A Review on Antioxidant Properties of Extracts Obtained from the Leaf, Stem, Bark and Flower of Calotropis Species

Introduction

Calotropis gigantea, a weed, is abundant in Asian and African countries. It is also commonly known as "crown flower," "giant milkweed," and "shallow wort". In India, it is known as "aak," "akauwa," or "arka".¹ This plant is easily identified by its thick, oval leaves and white or purplish-coloured, odourless blooms. Flowers of Calotropis gigantea are white in colour whereas flowers of Calotropis procera are purplish coloured. Calotropis procera L., is said to have therapeutic properties. In local or tribal culture, Calotropis gigantea is utilized to treat leprosy, leukoderma, rheumatism, diarrhoea, fever, indigestion, cold and cough. It has been used in Ayurvedic, Chinese, and homoeopathic medicines. It is prescribed to cure toothaches, elephantiasis, vomiting, and purging, according to the homoeopathic Materia Medica. It was observed that the permeability of skin increases on using dipeptide-2 (DIP-2), a product made by Defence Institute of Physiology & Allied Sciences (DIPAS), India, from the alcoholic extract of Calotropis gigantea.² This formulation has been investigated as a potential auto-debridement and tissue regenerating agent in conjunction with DIP-1 as one of its active ingredients. Numerous benefits of DIP-2 include anti-inflammatory, antioxidant, antibacterial, vasodilation, and wound healing properties.³

Through phytochemical tests, the latex was observed to have terpenes, phenolic compounds, cardenolides, flavonoids, and saponins. But, tannins, alkaloids, and resin were lacking in moderate to high concentrations. According to studies on the latex's antioxidant and antiapoptotic properties, crude Calotropis procera latex demonstrated antioxidant and antiapoptotic actions against the toxicity of 4-Nonylphenol.⁴ In addition to protecting cells from damage and exhibiting antidiabetic properties, when used against reactive oxygen species created by hyperglycaemia, Calotropis gigantea fortifies the antioxidant defence.⁵ The antioxidant activity, total phenolic content (TPC), and total flavonoid content (TFC) of the crude extracts produced from Calotropis procera were evaluated.⁶ The phytochemical makeup of the ethanolic extract of Calotropis gigantea (L.) dry leaves was determined using the total bioactive (total phenolic and total flavonoid) contents and ultra-high performance liquid chromatography- mass spectrometry UHPLC-MS secondary metabolites analysis. To study the phytopharmacology, in vitro antioxidant activity [containing diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), Ferric Reducing Antioxidant Power (FRAP), Cupric Reducing Antioxidant Power (CUPRAC), phosphor molybdenum, metal chelation antioxidant assays), and enzyme inhibition potential [against Acetylcholinesterase (AChE), butyrylcholinesterase (BChE), -amylase, and tyrosinase enzymes] were studied.⁷ The high molecular weight protein fraction recovered from Calotropis procera latex caused a dose-dependent suppression of oedema formation, levels of oxidative stress markers [glutathione (GSH) and thiobarbituric acid reactive substances (TBARS)] and Myeloperoxidase (MPO), a marker for neutrophils in the paw tissue also reverted to normal. High molecular weight protein in Calotropis procera latex, also serves as an antioxidant, lowers oxidative stress, inflammation, and acute inflammation in the paws.⁸

The hepatoprotective properties of 70% ethanolic extract of Calotropis procera flowers against CCl₄ -induced liver injury in albino rats and mice were investigated. In vitro models were also employed to investigate antioxidant activity. The powerful antioxidant and its corresponding hepatoprotective activity of the methanolic extract and separated ingredient dehydro-abietylamine is therefore attributed to its antioxidant and free radical scavenging activities because they are crucial in the prevention of CCl4-induced liver injury. Spectrophotometric measurements were performed to determine the impact of the extracting solvent on the total phenolic and flavonoid contents. Reversed-phase HPLC (RP-HPLC) was used to assess the flavonoid and other polyphenolic components of the methanol extracts. The DPPH radical scavenging potential of different extracts was determined. The findings showed that the tested extract had sizable DPPH (67.90 mg TE/g extract) and ABTS (89.67 TE/g extract) capabilities. Through the use of in vitro techniques such as 2,2-diphenylpicrylhydrazyl (DPPH) and deoxyribose degradation tests, the antioxidant potential and effects of an ethanolic extract of Calotropis procera bark on wound healing were examined.

Calotropis procera's dry latex (DL), which has strong anti-inflammatory properties, was tested for its antioxidant and anti-hyperglycaemic activity against rats with alloxan-induced diabetes. After daily oral treatment for decreased blood glucose levels in a dose-dependent manner with dry latex at dosages of 100 and 400 mg/kg, the hepatic glycogen content increased. Additionally, dry latex stopped diabetic animals from losing weight and reduced their daily water intake to levels equivalent to normal rats. In addition, dry latex increased the hepatic levels of endogenous antioxidants like catalase, glutathione, and superoxide dismutase (SOD), while lowering thiobarbituric acid-reactive substance (TBARS) concentrations in rats with alloxan-induced diabetes.⁹

Habitat

Originally, it is from India, Southern China, Malaysia, and Indonesia. It can be a large shrub or a small tree and has been widely cultivated in tropical regions all over the world.¹⁰

Taxonomy of Calotropis gigantea

• Kingdom – Plantae

• Order – Gentianales

• Family – Apocynaceae

• Subfamily – Asclepiadaceae

• Genus – Calotropis

• Species – Calotropis gigantea

Morphological description

This is a member of the milkweed family and normally develops to be 8 to 15 feet tall. It is distinguished by its upright growth, milky sap, pale purple or white blooms that resemble crowns, as well by its white woolly branches and leaves that develop into subglabrous leaves. Its flowers in clusters (umbellate cymes) throughout the year, but in summer, it flowers more. In Hawaii, leis are frequently made out of these flowers (both purple and white). The bases of the leaves are up to 4-8" long, elliptic to oblong. The leaves are cordate. In arrangement, the fruits hardly ever develop. This enormous milkweed serves as a crucial monarch butterfly larval host plant. In relation to the blooms, the Greek word ‘kalos’ meaning beautiful, and ‘tropos’ meaning boat, were used as the genus name.¹¹ Figure 1 shows the flower of Calotropis gigantea and Figure 2 shows a twig of Calotropis gigantea.

Methodology

Keywords like Calotropis, antioxidant, extraction were searched across Pubmed, Science Direct and Google websites. The published articles were referenced appropriately. 

Discussion

Plant material The phytochemical analysis, anti-inflammatory, and antioxidant activity of Calotropis gigantea and its therapeutic uses have been reported. The dried plant parts were used to extract the phytoconstituents using the Soxhlet extraction method. Investigations of alkaloids, flavonoids, terpenoids, and phenols were performed, and qualitative analysis was used to confirm the presence of the phytoconstituents in all three Calotropis gigantea leaf extracts. To assess the ability of crude Calotropis gigantea extracts to neutralise free radicals, nitric oxide scavenging tests were conducted. The ability of the extracts to control protein denaturation and to inhibit essential enzymes that harm tissue was examined using protein denaturation and proteinase inhibition tests. Additionally, the efficiency of plant extracts at stabilising membranes was tested using the heat induced haemolysis technique. The DPPH and FRAP tests were carried out to evaluate the antioxidant activity of phytoconstituents extracted using different solvents. The Calotropis gigantea plant's methanolic, aqueous, and petroleum ether extracts were analysed using Gas chromatography–mass spectrometry (GC-MS), and a range of compounds with beneficial medicinal properties were found.¹²

Phytochemistry of Calotropis gigantea

Numerous compounds isolated from the plant Calotropis gigantea were found to have biological activities. From the aerial parts of Calotropis gigantea, lupeol, g -Taraxasterol, Isorhamnetin were obtained.¹³ Anhydrosorphoradiol-3-acetate was produced from the flowers of Calotropis gigantea by di-(2-ethylhexyl) phthalate.¹⁴ From the roots of Calotropis gigantean, calotropone,¹⁵ gofruside, asclepin were obtained. From the roots and bark of Calotropis gigantean, a-Taraxerol, Lupenyl-1-acetate, stigmasterol, b- Sitosterol, b-Sitosterol acetate¹⁶ b- Amyrin, b-Amyrin acetate, and Isovaleric acid were obtained.¹⁷ A rich variety of the phytoconstituents have been isolated from Calotropis gigantea.¹⁸

Pharmacological effects reported in Calotropis gigantea

Calotropis gigantea has been the subject of numerous investigations to identify it s chemical constituents and potential biological activities, which is one of the reasons it was once regarded as an important weed. The pharmacological effects of numerous extracts, fractions, and isolated compounds from distinct plant regions were investigated which show many pharmacological effects like antibacterial, antifungal property,¹⁹ anti-diarrhoeal,²⁰ hepatoprotective activity,²¹ antitumor property,^22,23 anthelmintic,²⁴ anti-hyperglycemic,²⁵ anti-ulcer activity,²⁶ anticonvulsant and sedative property,²⁷ anti-microbial,²⁸ and wound healing property.²⁹

Toxicity reported in Calotropis gigantea

Calotropis gigantea has been used for many years in a variety of conventional therapies. However, the market value of the formulations will be very low without adequate toxicity profiling. To test the ethanolic flower extract for acute and subacute toxicity in rats, the species' toxicity profile was created using an in vivo method. Various blood indicators (white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular hemoglobin concentratio (MCHC), platelet ratio (PLT), lymphocyte (LYM), and neutrophil (NEUT)] and biochemical markers [aspartate transferase (AST), Alanine transaminase (ALT), alkaline phosphatase (ALP), albumin blood test (ALBN), tuberculosis (TB)] were examined.¹⁷

Different methods of extraction

Procedures frequently employed in the extraction of phytoconstituents from medicinal plants are:

A. Maceration: This is an extraction technique in which menstruum is poured on top of coarsely powdered plant material, such as leaves, until the drug material is completely covered. The container with extract is sealed and stored for at least three days.^30-32 The mixture is occasionally stirred, and stored inside a container. It should be shaken occasionally to guarantee thorough extraction. Micelle and marc are separated after extraction using filtration or decantation. The micelle is then extracted from the menstruum by evaporation in a water bath or an oven. This approach is practical and excellent for thermo labile phytoconstituents.^33-35

B. Infusion: In this process, the raw dried plant material is ground into a fine powder and then put inside a clean container. The powdered plant parts are covered with the hot or cold extraction solvent, which are absorbed and held for a brief time. This approach is practised for extracting readily soluble phytoconstituents.^30-32,34

C. Digestion: This extraction method uses a moderate amount of heat to carry out the extraction. The powdered plant material is mixed with solvent, later the mixture is placed in an oven at a temperature of about 50 or 60 degrees Celsius or over the water bath. The viscosity of the extraction solvent is reduced during this extraction procedure, which improves the removal of secondary metabolites. This method is suitable for the extraction of phytoconstituents which are readily soluble.^30,32,34

D. Decoction: In this method of extraction, the plant material is dried, grounded and powdered before depositing in a clean container. After that, an appropriate solvent is poured and continuously agitated for few minutes before applying heat to it. The process normally lasts 20 minutes. Usually 4:1 or 16:1 is the solvent to crude drug ratio used. Plant material, that is both heat and water-soluble is extracted using this method. This approach is suitable for the extraction of readily soluble phytoconstituents and heat stable plant materials.^30-32,34

E. Percolation: In this extraction procedure, a percolator (which has an opening at both ends of a thin cone-shaped glass vessel) is utilized. Throughout the extraction process, more solvent is added, and the mixture is kept for four hours. The extracted mixture is then transferred to a percolator with the lower end closed and let to stand for 24 hours, while the extraction solvent is poured from the top until the plant material is completely saturated. The addition of solvent is halted when the solvent volume reaches 75% of the total volume intended for the preparations. Filtering and decantation are then used to separate the extracted liquid. The appropriate volume is then attained by adding the last bit of solvent after reaching the mark.^31,32,34

F. Microwave-aided extraction: This approach is one of the most advanced extraction procedures for many medicinal plants. To eliminate charged ions that are present in the solvent and drug material, it uses dipole rotation and ionic transfer. It requires using electromagnetic radiation with frequencies between 300MHz and 300GHz and wavelengths between 1cm and 1m. It is more suitable for polar solvent extracts because dipole rotation and ion migration occur more easily and aid in the extraction process. This approach is appropriate for extracting flavonoids from plant sources. It is advantageous as it involves less solvent usage, a shorter extraction process, and improved extraction results. The disadvantage is that it is only suitable for extracts containing phenolic chemicals and flavonoids.^30,33,36,37 Tannins and anthocyanins are destroyed due to their high concentration in the microwave.

Extraction of phytoconstituents from Calotropis gigantea

Extraction of Calotropis gigantea leaves, stem, bark and flower

The leaves of Calotropis gigantea were harvested and dried. The extraction was done for 16 hours using a Soxhlet unit utilising about 50 g of the powdered leaves and 400 mL of ethanol. Distillation was used to concentrate the resulting extract and evaporate it into dryness at a low temperature. The extract was then weighed, and the extractive % was calculated using the weight of the air-dried plant material.³⁸

Extraction of latex found in Calotropis gigantea

The latex was first aseptically collected from the Calotropis gigantea plant and baked for 12 hours at 60 °C. To extract the dried latex material, 1.0 mL of organic solvents were utilised for 100 mg. There were six different solvents (chloroform, distilled water, dimethyl sulfoxide, ethyl acetate, hexane, and methanol) used. The finished mixture was centrifuged for 10 minutes at 3000 rpm while being vortexed. The supernatant was used for its antimicrobial properties.³⁹

Antioxidant activity reported in Calotropis species

DPPH assay

Antioxidants have been widely estimated using the stable, nitrogen-centred free radical known as DPPH. Reduction of the DPPH radical is a quick and simple method to assess the antioxidant potential of a molecule, especially for those possessing phenol groups. It is known that substances with weak N-H or O-H bonds cause DPPH to react quickly. Electron transfer is a key factor in its decline as well. It is reduced, reversible, and heavily coloured because it has an unpaired electron. This property makes it perfect for spectrophotometric analysis. To test the fractions' ability to scavenge free radicals, a DPPH solution in methanol was decoloured to evaluate the potential of free radical scavengers. 0.1 mL of dimethyl sulfoxide (DMSO) was used to dilute different leaf extracts.²⁸ 0.1 mL of 0.1 mM DPPH in methanol was added, and the compound’s antioxidant and free radical scavenging properties were tested. After carefully mixing the ingredients, the mixture was allowed to stand at room temperature in the dark for 10 minutes. The absorbance of DPPH was measured using a spectrophotometer at 565 nm. Butylated hydroxy toluene was provided as a positive control. Owing to a significant concentration of several phytochemicals, it is discovered that Calotropis gigantea possesses antioxidant activity by the DPPH assay and reducing power assay, with its extracts having higher antioxidant activity than conventional ascorbic acid as shown in Table 1.^40,41

Reducing power assay

The process was carried out to ascertain the Calotropis plant extracts capacity for reduction as shown in Table 2. Methanolic extracts were prepared for each sample at concentrations of 0.1 mg/mL, 0.5 mg/mL, 1 mg/mL, and 2 mg/mL. These concentrations were combined with 2.5 mL of 0.2 mM phosphate buffer solution at pH 6.6 and 2.5 mL of 1% potassium ferrocyanide. The mixture was incubated in a water bath at 50 °C for 20 minutes. The mixture was centrifuged at 3000 rpm for 10 minutes after 2.5 mL of 10% trichloroacetic acid was added. 2.5 mL of the supernatant from centrifugation was combined with 0.5 mL of 0.1% FeCl3 and 2.5 mL of distilled water. Using a UV-30 spectrophotometer, the absorbance at 700 nm was calculated.⁴²

Nitric oxide scavenging activity

The Griess reaction was used to determine the amount of nitrite ions produced by the interaction of oxygen and nitric oxide produced from sodium nitroprusside in aqueous solution at physiological pH. About 3.0 mL of phosphate buffered saline (0.5 M) and sodium nitroprusside (10 mM) were combined. The medication was dissolved at 100 µg/mL in the appropriate solvent systems and incubated for 150 minutes at 25 ºC. 0.5 mL of Griess reagent (1% sulphanilamide, 0.1% naphthyl ethylene diamine dihydrochloride in 5% H3PO4) was added to the samples from the previous step. At 546 nm, the chromophore's absorbance was quantified. As a control, the identical reaction mixture was used, but with no ethanolic plant extracts and an equivalent amount of 0.5M phosphate buffer. By contrasting the absorbance values of the control and test groups, the percentage inhibition of the nitric oxide production was calculated as shown in Table 3. Curcumin was employed as a benchmark substance.^43,44

% Inhibition = A0 – A1/A0 × 100

Where A0 - the absorbance of the control (blank);

           A1- the absorbance in the presence of extract.

Ferric Reducing Antioxidant Power (FRAP)

In the FRAP assay, an antioxidant that donates electrons causes the colourless oxidised FeIII form of the FeII - tripyridyl triazine molecule to turn blue. At 593 nm, the absorbance change was observed. Briefly, 1.5 mL of freshly made FRAP reagent (300 mM acetate buffer, pH 3.6; 10 mM TPTZ in 40 mM HCL and 20 mM FeCl₃ 6H₂ O in the ratio of 10:1:1) was added to 30 µL of the standard (ferrous sulphate) or 50 µL of the sample. The absorbance was measured at 593 nm following a 10-minute incubation period at 37°C. The results are given in Table 4 in terms of ferrous sulphate equivalents per gram of material.^43,45 The significance of the studies on Calotropis species is depicted in Table 5. 

Conclusion

Extraction from different parts of Calotropis species can successfully be done by using solvents like methanol and ethanol. Various extracts and their pharmacological efficacy have been reported in many studies. The antioxidant properties may be utilized for managing various diseases that arise due to oxida tive stress. The exploration on phytochemistry opens the door for more detailed studies on effects of phytoconstituents of Calotropis species. As this herb can be grown easily in almost all parts of India, the research on this plant can be promoted. However, more in vivo research and clinical tests using isolated phytoconstituents are needed to support the scientific data.

Conflicts of Interest

Nil

Acknowledgement

The authors acknowledge Acharya & BM Reddy College of Pharmacy, Karnataka-560107, India for the physical facilities. The authors acknowledge Rajiv Gandhi University of Health Sciences, Karnataka560041for the financial support through UG research grant (UG22PHA423).