Natural remedies offer alternative or complementary treatments for various diseases and are usually considered to be safe. For specific diseases that require lifelong pharmaceutical medication, therapeutic safety is important. For example, diabetes mellitus can be a lifelong disease and is globally of serious concern due to its complications. Several medicinal plants have been studied and scientifically proven to be beneficial for the treatment of diabetes mellitus [1
]. Plant-based natural medicines are safe and cost-effective. In fact, in addition to bioactive phytochemicals, medicinal plants do have bioactive elements in appropriate ratios, and thus, they are helpful in maintaining a balance of trace elements in disturbed metabolic processes diabetic mellitus [5
]. However, medicinal plants need to be scientifically explored for assessment of efficacy and safety. In addition to phytochemical analysis, the detection of phyto-elemental composition and elemental ratios of medicinal plants is expected to play a pivotal role in evaluation of probable medicinal activity [6
Recently, laser-induced breakdown spectroscopy (LIBS) has emerged as a promising analytical spectroscopy for the elemental analysis of samples of interest [20
]. Although other conventional analytical spectroscopic techniques such as inductively-coupled plasma mass spectroscopy (ICP-MS), X-ray photoelectron spectroscopy (XPS) [29
], atomic absorption spectroscopy (AAS), inductively-coupled plasma optical emission spectrometry (ICP-OES), and flame atomic absorption apectroscopy (FAAS) are available for elemental analysis, LIBS shows several advantages. Particularly, LIBS can be rapid, shows minimal environmental loads, requires minimal efforts in sample preparation, and provides in situ real-time analysis of the sample in solid, liquid, or gas phases [30
]. Furthermore, LIBS is capable of simultaneous multi-elements detection and evaluation of their concentration and intensity ratios, which help for establishing a correlation of a particular bioactivity of any part of a plant with their specific elemental composition.
The current study addresses the generation of the bio-active profile of leaves of thrichosantes dioica
from the evaluation of elemental ratios with laser-plasma spectroscopy and the interpretation of the results in view of the anti-diabetic potency for in vivo models. Trichosanthes dioica
, or t. dioica
, is a vegetable of the cucurbitaceae
family. It is widely grown throughout India and to a lesser extent in other parts of South Asia [31
]. It is also known as ’Parwal’ in Hindi, or colloquially, the pointed gourd is called a green potato. In addition to the fruit, its leaves are also used as a vegetable. Leaves of t. dioica
of weight 100 g contain 9.0 mg of magnesium, 2.6 mg of sodium, 83.0 mg of potassium, 1.1 mg of copper, and 17.0 mg of sulfur [32
]. Its pointed gourd leaves and delicate shoots are applied in traditional medicinal therapy. The literature reveals that the effect of feeding shade-dried leaves to animals impacts their blood glucose level and lipid profile [33
]. The plant seeds are commonly used to treat acid reflux symptoms and display antifungal and antibacterial activities [35
]. The present study was conducted to evaluate the glycemic potency of aqueous extract of t. dioica
leaves on blood glucose levels (BGLs) of normal and streptozotocin-induced subdiabetic, mildly diabetic and severely diabetic models. For a complete biomedical profile of the aqueous extract of t. dioica
leaves, several other parameters were monitored, including TC, HDL, LDL, VLDL, TG, AST, ALT, ALKP, and CRTN. Based on the parameter ranges, and especially the LFT (liver function test) and KFT (kidney function test), one can infer that t. dioica
leaves should be developed as a promising anti-diabetic agent for the management of diabetes type II and associated complications.
The present study includes the LIBS-based detection of the elemental composition of t. dioica leaves. A calibration-free LIBS, or CF-LIBS, algorithm was applied for the recorded LIBS spectra of t. diocia leaves to determine their elemental ratios of Na/K, Mg/Na, and Mg/K. The anti-diabetic behavior of T. diocia leaves has been correlated with these elemental ratios. The same intensity ratios of the elements Na, K and Mg were observed in LIBS spectra as for ayurvedic medicines prescribed for the treatment of diabetes mellitus. The anti-diabetic effect of t. diocia leaves extract was reconfirmed by conducting in vivo studies on normal, subdiabetic, mildly diabetic and severely diabetic models. Variable doses of the extract were given orally, and the impact on the BGL of selected animals is investigated in this work. The novelty/innovative aspects of this research work include (i) the herbal medicine, t. diocia leaves, contains specific a elemental ratio of sodium, Na, potassium, K, and magnesium, Mg, that is desirable for diabetes type II management, and (ii) the in vivo study on rats and biochemical analysis yield effective results for the management of diabetes type II.
2. Materials and Methods
2.1. Experimental Arrangement for LIBS Instrumentation
illustrates the design of LIBS experiments. The Nd:YAG laser device (Continuum Surelite III-10, San Jose, CA, USA) is operated at a repetition rate of 10 Hz, delivering pulses of full-width-half-maximum 4 ns and maximum laser energy up to 425 mJ at the second harmonic wavelength of 532 nm. The laser beam is focused onto the sample with a 30 cm focal length lens to achieve an irradiance of the order of 1 TW/cm2
. The light emitted from the plasma was collected by a small lens on the tip of the optical fiber that is connected to the entrance slit of an echelle-grating spectrometer equipped with a charge-coupled device (CCD). The lens at the tip of the fiber is positioned 1 cm distance from the laser–plasma. To avoid craters formed by an individual laser–sample interaction, the sample is mounted on a translational stage and moved so that a fresh sample surface is available for each laser–plasma generation.
2.2. Sample Preparation
Fresh leaves of t. dioica
in the amount of 5 kg were purchased at a local market in Allahabad, India, and were subsequently verified by Prof. Satya Narayan, Taxonomist, Department of Botany, University of Allahabad. A voucher specimen (Allahabad University 321) was also submitted. Leaves were washed, chopped, shade dried, and then crushed into powder. The Soxhlet technique [36
] was employed with distilled water for 36 h for the extraction of dried powder at 100 °C. The aqueous extract was filtered and concentrated in a rotatory evaporator at a temperature of 35 °C under reduced pressure to produce semi-solid material, which was subsequently lyophilized to obtain the powder with a 25.1% weight-to-weight yield.
2.3. Animals for the Study
Healthy male albino, Wistar rats at an age range of 6 to 8 weeks and at a weight in the range of 0.15 to 0.2 kg served as the subjects in the experiments. Animals were obtained from the Luknow, India, Central Drug Research Institute. The rats were housed in standard ambient conditions, namely, 25 °C temperature, 50% humidity, 12 h of darkness and light per day, and with free access to water and standard laboratory diet composed of carbohydrates: 30%, proteins: 22%, lipids: 12%, and vitamins: 3%). The study was approved by the Allahabad University Institutional Ethical Committee, 83a/a/04/CPCSEA—Committee for the Purpose of Control and Supervision of Experiments on Animals. Prior to the animal work, in vitro studies have been performed: only enzymatic studies were carried out on certain enzymes that are involved in the carbohydrate metabolism. The administration of variable doses to each group of animals was based on our previous studies of t. dioica fruit extract.
2.4. Induction of Diabetes
In a group of overnight fasted rats, a single intra-peritoneal injection of freshly produced streptozotocin (bought from Sigma Aldrich Chemical Company, St. Louis, MO, USA) at a dose of 55 mg per kg body weight was given to induce diabetes [37
]. The FBG level was checked after 3 days, and the PPG was continuously monitored up to stable hyperglycemia, which occurred a week later. Animals with significant hyperglycemia (FBG > 250 mg/dL) were selected for the study.
2.5. Blood Glucose Level Measurements
Standard kits from Bayer Diagnostics India, Limited were used to estimate blood glucose level (BGL) based on the glucose oxidase method [38
] and serum levels of TC (total cholesterol), TG (triglycerides), and HDL (high-density lipoprotein) spectrophotometrically in accordance with the manufacturer’s instructions based on enzymatic studies [39
]. However, Friedwald’s formula, VLDL = TG/5, was used to determine the amount of VLDL (very low-density lipoprotein), and similarly, another Friedwald’s formula, LDL = TC − VLDL + HDL, was used to determine the amount of LDL (low-density lipoprotein) [41
]. Serum levels of LFT (liver function tests) [42
] viz. AST (aspartate transferase), ALT (alanine transferase), ALKP (alkaline phosphatase) [43
] and KFT (kidney function tests) viz. CRTN (creatinine) [44
] and TPR (total protein) [45
] were also estimated using standard kits from Bayer Diagnostics India Limited. Total Hb (total hemoglobin) was also measured before and after the therapy [46
]. Reagent-based Uristix from Bayer Diagnostics was used to identify US (urine sugar). All parameters were assessed regularly throughout the long-term treatment.
2.6. Experimental Design
FBG and GTT experiments were performed with variable doses of aqueous extract of leaves in normal, subdiabetic, and mild diabetic rats to assess hypoglycemic and anti-diabetic effects. The most effective dose identified was used for the long-term treatment of severely diabetic rats to assess the hypo-lipidemic-, hepato-protective-, and renal-protective efficacies of the extract.
2.6.1. Assessment of Hypoglycemic Activity in Normal Healthy Rats
Five groups of six rats each were employed in the experiments. Animals in groups II, III, IV, and V received variable doses of aqueous extract viz. 500, 750, 1000, and 1250 mg/kg bw, respectively. In contrast, group I served as the control group and received vehicle distilled water only. After administering the extract, blood samples were taken from the tail vein after 1.5, 3, 4.5, and 6 h.
2.6.2. Assessment of Hypoglycemic Activity with the Glucose Tolerance Test in Normal Healthy Rats
A different group of healthy, normal animals received the aqueous extract orally in the same manner as above, and the effects on FBG were observed hourly for up to two hours. The BGL value at 2 h was treated as ‘0’ h value for the Glucose Tolerance Test (GTT). The animals were then orally administrated with 4 g/kg of glucose, and their glucose tolerance was studied at a 1 h interval for another 3 h. Thus, the total time for collecting the blood was up to 5 h.
2.6.3. Assessment of Anti-Diabetic Activity with the Glucose Tolerance Test in Sub- and Mild-Diabetic Rats
The improvement of glucose tolerance in subdiabetic and mildly diabetic rats was another way to measure the anti-diabetic impact of the aqueous extract of t. dioica leaves. The rats were divided in to six groups. Group I served as the control and received the vehicle (distilled water) only, whereas groups II, III, IV, and V received variable doses of the leaf extract—500, 750, 1000, and 1250 mg/kg bw, respectively. Blood glucose levels were checked firstly after 90 min of treatment, which was considered as the ‘0’ h value, and then, 2 g/kg glucose was given orally to all the groups. Blood glucose levels were further checked up to three hours at regular intervals of 1 h each, which were considered as 1 h, 2 h, and 3 h values. The results were compared with the group IV rats, which were treated with 250 mg/kg of tolbutamide (hypoglycemic agent).
2.6.4. Assessment of Anti-Diabetic Activity in Severely Diabetic Rats
For normal, subdiabetic and mild diabetic models, the most effective dose was determined to be 1000 mg/kg. A number of biochemical parameters related to antidiabetic attribute viz. FBG, PPG, Lipid profiles, Total Protein, Haemoglobin and Enzymatic assays were taken into consideration for obtaining a complete biomedical profile of aqeous extract of t. dioica leaves. Two groups of 6 rats each were used in the experiment. Group I served as the control group, whereas group II was treated daily with a single oral administration of the above-mentioned dose. Body weight and urine sugar were also estimated weekly along with other biochemical parameters up to 30 days.
2.6.5. Lethal Dose 50 Experiment
The lethal dose 50, LD50, experiment was designed to assess the extract’s toxicity. Two variable doses of 10 g and 15 g of the aqueous extract of t. dioica leaves were given once, orally to two different groups of rats of both sexes, weighing between 180 and 200 g. Afterwards, rats were continuously monitored for gross behavioral, neurologic, autonomic, and toxic consequences. Food intake, face, and urine were also analyzed every two hours for the first six hours during a period of 24 h.
2.7. Statistical Analysis
The one-way analysis of variance, ANOVA, was utilized in the statistical analysis of the data. The Statistical Package for the Social Sciences, SPSS, version 7.5 assessed the post hoc Scheffe test. When the p values were less than 0.05, p < 0.05, results were deemed significant.
The application of LIBS delivers the elemental composition of t. diocia
leaves. These leaves are instrumental for the lowering of an elevated BGL, and consequently, they can be used for the therapy and management of diabetes. Elements including Na, K, Ca, Mg and Fe, and elemental ratios play a vital role in managing diabetes. It is noteworthy that the ratios of Mg/Na and Mg/K, determined by CF-LIBS in aqueous extract of t. diocia
leaves, indicate similar trends as observed for other anti-diabetic medicines. The t. diocia
plant is traditionally used as a vegetable in today’s Indian food system and as well in the ayurvedic system of medicine [57
The communicated work demonstrates the considerable hypoglycemic and anti-diabetic efficacy of t. diocia
leaves corroborated by short- and long-term studies. FBG studies on normal rats show the greatest hypoglycemic impact (23.8%) in less than 6 h; see Table 3
. In contrast, the GTT studies on normal rats demonstrate that the hypoglycemic impact was initiated within 1 h and intensified later; see Table 4
. This implies that within about one hour, the active components of aqueous extract induces the hypoglycemic effect after reaching the target tissues through circulation. The data also confirm that a significant effect persists for the next three hours after the glucose administration.
The GTT results reveal an improvement in the glucose tolerance of subdiabetic and mildly diabetic animals, reflecting a significant drop in high blood glucose levels. The results with a dose of 250 mg/kg bw of tolbutamide, a synthetic drug, are comparable with the dose of 1000 mg/kg bw of the aqueous extract. This would indicate similar mechanisms of action. STZ induces type II diabetes in which pancreatic insulin-secreting cells are destroyed [58
]; consequently, the elevated blood glucose levels in the diabetic animals used in this study are consistent with type II diabetes. Hence, t. diocia
aqueous extract, which supposedly acts as an insulin secretion enhancer by activating beta cells, could be considered an important and useful treatment in the context of type II diabetes.
In cases of normal, subdiabetic, and mildly diabetic animals, the dose of 1000 mg/kg bw was found to be the most effective dose; therefore, this dose was used to treat the animals with severe diabetes for further studies. It has been noted frequently that hyperlipidemia is always associated with hyperglycemia. As indicated in the medical literature, hypertriglyceridemia is the most prevalent lipid abnormality [59
]. Numerous studies have linked elevated blood triglyceride levels to a higher risk of stroke. If high levels of TC and LDL cholesterol persist, then LDL has a propensity to adhere to the blood vessel walls, stimulating atherosclerosis, which in turn causes heart attacks and strokes. Consequently, high levels of LDL cholesterol increase the danger of developing heart disease that may induce stroke [60
]. There is currently a volley of data supporting the notion that HDL is a beneficial lipid, as high levels are linked to low rates of heart disease while low levels are linked to higher rates. After 30 days of treatment, the animals in the current study with severe diabetes had increased HDL cholesterol levels and considerably decreased levels of TC, LDL, VLDL, and TG.
Other biochemical parameters taken into consideration for assessing the improvement in LFT and KFT of severely diabetic animals include AST, ALT, ALKP, CRTN, and TPR. AST is located in the liver, and it is released into the serum when the liver suffers from damage. Increased levels of AST in serum cause heart attacks and muscle disorders. Although ALT is not just found in the liver, liver damage causes it to be released into the bloodstream. As a result, it acts as a pretty accurate measure of liver health. Although ALKP is present in a variety of organs, its significant presence in the liver cannot be ignored. Hence, elevated levels of ALKP are indicative of liver disease or bile tract obstruction. However, high levels of CRTN cause renal failure. The reduction of raised levels of AST, ALT, ALKP, and CRTN by the treatment of aqueous extract of t. dioica leaves suggests that it could be explored as an anti-hyperglycemic agent and as an hepato-protective and renal protective agent to manage diabetic complications as well. The additional advantageous results in this study include a reduction in urine sugar level, an increase in body weight and total protein level. It has considerable value for human subjects too due to its traditional use as an Indian vegetable and its high LD50, showing a great margin of safety.