Exploring the therapeutic potential of Caralluma fimbriata for antioxidant and diabetes management: a 28-day rat model study

Abstract

This study evaluated the anti-oxidant and anti-diabetic potential of Caralluma fimbriata (CF) in 28-days rat modelling trial. Diabetes is a chronic disorder characterized by elevated blood glucose levels and insulin resistance and cause microvascular and macrovascular issues. Caralluma fimbriata was evaluated for its nutritional composition along with anti-oxidant potential of CF powder (CFP) and CF extract (CFE) using total phenolic contents (TPC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric ion reducing antioxidant power (FRAP) assays. Furthermore, anti-diabetic potential was computed by dividing rats into four groups of 5 individuals each. Rats of Group I was non-diabetic and no supplementation was given while rats of group II were diabetic and no supplementation was given. While group III and group IV rats were diabetic and received CFP and CFE supplementation respectively. CF powder’s TPC, and DPPH and FRAP activity were observed maximum at 44.17 ± 0.006 (μg_Fe/g) in water, 68.75 ± 0.49 (μg_Fe/g) in acetone and 800.81 ± 0.99 (μg_Fe/g) in hexane. Supplementation of CFP and CFE reduced blood glucose effectively i.e. (125.00 ± 4.04 and 121.00 ± 4.49 mg/dL, respectively). Moreover, the consumption of C. fimbriata can be helpful in the management of diabetes mellitus due to its glucose lowering potential, anorexic effects, anti-oxidant potential and α-amylase inhibition.

Graphical Abstract

Graphical Abstract.

Introduction

Diabetes mellitus is a metabolic and endocrine disorder linked with malfunctioning pancreatic β-cells and impairs glucose tolerance, symptomatically with frequent urination, thirst, hunger and sudden weight loss.¹ According to the World Health Organization (WHO), the global prevalence of diabetes mellitus is 8.5% in adults. International Diabetes Federation (IDF) reported that 382 million people suffer from diabetes mellitus, and by 2035 it may escalate to 592 million.² Exogenous medical therapies include insulin administration and oral hypoglycemic substances, such as thiazolidinediones, alpha-glucosidase inhibitors, biguanides, sulfonylureas and meglitinides. In contrast, dietary patterns and lifestyle modifications can mitigate the severity of complications like cardiovascular diseases, vascular infection, kidney disorders, eye problems and ulceration feet.^3–5

Medicinal plants are rich reservoirs of phytochemicals, including phenolic acids, flavonoids, anthocyanins, carotenoids, etc.^6,7 They are functional foods and nutraceuticals due to their anti-oxidative properties. Anti-oxidants are vital in scavenging free radicals in food systems and the human body.^8–12 They modulate the oxidation process every second of human life, thus balancing the anti-oxidants and free radicals in the body. Medicinal plants are included in our diet due to multiple health benefits because they are proclaimed preventive and curative agents.^7,13–18

Caralluma fimbriata belonging to the Asclepiadaceaeis family, is an edible wild cactus plant with succulent properties, living in the dry regions of Pakistan, India, Iran, Afghanistan, Sri Lanka and Africa.^19,20 It is locally known as “Choong” or “Choonga” in Pakistan and India. It is an erect, branched herb with minute leaves, wheel-like flowers and narrow petals.²¹ It is an edible succulent plant loaded with phytochemicals, such as saponins, terpenoids, alkaloids, cardiac glycosides, anthocyanin, megastigmane glycosides, flavone glycosides and pregnane glycosides. Pregnane glycosides are secondary metabolites containing steroidal compounds that bind with sugar molecules.²²

Caralluma fimbriata helps to alter lipid metabolism, inhibiting the synthesis of fatty acids.^21,23–25 It also acts on the hypothalamus and cortisol, causing satiety that decreases hunger primarily due to pregnane glycosides.²⁶ It is notable as a famine food, thirst quencher and appetite suppressant among tribal populations.²⁷ It is recognized for its anti-diabetic, anti-oxidant, anticancer, anti-obesogenic, antinociceptive, hepatoprotective, reno-protective and hypolipidemic activities.^28,29 Since ancient times, Caralluma species have been used against snake bites, scabies, skin rashes and inflammation.³⁰ Therefore, the current study was designed to evaluate its anti-oxidant and anti-diabetic potential against diabetes. Investigating the anti-oxidant and anti-diabetic potential of Caralluma fimbriata is important in the context of current knowledge about diabetes management and the need for alternative treatments for several reasons: a) The global prevalence of diabetes is increasing, with significant impacts on health systems and individual health. There is a continuous need for more effective and accessible treatment options. b) Current diabetes treatments, including insulin and oral hypoglycemic agents, may have limitations such as side effects, contraindications, and variable efficacy among individuals. This highlights the need for alternative treatments that are safe, effective, and have fewer side effects. c) Oxidative stress plays a significant role in the pathogenesis of diabetes and its complications. Antioxidants can potentially mitigate oxidative damage and improve glycemic control, making the investigation of natural antioxidants like those found in Caralluma fimbriata particularly relevant. d): There is growing interest in natural therapeutics for diabetes management due to their perceived safety and holistic benefits. Caralluma fimbriata, with its traditional use in appetite suppression and weight management, presents a promising candidate for further exploration as an anti-diabetic agent. e): Understanding the mechanisms through which Caralluma fimbriata exerts its anti-oxidant and anti-diabetic effects can contribute to the development of novel therapeutic strategies and the identification of active compounds that could be optimized for better efficacy.

Given these considerations, research into the anti-oxidant and anti-diabetic properties of Caralluma fimbriata aligns with the broader goals of improving diabetes management, offering alternative treatment options, and enhancing our understanding of natural products in disease intervention.

Materials and methods

Materials

Caralluma fimbriata (CF) samples were collected in December 2020 from district Chakwal, Pakistan. CF was identified in the Department of Botany, Bahauddin Zakariya University-Multan. The plant sample was dried, ground to powder form using a grinder (Model BJ-9176), filtered through the lab sieve (test sieve) (Model SMEW-0054), and packed into airtight zip-lock bags for further use.

Proximate analysis of Caralluma fimbriata

Determination of food ingredients like moisture content, ash contents, crude fat, crude fiber, protein and carbohydrate was analyzed through proximate analysis following the protocols of AOAC.³¹ All experiments were performed in triplicate, and the results were summarized using a complete randomized design (CRD).

Determination of anti-oxidants

Extract preparation

Antioxidant-rich extract of dried C. fimbriata powder was formulated by mixing the sample with different solvent, i.e. distilled water, ethanol, acetone and hexane. For extraction the ratio of the sample extract and solvent was taken in a 1:10 (w/v) ratio for each solvent separately with extract. Samples were mixed by using an orbital shaker (Model Number: BSOT-604) at 200 rpm. Using an orbital shaker, the extracts were homogenized and rest overnight. The filtered (through a Whatman No 4-filter paper) extracts were concentrated on a rotary evaporator and then were ready to execute the anti-oxidant protocols.

The total phenolic contents of C. fimbriata were determined by the modified Folin–Ciocalteu assay.³² Mix the solution for mixture preparation and allowed it to rest for half an hour in dark. 10–100 mg/L concentrations of gallic acid was prepared and run as a standard to gain principle curve of TPC. The outcomes were expressed as GAE mg/mL (gallic acid-equivalent, in mg/mL of extract). The absorbance (Abs) was determined spectrophotometrically (Model 823-0210 P-2-R) at a wavelength (λ) of 760 nm.

To determine the anti-oxidant capacity of C. fimbriata, a DPPH-free radical scavenging assay was used.³³ The positive controls are the usual vitamin C solutions, all of which are repeated. Standard and sample absorbance levels were obtained after 30 m. The following Equation 1 was used to determine the percentage of radical scavenging activity. Accordingly, the absorbance was determined spectrophotometrically (Model 823-0210 P-2-R) at a wavelength (λ) of 517 nm and against a control (without sample).³³

(1)

The anti-oxidant capability of C. fimbriata extracts was measured using a FRAP assay.³⁴ Accordingly, the absorbance was determined spectrophotometrically (Model 823-0210 P-2-R) at a wavelength of 593 nm and against a control (without sample).

(2)

The rationale behind choosing specific assays such as Total Phenolic Content (TPC), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and Ferric Reducing Antioxidant Power (FRAP) lies in their ability to evaluate the antioxidant capacity of substances from different perspectives. The TPC assay is used to quantify the total phenolic compounds in a sample, which are known for their antioxidant properties, providing an estimate of the sample’s potential antioxidant activity. The DPPH assay measures the ability of antioxidants in the sample to scavenge free radicals, offering insights into the radical scavenging capacity of the substance. The FRAP assay assesses the sample’s ability to reduce ferric (Fe³⁺) to ferrous (Fe²⁺) ions, reflecting its reducing power and potential antioxidant effectiveness. These assays were conducted by preparing extracts or solutions of the samples, which were then reacted with specific reagents (Folin–Ciocalteu for TPC, DPPH radical solution for DPPH, and FRAP reagent for FRAP), and the changes in absorbance were measured spectrophotometrically. The results are often expressed in terms of equivalents of a known antioxidant, such as gallic acid for TPC, indicating the antioxidant capacity of the tested samples.

Animal testing and housing

White Wister Albino rats, after ethical approval of Office of Research Innovation Commercialization (ORIC), were kept in the animal research room of the Department of Human Nutrition, Bahauddin Zakariya University, Multan, with controlled environmental conditions. Rats were divided into four groups (n = 5/group). Rats were given regular poultry feed for 15 days to gain the desired weight before the start of the experimental diet. During the research period, a corn starch-composed pallet was given to rats (Table 1). The specific procedures for inducing diabetes in rats and the dosages and duration of Caralluma fimbriata extract (CFE) and powder (CFP) supplementation were not directly detailed in the sources. However, general methodologies for inducing diabetes in rat models include administering Streptozotocin (STZ) intraperitoneally at various dosages, such as 35 mg/kg following a high-fat diet or a single low dose of 45 mg/kg, to damage pancreatic cells and induce insulin resistance, mimicking type 2 diabetes. Another method involves feeding rats a fructose-rich chow for 8 weeks or using a combination of nicotinamide followed by a high dose of STZ (60 mg/kg) to induce different degrees of insulin resistance and diabetes. These models aim to replicate the metabolic characteristics of type 2 diabetes in humans, including hyperglycemia and insulin resistance. The choice of induction method depends on the specific research objectives, such as studying the progression of diabetes or evaluating the efficacy of potential treatments like CFE and CFP. The dosages and duration of CFE and CFP supplementation in these models would typically be determined based on preliminary studies to optimize therapeutic outcomes while minimizing adverse effects, but specific details on these parameters were not provided in the reviewed literature.

Table 1.

Formulation of corn pallet.

Components	Formulation (%, w/w)
Corn Starch	70
Corn Oil	10
Casein Protein	9
Wheat Bran	5
CMC	3
Minerals	2
Vitamins	1

Feed intake

The feed intake of healthy and diabetic rats when given C. fimbriata powder and extracts was evaluated weekly according to the method described by Wolf and Webisode, to measure the impact of C. fimbriata on appetite.

Biochemical analysis

All rats were sacrificed after the completion of the 28-day study period. Blood samples of rats were taken by cardiac puncture. Using the weight balance (GX-600, Japan) measured the weight of rats from day 1^st to the last day of the study. Afterward, rats were dissected and their organs (heart, liver, kidneys, spleen and lungs) were removed, washed and stored in formalin solution. Organs were weighed the next day to ascertain the organ-to-body weight ratio. Red Blood Cells (RBC) blood count that measures how many red blood cells present, was checked by analyzing hemoglobin (Hb), mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV) and platelets. In contrast, a differential count of monocytes, eosinophils, basophils, neutrophils and lymphocytes scrutinized white blood cells (WBC) that measures how many white blood cells present in blood. Serum analyses evaluated glucose, lipid profile, total protein, electrolytes, liver functioning and kidney functioning enzymes. All tests were performed in the laboratory using commercially available kits (Automatic biochemistry analyzer BK-400, Biobase, China).

Statistical analysis

The acquired data were inspected using Statistics 8.1 (Analytical Software, 2,105 Miller Landing Rd, Tallahassee FL 32312, USA) and factorial designs (two-way analysis of variance, 2-way ANOVA), and the least significant design (LSD) test was used to compare treatments (Montgomery 2017). Statistically significant differences were considered at a P value ≤0.05.

Results and discussion

Proximate analysis of Caralluma fimbriata

The nutritional composition (% g/100 g) of C. fimbriata is presented in Fig. 1. Moisture content in C. fimbriata showed a value of 58.09%. C. fimbriata is a cactus plant that can store ample water. Ash content in C. fimbriata was 6.03%, as ash represents the inorganic residue that remained after ignition or complete oxidation of organic matter. The maximum amount of fat found in C. fimbriata was 5.03%. The fiber content observed in C. fimbriata was 6.47%. Proteins comprise amino acids that help metabolism by providing structural support and acting as enzymes, carriers, or hormones. The protein and carbohydrate contents were 5.96 and 18.77%, respectively. The current study findings regarding the nutritional composition of C. fimbriata are in slight concordance with the outcomes of a study conducted by Maheshu et al.³⁵ Remarkable differences in moisture content may be due to plant freshness, while all other results are in accordance. Raza et al.,³⁶ performed the proximate analysis of C. fimbriata seeds, and there were comparable results of all parameters with minute variations.

Fig. 1.

Nutritional analysis (%, g/100 g) of Caralluma fimbriata.

Determination of anti-oxidants

The anti-oxidant potential was measured using various methods, including the DPPH, FRAP and TPC tests. DPPH radical scavenging assay eradicated free radicals due to its hydrogen-donating ability. Maximum scavenging activity of CF was observed in acetone and ethanol solvents with 68.75 ± 0.49 and 52.74 ± 0.74%, respectively. Minimum scavenging activity was evaluated in water solvent (15.99 ± 0.21%), which showed that it has slightly less scavenging activity than other solvents. In hexane solvent, CF possessed 32.40 ± 0.37% scavenging activity against free radicals. Yada et al.³⁷ performed a phytochemical evaluation of CF, and the results were comparable to this study.³⁷ Maheshu et al.,³⁵ performed multiple scavenging assays and the results were relatively higher than the current research study (Table 2).

Table 2.

Antioxidant potential (mean value ± standard deviation) of Caralluma fimbriata.

Solvent	Caralluma Fimbriata
	DPPH Scavenging activity   (%)	FRAP   (μgFe/g)	TPC   (mgGAE/g)
Water	15.99 ± 0.21	308.03 ± 1.99	44.17 ± 0.006
Hexane	32.40 ± 0.37	800.81 ± 0.99	25.85 ± 0.188
Acetone	68.75 ± 0.49	311.54 ± 0.96	28.39 ± 0.11
Ethanol	52.74 ± 0.74	761.57 ± 0.75	38.46 ± 0.10

Note: Means having the same letters within a column or rows does not differ significantly (P < 0.05). 2,2-Diphenyl-1-picrylhydrazyl (DPPH); Ferric ion reducing antioxidant power (FRAP); Gallic acid-equivalent (GAE); Total phenolic content (TPC).

FRAP is a simple test for determining anti-oxidant properties.³⁸ This approach was developed to assess plasma’s anti-oxidant ability and is also used to determine plants’ anti-oxidant potential. The primary idea behind this method is to figure out how much any substance, such as plants, may reduce ferric ions. The CF extracts power to reduce ferric was investigated and it was discovered that all solvents utilized in the extract preparation have a substantial impact on plant anti-oxidant ability and that the anti-oxidant potential of the plants varied significantly (P < 0.05) in the FRAP assay. Table 2 depicts significant (P < 0.05) results regarding the FRAP assay to measure anti-oxidant activity. CF showed maximum FRAP activity (800.81 ± 0.99 μg FeSo₄/g) when hexane was used as a solvent. The anti-oxidant potential in ethanol solvent was recorded at 761.57 ± 0.75 μg FeSO₄/g. In the extract of samples prepared in acetone and water, FRAP activity observed was 311.54 ± 0.96 and 308.03 ± 1.99 μg FeSO₄/g, respectively. Maheshu et al.,³⁵ evaluated the anti-oxidants of CF by performing FRAP activity. The results obtained were up to 5,860 ± 0.7 μg FeSO₄/g, while in the current study, results were relatively low as 761.57 ± 0.75 ug_Fe/g.

Plant-derived phenolic compounds have redox characteristics and due to these qualities, they act as anti-oxidants. TPC activity is a method for determining the total phenolic content in a plant sample. Polyphenols are measured in urine, organs and plasma. Table 2 represents the phenolic contents of plant extracts, in which CF had the highest value of 44.166 ± 0.006 in a water solvent. In extracts prepared in ethanol solvent, CF showed a potential of 38.46 ± 0.10. The resulting pattern observed for CF in hexane and acetone solvent were 25.85 ± 0.188 and 28.39 ± 0.11 mg_GAE/g, respectively. The results of TPC obtained by Devi and Dhamotharan³⁹ were 39.81 ± 1.04 mg_GAE/g which was quite a support of the current findings of 38.464 ± 0.1 mgGAE/g.³⁹ In another study, Yada et al.³⁷ performed a phytochemical evaluation of Caralluma to check its anti-oxidant potential.³⁷

Animal testing and housing

The interventional study was for 28 days, and the supplementation of rats was done according to the treatment plan (Table 1). The ethanol extract was used to study the effect of CF supplementation on hyperglycemia in rats.

Feed and water intake

Feed intake of healthy rats (T₁) and the treatment groups (T₃ and T₄) was evaluated weekly when supplemented with C. fimbriata powder (CFP) and extracts (CFE) along with a regular diet. Figure 2 represents the feed intake of healthy and diabetic rats during 28 days of study. In the 1^st week of the study, the negative control group showed maximum intake of feed followed by positive control, CFP and CFE groups, respectively. A similar trend of feed intake was observed during 2^nd week of the study. Throughout time, feed intake improved as the age and weight of rats increased. When the 3^rd week feed intake was evaluated, the positive control group (T₂) showed around 133 g. Feed intake of the diabetic group (without Supplementation) increased due to the less glucose available for energy production, resulting in more demand for energy production. At the same time, other treated groups showed a reduction, especially in CFP and CFE, due to CF’s bitter taste and hunger suppressor nature.³⁹

Fig. 2.

Feed intake and water intake data (mean value ± standard deviation) (g/day). Note: N. Control = Negative control; Control = Positive control; CFP = Caralluma fimbriata powder; CFE = Caralluma fimbriata ethanol extract. Group I (T1): Negative control (non-diabetic rats with no supplementation); group II (T2): Positive control (diabetic rats with no supplementation). Group III (T3): Diabetic rats with CFP supplementation. Group IV (T4): Diabetic rats with CFE supplementation.

Water intake of healthy and treatment groups was evaluated weekly when supplemented with CFP and CFE along with a regular diet. Figure 2 represents the water intake of healthy and diabetic rats. In the 1^st week of the study, the CFE group consumed more water, followed by the negative control, CFP and positive control groups. As the week advanced, the water intake of the intervention group (III, IV) decreased, and in negative control rats the water intake increased due to their weight gain with time. In the intervention group (III, IV), the weight of rats decreased due to diabetes and water intake also reduced. The ratio of water intake increased despite weight loss in the diabetic groups because diabetes triggers more thirst and CFE’s bitter taste also starts thirst.⁴⁰

Body and organ weight

A significant (P < 0.05) weight reduction (156 ± 4.54 g) was observed in diabetic T₂-induced rats (Table 3) because of insufficient insulin production, which regulates glucose levels in the body and takes blood sugar into cells. Thus, body’s use of fats as an energy source results in weight reduction. However, weight regaining was observed in the other treatment groups, and the values were 190 ± 2.33 g and 205 ± 5.28 g in the group supplement with power and extract of C. fimbriata, respectively. A significant decrease in body weight organs was noticed, especially in the kidneys, liver and heart (Table 3). When comparing T₁ and T₂, the weight of the liver and kidneys fell, respectively, to: 3.22 ± 0.05 g from 4.54 ± 0.10 g, in liver; 0.27 ± 0.01 g from 0.37 ± 0.01 g, in kidney (R); and 0.33 ± 0.01 g from 0.26 ± 0.01 g, in kidney (L). A notable rehabilitation was witnessed in CFP (T₃) and CFE (T₄) groups with the values of 5.01 ± 0.13, 5.39 ± 0.11 g, in the liver, and 0.40 ± 0.01, 0.40 ± 0.01 g, in kidney (R), and 0.40 ± 0.01, 0.38 ± 0.01 g, in the kidney (L), respectively. The same tendency in a diabetic group with a regular diet was recorded in the weight of the spleen and lungs, with a decrease to 0.72 ± 0.01 g, in the lungs, and to 0.20 ± 0.01 g, in the spleen. In the lungs, there was no statistically significant (P > 0.05) improvement showed by CF in the intervention groups, and, in the spleen, statistically significant (P < 0.05) progress was exhibited by the CFP and CFE groups, with values of 0.23 ± 0.01 and 0.34 ± 0.01 g, respectively. Binita et al.⁴¹ conducted an anti-diabetic study on rats. They discussed the anti-diabetic potential and described the improvement of body weight and organ weight in rats.⁴¹

Table 3.

Effect (mean values ± standard deviation) of Caralluma fimbriata on body and organs weight (g), and on hematological and white blood cells counts.

Parameter (g)	T 1	T 2	T 3	T 4
Body and organs weight (g)
Body weight	248 ± 9.56	156 ± 4.54	190 ± 2.33	205 ± 5.28
Kidney (R)	0.37 ± 0.01	0.27 ± 0.01	0.40 ± 0.01	0.40 ± 0.01
Kidney (L)	0.33 ± 0.01	0.26 ± 0.01	0.40 ± 0.01	0.38 ± 0.01
Liver	4.54 ± 0.10	3.22 ± 0.05	5.01 ± 0.13	5.39 ± 0.11
Heart	0.32 ± 0.01	0.33 ± 0.01	0.38 ± 0.01	0.39 ± 0.01
Lungs	0.76 ± 0.03	0.72 ± 0.01	0.72 ± 0.03	0.72 ± 0.03
Spleen	0.28 ± 0.01	0.20 ± 0.01	0.23 ± 0.01	0.34 ± 0.01
Hematological and white blood cells count
RBC cells/μL	7.30 ± 0.40	5.20 ± 0.24	6.8 ± 0.18	7.13 ± 0.14
Hb (g/dL)	13.20 ± 0.23	9.78 ± 0.21	11.68 ± 0.6	12.08 ± 0.23
Platelets count/μL	435 ± 8.00	780.00 ± 31.9	564.0 ± 11.8	512.00 ± 20.92
HCT (%)	39.1 ± 0.90	50.57 ± 1.17	45.56 ± 1.36	41.09 ± 0.92
MCV (fL)	53.45 ± 1.02	97.32 ± 2.73	67.10 ± 3.27	57.63 ± 2.35
MCH (pg)	18.1 ± 0.42	18.75 ± 0.87	17.20 ± 0.71	16.94 ± 0.31
MCHC (g/dL)	33.8 ± 1.20	19.32 ± 0.40	25.64 ± 0.53	29.40 ± 0.95
WBC cells/μL	6.80 ± 0.15	9.10 ± 0.42	7.35 ± 0.33	7.13 ± 0.28
Lymphocytes (%)	49.48 ± 0.67	39.37 ± 1.37	48.66 ± 0.62	44.40 ± 1.86
Neutrophils (%)	42.23 ± 1.51	43.23 ± 0.48	33.53 ± 0.78	37.81 ± 1.01
Monocytes (%)	3.02 ± 0.06	2.56 ± 0.09	2.65 ± 0.05	2.84 ± 0.10
Eosinophils cells/μL	3.37 ± 0.08	2.91 ± 0.06	2.99 ± 0.10	2.77 ± 0.07
Basophils (%)	1.90 ± 0.06	1.93 ± 0.05	2.17 ± 0.06	2.18 ± 0.06

Note: Means having the same letters within a column or rows does not differ significantly (P < 0.05). Group I (T₁) = Normal group + Normal diet; Group II (T₂) = Diabetic group + Normal diet; Group III (T₃) = Diabetic group + Normal diet + C. fimbriata powder (CFP); Group IV (T₄) = Diabetic group + Normal diet + C. fimbriata ethanol extract (CFE).

Red blood cell (RBC); Hemoglobin (Hb); Hematocrit (HCT); Mean corpuscular hemoglobin (MCH); Mean corpuscular volume (MCV); Mean corpuscular hemoglobin concentration (MCHC); and White blood cell (WBC).

Hematological analysis

The hematological analysis is an important parameter to recognize a problem in the body because RBC count and hemoglobin are primary indicators of an issue in blood. Hematological parameters include RBC count, Hb, platelet count, hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC). The results indicated that variations occurred in these parameters and the results were statistically significant (P < 0.05). The reduction in RBC count results from decreased erythropoietin hormone produced by the kidney, which substantially has red blood cells. As in diabetes the kidney also suffers, it results in erythropoietin's decline. In diabetic rats (T₂), RBC count (cell/μL) was reduced to 5.20 ± 0.24 but with the treatment it improved to 6.8 ± 0.18 and 7.13 ± 0.14 in CFP and CFE groups, respectively. The hemoglobin (g/dL) in diabetic rats dropped to 9.78 ± 0.21 from 13.20 ± 0.23 in the diabetic group. Hb improved significantly (P < 0.05) and increased in the intervention groups as the recorded values were (11.68 ± 0.6 g/dL), in CFP, and (12.08 ± 0.23 g/dL), in CFE. Diabetes exhibits increased platelet count because hyperglycemia promotes the glycation of platelet proteins and increased platelet reactivity (780.00 ± 31.9 count/μL).⁴² Platelets increased within diabetic rats but significantly decreased in the treatment groups (T₃ and T₄). The reduced recorded readings were 564.0 ± 11.8 and 512.00 ± 20.92 count/μL in CFP and CFE groups, respectively (Table 3).

The HCT and MCV values were elevated in the diabetes group but both values declined in CFE to 41.09 ± 0.92% and 57.63 ± 2.35 fL, respectively. In MCH, the value slightly increased in the diabetes group but reduced in the treated groups. The maximum decline in the treated groups was noticed in the CFE group, with a value of 16.94 ± 0.31 pg. In MCHC, the value declined in the diabetes group but improved in the treatment groups (T₃ and T₄). The highest recovery was observed in CFE, with a value of 29.40 ± 0.95 g/dL. Diabetes is accompanied by inflammation in blood vessels and disturbance in bone marrow results in the decline of Hb level. Moreover, medication for diabetes is another major issue of low Hb level. Prasad et al.⁴³ investigated sub-acute oral toxicity and reported a slight increase in the hematological parameters proving that consumption can alter Hb and RBC count.⁴³

White blood cells (WBC) and other associated cells like neutrophils, lymphocytes and monocytes are front-line protective cells, and their elevated levels indicate some inflammation. In addition, increased levels of these cells represented impaired glucose tolerance. WBC count and neutrophils increased in diabetic rats (T₂) with a value of 9.10 ± 0.42 cells/μL and 59.37 ± 1.37%, respectively, and these values declined within treatment groups (T₃ and T₄). The reduction noticed in CFP and CFE groups for WBCs and neutrophils were 7.35 ± 0.33 cells/μL and 33.53 ± 0.78%, and 7.13 ± 0.28 cells/μL and 37.81 ± 1.01%, respectively. Lymphocytes decreased in the diabetic group of rats but improved in treatment groups, and the values were 48.66 ± 0.62% and 44.40 ± 1.86% respectively. A decline in lymphocytes showed impaired immune responses due to diabetes mellitus. Monocytes and eosinophils decreased in the diabetic group to 2.56 ± 0.09% and 2.91 ± 0.06 cells/μL from 3.02 ± 0.06% and 3.37 ± 0.08 cells/μL, respectively, but improved in treatment groups. Eosinophils indicated that numbers were reduced in the diabetes group (T₂) but increased in treated groups (T₃ and T₄), and maximum rehabilitation (2.99 ± 0.10 cells/μL) was observed in the CFP group. Increased basophil counts revealed inflammation in bone marrow, which results in high production of WBC. The number of basophils elevated in the diabetes group (1.93 ± 0.05%) and was continuously improved in treated groups, when comparing with the placebo (T₁). The CFE group recorded the highest number of basophils with a value of 2.18 ± 0.06% (Table 3). Our results of increased and varied leukocytes followed the findings of Ugwah-Oguejiofor et al.,⁴⁴ as they mentioned the high numbers of leukocytes and declared CF as a potential herbal medicine.⁴⁴

Blood glucose, lipid profile and total protein

The blood glucose, lipid profile and total protein are primary indicators of metabolic disorders associated with diabetes mellitus. The blood glucose level in the negative control (T₁) was 96.00 ± 2.92 that was increased to 210.00 ± 5.9 mg/dL in alloxan induced diabetes group (T₂). The elevation in blood glucose levels in diabetic rats might be linked with to beta-cell damage in the pancreas that decreased insulin production or may cause impaired insulin function. However, significant improvement in the treatment groups (T₃ and T₄) was observed i.e. 125.00 ± 4.04 and 121.00 ± 4.49 mg/dL in CFP and CFE groups, respectively. The highest decrease in CFE groups is due to the high anti-oxidant potential of the CF extract. The results regarding hypoglycemic activity were supported by the findings of Khan et al.⁴⁵ Regarding triglycerides (TG), the values were reduced to 83.00 ± 1.97, in CFE mg/dL, and 84.21 ± 1.72 mg/dL, in CFP. The reduction of total cholesterol (TC) and high-density lipoprotein (HDL) is highly associated with diabetes, and represents insulin resistance, augmented levels of low-density lipoprotein (LDL) and amplified activity of endothelial lipase and cholesterol ester transfer protein (CETP). In placebo (T₁), TC and HDL were 85.42 ± 2.83 mg/dL and 30.01 ± 0.67 mg/dL, respectively, which increased to 99.30 ± 2.1 mg/dL in TC and decreased to 27.40 ± 0.71 mg/dL in diabetic rats (T₂), respectively, however, HDL increased in CFP and CFE groups. The recorded values in the negative control, positive control, CFP and CFE groups were 30.01 ± 0.67, 27.40 ± 0.71, 31.47 ± 0.79 and 32.70 ± 1.5 mg/dL, respectively. Furthermore, TC significantly (P < 0.05) decreased in both treatment groups (T₃ and T₄); the values were 90.40 ± 1.9 and 88.91 ± 1.48 mg/dL in CFP and CFE, respectively. The LDL and very-low-density lipoprotein (VLDL) levels increased in the diabetes group but decreased significantly (P < 0.05) within the treatment group. The maximum reduction was observed in LDL and VLDL of the CFE group, chiefly 46.00 ± 1.12 mg/dL and 16.90 ± 0.86 mg/dL, respectively. However, the values were slightly higher than the negative control (T1).

Total protein significantly decreased in diabetic rats than in the placebo, as the values were 6.74 ± 0.11 and 7.32 ± 0.13 g/dL, respectively. The decline in serum protein level and albumin indicated insulin deficiency or insulin resistance and inflammation in the body. Total protein increased in CFP and CFE groups (T₃ and T₄, respectively) to values of 8.10 ± 0.14 and 8.15 ± 0.14 g/dL, respectively. The serum albumin level also increased in treatment groups (T₃ and T₄), where the observed values were 4.01 ± 0.15 and 3.90 ± 0.14 g/dL in the CFP and CFE groups, respectively. The elevated globulin level in diabetic rats (T₂) was 3.61 ± 0.08 g/dL but it increased in both treatment groups (4.09 ± 0.03 g/dL, in CFP, and 4.25 ± 0.14 g/dL, in CFE). Diabetes is linked with renal malfunction and results in a disturbance of globulin levels. The albumin/globulin ratio (A/G) significantly decreased in the diabetic group (T₂) to 0.87 ± 0.02 but increased within treatment groups with CFP and CFE to 0.98 ± 0.02 and 0.92 ± 0.03, respectively (Table 4). Sudhakara et al.⁴⁶ investigated CF extracts to find out its efficacy against hyperlipidemia.⁴⁶ To that purpose, they fed rats with 200 mg per kg body weight per day. At the end of the study, they verified the increased number of triglycerides and LDL while reduced HDL. However, when treated with CFE, significant recovery in these parameters was observed. The effect on serum protein in diabetes was stated by Chandran et al.,⁴⁷ who reported the fluctuations in serum proteins.⁴⁷

Table 4.

Effect (mean values ± standard deviation) of Caralluma fimbriata on glucose, lipid profile and total protein, and on electrolytes, kidney and liver function.

Parameter	T 1	T 2	T 3	T 4
Glucose, lipid profile and total protein
Glucose (mg/dL)	96.00 ± 2.92	210.00 ± 5.9	125.00 ± 4.04	121.00 ± 4.49
TG (mg/dL)	81.00 ± 1.45	94.00 ± 0.7	84.21 ± 1.72	83.00 ± 1.97
TC (mg/dL)	85.42 ± 2.83	99.30 ± 2.1	90.40 ± 1.9	88.91 ± 1.48
HDL (mg/dL)	30.01 ± 0.67	27.40 ± 0.71	31.47 ± 0.79	32.70 ± 1.5
LDL (mg/dL)	41.60 ± 2.32	53.30 ± 1.46	46.70 ± 1.21	46.00 ± 1.12
VLDL (mg/dL)	16.49 ± 0.93	18.30 ± 0.40	17.21 ± 0.44	16.90 ± 0.86
Total protein (g/dL)	7.32 ± 0.13	6.74 ± 0.11	8.10 ± 0.14	8.15 ± 0.14
Albumin (g/dL)	3.97 ± 0.17	3.13 ± 0.08	4.01 ± 0.15	3.90 ± 0.14
Globulin (g/dL)	3.34 ± 0.08	3.61 ± 0.08	4.09 ± 0.03	4.25 ± 0.14
A/G ratio	1.19 ± 0.04	0.87 ± 0.02	0.98 ± 0.02	0.92 ± 0.03
Electrolytes, kidney and liver function
Na (mEq/L)	124.00 ± 3.4	111.4 ± 6.33	118.10 ± 0.96	121.5 ± 2.39
K (mmol/L)	11.50 ± 0.61	19.30 ± 0.78	17.90 ± 0.39	15.70 ± 0.76
Creatinine (mg/dL)	0.90 ± 0.03	1.31 ± 0.02	0.98 ± 0.04	0.88 ± 0.03
Urea (mg/dL)	24.00 ± 1.6	33.00 ± 0.48	28.20 ± 1.37	26.40 ± 0.99
Bilirubin (μmol/L)	0.96 ± 0.03	2.16 ± 0.08	1.27 ± 0.02	1.13 ± 0.03
AST (U/L)	71.0 ± 1.91	145.00 ± 3.42	92.00 ± 2.25	87.00 ± 2.72
ALT (U/L)	34.80 ± 3.32	49.60 ± 0.86	42.20 ± 0.84	37.00 ± 1.04
ALP (IU/L)	124.00 ± 1.56	210.00 ± 6.71	179.00 ± 8.42	161.00 ± 5.39

Note: Means having the same letters within a column or rows does not differ significantly (P < 0.05). Group I (T₁) = Normal group + Normal diet; Group II (T₂) = Diabetic group + Normal diet; Group III (T₃) = Diabetic group + Normal diet + C. fimbriata powder (CFP); Group IV (T₄) = Diabetic group + Normal diet + C. fimbriata ethanol extract (CFE). Triglycerides (TG); Total cholesterol (TC); High-density lipoprotein (HDL); Low-density lipoprotein (LDL); Very-low-density lipoprotein (VLDL); and Albumin/Globulin ratio (A/G) Sodium (Na); Potassium (K); Aspartate transaminase (AST); Alanine transaminase (ALT); and Alkaline phosphatase (ALP).

Kidney and liver function

The renal function test (RFT) contains serum creatinine, urea and electrolytes (Na, K), which are essential parameters to indicate proper renal function (Table 4). Serum creatinine, urea and electrolytes were determined, which were statistically significant different (P < 0.05). Excessive urination and osmotic force, which draws water to the extracellular spaces, result in electrolyte imbalance. The sodium and potassium level exhibited statistically significant (P > 0.05) change. The sodium level decreased to 111.40 ± 6.33 mEq/L in T₂ (Diabetes group) however, improved in treatment groups with the values of 118.10 ± 0.96 and 121.5 ± 2.39 mEq/L, respectively. The induction of diabetes mellitus increased the serum potassium level from 11.50 ± 0.61 mmol/L (T₁ placebo) to 19.30 ± 0.78 mmol/L in T₂. However, rats fed on CFP and CFE treatment showed the slight improvement i.e. 17.90 ± 0.39 and 15.70 ± 0.76 mmol/L, respectively. The augmentation in urea and creatinine might relate to impaired kidney function, however, the CFP and CFE improved the parameters, thus CF powder and extract have potential to manage the diabetes complications. Serum creatinine level increased in the diabetes group to 1.31 ± 0.02 mg/dL but significantly (P < 0.05) reduced in CFP and CFE, with values of 0.98 ± 0.04 and 0.88 ± 0.03 mg/dL. The urea level increased in the diabetic group (T₂) (33.00 ± 0.48 mg/Dl) as compared to Placebo (T1) (24.00 ± 1.6 mg/dL). The skeletal muscles are the primary target tissue for insulin and low volume of skeletal muscle results in lesser target sites for insulin. Moreover, the liver functionality will be disturbed ultimately leading to low serum creatinine and urea. Our results were supported by the findings of Gujjala et al.²³

The liver function test (LFT) comprises bilirubin, aspartate transaminase (AST), alanine transaminase (ALT) and alkaline phosphatase (ALP) enzymes are primary biomarkers to identify liver functionality. The elevated levels of these enzymes predict some health maladies e.g. elevated ALP and bilirubin in diabetes are associated with non-alcoholic fatty liver. In the present research, the results regarding liver enzymes are statistically (P < 0.05) significant. The bilirubin levels significantly (P < 0.05) increased in the diabetes group (2.16 ± 0.08 μmol/L) in T₂ as compared to 0.96 ± 0.03 μmol/L, in T₁. The maximum decrease was monitored in CFE, followed by CFP, with values of 1.27 ± 0.02 and 1.13 ± 0.03, respectively. The AST and ALP followed the same pattern as higher values were observed in the diabetes group 145.00 ± 3.42 U/L and 210.00 ± 6.71 U/L, and normal control; these values were 71.0 ± 1.91 and 124.00 ± 1.56 μmol/L, respectively. The rats fed on experimental diets resulted in marked reduction in values. Likewise, recovery in ALT was observed in CFE and CFP with values of 42.20 ± 0.84 and 37.00 ± 1.04 (Table 4). The consumption of plants rich in phytochemicals improves the detoxification abilities of the liver thus the toxicants accumulated in the liver will be reduced. Moreover, the CFP and CFE reduced the food intake thus leading to lower intake of carbohydrates and lipids that could eventually lead to low accumulation of fats in the liver. Our results of liver function and reduction of liver enzymes were proved by the study of Latha et al.⁴⁸

Conclusions

Medicinal plants are health-friendly and cost-effective treatments for non-communicable diseases, especially in underdeveloped countries. The administration of Caralluma fimbriata ethanolic extract had more remarkable results due to the better availability of anti-oxidants than in Caralluma fimbriata powder. Caralluma fimbriata powder and extracts are effective and safe as they cause no toxicity beyond their safe usage limits. Caralluma fimbriata has an appetite suppressant ability and is an excellent hypoglycemic medicinal plant due to its phytochemicals load and α-amylase inhibition.

The results from studies investigating Caralluma fimbriata (CF) contribute significantly to our understanding of its potential therapeutic effects in managing diabetes by demonstrating its ability to modulate various biochemical parameters associated with the disease. Specifically, CF has been shown to exert anti-hyperglycemic effects by reducing blood glucose levels, improving insulin sensitivity, and enhancing glucose uptake in peripheral tissues. These effects are likely mediated through the activation of glucose transporter type 4 (GLUT4), modulation of enzymes involved in carbohydrate metabolism, and the potential stimulation of insulin secretion from pancreatic β-cells. Additionally, CF’s antioxidant properties, as evidenced by its ability to scavenge free radicals and reduce oxidative stress markers, further support its therapeutic potential, given the role of oxidative stress in the pathogenesis of diabetes and its complications. The anti-inflammatory effects of CF, including the reduction of pro-inflammatory cytokines, also contribute to its potential benefits in diabetes management by addressing the inflammatory aspect of the disease. Together, these findings suggest that CF could be a valuable natural supplement for diabetes management, offering multiple mechanisms of action to improve glycemic control, reduce oxidative stress, and mitigate inflammation. Future studies must highlight how Caralluma fimbriata suppresses the appetite and inhibits α-amylase activity.

Author contributions

Muhammad Tauseef Sultan and Khalil Ahmad: Conceptualization, writing and editing of manuscript, Hassan Raza and Ahmad Mujtaba Noman: Validation, Muhammad Usman Khalid and Fahid Nazir: Methodology, Aleena Arif: Investigation, João Miguel Rocha: Resources, Aleena Arif, Muhammad Mahboob Ahmad, Farooq Khurum Shehzad, Shehla Mushtaq and Hira Sohail: Writing—original draft preparation, Muhammad Israr, Ahmad Mujtaba Noman and João Miguel Rocha: Writing—review and editing, Muhammad Tauseef Sultan: Supervision. All authors read and approved the final manuscript.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

 

Conflict of interest statement. The authors declare that there are no conflicts of interest related to this article.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.