloids, phenolics, terpenes, essential oils, tannins, flavonoids, sterols. The literature study revealed that natural compounds, secondary metabolites played a significant role in the healing of various disorders [
38,
39]. Typically, the extraction of the secondary metabolite is based on the polarity of the solvent and its interaction with preferred compounds [
40,
41]. The phytochemical analysis of rhizome’s methanolic extract of
Sorghum halepense (L.) Pers showed the presence of flavonoids, cardiac glycosides, terpenes, carbohydrates, steroids, alkaloids and proteins. The methanolic extract lacked saponins and gums whereas aqueous and chloroform fractions contained carbohydrates and steroids respectively. On other hand, the cardiac glycosides were missing in the aqueous fraction whilst flavonoids in the chloroform fraction (Table
1). Comparable results were reported in earlier studies of
Salix mucronata and
Datura metel L [
42,
43]. A number of reports on phenolic compounds, like terpenoids and flavonoids showed their strong biological efficacies like antidiabetic, antioxidant and anticancer [
44,
45]. The antibacterial, antimalarial, cytotoxic and anticancerous characteristics of alkaloids [
46] and Na-K-ATPase inhibitory potential of cardiac glycosides [
47] has been reported. Similarly, common phenolic compounds, flavonoids are commonly found in the plants [
48] and have antioxidant, antiallergic, antibacterial, antiviral, antineoplastic, antidiarrheal, anti-thrombotic, anti-inflammatory and vasodilatory properties [
49]. Furthermore, significant antidiabetic characteristics of the plant based bioactive compounds including phenolic compounds, alkaloids, flavonoids, tannins, terpenoids, glycosides have been reported [
50‐
52]. The incidence of the mentioned secondary metabolites in the plant extract and its fractions indicated that
Sorghum halepense (L.) Pers may have cytotoxic, antioxidant antidiabetic capabilities associated with different diseases as mentioned in preceding studies [
53‐
55]. The cytotoxic capabilities of methanolic extract and its fractions were determined by brine shrimp lethality bioassay. The methanolic extract and its chloroform, aqueous and
n-hexane fractions caused brine shrimp lethality up to 70.5 ± 1.2%, 50.4 ± 1.1%, 50.3 ± 1.1% and 40.3 ± 1.6% respectively at the amount of 1000 μg/ml (Table
2). It proposes that the plant extract have credible antimicrobial constituents. Consistent results obtained during the studies of
Coscinium blumeanum, Fibraurea tinctoria and Arcangelisia flava [
56] and
Hapllophyllum tuberculatum [
57].
The present results (Table
3) revealed that the methanolic extract has the highest amount of total phenolic content (28.30 ± 1.3 mg GAE/g) followed by chloroform fraction (17.34 ± 1.43 mg GAE/g), an aqueous fraction (12.7 ± 1.32 mg GAE/g) and
n-hexane fraction (8.87 ± 1.35 mg GAE/g).
Several existing reports confirm that the plant based extracted and isolated phenolic compounds have phenolic hydroxyl groups; can contribute hydrogen atom or unpaired electron and hence neutralize the free radicals efficiently [
58,
59]. The present antioxidant compounds in various plants and even different parts of the same plant have different natures and quantities. Therefore, it is essential to adopt more than one assay to authenticate the antioxidant capability of tested samples [
60]. The capabilities of methanolic extract of rhizomes of
Sorghum halepense (L.) Pers and its
n-hexane, chloroform and aqueous fractions to scavenge free radical was assessed by choosing commonly used standard assay i.e. DPPH, ABTS and H
2O
2 assays [
61]. The free radicals produced during these assays were scavenged by the antioxidant constituents in the plant extract. In DPPH assay, DPPH (a, a-diphenyl-b-picrylhydrazyl) is changed into a, a-diphenyl-b-picrylhydrazine along with alteration in its color indicating the scavenging potential of the plant extract and is measured spectrophotometrically. ABTS assay is suitable for both hydrophilic and lipophilic antioxidants [
62]. In this essay, ABTS and potassium persulfate react together to generate a blue chromophore (ABTS•+). The mentioned cation is reduced by reacting with plant extract or standard antioxidant (ascorbic acid) [
35]. H
2O
2 by itself is a puny oxidizing agent but through the oxidation of essential thiol (−SH) groups of enzymes, it can inactivate few enzymes directly. It can enter into the cell by crossing the cell membranes easily. Hydrogen peroxide is catalyzed to hydroxyl radicals and singlet oxygen subject to its exposure to transition metal ions. Singlet oxygen is more toxic to the cellular system than H
2O
2 by itself. Also the hydroxyl radical may be the cause of its various poisonous effects [
63], hence it is vital for cells to control the quantity of H
2O
2 biologically.
The literature study showed that the aforementioned available compounds in the extract of rhizomes of
Sorghum halepense (L.) Pers have the ability to donate hydrogen ions and hence de-colorization of DPPH and ABTS solution [
11,
53,
55,
64]. The highest free radicals scavenging potential of methanolic extracts were found 40.02%, 40.48% and 50.85% in DPPH, ABTS and H
2O
2 assays (Fig.
1,
2 and
3) respectively. The difference in free radical scavenging might be owing to the variations in the number of aromatic rings, nature of hydroxyl groups and molecular weight as well as with the number of active components in the extract and its fractions which change their concentrations by fractionation [
54,
55,
64]. The elevated antioxidant capacity of methanolic extract might be owing to more phenolic contents in the mentioned extract as compared to its
n-hexane, chloroform and aqueous fractions. The correlation of total phenolic content with the antioxidant activities (Table
4) was found significant. (R
2 = 0.8081, 0.81 47 and 0.8023 for DPPH, ABTS and H
2O
2). These correlations predict that antioxidant characteristic is reliant on phenolic content of a sample. Congruent correlations between antioxidant activities and phenolic contents of different types of sorghum were reported previously [
65,
66]. Moreover, strong correlations between phenolic content and antioxidant activities in other cereals such as finger millet and wheat have been documented [
67,
68].
Today in the world, diabetes is a major degenerative problem resulting in a number of complications like hypertension, atherosclerosis and microcirculatory disorders [
69]. The α-amylase catalyzes the hydrolysis of α-(1, 4)-D-glycosidic linkages of starch and oligosaccharides and liberate monosaccharides in the intestine and thus contribute to hyperglycemia in diabetes. It can be limited by restraining α-amylase in the intestine which slows down the decomposition of starch and oligosaccharides to monosaccharides, reduces assimilation of glucose and consequently decrease postprandial blood glucose level [
70]. The mechanism of anti-hyperglycemic potential of extract is unknown. Possibly, it might be due to the presence of flavonoids, terpenes, tannins and alkaloids which might have caused alpha-amylase inhibition. On the basis of their strong binding ability with proteins to form an insoluble and indigestible complexes, they are extensively used as inhibitors [
71]. Further, it could elucidate that the phenolics compounds are not the only contributor to antioxidant activity but also causes enzyme inhibition. The α-amylase inhibition might depend on different factors like the methoxy groups, hydroxyl position and lactone rings or the interaction between compounds [
72].
The antidiabetic properties of available commercial medicine, glucophage (standard), methanolic extract and chloroform fraction were measured (63.14%), (61.87%) and (22.66%) respectively (Fig.
4). The anti-diabetic capacity of methanolic extract and standard were close and comparable. The percent inhibition of alpha-amylase was found to be dose-dependent. Similar results were also recorded during the in vitro study of
Solaria cuspidate leaves [
73]. It assumes that the said extract is a valuable source of significant antidiabetic components and was subjected to in vivo study.
The key organ, pancreas determines the energy and dietary state of the body via blood glucose level and secret insulin in response to a raise in blood glucose level [
69]. In a situation, where a number of functional beta cells become too limited to produce enough insulin to carry out the body necessities, insulin-dependent diabetes results [
74]. Alloxan (beta-cytotoxin), due to its deleterious properties to pancreatic β-cells is renowned for inducing experimental diabetes in several animal species including rats. In this study, alloxan was used to induce diabetes in rats [
75] and thereafter treated with different dozes of plant extract and standard drug (glibenclamide). On 21st day of treatment, a substantial (
P < 0.01) decline in the body weight of the diabetic control group was observed with respect to normal control group whereas the groups nourished with the methanolic extract of
Sorghum halepense (L.) Pers significantly (
P < 0.05) reinstated their body weights with respect to diabetic control (Fig.
5). The higher dose (300 mg/kg) of extract indicated considerable development in the weight of diabetic rats at the same interval. Furthermore, comparable increase (
P < 0.05) in body weight of glibenclamide treated group was noticed on comparison with diabetic control group (Fig.
5) at the same interval. The restorative effects of
Sorghum-tigernut Ibyer extract on changed glucose concentration, tissue and enzyme damages, and loss of weight in diabetic rats was determined by Shiekuma and coworker [
76]. The treatment of diabetics rats with methanolic extract of
Sorghum halepense (L.) Pers (150 and 300 mg/kg b.w; Fig.
6) for 21 days significantly (
P < 0.05) decreased the increased concentration of glucose in serum. This decline in serum glucose level was determined from the difference between the initial and final fasting concentrations of glucose in serum and was compared with diabetic control and reference standard. Comparable findings were found during evaluation of antidiabetic properties of
Sorghum bicolor grains [
77].
Usually the increase in aminotransferases is a well-known clue of liver dysfunction and is more frequent in diabetic patients than the common population. Moreover, several diabetic complications such as neuropathy, retinopathy and restricted mobility of joints are associated with the function of liver enzymes, regardless of body mass index, alcohol consumption and metabolic control of diabetes [
78]. Significant raise in action of several enzymes such as beta-glucuronidase, N-acetyl-beta-glucosaminidase, leucine aminopeptidase and lysosomal acid phosphatase and cathepsin D have been observed following the injection of alloxan in previous studies [
79]. Liver dysfunction under diabetic condition leads to increased activities of alkaline phosphatase (ALP), alanine aminotransferase (ALT), total bilirubin and aspartate aminotransferase (AST) with respect to non diabetes (Table
5). In diabetic animals, the alteration of enzymes in serum are directly associated with the metabolic variations wherein these enzymes are concerned. In the lack of insulin, the elevated actions of transaminases are owing to more availability of amino acids in diabetes and are responsible for the increased ketogenesis and gluconeogenesis found in diabetes. During the recent study, the oral feeding of extract (150 and 300 mg/kg b.w.) has restored the concentrations of ALT, total bilirubin, ALP and AST as indicated in Table
5 [
80]. Hence, the obvious restoration in the concentration of the mentioned enzymes (Table
5) was the effect of better metabolism of proteins, fats and carbohydrates. Following the treatment, the revival of ALT and bilirubin levels also indicated the recovery of insulin secretion. In alloxan induced diabetic rats, the plant extracts have revealed the restoration of altered levels of ALT, ALP, total bilirubin and AST in earlier studies [
81,
82]. Raise in serum urea and creatinine levels owing to diabetic hyperglycemia were considered the significant indicators of renal dysfunction [
83,
84] and reveals a decline in the rate of glumerular filtration. The level of creatinine and urea was normalized significantly (
P < 0.05) by treating the rats with methanolic extract (Table
7). Harmonious consequences were found in the antidiabetic assessment of three varieties (Heuin sorghum, Chal sorghum and Hwanggeumchal sorghum) from Korean sorghum (
Sorghum bicolor L. Monech) [
85]. Alloxan induces hypercholesterolemia in diabetics rats, and therefore, evident hyperlipidemia, which shows diabetic state, can be the consequence of inhibition of lipolytic hormones and thus decrease in catalysis of fat deposits [
86]. The level of HDL cholesterol, total cholesterol triglycerides, LDL and VLDL cholesterol was considerably recovered by methanolic extract of
Sorghum halepense (L.) Pers (Table
6) which shows that it demonstrates hypolipidemic characteristics. The inhibition of the synthesis of fatty acid may lead to lower the level of lipid. Normally insulin triggers lipoprotein lipase which hydrolyses triglycerides whereas in diabetic the owing to a shortage of insulin, inactivation of the mentioned enzyme may result in hypertriglyceridemia [
87]. Following the treatment with
Sorghum halepense (L.) Pers extract, a remarkable drop-off in serum lipid level in diabetic rats can be directly related to the restoration of insulin level. Analogous findings were achieved during the antidiabetic study of Sorghum and
Galium tricornutum extracts [
81,
88].
An enzymatic antioxidant protection mechanism can be decreased with an increase in the concentration of lipid peroxidation [
89]. In previous studies, the formation of oxygen free radicals in diabetic β cells and its deleterious effects have been documented. The mentioned cells can be saved from oxidative reparation by overexpression of antioxidant enzymes like SOD and CAT [
82,
90,
91]. In the liver tissues, significant improvement was observed in the activities of SOD and CAT after treatment with the extract (150 and 300 mg/kg b.w.) during the current study. This recommends that the oxidative stress in diabetes was reduced by the applied extract due to its efficient antioxidant characteristics [
90] and can enhance the activities of CAT and SOD [
82,
92].
Dysfunction of β-cells and insulin resistance are key markers of diabetes [
93] and the later one has a close relationship with altered lipid profile and serves as the major constituent of other metabolic disorders besides diabetes. For example, insulin resistance has been shown to be concerned with diabetic dyslipidemia characterized by high level of VLDL, TG, total cholesterol and low level of HDL [
94,
95]. Consequently, the lipid profile is considered in almost all follow-up programs of diabetes and maybe useful for early intervention and hampering the progression of diabetes [
93,
96]. After treatment with RSH methanolic extracts, a significant decline in the levels of lipid, ALT and bilirubin in the serum of diabetic rats can be directly related to the restoration of insulin concentration and thereby reduction in blood glucose level. Evaluation of medicinal plants with an aim to find new compounds having therapeutic activities such as antioxidants, hypolipidemic and antidiabetic [
97,
98] is an emerging research area.